CN102988336A - Use of TPCK (N-tosyl-L-phenylanyl chloromethyl ketone) in preparation of anti-lenticular opacity products - Google Patents

Abstract

The invention relates to a new application of N-tosyl-L-phenylanyl chloromethyl ketone (TPCK) and a composition thereof in the treatment of lenticular opacity. The efficacy of the TPCK in the aspects of preventing and treating the lenticular opacity and the like is mainly as follows: the anti-lenticular opacity action is realized by closing a channel which is positioned between nonessential mercapto of crystallin and profilin and can form a disulfide bond for causing nonspecific accumulation and further contain the formation of the macromolecular crystallin, a new medicament source and an application formulation are provided for chemical control of cataract, and the important reference value is further realized for preventing and treating other age-related eye diseases. The TPCK disclosed by the invention has the advantages of safety, low price, significant efficacy and stable nature, is suitable for large-scale production and commercial application in medicines, reagents and other industries and easy to popularize and apply, and further has good application prospects and significant social benefits and economic benefits.

Description

TPCK is for the preparation of the purposes of anti-lenticular opacity product

Technical field

The present invention relates to the technical fields such as medicine, food, beverage and reagent, specifically relate to the sulfydryl of protein is carried out the new purposes of alkylating reagent, more particularly relate to N-to toluene sulfo group-L-phenethylamine acyl chlorides methyl ketone (N-tosyl-L-phenylalanyl-chloromethyl ketone, be called for short: a kind of new purposes TPCK) specifically relates to the new purposes that TPCK can be used in the muddy product of the anti-crystalline lens of preparation again.

Background technology

Cataractous research overview

1, general introduction

Cataract is the commonly encountered diseases that affects in the world human health at present, is commonly encountered diseases, the frequently-occurring disease that has a strong impact on human health, also is one of modal causes of blindness, ranks the disease of first blinding of China, and its pathogenesis is still not fully aware of.

The cataract clinical research shows, in all kinds of cataract cases, is occupied the ratio of the overwhelming majority by aging caused senile cataract.Senile cataract is that eyes are sick, but two morbidities can have successively.The position that begins to form according to cataract, senile cataract is divided into cortical cataract (corticsal cataract), nuclear cataract (nuclear cataract) and capsular cataract (subcapsular cataract), and wherein cortical cataract is the modal type of senile cataract.

Senile cataract is the main cause of old people's visual deterioration, and this disease main manifestations is muddy for engender degeneration in the crystal ageing process, claims again the degeneration cataract.In the old people of the U.S. more than 85 years old, prevalence accounts for 95%.

The whole world is China particularly, face the immense pressure of population fast growth and the severe situation that aging trend increases the weight of, more need to strengthen energetically the research to cataract pathogeny and influence factor, to make great efforts control and to slow down the trend that the cataract incidence brought thus rises.

2, cataractous formation principle and Advances in study of etiology thereof

Cataractous generation is relevant with many factors, is the result of many factors comprehensive function.Suffer dysbolismus, the lipid peroxidation injuries etc. such as oxidative damage, radiation damage, aging degeneration, glucose overstimulation, galactose all can cause in various degree damage to lenticular normal function such as gene mutation, the crystalline lens of crystallin, it is muddy that the result causes crystalline lens to occur, affect crystalline lens to optical transmission and refraction, thereby cause blurring of vision.

Lenticular function is to focus light rays to produce vision on the retina.Lenticular main component is water and protein, 80%～90% of crystalline lens dry weight, protein, the structure of every kind of protein and their interactions each other are the molecular basises of keeping the crystalline lens transparency, and described protein comprises α, β and γ three big nation's crystalline proteins.

Lenticular transparency is to be kept by the protein of constant moisture, high concentration and complicated metabolism, and namely the structure of every kind of protein and their interactions each other are the molecular basises of keeping the crystalline lens transparency; That is to say that the ordered arrangement of crystalline protein is the material base of keeping the crystalline lens space structure, if namely the crystalline protein composition changes, then can affect, destroy this kind normal configuration.

The breviary vocabulary

English abbreviation	English full name	Chinese
			HMW	High?molecular?weight	The high molecular polymerization state
PTM	Post?translational?modification	Post translational modification
			RSL	Reactive?site?loop	The circulus district
Ts	Tosyl	P-toluenesulfonyl
			DTT	Dithiothreitol	Dithiothreitol, DTT
TAB	Tert-butyl?alcohol	The tert-butyl alcohol
			Ts	Tosyl	To methyl sulphonyl
UPP	Ubiquitin-proteasome?pathway	Ubiquitin-proteasome pathway
			PTM	Post?translational?modification	Post translational modification
LEC	Lens?epithelial?cells	Lens epithelial cells
			PBS	Phosphate-Bufer-Solution	Phosphate buffer
GO	Glucose?oxidase	Glucoseoxidase
			OS	Oxidase?Stress	Oxidative stress
ROS	Reactive?oxygen?species	Active oxygen
			RNS	Reactive?ntrogen?species	Active nitrogen
G3PD	Glyceradehyde-3-phosphate?dehydrogenase	Glyceraldehyde-3-phosphate dehydrogenase
			NOS	Reactive?Ntrogen?Species	The active nitrogen free radical
TTase	Thioltransferase	Thiol transferase
			Trx	Thioredoxin	Thioredoxin
TR	Thioredoxin?reductase	Thioredoxin reductase
			Tr	Tris?HCL-Bufer-Solution	Tris HCL buffer
GR	Glutathione?reductase	Glutathion reductase
			Cys	Cysteine	Cysteine
EB	Ethidium?bromide	Ethidium bromide
			CF	Cystic?fibrosis	Cystic fibrosis
TEMED	Tetramethylethylenediamine	N, N, N', N'-tetramethylethylenediamine

[0014]?

DMSO	Dimethyl?sulfoxide	DMSO
			CK-19	Cytokeratin-19	Cyfra21-1
dsRNA	Double-stranded?RNA	Double-stranded RNA
			siRNA	Small?interfering?RNA	SiRNA
NC	Negative?control	Negative control
			RT	Reverse?transcription	Reverse transcription
FBS	Fetal?bovine?serum	Hyclone
			FAM	Carboxyfluorescein	CF 5(6)-Carboxyfluorescein
Abs	Absorption?wavelength	Absorbing wavelength
			Em	Emission?wavelength	Emission wavelength

Lenticular light transmission mainly is the quality and quantity that depends on water-solubility protein component in the crystalline lens, especially several main structural protein such as β B ₂(definition can be referring to document: Aarts HJ, Lubsen NH, Schoenmakers JG..Crystallin gene expression during rat lens development.Eur J Biochem, 1989,183 (1): 31-36), γ _5,6(γ C, γ D), γ _{Isosorbide-5-Nitrae}, γ _2,3With chaperone α A ₂And α B ₂The change of the quality and quantity of water solublity crystalline protein directly affects light transmission and the cataractous formation of crystal, with β B ₂For the crystal structure albumen of the high-load of representative also plays a part very important to lenticular structure and normal light transmission thereof.

α group crystal albumen has special molecular chaperones (Molecular Chaperone) effect, plays pivotal role for keeping lenticular normal configuration; Content variation tendency in ageing process of α group crystal albumen directly affects the change of its chaperone activity; Along with ager process, α group crystal protein molecular companion effect weakens usually, and in the cataract crystal, then manifests a large amount of α group crystal albumen and change non-water-soluble high molecular weight crosslinked polymer into.

So-called molecular chaperones refers to that a class can mediate the correct folding and assembling of other oroteins, and protecting the activity of this protein, but itself is not the constituent that the final assembling product of function is arranged.But α group crystal albumen is a kind of special albumen with molecular chaperones effect, and it not only has chaperone activity, and is one of most important structural protein in the crystal.

The light transmission of crystal not only needs the water-soluble components of q.s to keep, but also need to consider lenticular special metabolic way: from the crystalline protein self-generating, constantly pushed by the core of the crystalline protein of rear generation to crystal, and be stored in different crystal structure layers.Therefore, no matter water-solubility protein component or water-msoluble ingredients, all can't metabolism to external.But the accumulation of water-soluble components helps to make up the from inside to outside normal configuration of layer of crystal; And the gradually accumulation of water-insoluble composition, the light scattering that then can increase gradually crystal is unfavorable for light transmission, that is to say, it is the material base that senile cataract forms.

Mammal is after birth, because outside crystal was exposed to, in crystalline lens growing process, crystalline protein was subjected to the aging impact that reaches many extraneous factors of body own nature, be subject to the impact of inside and outside many factors and morph, different modification degeneration can occur.The quality and quantity of crystalline protein can change, and water-soluble components constantly changes water insoluble active ingredient into, affects its water solublity, and then affects lenticular normal configuration and light transmission, finally causes the formation of senile cataract.That is to say that in the process of human senility, in case the protein structure in the crystalline lens is damaged, then crystalline lens becomes muddy opaquely by as clear as crystal, and then affects lenticular normal configuration and light transmission, and progressively develops into senile cataract.

The construction features of the relevant crystalline protein of comprehensive literature; the principal element that the research discovery affects its protein structure is post translational modification (posttranslational modification; be called for short: PTM); be that many crystalline proteins can or react the modification that occurs by the catalysis of enzyme after the translation under enzyme control, it mainly comprises glycosylation, oxidation, carbamylation, phosphorylation, acetylation, hydroxylating, deacylated tRNA amine, racemization and excision effect etc.Have many bioactive molecules around the crystallins, comprise sugar, derive from activated other metabolites of cyanate, glucocorticoid and tool in the urine etc., these all can attack protein under non-enzyme environment.

Therefore, keep lenticular light transmission, on the one hand, need the amount of water-solubility protein enough in the maintenance crystalline lens, this gene that depends primarily on the internal body crystalline protein is not suddenlyd change, and can implement normal expression and get final product, and carries out high expressed or low the expression such as certain crystalline protein of needs, then can adopt gene engineering method, activate or suppress corresponding gene; On the other hand, need to contain as possible, slow down crystalline protein by the transformation of water-solubility protein to water-insoluble protein.

3, cataractous treatment present situation

Cataract is worldwide blinding disease.The clinic diagnosis aspect is surgical operation for cataractous unique effective treatment means at present.Although cataract operation becomes better and approaching perfection day by day, inevitably postoperative complication and socioeconomic problem are still very serious, as the incidence rates such as after cataract, secondary glaucoma or corneal injury are higher in the rear several years of performing the operation, and usually are difficult to eradicate.

Seek effectively prevention and drug treatment, control in early days cataract, remain the study hotspot in this field.Prevention and Drug therapy cataract mainly contain two kinds of approach, namely reduce the risk factor of pathogenesis of cataract and use anti-cataract medicine.

The Major Risk Factors relevant with pathogenesis of cataract has diabetes, glaucoma, myopia, prolonged application corticosteroid, severe diarrhea, ultraviolet radiation, heavy drinking and smoking etc.Taylor etc. are summarized as 6 " D ", i.e. solar irradiation, diet, medicine, diabetes, dehydration and agnogenio.Reducing these possible risk factors is a kind of positive preventive means.

Aspect medicinal application, now to have developed tens of kinds of medicines and be used for clinically, the light transmission that is used for keeping crystal delays or treats cataract.Owing to lack the perfect foundation for the cataract formation mechanism, still there are many defectives in development and still at the many medicines that use, and is lower such as effective percentage, specific aim is poor etc.Therefore, although the anti-cataract medicine of a multitude of names in the countries in the world extensive use, remains in a lot of problems.Cataractous Drug therapy still is faced with stern challenge: although tens of kinds of anti-cataract medicines are arranged by extensive clinical use, prove that the positive evidence of its effectiveness is very few.

Because cataractous is the comprehensive result of various factors, its pathogenic factor is not also come to a conclusion.Current, mostly each scientific research organization is for a certain pathogenic factors, or the research and development of anti-cataract medicine are carried out in some Physiology and biochemistry variation of eye in the antagonism pathogenic process.Nowadays the medicine that is used for cataract therapy mainly contains following a few class:

1. aldose reductase inhibitor (is called for short: ARlS) be the anti-cataract medicine that clinical experiment was systematically studied and carried out to the first kind, the osmotic pressure theory of using this medicine to be based on to propose in 1962 and early diabetes rat lens and high sugar are hatched under the environment evidence of a large amount of sorbitol depositions in the crystalline lens.

Represent medicine catalin (Catalin) and Phacolysin (Phacolysin) are arranged, by suppressing the accumulation of polyhydric alcohol in the crystalline lens, play the effect of alleviating sugared cataract, simultaneously the adhesion of the two and crystalline protein is all very strong, can protect crystalline protein not to be subjected to the damage of the quinone imidic acid that unusually produces because of tryptophan metabolism; But clinical practice and laboratory proofing show that ARIS treats sugared cataract and non-effective, and the side effect of ARIS is serious, and its drug Mechanism " falls short of the reality ".

2. the free-radical oxidation theory that occurs for cataract and the anti-oxidation medicine antioxidative types of drugs developed is a lot of comprises glutathion, taurine, vitamin E, vitamin C, bata-carotene, and with the classes of compounds of reproducibility coloured glaze base etc.

Glutathion and prodrug: along with aging and cataractous formation, the level of crystalline lens GSH-PX activity reduces, and can be of value to the treatment cataract so increase glutathion.Glutathion is a kind of tripeptides (γ glutamine, cysteine, glycine), and its level in crystalline lens will activate by tripeptides self or a kind of prodrug such as ester or by amino acid composition.Glutathion can suppress glycosylation.The ester of glutathion and its dipeptides precursor are adopted in many experiments, suppress the formation of various Experimental Cataracts, but generally use simultaneously or in advance than bringing out the cataractous factor, possible reason is the absorption that reduces simply the attack of incitant or affect this factor.For example, glutathion list isopropyl esters or used glutathion and γ glutamine cysteine ester in a hour in advance can suppress the cataractous generation that Buthionine Sulphonimine induces; Use glutathion and single isopropyl esters in 30 minutes in advance than sodium selenite, can reduce the generation of selenium cataract; Before the X-radiation rat, inject cysteine, thiourea, glutathion and cysteamine and can reduce the side effect of radiation; Behind the X-radiation rat one day, the Cataractogenesis that glutathion list isopropyl esters can protect X-radiation to induce.

Cysteine is the restricted substrate of glutathion, so use cysteine (or prodrug) can suppress cataract, if can reduce sulfenyl or disulfide group group, two sulphur compounds are anti-cataract medicines preferably.But at present clinical trial is very few.

Taurine: taurine can suppress galactose cataract, stops glycosylation and the oxidation of crystallin.If increase the taurine succedaneum in the diet, can reduce because the γ crystalline protein that the infringement of lens cell causes to Vitrea seepage, is considered to a kind of preferably antioxidant and anti-cataract medicine.

Taurine is a kind of sulfo group aminoacid, exists in a large number in human body, and it has antioxidation, and energy Cell protection and tissue are avoided oxidative damage.Crystalline lens has the ability of accumulating taurine, and taurine accounts for 50% of nonprotein hydrolysis amino acid in crystalline lens, is aminoacid important in the crystalline lens.Along with the development of the cataract course of disease, the content of crystalline lens sulfonic acid at noon significantly reduces.Some research groups of countries in the world have carried out a series of systematic studys in recent years, the inhibitory action of finding Taurine on Sub sodium selenate cataract is relevant with its anti-oxidation characteristics, and taurine can prevent or delay the mechanism of action of diabetic cataract and is mainly: antioxidation; Membrane stabilizing action; Hypoglycemic activity; The osmotic pressure regulating action.The antioxidation of taurine and osmotic pressure regulating action have certain feasibility to cataractous control.

The extensive age related eye diseases research of being subsidized by the American National institute of ophthalmology (is called for short: AREDS), 5000 examples are carried out the by a definite date follow-up investigation in 5～10 years, and purpose is that the absorption that discloses heavy dose of antioxidant and zinc additive comprises cataractous preventive and therapeutic effect to age related eye diseases.Nearest report is to 3640 examples at 55～80 years old age, the situation of long-term taking vitamin medicaments is investigated, follwing-up in average 6.3 years, the result advises that suspicious age-related macular degeneration was arranged more than 55 years old, should take antioxidant, such as vitamin C, vitamin E, bata-carotene and zinc, only can provide very limited anti-cataract effect but take in a large number nutrition.

Antioxidant is more and more tested and has been confirmed, in the cataract genesis, oxidative injury is the hinge that upper Physiology and biochemistry at all levels changes, so the research of various antioxidants is become the focus of anti-cataract medicine exploitation.Japanese scholars has been studied a series of reducible sulfur 01 derivatives, find they can with the free radical fast reaction, the blocking-up glutathion oxidized, recover lenticular normal condition.Wherein, DISULFIRAM (Disulfiram, be called for short: DSF) be a kind of prodrug that has greater activity in this class medicine, after fat-soluble DSF sees through corneal epithelium, can be converted into monomer whose diethyldithiocarbamic acid (Diethyldithjocarbamateacid, be called for short: DDC) performance anti-cataract effect has the effect that significantly delays the cataract genesis for acute selenium rat cataract model, and can alleviate the damage that oxidation causes.

But, use merely vitamins for the prevention of antioxidant and to treat cataractous effect limited, clinical data lacks comparability.

The energy supplement agent of 3. developing for the cataract that causes because the ocular tissue imbalance of energy metabolism, particularly manufacturing are not enough mainly contains vitamin B ₁, B ₂, B ₆, the composition such as nicotinoyl ammonia, ribosidoadenine, succinic acid, pantothenic acid, vitamin C, vitamin E, ATP, catechol, cytochrome C.

4. the cataract that too much causes for calcium ion and be applied to the Ca antagonism of ions agent verapamil of cataract therapy can be blocked the interior stream of metabolic defect in cellular calcium ion, reduces the concentration of the calcium ion in the cataractous eye tissue.

5. reaction is pathogenetic medicine for Mallard, such as ibuprofen, DETAPAC etc.

6. Chinese herbal and crude drugs preparations pushes the leachate of juice and U.S. Radix Hamamelidis Mollis etc. such as Rehmanniae Bolus of Eight Ingredients, Margarita liquid tomorrow and dustymiller;

7. amino acid preparation Phakan contains glutamic acid, cysteine, glycine, arginine, inositol, pyridoxal hydrochloride etc.

8. aspirin

Aspirin is one of greatest invention of twentieth century, constantly has new purposes to occur as medicine.Studies show that aspirin also has the effect of anti-cataract.The mechanism of action by the animal experiment study aspirin, think that aspirin anti-cataract mechanism has 4 kinds: (A) suppress the non-enzymatic protein glycosylation, be that aspirin lysine residue in crystalline protein is combined, stop the non-enzymatic glycosylation of glucose and lysine, and can prevent the crystalline protein polymer formation; (B) carbamyl of inhibition crystalline protein, the acetyl group of aspirin is combined with the amino acid group of crystalline protein, blocked the amino acid residue formylation reaction, made the normal electric charge on protein molecular surface join cloth and exempt from destruction, thereby kept the normal configuration of crystalline protein; (C) suppress lipid peroxidation, the acetyl group of aspirin can preferentially be captured the active amino site of crystalline protein, the infringement of having avoided lipid peroxidation to bring to crystal; (D) aspirin makes calcium channel inactivation on the crystal cell film by suppressing Cycloxygenase on the film, thereby prevents the polyreaction of crystalline protein polypeptide chain.

Aspirin class medicine is cheap convenient, side effect is less, the clinical value that potential treatment senile cataract is arranged, but this medicine indication is very wide, there is the part patient that this medicine is had dependency, long-term larger dose oral also has side effect, finds that such as clinical case long-term, high-dose is used aspirin can cause gastrorrhagia.

9. glycosylation inhibitor

Protein glycosylation theory-glycosylation.The core of this theory is the (lysine or arginic of free amino group in reducing sugar aldehyde radical and the protein.NH ₂Or the N end dissociative of protein molecule-NH ₂) generation non-enzymatic glycosylation (Maillard reaction), generation contains the intermediate product of SchiffShi base pair, behind a series of slowly Amadori structural rearrangements of process, be formed with the various Amadori products of ketoamine architectural feature, behind dehydration and molecular rearrangement, the various advanced glycation end products that finally are formed with fluorescence and protein cross characteristic (are called for short: AGE) again.

Glycosylation inhibitor---aminoguanidine: aminoguanidine (aminoguanidine) is a kind of nucleophilic hydrazine derivative, thinks the formation that can suppress the glycosylation end products and suppress diamine oxidase, nitric oxide synthetase and catalase activity the seventies.Studies show that in recent years, aminoguanidine, 1,3-diaminoguanidine and methylamino guanidine can suppress aldose reductase.But in the galactose cataract model, these 3 kinds of chemical compounds there is no the effect of aldose reductase inhibitor.Aminoguanidine is glycosylation inhibitor, is used to the treatment of diabetic complication, comprises cataract, and it can be combined with glycosylated chemical compound, suppresses in early days and the terminal glycosylation product each period in glycosylation.Aminoguanidine can suppress the muddiness of slight diabetic rat lens, can reduce fluorescent value in Streptozotocin-Diabetic Rats aorta, glomerule and the renal tubules blood, reduce albuminous secretion in the urine, but can not reduce the sorbitol level that renal cortex has raise.Aminoguanidine Main Function be to suppress glycosylation, rather than change Biochemical processes.Aminoguanidine also can suppress calcium dependent Calpain(endopeptidase) hydrolysis of the crystallins of proteolytic enzyme mediation, stop heritability Shumiya rat cataract.This medicine has been used for clinical trial, and its derivant and the crosslinked factor of removing glycosylation are just under study for action.

10. Calpain inhibitor

The selenium cataract is cataract model quick, effective and that repeatability is good, has been widely used in the research of pathogenesis of cataract mechanism and evaluation anti-cataract medicine.The excision effect of the crystalline protein of proteolysis enzyme induction, particularly the activation of calcium dependent Calpain proteolytic enzyme LP82 and M Calpain plays an important role in this pathogenesis of cataract.Calpain proteinase inhibitor E64 can postpone the formation of rat selenium cataract, but must be before sodium selenite is used; E64 can suppress lenticular muddiness stripped hatching in the crystalline lens, and other also comprises E64D and S JA6017 etc.But important problem is the hydrolysis of localised crystal's albumen of finding not yet that in cataractous lens Calpain induces.The content of calcium dependent Calpain proteolytic enzyme in young Mus crystalline lens is considerably beyond people's content, so there is different mechanism in it from the morbidity of human cataracts.The Calpain inhibitor mainly is results of laboratory, there is no clinical data.

Though the research of glycosylation inhibitor, glutathion and prodrug, phase separation inhibitor and Calpain inhibitor etc. has greater advance, but still needs the further checking of clinical research.In addition, also have many medicines of anti-cataract that promise to be just in development.

4, the development trend of cataract therapy drug research

At present, operation is direct, the most effective cataractous Therapeutic Method.Although cataract operation is ripe, corresponding postoperative complication is inevitably arranged, and the operation period of needs of patients selection the best, cross best average of operation periods and just do not had very good effect.Therefore, study cataractous paathogenic factor, pathogenesis, and effectively anti-cataract medicine is of crucial importance.

It is generally acknowledged that radical damage is to cause the various common pathways that cause the effect of cataract factor, lens epithelial cells excessive Apoptosis and crystallin damage also are the key factors in the cataract mechanism.Along with to the pathogenetic further investigation of cataract, although increasing researcher is recognized different damage factors and is caused cataractous approach and machine-processed different, but they are mostly by a common end product---(High molecular weight is called for short: HMW) cause lenticular opacity high molecular weight material.People have carried out large quantity research to the cataractous cause of disease and mechanism for many years, use different Drug therapy cataract for different etiologies, although the clinically extensive use of medicine of nearly more than 40 kinds of anti-cataracts is worldwide arranged at present, but its curative effect is very indefinite, and operative treatment remains various cataractous Main Means.Therefore study the curative effect of novel anti-cataract medicine, the mechanism of inquiring into its intervention is the important content of cataract research field.

Cataractous is multifactor combined effect and the synthesis result that causes, and its pathological change is multilevel multi-level.Along with improving constantly of research means, pathogenetic research also will be goed deep into day by day to cataract, and the exploitation specific aim to the treatment cataract medicine is also just stronger simultaneously.Desirable cataract therapy medicine should be able to recover the homergy of crystal and promote the absorption of lenticular opacity, but there is no up to now a medicine and can reach above-mentioned requirements, however, the measure of any inhibition pathogenesis of cataract likely all demonstrates the value of research, and traditional treasure-house of world medicine be still waiting the inventor go the exploitation.

Now multiplely clinically be used for the treatment of cataractous medicine, owing to lack the perfect foundation for the cataract formation mechanism, make still to have many defectives at widely used many medicines: some efficient is lower, specific aim is poor, and some also has larger side effect.For this reason, demand further verifying pathogenesis of cataract mechanism urgently, find out the susceptible protein that affects the crystal light transmission, as treatment and the Effective target site of prevention, be designed with targetedly medicine, to delay crystal aging and prevent, treat cataractous purpose to reach.

Along with the pathogenesis of cataract research on mechanism constantly is tending towards perfect, the application thinking and the practicable method that also are bound to produce more heterogeneous pass are served clinical treatment, also provide a new strategy for research control crystalline lens muddiness simultaneously.

Therefore, it is still imperative to seek the little muddy product of anti-crystalline lens of novel, definite ingredients, determined curative effect, untoward reaction, and particularly medicine is significant, and has significant Social benefit and economic benefit.By literature search etc., up to now, not yet find to utilize TPCK as the report of the muddy medicine of the anti-crystalline lens especially anti-crystalline lens of muddy product.

Summary of the invention

This finds that the technical problem of required solution is to disclose the new purposes that a kind of sulfydryl to protein carries out alkylating reagent, it is the new purposes that TPCK has the muddy effect of anti-crystalline lens, can be used in the muddy product of the anti-crystalline lens of preparation, the defects that exists to overcome prior art.

That is to say, the present invention is directed to the deficiencies in the prior art, by experiment research and theory study, purpose is intended to the purposes of TPCK, namely provides to contain said medicine or its compositions as the application of the muddy product of anti-crystalline lens aspect.

The muddy product of anti-crystalline lens of the present invention refers in the technical fields such as medicine, food, beverage and reagent, a kind ofly directly or indirectly is used for prevention, diagnosis, detects, protection, treatment and the research crystalline lens is muddy and the product of directly related disease;

Preferably be directly used in the product of prevention, diagnosis, detection, protection, treatment and research crystalline lens muddiness and directly related disease thereof, described lenticular opacity disease comprises one or more in all kinds of lenticular opacity diseases.

The muddy product of described anti-crystalline lens is to comprise in medicine, the reagent etc. one or more, preferred agents.

One, technical conceive

Independent development innovation medicine is a present urgent task of China, Chinese medicine has a long history, also accumulating rich experience aspect prevention and the treatment disease, seek effective active component or its compositions, the new combination of discovery novel drugs, existing medicine or new purposes etc. all are effectively quick approach, the new purposes of particularly expanding existing chemical compound or existing medicine is efficient, quick, economic means especially, also are the places of the advantage of Chinese original new drug development.

The control of crystalline lens muddiness is a study hotspot in recent years, yet relevant cataractous Study of Etiology still lags behind clinical needs far away.Aspect the Study of Etiology of cataract disease, particularly be subjected to the change of crystalline protein generation quality and quantity, scarcely to segment, such as macroscopic description β crystalline protein aspect the research that different extraneous factors affect at relevant crystalline protein in the past.But the inventor thinks that crystalline protein is with ager process, and different component should have different expression trend, Fluctuation to differ, and does not segment and implements to concrete albumen, then is difficult to the separately characteristic of crystalline protein of decision-orientated study.

Lenticular wearing out is a kind of inevitable natural physiology process, and namely with advancing age, crystalline lens is aging gradually.But the inventor still can adopt rational control method or treatment means by inquiring into the formation mechanism of senile cataract, delays lenticular ager process.But the mechanism of crystalline lens muddiness is complicated, be mutual, the coefficient result of many factors, and the research of single mechanism often can not reach satisfied effect, so comprehensive study number of mechanisms, that the multiple medicines thing is united utilization remains further to be carried out.

Oxidative stress be by active oxygen (be called for short: a kind of and aging closely-related type of impairment of ROS) inducing, relevant with the formation of senile cataract, be considered to the cataractous factor that starts.The main active oxygen species that crystalline lens produces comprises superoxide anion, hydroxy radical and H ₂O ₂The structure of the active oxygen that endogenous produces comprises nadph oxidase, mitochondrion and peroxisome, and two kinds of main approach that produce the endogenous active oxygen are ultraviolet and ionizing radiation.The ROS molecule that produces by these approach in the crystalline lens can be via antioxidant and the neutralization of oxidation system of defense, so possesses this two large repair system in the crystalline lens and reinvent molecular injury or alleviate degree of injury.Thiol transferase (thioltransferase, be called for short: TTase), thioredoxin (thioredoxin, be called for short: Trx) and thioredoxin reductase (thioredoxin reductase, be called for short: TR) and glutathion reductase (glutathione reductase, abbreviation: GR) system is considered to strong albumen repair system in the mammalian tissues.In recent years studies show that TTase/GR and Trx/TR all are present in the crystalline lens, to keeping the lenticular transparency important effect are arranged.

The β crystalline protein becomes study hotspot in recent years, and inventor's early-stage Study has shown that it has certain effect to keeping the lenticular transparency and diopter.Cysteine (except the Cys, α B), the especially β, the γ class content that all contain some in the crystalline protein are higher, and it is (SH), very responsive for oxidation that Cys contains a sulfydryl that dissociates; Sulfydryl forms the disulfide bond mixture through Oxidation, is the main driver that crystalline protein unfolding, gathering strengthen.Produce thus conflict: oxidative stress can make sulfhydryl oxidase, and in order to keep lenticular light transmission, crystalline protein must keep q.s reducing condition sulfydryl (SH).

The post translational modification of crystalline protein (is called for short: PTM) be the main inducing of pathogenesis of cataract.Crystalline lens is the very special organ of a kind of structure: transparent, shortage blood confession, namely follow lifelong after the crystalline protein shortage transforms and upgrades, forms.Make crystalline protein suffer the probability of PTM large far beyond other albuminoid.PTM to the major effect of crystalline protein is: very easily cause the crystalline protein unfolding, change the original conformation of albumen; Non-specific crosslinked between disulfide bond formation, the crystalline protein, the albumen water solublity descends; Assemble to strengthen and form high molecular weight material (High molecular weight, abbreviation: HMW), reach 1 * 10 when HMW assembles ⁷When Da is above, light scattering can occur, crystal devitrification, generation cataract.

So how keep in the crystalline protein-SH is not oxidized crosslinkedly to be disulfide bond, and then block follow-up protein molecular crosslinked, assemble, precipitation, play a part crucial to keeping the crystal light transmission and preventing and treating senile cataract.

Inventor's achievement in research has in earlier stage shown that (dithiothreitol, be called for short: DTT, molecular formula is C to dithiothreitol, DTT ₄H ₁₀O ₂S ₂) have an effect of good anti-denaturation of lens proteins; DTT is the Reducing agent of a class chemical property; it can untie the disulfide bond between protein molecule inside and protein molecule, as the protective agent of sulfydryl, can suppress thermal denaturation and the non-specific aggregation precipitation of crystallin.Inventor's early-stage Study demonstrates at the DTT eye drop with voluntarily preparation the degree of selenium cataract mouse model lenticular opacity is alleviated.And inventor research also demonstrates DTT ultraviolet cataract rat model is had good inhibitory action.DTT not only can prevent from forming between protein molecule disulfide bond, and can with protein competitive binding-SH, therefore all bringing into play certain effect aspect prevention and the treatment.

Previous experiments also preliminary identification the alkylating of isopropyl alcohol to the crystalline protein sulfydryl, by the enclosure portion sulfydryl, suppressed the formation of disulfide.Can carry out alkylating reagent to the sulfydryl of protein has a lot, and the inventor has filtered out

Some chemical compounds; the tert-butyl alcohol (Tert-butyl alcohol for example; be called for short: TAB); N-is to toluene sulfo group-L-phenethylamine acyl chlorides methyl ketone (N-tosyl-L-phenylalanyl-chloromethyl ketone; Chinese: N-toluene sulfo group-L-phenethylamine acyl chlorides methyl ketone; the p-tosyl phenylalanyl of N-chloromethyl ketone; Na-tolysulfonyl-L-Phe chloromethyl ketone or: L-1; 4'-methyl sulphonyl-2-phenylethyl chloromethyl ketone etc.; be called for short: TPCK); toluene nefrosulfin methyl ketone etc., wherein the tert-butyl alcohol has also obtained the preliminary confirmation of inventor to the therapeutical effect of selenium cataract.

The present invention research has confirmed that TPCK can suppress the effects such as the apoptosis of cell and antiinflammatory cerebral ischemia is had certain therapeutic effect and Preliminary Applications in clinical.P-toluenesulfonyl (Tosyl, be called for short: Ts) be a substituent group that is derived by p-methyl benzenesulfonic acid, it is an electrophilic base, is commonly used for the protecting group of alcoholic extract hydroxyl group; And the sulfydryl that methyl ketone can enclosed crystal albumen exposes as alkylating agent, therefore the polymerization between the Profilin can stop and delay lenticular muddy degree.

Based on the previous experiments result, the inventor is at first with the isolated culture crystalline lens and simulate the environment of oxidative stress, after adding the TPCK intervention, observe TPCK to the inhibitory action of lenticular opacity degree, detect the wherein variation of enzyme content, the situation of observation of cell apoptosis, the effect of research TPCK anti-oxidative damage is studied its mechanism.

In addition, Ubiquitin-proteasome path (ubiquitin-proteasome pathway, be called for short: UPP) be the high efficiency protein degradation systems that Hershko etc. finds, at present countries in the world mainly concentrate on pathogenesis and the therapeutics aspect of tumor for the research of UPP, are very important mechanism in the degradation selectivity of protein; Research finds that the cell protein degraded of this path mediation is a very complicated and careful regulation process, is the important mechanisms of cell regulate and control, the inhibitive factor by each path of degradation of cell and (or) activity factor bringing into play rise or downward modulation effect.Up-to-date have experimentation to go out all constituents that also contains the UPP system in the lens epithelial cells, and nearly 40～50% impaired crystalline proteins can be degraded by UPP.The UPP system is the protein quality hierarchy of control main in the crystalline lens, it can be by the impaired crystallin of optionally degrading, the effect of the conditionally degraded modulin relevant with multiple bioprocess in the cell be can also bring into play, lenticular growth and differentiation participated in.In recent years, UPP has been subject to people's pay attention to day by day as the main path of the non-dissolve body degraded of intracellular protein, but UPP still is in the starting stage at present in cataractous research.

The inventor studies UPP to the impact of impaired protein level in the lens epithelial cells, inquires into it and whether can remove the crystalline protein of impaired degeneration.The inventor assesses the relation between UPP activity and Cataractogenesis and the impaired albumen by the lens epithelial cells of former culture, inquires into the mechanism of action of anti-cataract.

The inventor is by carrying out the anti-muddy experiment of system to crystalline protein, infer that TPCK is in the drug effect of the aspects such as control crystalline lens muddiness, should be mainly by causing the path of non-specific aggregation and then the formation of realization containment macromolecule crystal albumen to bring into play anti-protein haze effect by forming disulfide bond between the nonessential sulfydryl of the part of enclosed crystal albumen own, the Profilin, result of study also proves and has confirmed that TPCK has the pharmacological action of significant anti-crystalline lens muddiness.

In a word, inventor's experiment has found that this chemical compound has the muddy effect of good anti-crystalline lens, the preclinical phase research that prevents and treats for cataract medicine provides reliable theoretical experimental basis, and this will also have important reference value for the control of other age related ocular disease.

According to this idea and thinking, the inventor passes through experimentation and analysis and theory study repeatedly, successfully obtains result of study and the application product of expecting.

Two, determine that TPCK can be used in theory vision and the Research foundation of the muddy product of the anti-crystalline lens of preparation

1, general introduction

Crystalline lens has unique metabolic way, its central authorities are extremely low without significant protein synthesis, oxygen concentration, suffer for a long time environment and ophthalmic various stress stimulation, brought chance to protein post-translational modification, so crystallin is the aging fabulous organ of researching human body.Cataractous formation is the aging final result of crystalline lens.Crystalline lens is the tissue of rich in proteins, crystallin accounts for 34% of whole crystalline lens weight in wet base, and the transparency is because the arrangement mode, particularly α crystallin of these protein shortrange orders, along with age ageing, or suffer oxidative stress, the arrangement mode of crystalline protein shortrange order, particularly α crystallin, along with age ageing, or suffer oxidative stress, when the structural arrangement mode of crystallin changes, all can affect lenticular normal function.

(post translational modification is called for short: PTM) comprise the excision of β B1, β B2 and β A3/ β A1 crystallin N end, the terminal degraded of α crystallin part phosphorylation and C-in the various post translational modifications of crystallin; Cystine linkage formation and the exposure of γ s crystallin sulfydryl etc. can cause the decline of α crystallin chaperone activity in various degree in the deacylated tRNA amine of α A and γ s crystallin, α A crystallin, and the dissolubility of β crystallin reduces.So how effectively to repair or remove impaired crystallins, prevent its polymerization precipitation in crystalline lens, play pivotal role to keeping the lenticular transparency and preventing and treating senile cataract.

TPCK can regulate many important life processes in the organism, such as folding, the inflammatory reaction of protein, cellular matrix is rebuild and tumor suppression etc.It is comprised of 240 amino acid residues, and the crystal structure under X-ray is globular preteins and contains 9 alpha-helixs and 3 beta sheets.Has a circulus district RSL(Reactive site loop at about 20 the residue places of its surface distance C-terminal), this structure is the significant structure of serpin family, exist in this structural area and can by the restriction enzyme site of the substrate recognition site of target enzyme identification, comprise first aminoacid (P that closes on the N-end ₁), and close on C-end and P ₁Adjacent first aminoacid (P ₁').TPCK will lose about 37% architectural feature in reaction conformational change process, the ability of its opposing thermal denaturation and chemical modification is improved greatly.Inventor's experimental result has in earlier stage shown dithiothreitol, DTT (dithiothreitol, be called for short: DTT) and the tert-butyl alcohol (Tert-butyl alcohol, be called for short: TAB) and isopropyl alcohol lenticular opacity is had certain inhibitory action, in the TPCK chemical constitution to methyl sulphonyl (Tosyl, be called for short: Ts) as an electrophilic group, can protect alcoholic extract hydroxyl group, performance similarly acts on DTT indirectly; Methyl ketone in the structure is as alkylating agent in addition, the same sulfydryl that seals with isopropyl alcohol, can substitute the sulfydryl that protein exposes, reduce the probability of non-specific aggregation degeneration between the albumen, stop the formation of albumen high molecular polymer, and then stop its albumen to become water-insoluble by water solublity, have the antioxidation that contains sulfydryl albumen in the good inhibition crystallin.

This research not only can make the inventor understand the effect of the anti-lens damage muddiness of TPCK by the research to TPCK and crystallin oxidative damage Relations Among, also will provide new thinking for the control of cataract and other age related ocular disease.And provide experimental basis to the further the Molecular Biology Mechanism of exploring the anti-Oxidative Damage of Lens of TPCK.

In sum, the present invention sets about from the biological chemical structure feature of this target protein, and the factors such as physics, chemistry of having studied are emphatically induced the mechanism of crystalline protein degeneration; And develop accordingly the chemical compound of anti-target protein degeneration, this chemical compound can be used as the external used medicine that can protect crystalline protein not to be subjected to or affected by short variation; This chemical compound can adopt liposome as the importing body of crystal cornea; And in advance for selenium (sodium selenite Na ₂SeO ₃) the SD rat cataract model of inducing, the pathological change of simulation senile cataract, slit lamp is observed every day, record crystal light transmission changes process, observe the curative effect; And then inquire into the prospect of clinical practice, expect that this chemical compound can be for delaying and treat senile cataract, especially for the senile cataract treatment generation positive impact of most of initial phase.

2, the basic condition of experimentation

The present invention is based on pathogenetic the understanding in depth of lenticular opacity is starting point, the newtype drug of the anti-lenticular opacity that design, screening are suitable, and verify its action effect, and then inquire into the mechanism of action of medicine.Experimentation of the present invention mainly comprises following two parts content:

(1) first: TPCK is on the impact of muddy degree and heat stability under the crystalline lens oxidative stress

The research contents of first of the present invention is to develop clinical existing medicine N-toluene sulfo group-L-phenethylamine acyl chlorides methyl ketone (is called for short: novel clinical use TPCK).The ability of discussion TPCK opposing oxidative stress in the isolated rat crystalline lens of cultivating reaches the impact on the crystallin heat stability.

TPCK is the known medicine that is used for the anti-cerebral ischemia secondary injury.The ingenious special construction characteristics of utilizing this medicine, it is multiplex to attempt a medicine, inquires into this medicine for the intervention effect of lenticular opacity, and this research contents is that the inventor takes the lead in carrying out, and countries in the world are not reported.

At first, the inventor is by the environment of oxidative stress damage in isolated culture rat lens and the analogue body, and the TPCK that adds various dose in different time points intervenes.

Method: take whole crystalline lens H ₂O ₂Induce its oxidative damage, observe complete crystalline lens in the oxidative damage group with the muddy degree of TPCK intervention group at different time points respectively, detect not the variation of the heat stability of crystallin on the same group.

Result: do not add TPCK and only use H ₂O ₂The lenticular opacity degree of induced oxidation damage is higher than TPCK medication group; The crystallin absorbance be lower than the oxidative damage group with TPCK medicine group and the rate of climb slow.

Muddy development degree when the result observes TPCK and can effectively delay crystalline lens and be subject to oxidative damage, and help to improve the heat stability of crystallin.This is an innovative point of the present invention, and is the clinical conversion of cataract medicine control, and new approaches and experimental basis are provided.Further research also confirms; this medicine is brought into play anti-lenticular opacity and realized by following mechanism: namely TPCK is by discharging active tosyl; act on the very easily oxidized aminoacid ingredient of crystallin; especially the sulfydryl position of cysteine; form sulfhydryl protected group; thereby stoped sulfydryl by the Oxidation of the oxidants such as free radical; therefore effectively stoped crystallin to form disulfide bond because of oxidated effect; bring out albuminous degeneration; further have influence on normal biology of the configuration of crystallin; so that the crystalline lens transillumination weakens; refracted light strengthens, and biological action shows as blurring of vision; the final cataract that produces.

Conclusion: TPCK can alleviate generation and the stable crystallin of muddy degree under the crystalline lens oxidative stress status to a certain extent.

(2) second portion: TPCK is on the impact of antioxidase and apoptosis in the crystalline lens oxidative stress

Second portion research inventor carries out for Antioxidant Enzyme Systems in the body.Inquire into H ₂O ₂After inducing Oxidative Damage of Lens to intervene with TPCK on the impact of antioxidase content and TPCK to apoptotic effect.

Since first studies show that oxidation is most serious for lenticular damage, it is then most important for lenticular protective effect to inquire into so the interior effective antioxidation of body.

For this reason, the inventor has detected crucial antioxidase TTase in the oxidative stress damage crystalline lens and the variation of Trx.The result shows that the expression of TTase and Trx activity raises under the crystalline lens oxidative stress status, and has ageingly, raise gradually in 3 hours, and along with the damage time lengthening, 6 hours enzyme contents, slowly depleted later on; Contrast has added the crystalline lens of TPCK group, and 6 hours TTase compare the matched group reduction and still are higher than the oxidative damage group with Trx.Simultaneously the anti-apoptotic effect of TPCK is studied, got respectively lens epithelium tissue and lens epithelial cells and carry out TUNEL and the detection of streaming apoptosis, results suggest TPCK can effectively reduce the apoptosis of cell.This part experimental result reasonable dismissal crystalline lens enough oxidation defence capabilities are arranged in order to resist hydrogen peroxide (H in the starting stage ₂O ₂), and the existence of capacity TTase, Trx alleviated the isomery of mercaptan in crystallin and the metabolizing enzyme, thus have effective oxidation resistance.But along with increase or the degree of oxidative stress time increases the weight of, the TTase of crystalline lens inherence and Trx lack enough amounts and activity is not enough to offset the harm that oxidative stress causes, and makes the crystalline lens normal function impaired.

Method: adopt the method for crystalline lens isolated culture to add H with first ₂O ₂Cause oxidative damage, other adds TPCK and intervenes, and the matched group crystalline lens is only used the TC-199 nutrient chemical.Get respectively crystalline lens in different time points and measure the variation of TTase, Trx and the situation of observing the TUNEL apoptosis; Former culture lens epithelial cells (Lens epithelial cells, LES) is observed the impact of TPCK behind the ultraviolet induction apoptosis in addition.

The result: TTase, the Trx of two groups of 3hr increases significantly behind the oxidative stress, the content of these two kinds of enzymes reduces in the crystalline lens of 6hr oxidative damage, TTase, the Trx content of TPCK group is lower than Normal group but apparently higher than the oxidative damage group, difference has statistical significance; The prompting of TUNEL apoptosis, apoptosis is at H in the lens epithelial cells of cultivating 3hr ₂O ₂The damage group is maximum, and the TPCK group is less; Cell culture lens epithelial cells streaming apoptosis detection display TPCK group cell born of the same parents die to count and are less than the oxidative stress group.

This result has explained at the beginning of the experiment increase of TTase and the Trx under the oxidative stress condition of crystalline lens in 3 hours.But prolong the oxidative stress time, crystalline lens self is not enough to resist lasting damage, and the expression of TTase and Trx begins to descend the minimizing of 6 hours these two kinds of enzymes among this corresponding result.But the level of this time point TPCK group TTase and Trx still is higher than the oxidative damage group, thereby illustrates that TPCK can induce the expression of these two genes to play a role in to antioxidant stress injury.Increase gradually the time of oxidative stress, the irreversibility muddiness has appearred in crystalline lens, himself or add in addition the degree that pharmaceutical intervention also can't be resisted damage, this just in time coincide with the significant depletion of 12 hours enzyme content of experimental result.And in apoptosis detects, the apoptosis analysis of tissue and cell all visible TPCK can reduce apoptosis quantity, the apoptosis that shows cell in the lenticular opacity process also is a factor of bringing out, so another effect of TPCK is to play the effect that delays lenticular opacity by reducing apoptosis.

Degree when conclusion: TPCK can reduce the oxidative damage of rat lens.

Three, the purposes of TPCK

1, general introduction

Non-specific aggregation is relevant between the mechanism of drug action that the present invention research relates to and enclosed crystal protein part sulfydryl, Profilin, and has carried out further animal experiment study and theory study.

The inventor through the latest find of research is: the inventor has elaborated the positive role of the sulfydryl of protein being carried out alkylating take the crystalline lens muddiness as research emphasis.As adopt TPCK to intervene crystalline lens, it is muddy because of oxidated stress to slow down crystalline lens, the namely clear and definite anti-oxidative stress of TPCK.

Although also have a lot of problems to remain to be explained and solve, cataractous mechanism of action provides experimental basis for TPCK prevents and treats in inventor's experiment, provides brand-new thinking to next step drug development and the therapeutic scheme that applies to preclinical phase.

That is to say that TPCK or its compositions can be used in the muddy product of the anti-crystalline lens of preparation, can be used for the application such as protection, treatment of related crystalline lens muddiness, most preferably the muddy medicine of anti-crystalline lens.

Show the external development that can significantly delay the muddy relevant disease of crystalline lens of TPCK through experimentation.Completed acute toxicity testing proves, the mouse peritoneal drug administration by injection surpasses 1mg/kg to the maximum tolerated dose of TPCK, be equivalent to more than 20 times of clinical recommended drug dosage, show that this effective site is safe and reliable, solved the problem in such drug dose use taboo.

In sum; the inventor resists the muddy product of crystalline lens and has carried out theory study; comprise long-term pharmacology test through a large amount of experimentatioies; find that the TPCK that addresses has the activity of significant prevention, diagnosis, detection, protection, treatment and Effect of Anti crystalline lens muddiness; be the new source of having developed the muddy drug provision of the anti-crystalline lens especially anti-crystalline lens of muddy product, for the further existing drug provision of development and use China scientific basis.

Therefore, TPCK or its compositions especially pharmaceutical composition can be used for preparing the muddy product of anti-crystalline lens, the muddy medicine of the anti-crystalline lens that preferably is prepared from take TPCK of the present invention as raw material.

2, the using method of TPCK and requirement

TPCK of the present invention can unite use with other active component separately or further, comprises for the preparation of being used for the muddy product of prevention, diagnosis, detection, protection, treatment and Effect of Anti crystalline lens, comprises medicine or reagent etc., especially medicine.

In concrete use, TPCK of the present invention can directly use separately, can also use with other many chemical substances.No matter whether these chemical substances have biological activity or have the function for the treatment of disease, comprise miscellaneous function such as collaborative amplification, antagonism or alleviate the side effect etc. of TPCK that these chemical substances are to comprise in pharmaceutically acceptable carrier, natural product, chemical synthetic drug or the human medication etc. one or more; Preferably include in pharmaceutically acceptable carrier etc. one or more.

" pharmaceutically acceptable carrier " used herein comprises one or more in any He all physiology applicable solvent, disperse medium, afterbirth, antibacterial and antifungal, isotonic agent or the absorption delay agent etc.The example of pharmaceutically acceptable carrier comprises one or more water, saline, phosphate-buffered saline, glucose, glycerol or ethanol etc. and in the compositions one or more thereof.In many cases, in said composition, preferably include isotonic agent, for example, sugar, such as in the polyhydric alcohol of mannitol, sorbitol, sorbitol or the sodium chloride etc. one or more.Pharmaceutically acceptable carrier can also comprise a small amount of auxiliary substance, and such as in wetting agent or emulsifying agent, antiseptic or the buffer etc. one or more, they have strengthened effect duration or the effectiveness of TPCK.

From concrete classification, said pharmaceutically acceptable carrier refers to the pharmaceutical carrier of medicine and pharmacology field routine, comprises lubricant, such as in Pulvis Talci, Polyethylene Glycol or the magnesium stearate etc. one or more; Disintegrating agent is such as in microcrystalline Cellulose, sodium carboxymethyl cellulose or the low-substituted hydroxypropyl cellulose etc. one or more; Filler is such as in starch, dextrin or the lactose etc. one or more; Binding agent is such as in pregelatinized Starch, cellulose derivative, alginate, gelatin, polyvinylpyrrolidone, polyvinylpyrrolidone or the hydroxypropyl cellulose etc. one or more; Osmotic pressure regulator is such as in sodium chloride, glucose, sucrose, sorbitol or the mannitol etc. one or more; PH adjusting agent, one or more in the acid such as example hydrochloric acid, sodium hydroxide or the alkali; Solvent, as in water, buffer, ethanol or the propylene glycol etc. one or more etc.; Antioxidant and chelating agent are such as among sodium sulfite, the EDTA etc. one or more; Surfactant is such as quaternary ammonium compound, hexadecanol etc.; Absorption carrier is such as in Kaolin or the soap clay etc. one or more; The macromolecular scaffold agent is such as in cyclodextrin, Polyethylene Glycol, the poloxamer etc. one or more; In diluent such as starch, Icing Sugar, dextrin, microcrystalline Cellulose, mannitol, lactose and the Semen sojae atricolor wet goods one or more; In stabilizing agent such as sodium carboxymethyl cellulose or the cyclodextrin etc. one or more; In antiseptic such as ethylparaben or the sodium benzoate etc. one or more.In addition, can also in TPCK, add other adjuvant, such as in flavouring agent and/or sweeting agent such as sucrose, fructose and the aspartame etc. one or more.

For example, TPCK is dissolved, suspendible or (for example be emulsifiable in the suitable aqueous solvent, distilled water, in normal saline or the Green's solution etc. one or more) or in the oil-based solvent (for example, vegetable oil is olive oil for example, Oleum sesami, Oleum Gossypii semen, in Semen Maydis oil or the propylene glycol etc. one or more) in, can make ejection preparation, wherein (for example can contain solubilizing agent in the solvent, polyoxyethylene sorbitan monoleate, polyoxyethylene hydrogenated Oleum Ricini, polyvidone, cyclodextrin, poloxamer, Polyethylene Glycol, benzyl alcohol, in chlorobutanol or the phenol etc. one or more), osmotic pressure regulator (for example, sodium chloride, glycerol, the D9-mannose, in D-glucitol or the glucose etc. one or more).In this case, if necessary, can add additive, for example stabilizing agent (for example, human serum albumin etc.), analgesic (for example, one or more in procaine hydrochloride or the lignocaine etc.) etc.

Of the present invention and TPCK can also unite use, particularly with other chemical substance such as medicine animal especially mammal is comprised that people or other animals treat used compositions or similar compositions.Described mammal, comprise in people, mice, rat, sheep, monkey, cattle, pig, horse, rabbit, dog, chimpanzee, baboon, Adeps seu carnis Rhiopithecus roxellanae, macaque or the Rhesus Macacus etc. one or more, in preferred people, mice, rat, monkey, pig, rabbit or the dog etc. one or more, one or more in further preferred people, rat or the monkey etc.For example, TPCK of the present invention can be added be suitable for to curee's Pharmaceutical composition in.Usually, this Pharmaceutical composition comprises TPCK of the present invention and pharmaceutically acceptable carrier.

The compositions of TPCK particularly pharmaceutical composition can have various forms, comprises in the dosage forms such as liquid, semisolid and solid one or more; Wherein said pharmaceutical composition comprises that the TPCK that treats effective dose is active component, and one or more pharmaceutically acceptable carriers.

The compositions of TPCK of the present invention especially pharmaceutical composition can adopt conventional production method well known in the art to make any dosage form that is suitable for testing, study or uses clinically, comprise solid preparation such as capsule, tablet, granular preparation etc., liquid preparation such as oral liquid or injection etc.

Active component is mixed with one or more carriers, then be made into required dosage form.Described dosage form comprises one or more in tablet, capsule, granule, suspensoid, Emulsion, solution, syrup or the injection etc., takes one or more route of administration in oral or injection (comprise in intravenous injection, intravenous drip, intramuscular injection or the subcutaneous injection etc. one or more), the mucosa dialysis etc. to carry out prevention, diagnosis, detection, protection, treatment or the scientific research of anti-crystalline lens muddiness.

The compositions of TPCK especially pharmaceutical composition generally must be aseptic and stable under the production condition of storage.Said composition can be mixed with solution, microemulsion, dispersion liquid, liposome or other is suitable for the ordered structure of high drug level.By with a kind of of the TPCK of aequum and required mentioned component or combine to add in the suitable solvent and then carry out aseptic filtration and prepare aseptic parenteral solution.Generally speaking, prepare dispersion liquid by TPCK being added in the aseptic solvent contain basic disperse medium and required above-mentioned other composition.In the situation for the preparation of the sterile powder of aseptic parenteral solution, the preparation method of recommendation is vacuum drying and lyophilized preparation.For example, by passing through to keep required granular size such as the coating of lecithin, in the situation of dispersion liquid and by using surfactant, can keeping the adequate liquidity of solution.

Can comprise the medicament that postpones absorption in the said composition, for example Monostearate or gelatin absorb with the prolongation that reaches injectable composition; Can comprise the high molecular polymer carrier, such as hydroxypropyl methylcellulose or polyoxyethylene, discharge with the prolongation that reaches Orally administered composition.

When being used for the patient, TPCK dosage of the present invention is 0.005 ~ 0.05mg/kgd, can use one or more times, and this dosage or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.

The compositions of TPCK of the present invention especially pharmaceutical composition can comprise the TPCK of the present invention of " treatment effective dose " or " prevention effective dose "." treatment effective dose " refers at the dosage of necessity and effectively reaches the amount of required therapeutic effect under the time.The treatment effective dose of TPCK can cause that at this individuality the factors such as ability of required reaction change according to the patient's condition, age, sex and body weight and the TPCK such as individuality.The treatment effective dose also refers to that the useful therapeutic effect of TPCK surpasses the amount of its any toxicity or harmful effect.

" prevention effective dose " refers to effectively reach the amount of required preventive effect under necessary dosage and time.Because preventive dose is used for the ill front or early stage curee of disease, the prevention effective dose is usually less than the treatment effective dose.The typical non-limiting scope of the treatment of TPCK of the present invention or prevention effective dose is 0.005 ~ 0.05mg/kg, more preferably 0.01 ~ 0.02mg/kg.Should note, dose value will change according to the disease type of wanting to alleviate and seriousness, when that is to say for the patient, TPCK dosage of the present invention or consumption decide according to the age of patient or user and the situation of body weight and health or patient's symptom usually.

In addition; should understand; for any specific curee; should along with the time according to individual need and give with or supervision to adjusting the given dose system with the people's of described compositions professional judgement; and the dosage range that this paper sets only be illustrative, scope or the practice of the compositions of can't requirement for restriction protecting.

That is to say, need to be according to object, route of administration, institute's disease of treat and the situation etc. for the treatment of, variation TPCK of the present invention at every turn and/or dosage or the consumption of every day.For example, give mammal through vein, adult (such as body weight 60kg) especially, the single dose of described TPCK is about 0.3 ~ 3mg, preferred about 0.5mg, preferred administration every day 1 time.Can adjust dosage unit, to propose the best required reaction of arch (for example, treatment or prevention are replied).

For example, can the single-bolus high-dose administration, can within a period of time, give several divided doses or reduce in proportion or increase dosage according to the urgency for the treatment of situation.The non-intestinal compositions that preparation is easy to the unified dosage unit form of administration and dosage is especially favourable.Dosage unit form used herein refers to be suitable for the physical separation unit of dosage unit of the mammalian subject of wish treatment; The calculating that each unit contains scheduled volume is used for together producing with required pharmaceutical carrier the active matter TPCK of required therapeutic effect.The specification of dosage unit form of the present invention, determine and directly depend on the specific characteristic of following (a) TPCK and particular treatment or the preventive effect of wanting to reach by following, and (b) interior in restriction in mixing this technology that is used for the treatment of individual sensitivity TPCK.

3, the pharmaceutical dosage form of TPCK and route of administration

The compositions of TPCK of the present invention especially pharmaceutical composition preparation be used for the muddy product of prevention, diagnosis, detection, protection, treatment and Effect of Anti crystalline lens, it is muddy wherein to can be used in diagnosis, detection, Effect of Anti crystalline lens according to the product of the requirement preparation of reagent technical field; Can be used in patient's treatment, diagnosis, prevention or research according to the product of the requirement of medical technical field preparation, can either be directly used in separately the medicine of preparation treatment, prevention or research, also can mix with many chemical substances or make up, directly or indirectly for the preparation of the medicine for the treatment of, preventing or studying.Chemical substance described here is above described identical with this section.

In the present invention, required material comprises raw material of the present invention, above-mentioned matching used chemical substance etc., all should according to practical situation and needs, adopt the material of SILVER REAGENT or pharmaceutical grade.

The compositions of TPCK of the present invention is pharmaceutical composition especially, can be with the whole bag of tricks administration known in the art, although route of administration/administering mode of recommending in many therapeutic use is spray or oral administration.But the technical staff will appreciate that route of administration/administering mode changes with required result.In some implementation, this reactive compound can avoid the quick carrier that discharges together preparation example such as controlled release preparation with this chemical compound of protection, comprises that graft transmission system, transdermal paste one or more in transmission system or the microcapsule transmission system etc.In addition, can also use biodegradable, biocompatible polymer, such as in ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or the polylactic acid etc. one or more.Prepare the equal patent applied for of many methods of this preparation or general (Sustained and Controlled Release Drug Delivery Systems for example known to those skilled in the art, J.R.Robinson edits, Marcel Dekker, Inc., New York, 1978).

The compositions of TPCK of the present invention especially in the pharmaceutical composition, can contain pharmaceutically acceptable carrier well known in the art and other optional member.Carrier comprises carboxymethyl starch, starch, cellulose, gelatin, sodium bicarbonate, propylene glycol or Tween 80 etc.Optional member is such as being coloring agent, sweeting agent, antioxidant etc.

The compositions of TPCK of the present invention is pharmaceutical composition especially, usually by one or more modes in oral, rectum or the parenteral etc., is applied to the patient who needs this treatment.

The compositions of TPCK of the present invention especially pharmaceutical composition can be made any dosage form that is suitable for using clinically, comprise solid preparation, such as capsule, tablet, granular preparation etc., semi-solid preparation such as ointment etc., liquid preparation such as oral liquid, suspensoid, Emulsion etc., perhaps injection.Take one or more route of administration in oral or injection (comprise in intravenous injection, intravenous drip, intramuscular injection or the subcutaneous injection etc. one or more), the mucosa dialysis etc. to carry out prevention, diagnosis, detection, protection, treatment or the scientific research of anti-crystalline lens muddiness.

Be used for when oral, can be made into conventional solid preparation such as in tablet, powder, granule or the capsule etc. one or more.When implementing, TPCK of the present invention can be together oral with for example inert diluent or assimilable edible carrier.TPCK(and other composition if necessary) can also be wrapped in hard or soft shell gelatin capsules, be pressed into tablet or directly add in curee's the meals.About oral therapeutic administration, described TPCK can be added with excipient and use with one or more forms in edible tablet, buccal tablet agent, lozenge, capsule, suspension, syrup or wafer etc.

For to give TPCK of the present invention outside the parenterai administration, may need with preventing that the material of its inactivation from together giving to the TPCK coating or with TPCK.The reactive compound that replenishes can also be added in the said composition.In the specific implementation, TPCK of the present invention and one or more other medicines that can be used for the treatment of disease are prepared altogether and/or given altogether.Thisly unite use, can utilize primely this medicine that gives than low dosage, therefore avoid possible toxicity or the complication relevant with various monotherapies.

Make in liquid preparation such as water preparation, oil-suspending agent or other liquid preparation one or more, such as in syrup, tincture or the elixir etc. one or more; When being used for parenteral, can be made in solution, water preparation or the oiliness suspending agent etc. of injection one or more.

Above medicine or pharmaceutical composition can use various approach, in described type of service, preferred form is that oral formulations (such as in tablet, coated tablet, capsule, solution or the suspension etc. one or more), non-intestinal give one or more in the dosage form (such as in injection, ointment or the patch etc. one or more) etc., further one or more in preferred tablet, capsule or the injection etc., particularly preferably a kind of in tablet or the injection.

In sum, the compositions of TPCK of the present invention especially pharmaceutical composition can be used for prevention, diagnosis, detection, protection, treatment and the muddy product of Effect of Anti crystalline lens, preferred agents and food, further preferred agents.

Four, technology speciality

The present invention provides a kind of new medicament sources and application forms for prevention, diagnosis, detection, protection, treatment and Effect of Anti crystalline lens muddiness; thereby to the muddy product systems of existing anti-crystalline lens particularly medicine carried out improvement, improved, thereby the application of having expanded existing medicine.

The present invention is safe and effective, and practicality is stronger, and is inexpensive, convenient and swift, and easy, the easy operation of its preparation technology is easy to use, evident in efficacy, can be used for preventing, diagnoses, detects, protects, treatment and the prevention for the treatment of and study of various crystalline lens muddiness.

The present invention studies the crystalline lens muddiness targetedly, has found the muddy product of a kind of new anti-crystalline lens, has made beyond thought achievement; Simultaneously, the present invention also studies the activity of TPCK targetedly, and its pharmacological action is stronger, uses safety, has brought into play to greatest extent effect.The present invention has expanded new medical usage to TPCK, also provides a kind of new medicament sources for prevention, diagnosis, detection, protection, treatment and Effect of Anti crystalline lens muddiness.

It is muddy that TPCK is used for the treatment of crystalline lens muddiness, particularly crystalline lens, can obviously improve the apoptosis that the crystalline lens muddiness causes, therapeutic effect is remarkable, and toxic and side effects is little, has overcome the side effect that existing common drug causes.The present invention has expanded the new medical usage of existing TPCK, also provides a kind of new pharmaceutical intervention means for control crystalline lens muddiness.

TPCK pharmacological action of the present invention is stronger, successful, and its raw material sources are abundant, inexpensive, stable in properties, preparation technology is simple, and convenient quality control is more suitable for large-scale production and commercial application of the industry such as medicine, reagent and industry; The scope of application is wide especially, therefore applies easily, can have a tremendous social and economic benefits in the short period of time.

In a word; active adaption of the present invention modern medical service and the job demand of scientific research field and the needs of human nature service; provide new medicine and preparation source for researching and developing the muddy product of new anti-crystalline lens; has important value to developing the existing medicine of China; be for the safe raw material of the aspects such as prevention, diagnosis, detection, protection, treatment and research crystalline lens muddiness, have important value to improving and improving existing medical level.

Description of drawings

Fig. 1: the SD rat lens isolated culture of not dosing group and adding TPCK medicine, all use H ₂O ₂The induced oxidation damage.The same time not the muddy degree of dosing rat lens be higher than TPCK group rat;

A, D are respectively not dosing and TPCK rat lens cultivation group 0hr, and B, C represent respectively not dosing group rat lens isolated culture 6,12hr, and E, F represent respectively TPCK group rat lens isolated culture 6,12hr;

Fig. 2: BCA canonical plotting;

Fig. 3: oxidative stress and TPCK group Rat Lens protein thermal denaturation;

Fig. 4: according to standard protein molecular weight Maker(as shown in Figure 4), downcuts gel piece corresponding to desired molecule amount, place transfering buffering liquid; Required antibody molecule amount 12kDa

Fig. 5: Trx is at the PCR product electrophoretogram of different time points on the same group not;

Fig. 6: TTase/TrxmRNA is the different time points variation tendency after isolated rat is cultivated Oxidative Damage of Lens;

Fig. 7: TTase and Trx are in the not on the same group expression of different time points;

Fig. 8-1: crystalline lens epithelial cell TUNEL apoptosis (* 100);

Fig. 8-2:H ₂O ₂Lens epithelial cells TUNEL apoptosis (* 100) behind the oxidative damage 3hr;

Fig. 9: the lens epithelial cells of former culture (* 100);

Figure 10: the lens epithelial cells of former culture (* 100);

The operating process of Figure 11: Trizol one step extraction process;

Figure 12: the operating process of RNA denaturing formaldehyde gel electrophoresis;

Figure 13: paraffin section TUNEL method operating process;

Figure 14: flow cytometry detects operating process.

The specific embodiment

The present invention has studied the muddy technology of existing crystalline lens, and a kind of new purposes of prescription of the muddy product of anti-crystalline lens newly is provided, and is convenient to the safe handling of the industries such as medical treatment, reagent.

The present invention finally need to be prepared into the muddy product of anti-crystalline lens and use, and the below will enumerate embodiment and further specify.If any problem, can contact directly 13386272938 with the inventor.Provide several prescriptions and using method and some experimental study contents by aforementioned summary of the invention in above-mentioned some experimental datas that provide and the following example, but the research contents that list in the place that should be appreciated that the present invention is not limited to this, should also be appreciated that term as used herein only is used for describing specific embodiment, and be not limitation of the invention.

The below further sets forth the new purposes of TPCK of the present invention take the concrete experimentation content of TPCK part as example.

(1) first: TPCK is on the impact of the muddy degree of crystalline lens oxidative stress and heat stability

1, general introduction

Cataract is first blinding disease of China, has had a strong impact on China people's health.The key factor that cataract occurs is that muddiness occurs crystalline lens, passes through the refracting media transmission, converges to retina thereby affected light, finally causes blurring of vision clear.Senile cataract is the disease of age related, also is the main cause of old people's visual deterioration and blinding, traces it to its cause and just be the degeneration muddiness that crystalline lens occurs in ageing process.Along with the gradually aging trend of China's population, its number of patients also can continue to increase.External research data shows (Bloemendal H, Jong W, Jaenicke R, Lubsen N H, Slingsby C, Tardieu A.Progress on the function and structure of serine protease inhibitor[J] .Prog Biophys Mol Biol, 2004,86:407-485.), the crowd person in middle and old age cataract prevalence more than 75 years old is up to 88%.Well known oxidative stress (oxidative stress) has been brought into play important function in the pathogenic process of senile cataract, oxidative stress and lenticular opacity and crystallin suffer post translational modification (the post translational modification of stress damage, be called for short: PTM) closely related (Quillen DA.Common causes of vision loss in elderly patients[J] .Am Fam Physician, 1999,60 (1): 99-108. and Sun W R, Zhang X H, Zhang S J, Zhou Z Y.Oxidative damage and enzyme histochemistry changes in senile cataract[J] .US Chin J Ophthalmol, 2000,1:1-3.).The crystalline lens of human eye is a more special organ, the lens epithelial cells of its ambitus (lens epithelial cells, be called for short: LECs) constantly be differentiated to form new lens fibers, and progressively move to lenticular nuclear district, in this transition process, fibrocellular nucleus and organelle are all constantly degenerated and then are disappeared, residual lower transparent Cytoplasm only, thus formed the nuclearity zone of the renewal that has hardly protein.Accumulation along with the time, the hardness of lens tissue significantly increases, there are some researches show (Heys KR, Cram SL, Truscott RJ.Massive increase in the stiffness of the human lens nucleus with age:the basis for presbyopia [J] .Mol Vis, 2004,10:956-963.), probably there is people in middle age the 50% originally protein meeting degeneration of solubility become insoluble protein, therefore so that lenticular hardness increases, the transparency also obviously reduces.

Growth along with human age, natural aging is an inevitable physiological process, people can't fully stop ager process for the negative effect of crystalline lens normal function, but the inventor can be by taking some safeguard procedures, delay or suppress to a certain extent lenticular aging.For senile cataract, if can adopt as early as possible the external used medicine that helps to prevent lenticular opacity, lay ahead of time the prevention basis, make great efforts to keep lenticular transparency, alleviate the occurrence degree of senile cataract, will be a large Gospel to Aged-related Cataract Patients as far as possible.The Therapeutic Method that cataract is traditional up to now still is the crystalline lens by the surgical removal muddiness, although that technology maturation still has is certain traumatic; The clinical application effect of the traditional treatment cataract medicines such as conventional eye liquid catalin, card woods excellent (Bernetine Sodium eye drop) is not fairly obvious yet in addition.Therefore, seek the mechanism of senile cataract, further demand outline urgently for the novel anti-cataract medicine of searching becomes the inventor.

The design of this part research is expectation by adopting the method for TC-199 culture fluid (ion component in vivo of composition and crystalline lens, pH value etc. are close) isolated culture rat lens, outer adding H ₂O ₂Stimulate the model that causes oxidative stress; come thus the condition that crystalline lens constantly suffers with advancing age extraneous oxidative damage and wears out gradually in the simulated in vivo environment; and carry out intervention by medicine TPCK simultaneously and regulate; thereby whether TPCK can have certain effect that delays or suppress to the lenticular opacity degree after the paired observation intervention; and the method that adopts the thermal induction denaturation of lens proteins detect the crystallin of analyzing the different disposal group and be heated after the change of its stability, with this preliminary clear and definite TPCK for the protective effect of crystalline lens under oxidative stress status.

2, materials and methods

(1) laboratory animal: rat

(2) key instrument and consumptive material: micro-surgical instruments, micro mirror, pipettor, filter paper, camera, balance, centrifuge, water bath, ultrapure water equipment, drying baker, disinfect box, ice machine, vortex mixer, refrigerator, PH adjusting apparatus;

(3) main agents: culture fluid, GO, H ₂O ₂, TPCK, chloral hydrate, determination of protein concentration test kit;

(4) experimental technique:

1. crystalline lens isolated culture

A, animal crystalline lens are prepared: get rat, put to death after the intraperitoneal anesthesia of employing chloral hydrate, around the alcohol wipe Dual-eyelid, take out eyeball, put into PBS and clean blood, rinsing is placed under the super-clean bench aseptic condition and cuts off partially afterwards at corneoscleral junction with micro-shears, the crystalline lens of giving birth to.

B, method: take out complete crystalline lens and put into PBS, remaining on every side vitreous body is carefully peeled off, crystalline lens with rinsing blots surface moisture and unnecessary impurity at filter paper again, afterwards crystalline lens is put into culture fluid, move into culture plate after soaking, add culture fluid, put into incubator, then add H ₂O ₂And GO; Experimental group adds TPCK again, and matched group only adds culture fluid.

C, grouping: H ₂O ₂Oxidative stress is respectively matched group and experimental group with the other TPCK treatment group that adds.

2. external evoked crystallin thermal denaturation

A, prepare before measuring: get and only add H ₂O ₂The rat lens of oxidative stress and adding TPCK treatment group crystalline lens move into respectively in the homogenate tube, add protein lysate, place and use homogenizer homogenate on the ice chest, centrifugal, get the rear mensuration of supernatant dilution protein concentration, and two groups of rat lens albumen supernatant are transferred to same concentration.

B, method: the equal serosity of the crystallin that concentration level is consistent moves into cuvette, the water bath preheating, and sample is at first measured initial absorbance, and distilled water is done reference, after the heating, regularly measures absorbance, does reference with distilled water.

(5) statistics and analysis

All data mean ± standard deviation

The t check is carried out in expression, applied statistics software, and p＜0.0l represents the significant difference highly significant, and p＜0.05 expression significant difference is remarkable, p＞0.05 expression no difference of science of statistics.

3, experimental result

(1) crystalline lens isolated culture

Crystalline lens is isolated culture in culture fluid, observes through H ₂O ₂The crystalline lens of induced oxidation damage and TPCK matched group crystalline lens are all without obviously changing; Afterwards, along with the prolongation of time, H ₂O ₂Induce damage group crystalline lens the muddiness that increases the weight of gradually to occur, develop into whole bark matter even full lenticular opacity from the front or rear cortex muddiness that begins, the muddy degree of rat lens that adds TPCK in the same time all is lower than in not dosing group rat.

(2) external evoked crystallin thermal denaturation

1. the BCA method is measured protein concentration: according to protein standard substance, measure absorbance, the drawing standard curve (is annotated: R in the formula ²Value is more better near 1).

2. thermal stability determination: H ₂O ₂The muddy degree of the supernatant of the crystalline lens mixed protein of induced oxidation damage increases very fast, and the anti-oxidative damage ability descends; H ₂O ₂The crystalline lens mixed protein supernatant that has added TPCK in the time of the induced oxidation damage becomes muddy gradually after water-bath, degree slowly increases the weight of.

4, discussion and conclusion

Oxidative stress and thermal induction degeneration are the risk factors of pathogenesis of cataract.(Oxidation Stress is called for short: the concept of a kind of Pathophysiology character that OS) to be U.S. RS.Sohal propose first in nineteen ninety oxidative stress.Oxidative stress is all being played the part of important role in various types of cataractous pathogenic processes.The damage of oxidative stress can be so that produce some oxygen-derived free radicals (Reactive Oxygen Species in the crystalline lens, be called for short: ROS), active nitrogen free radical (Reactive Ntrogen Species, be called for short: RNS), protein, lipid and nucleic acid that these all can damage in the lens cell bring out the lenticular opacity degeneration.When cell is under the higher temperature, cell membrane will produce the membranaceous structure of some monolayers, thereby destroyed cell membrane globality and permeability (Dowhan W. Molecular basis for membrane phospholipid diversity:why are there so many lipids [J] .Annu Rev Biochem, 1997,66:199-232.).Crystallin is changed by thermal property, forms insoluble protein, and the coagulation sedimentation reaction can occur, so that crystalline lens loses its transparency, and muddy the generation.(the Zhen-Yu H such as Heys KR, Rong Z, Pei-Hong S, Bo W, Cheng-Jian J.Progress on the function and structure of serine protease inhibitor[J] .Genomics and Applied Biology, 2011,30 (45): studies show that 1290-1298.), complete crystalline lens is placed under the heat stress of 50 ℃ of gentlenesses, be used for simulating relevant change of age that crystalline lens occurs in the environment in 35 ℃ of body temperature of human body, the decline of solubility crystallin content in the cell also can appear in crystalline lens inside in this model, albumen macromolecule form (High molecular weight, be called for short: HMW), the formation of HMW and final coagulation sedimentation and lenticular hardness increase etc. are similar to the lenticular change of age related.Therefore the method for inventor by oxidative stress and thermal induction degeneration can detect TPCK for the impact of the muddy degree of SD rat lens and to the effect of protein stability, thereby learns whether have certain protective effect when TPCK is damaged for crystalline lens.

Pointed out TPCK not only to help to protect rat lens albumen to avoid the progress of the muddy degree under the oxidative stress status according to the inventor's of first experimental result, and the crystallin heated denaturalization has been had certain resistivity.Receive extraneous H at the crystalline lens of isolated culture ₂O ₂The damage of oxidative stress the time, the protective effect of muddiness can be observed in front 3 hours rat lens isolated culture H when TPCK showed crystalline lens suffered extraneous oxidative damage ₂O ₂Oxidative damage group and TPCK intervention group are not seen obvious variation, in continuous 6 hours afterwards, can see obviously that the crystalline lens that has added the TPCK intervention group is than H ₂O ₂The muddy degree of oxidative damage group alleviates greatly.But after 12 hours, crystalline lens begins to occur the completeness muddiness of nuclearity, even TPCK also can't slow down or reverse the generation of muddy degree.Therefore the result according to this experiment can find out that TPCK is to have certain protective action for the crystalline lens that is under the oxidative stress status.

H ₂O ₂It is a kind of mode of oxidative stress, except causing increasing of free radical, outside the variation of protein structure and space conformation, can also activate the oxidation sensitive transcription factor NF-К B of lens epithelial cells, thereby cause abnormal gene expression, finally cause cataract.This is H ₂O ₂A mechanism of damage, TPCK protects the damage of crystalline lens under oxidative stress by which kind of mechanism, also needs the inventor further to inquire into.And for the crystallin that in the cataract generating process, has important function, the raising of its heat stability also is to delay one of reason of cataract generation, the inventor can find out in the experiment in this section, the external oxidative damage of protein stability when TPCK is subject to to(for) crystallin has certain resistant function, than the rat lens that does not add TPCK, can improve the rock-steady structure of crystallin, improve the ability of anti-damage.

As everyone knows, during Cataractogenesis, nucleus lentis becomes dark yellow and even brown, van Heyningen contains fluorochrome thing (glucose side's glycosides) (YAN Hong in reported first crystalline lens in 1971, HU I Yan-Nian.Ageing lens and development oftataract[J] .Fourth Mil Med Univ, 2005,26 (2): 97-100.).The kynurenin (Kyn) that comes from the development of tryptophan oxidation product is arranged in the lens fibers cell, wherein mainly comprises 3-hydroxykynurenine, 3-hydroxykynurenine glucose side glycosides and 4-(2-amino-3-hydroxyphenyl)-4-oxy butyrate O-L-D-glucose side glycosides.The oxidation of these materials has increased the increase of cohesion, pigmentation and the non-tryptophan fluorescent material of crystallin in the crystalline lens, and above-mentioned change and lenticular opacity are closely related.Kynurenin may form 3-hydroxyl glutathion kynurenin glucose side glycosides adduct with glutathion or nucleophilic aminoacid (such as cysteine).(the Gamer B such as Gamer, Roberg K, Q ian M, et al.Distribution of ferritin and redox-actinve transition metals in normal and cataractous human lenses[J] .Exp Eye Res, 2000,71 (6): 599-604.) find that the cattle crystallin can be modified generation coloured (absorbing at 365nm) by kynurenin and fluorescence (excites 380nm, the protein adduct of emission 450～490nm).These adduct cohesions have increased light scattering, have reduced the lenticular transparency.Oxidative damage causes that the rising of free radical not only can cause lipid peroxidation and produce cataract, can also improve the concentration of 3-hydroxykynurenine in the crystalline lens.Inventor's previous experiments studies show that to the excellent woods of anti-cataract medicine card it plays a role by chromophores such as this metabolism histidine, tryptophans.

If stop crystallin crosslinked, be gathered into high molecular weight material, just can suppress so coupling precipitation and consequent lenticular opacity between the impaired albumen.Thereby the inventor proposes a kind of hypothesis: use alkyl (methyl, ethyl etc.) to substitute to expose in the impaired crystallin-H among the SH reaches the effect of sealing sulfydryl, just can stop that crystallin is assembled, the inhibition lenticular opacity.Crystallin mostly contain the sulfydryl of some and in crystalline lens very high concentrations, can reach 35% of crystal weight in wet base; The nuclear district protein content of crystal is more up to 50%(Simpanya MF, Ansari RR, Suh KI.Aggregation of lens crystallins in an in vivo hyperbaric oxygen guinea pig model of nuclear cataract:Dynamic light-scattering and HPLC analysis[J] .Invest Ophthalmol Vis Sci, 2005,46 (12): 4641-4651.).Therefore, it is more to contain the albumen of free sulfhydryl groups in the crystal.By the control concentration of alkylating reagent and activity, partially enclosed sulfydryl, crystalline protein is no longer occured or that disulphide occurs less is crosslinked, should stop or reduce protein aggregation, maintenance crystal light transmission.And the inventor is according to chemical constitution and the biological activity of TPCK, infers that it has born this alkylating just.The inventor has a bold conjecture TPCK can also bring into play by following approach the effect of anti-oxidative damage in addition: after at first the TPCK protease that plays protective action when suffering oxidative damage in the crystalline lens is combined, a circulus will cracking falling its active site, make simultaneously its opposing thermal denaturation and the chemical property of self greatly improve (Heys KR, Friedrich MG, Truscott RJ.Presbypoia and heat:changes associated with aging of the human lens suggest a functional role for the small heat shock protein, alpha-crystallin, in maintaining lens flexibility[J] .Aging Cell, 2007; 6 (6): 807-815.).This may be that TPCK keeps lenticular light transmission with the self-constituent of continuous sacrifice.On the other hand, but methyl ketone in its structure is to have active the alkyl of neutral characteristics, can replace the sulfydryl that crystallin exposes and play the closure effect, thereby stop mutually combining between the albumen sulfydryl, may play a kind of active role aspect the non-specific aggregation of albumen.P-toluenesulfonyl in the chemical constitution of TPCK can be protected alcohols in addition, and the inventor imagines it can bring into play the antioxidative effect by the protection alcohol compound indirectly.The damage of protecting so crystalline lens to avoid oxidative stress is the primary content that keeps the crystalline lens light transmission, and the inventor infers that tentatively TPCK is just by having kept the lenticular transparency to the releasing effect after the defence before the crystallin non-specific aggregation and the combination.

The crystalline lens that any reason causes is damaged by the persistence oxidative stress, all can start the system of defense in the crystalline lens, and the expression of these systems of defense is (Meyer M, Schreck R, the Baeuerle PA.H that are subjected to the regulation and control of AP-1 transcription factor ₂O ₂And antioxidants have opposite effects on activation of NR-kappa B and AP-1 in intact cells:AP-1 as secondary antioxidant responsive factor[J] .EMBO, 1993,12:2005-2015. and Sen CK, Packer L.Antioxidant and redox regulation of gene transcription[J] .FASEB, 1996,10:709-720.), comprise glutathion reductase (Pinkus R, Weiner LM, Daniel V. Role of oxidants and antioxidants in the induction of AP-1, NF-kappaB, and glutathione S-transferase gene expression[J] .Biol Chem, 1996,271:13422-13429.), glutathione S-transferase (glutathione S-transferase) (Grant CM, Collinson LP, Roe JH, Dawes IW.Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for Yap-1 transcriptional regulation[J] .Mol Microbiol, 1996,21:171-179.), TTase, Trx(Kuge S, Jones N.YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides[J] .EMBO, 1994,13:655-664.) and TR(Morgan BA, Banks GR, Toone WM, Raitt D.Kuge S.Johnston LH.The Skn7 respnse regulator controls gene expression in the oxidative stress response of the budding yeast Saccharomyces cerevisiae[J] .EMBO, 1997,16:1035-1044.).Li, SpectorA(Li DW, Spector A.Hydrogen peroxide-induced expression of the proto-oncogenes, c-jun, c-fos and c-myc in rabbit lens epithelial cells[J] .Mol Cell Biochem, 1997,173:59-69. and Li WC, Wang GM, Wang RR, Spector A.The redox active components H ₂O ₂And N-acetyl-L-cysteine regulate expression of c-jun and c-fos in lens system[J] .Exp Eye Res, 1994,59:179-190.) etc. confirmed the lens epithelial cells H of rabbit ₂O ₂The Rotooncogene c-Jun of mediation, c-fos and c-myc change to be controlled by transcription factor.Under the oxidative stress condition that continues, TTase expresses and active increase is necessary for the protection lens cell.Antioxidant two sulfur enzymes can be repaired key enzyme and the albumen in the oxidative damage, glyceraldehyde-3-phosphate dehydrogenase (glyceradehyde-3-phosphate dehydrogenase such as the ATP generation, G3PD), hydrogen peroxide detoxication enzyme and glutathion peroxidase (Xing K, Lou MF.The possible physiological function of thioltransferase in cells[J] .FASEB, 2003,17:2088-2090.).In addition, the active increase of TTase can strengthen the recycling ability of Ascorbate to reduce accumulation (the Fernando MR of hydroascorbic acid in the cell of poisoning, Nanri H, Yoshitake S, Nagata-Kuno K, Minakami S.Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells[J] .Eur J Biochem, 1992,209:917-922.).The Trx and the TR that activate can help the cytothesis oxidative damage under oxidative stress, Trx passes to H to electronics from hydrogen-peroxide reduction albumen ₂O ₂Destroy the combination of disulfide bond, remove the hydrogen peroxide of toxicity, thereby albumen and enzyme (the Yoshitake S of regenerating oxidation damage, Nanri H, Yoshitake S, Fernando MR, Minakami S.Possible differences in the regenerative roles played by thioltransferase and thioredoxin for oxidatively damaged proteins[J] .Biochem(Tokyo), 1994,116:42-46. and Chae H, Robison K, Poole L, Church G, Storz G, Rhee S.Cloning and sequencing of thio-specific antioxidant from mammalian brain:alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes[J] .Proc Natl Acad Sci USA, 1994,91:7071-7072.).

But under strong oxidative stress effect, above-mentioned enzyme and protein function are impaired equally, can't stop protein aggregation subsequently.Experimentation shows, the simple crystalline lens that relies on is kept in the body self TTase, Trx concentration, still be difficult to effectively resist crystalline protein Cys-SH and avoid oxidation (Ganea E, Harding JJ Glutathione-related enzymes and the eye[J] .Curr Eye Res, 2006,31 (1): 1-11. and Yogorova S, Yegorov O, Lou MF.Thioredoxin induced antioxidant gene expression in human lens[J] .Exp Eye Res, 2006,83 (4): 783-792.).The experiment inventor of this part has observed the crystalline lens that TPCK can protect isolated culture and has avoided suffering the damage of oxidative stress, alleviates the lenticular opacity degree, and stablizes to a certain extent the crystallin heated denaturalization.Whether TPCK can affect the above-mentioned cytosol enzyme with antioxidation in the crystalline lens, by self and TTase, Trx the lenticular transparency is kept in the combination of crystallin disulfide bond? this is the starting point of second portion experimentation, for the known anti-apoptotic effect of TPCK, second portion detects TPCK impact on apoptotic cell under the damage of crystalline lens oxidative stress simultaneously in addition.

(2) second portion: TPCK is on the impact of crystalline lens antioxidase and apoptosis situation

1, general introduction

Many experiments have proved that crystalline lens betides before the cataractous lens muddiness the reaction of oxidative stress, and oxidative damage is first factor of Cataractogenesis.Oxidative stress is that (reactive oxygen species is called for short: ROS) induce by active oxygen.Lenticular enzyme system can produce endogenic active oxygen species in normal metabolic process, or crystalline lens suffers can produce ectogenic active oxygen after the stimulation of external environment.Active oxygen species main in the crystalline lens comprises superoxide anion, H ₂O ₂And hydroxy radical (Lou MF.Redox regulation in the lens[J] .Prog Retin Eye Res, the endogenous system that 2003 22:657-682.), can produce active oxygen in the crystalline lens comprises NAPDH oxidase, mitochondrion and peroxisome.Two kinds of main approach that produce exogenous active oxygen are ultraviolet and ionizing radiation in the crystalline lens.Have antioxidant and oxidative damage protection system to neutralize in the crystalline lens active oxygen species that these two kinds of approach produce (Lou MF.Redox regulation in the lens[J] .Prog Retin Eye Res, 200322:657-682. and M.McNamara, R.C.Augusteyn.The effects of hydrogen peroxide on lens proteins:A possible model for nuclear cataract[J] .Exp Eye Res, 1984; 38 (1): 45-56.).Although have in these strong antioxidants and the active oxygen and enzyme system, some active oxygen species still can be escaped protein, lipid and the DNA(Lou MF.Redox regulation in the lens[J in these defense systems and then the oxidative damage cell] .Prog Retin Eye Res, 200322:657-682. and M.McNamara, R.C.Augusteyn.The effects of hydrogen peroxide on lens proteins:A possible model for nuclear cataract[J] .Exp Eye Res, 1984; 38 (1): 45-56. and Lou MF.Thiol regulation in the lens[J] .Ocul Pharmacol Ther, 2000,16:137-148.).Therefore also possesses protease (the Wagner BJ that some can be repaired molecular injury or alleviate degree of injury in the crystalline lens, Margolis JW, Garland D, Roseman JE.Bovine lens neutral proteinase preferentially hydrolyses oxidatively modified glutamine synthase[J] .Exp Eye Res, 1986,43:1141-1143. and Jahngen-Hodge J, Cry D, Laxman E, Taylor A.Ubiquitin and ubiquitin conjugates in human lens[J] .Exp Eye Res, 1992,55:897-902.).Thiol transferase (Thioltransferase, be called for short: TTase), thioredoxin (Thioredoxin, be called for short: Trx), thioredoxin reductase (Thioredoxin reductase, be called for short: TR) and glutathion reductase (glutathione reductase, be called for short: GR) system be considered to the system that this class in the mammal has strong albumen repair ability (Holmgren A.Thioredoxin and glutaredoxin systems[J] .Biol Chem, 1989,264:13963-13966. and Yoshitake S, Nanri H, Fernando MR, Minakami S.Possible differences in the regenerative roles played by thioltransferase and thioredoxin for oxidatively damaged proteins [J] .Biochem (Tokyo), 1994,116:42-46. and Fernando MR, Nanri H, Yoshitake S, Nagata-Kuno K, Minakami S.Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells.[J] .Eur J Biochem, 1992,209:917-922.), in recent years studies confirm that TTase/GR and Trx/TR are present in the crystalline lens, and be in reducing condition keeping crystalline lens, keep its transparent aspect to play vital effect (Raghavachari N, Lou MF.Evidence for the presence of thioltransferferase in the lens[J] .Exp Eye Res, 1996,63:433-441. and Yoshitake S, Liu A, Lou MF.Human lens thioredoxin:molecular cloning and functional characterization[J] .Invest Ophthalmol Vis Sci, 2003,44:3263-3271.).

At present research is more be thiol transferase (be called for short: TTase) and thioredoxin/thioredoxin reductase (be called for short: Trx/TR) etc. (Lou MF.Redox regulation in the lens[J] .Prog Retin Eye Res; 200322:657-682. and Yoshitake S; Liu A; Lou MF.Human lens thioredoxin:molecular cloning and functional characterization [J] .Invest OphthalmolVis Sci; 2003; 44:3263-3271. and Holmgren A.Glutathione-dependent synthesis of deoxyribo nucleotides:characterization of the enzymatic mechanism of Escherichia coli glutaredoxin[J] .Biol Chem; 1979; 254:3272-3278.) with GSH metabolism and regulate closely related; everybody knows and knows; high (mmol level) (the Moon S of GSH content in crystalline lens; Rohan Fernando M; Lou MF.Induction of thioltransferase and thioredoxin/thioredoxin reductase systems in cultured porcine lenses under oxidative stress[J] .Invest Ophthalmol Vis Sci; 2005; 46:3783-3789.); help to protect that cysteine Cys-SH is in reducing condition in the crystalline protein; anti-oxidative defense barrier (Yoshitake S important in the crystalline lens; Liu A; Lou MF.Human lens thioredoxin:molecular cloning and functional characterization[J] .Invest Ophthalmol Vis Sci; 2003; 44:3263-3271. and Moon S; Rohan Fernando M; Lou MF.Induction of thioltransferase and thioredoxin/thioredoxin reductase systems in cultured porcine lenses under oxidative stress[J] .Invest Ophthalmol Vis Sci; 2005,46:3783-3789.).Result of study shows that also the GSH CONCENTRATION DISTRIBUTION is descended to the nuclearity district gradually by the crystal skin in the crystal, illustrates that the GSH antioxidation mainly plays a role in crystal outer cortex district.These enzymes or protein also have antioxidation, and its enzymatic activity or protein expression raise under the oxidative stress effect, impel dissociating of albumen two sulfur mixture, are conducive to keep the crystal light transmission.But along with these enzyme content of increase at age reduce gradually, and can not stop the disulfide bond between the follow-up crystallin to be assembled.

By the experiment of first, the inventor has observed the effect of the anti-lenticular opacity of TPCK, and based on its chemical constitution, the inventor infers that it passes through the enclosure portion sulfydryl, both can suppress the formation of a large amount of disulphide; The combination that also can be at war with to the disulphide polymer that has formed impels to reopen disulfide bond.Does thereby it play a role indirectly in the face of TTase, Trx have the rise effect? the inventor explores the effect of TPCK by the change that detects the content of these two kinds of enzymes in the crystalline lens of having intervened TPCK under the oxidative stress status, and this is the groundwork that this part will be done.

Had and studies confirm that TPCK can have clear and definite therapeutic effect (Sun Ming to nervous system by the effect of anti-apoptosis, Zhao Yumei, Xu Chao .N-toluene sulfo group-L-phenethylamine acyl chlorides methyl ketone treating cerebral ischemia [J]. apoplexy and sacred disease magazine, 2002,2 (19): 70-72.); Along with the increase lens epithelial cells at age apoptosis gradually, apoptosis is a certain amount of can to bring out cataractous formation.The epithelial apoptosis quantity of cataract patient anterior lens capsule is apparently higher than normal person (Ding Jianguang, Li Hanyu, Ceng Lingbai, Wang Jiacui, Yang Ceyao. lens epithelial cell apoptosis and hydrogen peroxide-induced Cataractogenesis research [J]. China's experiment ophthalmology magazine, 2001,3:202-204.).This part also will be detected the anti-apoptotic effect of TPCK so, gets the lens epithelium tissue and does the TUNEL apoptosis and the lens epithelial cells of cultivating is carried out the detection of streaming apoptosis, thereby analyze TPCK for the impact of lens epithelial cell apoptosis situation.

2, materials and methods

(1) laboratory animal: rat

(2) key instrument and consumptive material: operating theater instruments, microscope, pipettor, filter paper, microwave oven, balance, centrifuge, ultrapure water equipment, pcr amplification instrument, microplate reader, agitator, water-bath, shaking table, electrophoresis tank, electrophresis apparatus, imager, refrigerator, flow cytometer, pasteur pipet, culture bottle, super-clean bench

(3) main agents: the anti-Mus TTase of rabbit antibody, the anti-Mus Trx of rabbit antibody, β-actin antibody, HRP Conjugated goat-anti rabbit two resist, RNA extracts reagent Trizol, various DNA marker, 6 * DNA sample-loading buffer, agarose, PrimeScriptRT reagent Kit, SYBR-Green Master Mix, DAB colour reagent box, paraformaldehyde, TUNEL apoptosis test kit, E.C. 3.4.21.64, LEC primary cell line, DMEM F12 culture medium, hyclone, pancreatin

(4) experimental technique

1. the cultivation of crystalline lens isolated culture and lens epithelial cells

A, animal crystalline lens are prepared (same first): get rat, put to death after the employing chloral hydrate anesthesia, around the alcohol wipe Dual-eyelid, take out eyeball, put into PBS and clean blood, rinsing is placed under the super-clean bench aseptic condition and cuts off partially afterwards at corneoscleral junction with micro-shears, the crystalline lens of giving birth to.

B, crystalline lens isolated culture method: take out crystalline lens and put into PBS, remaining on every side vitreous body is carefully peeled off, again with crystalline lens suck dry moisture and impurity on filter paper, at last crystalline lens is placed culture fluid, move into culture plate after soaking, add culture fluid, put into incubator, reject the crystalline lens that becomes muddiness after cultivating, then transfer in the new culture plate, add H ₂O ₂And GO, experimental group adds TPCK again, and matched group only adds culture fluid.

C, lens epithelial cells cultural method: get cell, dissolve in the water bath, take into the interior wherein Cell sap of super-clean bench to be drawn onto in the centrifuge tube with suction pipe, add again culture fluid, centrifugal, discard supernatant, add culture fluid, be mixed into single cell suspension.Single cell suspension is added in the culture bottle of existing culture fluid, microscopically observation of cell state, the cell that recovery is good is put into incubator and is cultivated.The observation of cell growth conditions.Continue behind the cell attachment to cultivate, namely can be used for experiment.

D, lens epithelial cells oxidative stress damage method:

Lens epithelial cells is without H ₂O ₂The oxidative stress damage method, because lens epithelial cells is relatively more fragile, H ₂O ₂Oxidative damage may cause irreversible damage, even also can't reverse with interfering effects of drug.So the inventor adopts ultraviolet irradiation to cause the oxidative stress damage.With cultured cells uncap in super-clean bench, the Burdick lamp irradiation.

E, grouping

H ₂O ₂Oxidative stress is respectively matched group and experimental group with adding in addition the TPCK treatment group, and the crystalline lens that simple culture of ex vivo crystalline lens is not done any processing is blank group.Immunohistochemistry detects with the experiment of TUNEL apoptosis same as described above.Lens epithelial cells is cultivated and is divided into H ₂O ₂Oxidative stress and TPCK intervene, and are respectively matched group and experimental group.

2. RT-PCR detects the mrna expression of TTase and Trx

A, the total RNA of crystalline lens extract

The crystalline lens of getting isolated culture places homogenate tube, and every pipe adds homogenizer and carries out homogenate.

B, Trizol one step extraction process

The evaluation of C, nucleic acid samples

The purity testing spectrophotometric determination of nucleic acid samples.The reading of observation nucleic acid samples under two wavelength (is called for short: OD) be worth, judge the purity of nucleic acid samples according to ratio.Calculate the concentration (μ g/ μ l) of RNA sample according to formula:

Concentration=OD 260 * nucleic acid extension rate of RNA sample * 40/1000

Gel electrophoresis is adopted in the detection of RNA sample integrity.

D, design of primers

Utilize on the internet Pubmed to search the genes of interest total length, design and synthesize.Design result compares with Blast, guarantees its specificity.Total amount 2ODU ₂₆₀, adopt PAGE mode purification, effectively removed non-full length fragment, primer efficient is high.The employing distilled water redissolves, and then forward primer and downstream primer mix homogeneously add DEPC water again, deposit.

(be called for short: RT-PCR) E, reverse transcription-polymerase chain reaction

F, polymerase chain reaction

Before upper simple PCR machine, annealing temperature arranges four, carries out simple PCR, in the hope of groping optimum annealing temperature.

G, agarose gel electrophoresis

Glue: the TAE electrophoretic buffer of in conical flask, packing into, use dd H ₂O and agar Icing Sugar.Heating makes the agarose dissolving in microwave oven, makes the solution cooling, adds EB, mixes.Comb is placed in position at the distance base plate, warm agarose solution is poured in the rubber moulding, and checked under the tooth of comb or whether between cog has bubble.After gel solidifies fully, remove comb, gel is put into electrophoresis tank, add electrophoretic buffer.

Electrophoresis: after getting the PCR product and sample loading buffer mixing, with pipettor mixture is added in the sample cell, wherein the first swimming lane adds DNA ladder.Regulate energising voltage, make DNA anode (red line) mobile.Electrophoresis to bromophenol blue and the blue or green FF of dimethylbenzene moved out of suitable distance in gel.Cut off the electricity supply, take out glue observed result and taking pictures under uviol lamp.

The result judges: present white fluorescent under the ultraviolet light, can see the position of migrating of band, the gel images photograph determines that the mRNA of TTase/Trx and confidential reference items β-actin has the rear upper RT-PCR instrument of expression.

H、Real-time?PCR

Use SYBR Green fluorescent dye imbedding method to carry out the Real time pcr amplification of above-mentioned purpose gene.

RT-PCR is according to the optimal temperature conditions of above-mentioned electrophoresis: 95 ℃, and 2min → (95 ℃, 15s → 62 ℃, 30s → 72 ℃, 15s) circulation is 40 times → 72 ℃, 5s; Simultaneously with 95 ℃, 15s → 62 ℃, 30s → 72 ℃, the chain melt curve analysis of the condition monitoring amplified production of 15s, the result shows and all becomes overlapping unimodal, confirms that the specificity of primer is better.Analysis software is obtained mean CT-number, carries out quantitative analysis with the ratio of the mRNA relative expression quantity of purpose and reference gene.

3. Western Blot detects the expression of TTase and Trx

The extraction of A, sample total protein

The crystalline lens of getting isolated culture places homogenate tube, and every pipe adds the crystallin lysate, and sample carries out homogenate with homogenizer, and homogenate leaves standstill, and is centrifugal, gets its supernatant and transfers in another new EP pipe.

B, protein quantification

Adopt BCA determination of protein concentration test kit detection by quantitative protein content: the protein standard substance that configures in the test kit is added to 96 orifice plates, adds the deionization distilled water.Add successively sample in 96 orifice plates, last every hole all adds the BCA working solution, places.540nm wavelength place microplate reader is measured the OD value.The standard curve of making according to standard substance albumen calculates the sample protein concentration.Each sample adds the albumen sample-loading buffer, places the EP pipe, and water-bath saves backup.

C, SDS-PAGE gel electrophoresis

Encapsulating: in the gap, pour into rapidly separation gel, reserve concentrated glue space, cover one deck isopropyl alcohol with pipettor at separation gel solution, gel vertically is put in room temperature.Inclining the cover layer isopropyl alcohol, and the deionized water wash gel is for several times to take out unpolymerized separation gel, with napkin sucking-off residual liquid.

Add concentrated glue, the loading comb is installed.Gel vertically is put in room temperature, shifts out comb.With sample injector application of sample in order, the first hole adds the molecular weight of albumen standard.Order adds sample specimen, and every sample adds albumen and adds to the bottom, whenever adds one and should wash sample injector in the groove buffer.Positive pole connects lower groove, the beginning electrophoresis.Observe bromophenol blue and reach separation gel bottom, powered-down.

Transferring film: after transferring film is finished nitrocellulose filter is put into the plate that fills defatted milk powder solution, hatch in the shaking table, the sealing heterogenetic antigen.Nitrocellulose membrane is put into microscler glass container, add antibody, shaking table spends the night.Put into the glass dish excessive primary antibodie of TBST eccysis.Again film is changed in the microscler glass drying oven, add goat-anti rabbit two and resist.The room temperature shaking table is hatched.Take out film and wash film with TBST again.The BCIP/NBT colour developing, and preserve image.When experiment repeated, conditionally complete was consistent.

According to standard protein molecular weight Maker, downcut gel piece corresponding to desired molecule amount, place transfering buffering liquid.Cut nitrocellulose filter and filter paper, size fits like a glove with the gel size.Nitrocellulose filter is soaked in the transfering buffering liquid, and the expulsion bubble.Keep flat electrode stack filter paper, extrude bubble, nitrocellulose filter is placed on the filter paper, the lower left corner is cut off one jiao and is made marks Accurate align.Gel lies against on the film, the filter paper that superposes again, the side's of putting electrode.Energized, electrotransfer.

4. the TUNEL method detects the apoptosis of lens epithelial cells

The preparation of A, tissue specimen

Get the rat lens of isolated culture, the PBS rinsing sucks moisture at filter paper, then places to be placed with paraformaldehyde, and it is fixing to spend the night.

B, paraffin section TUNEL method operating process

5. flow cytometry detects lens epithelial cell apoptosis

The lens epithelial cells of cultivating is transferred in six orifice plates, be divided into two groups after the cultivation, add TPCK in one group, another group does not add, and then shines under uviol lamp, and is apoptosis-induced.Cultivate, the post-stimulatory cell of trypsinization is collected cell and is done streaming again.

6. statistics and analysis

All data mean ± standard deviation The t check is carried out in expression, applied statistics software, p＜0.01 expression significant difference highly significant, p＜0.05 expression significant difference is remarkable, p〉0.05 expression no difference of science of statistics.

3, result

(1) RT-PCR interpretation of result

The PCR electrophoresis result shows that the band brightness of respectively organizing crystalline lens Trx exists, and significant change is arranged.RT-PCR result carries out quantitative analysis with Imageanalyst software, and the mRNA of Trx has high expressed in the Normal group crystalline lens; 3hr H ₂O ₂The mrna expression of the crystalline lens Trx of oxidative damage is also higher, but 6hr is H ₂O ₂The mrna expression of the crystalline lens Trx of oxidative damage obviously reduces.Not medication group is H only ₂O ₂Oxidative damage group and TPCK group is compared with the blank group respectively, and TTase and the Trx expression of each time point after with TPCK all has statistical significance.

The relative expression quantity of the mRNA of TTase and Trx is compared with matched group at 3 hours and is obviously raised, 6 as a child expression reduce gradually.

(2) Western-Blot interpretation of result

Western Blot marking results of hybridization shows organizes respectively that cornea β-brightness of actin band is similar, and the band brightness of TTase and Trx has significant change.The protein expression level of TTase and Trx all is increased significantly than the crystalline lens group behind the processing 3hr, and TPCK medication group is higher than not medication group; Begin behind the 6hr to go down, but the TPCK group still is higher than not medication group but slightly is lower than Normal group.

(3) TUNEL apoptosis interpretation of result

The crystalline lens epithelial cell only has the positive of trace and dye.H ₂O ₂Oxidative damage 3hr, the visible positive expression of lens epithelial cells, apoptotic cell and dye the brown granular shape, compare with the oxidative damage group, and the apoptotic cell of TPCK processed group is less but still have a little positive to dye.

(4) fluidic cell apoptosis interpretation of result

1. lens epithelial cells cultivation results

After the former generation rat lens epithelial cell line recovery, the 3rd day cellular morphology, cell is almost all adherent, and without the suspension dead cell, the iuntercellular free from admixture has no pollution condition, and lens epithelial cells is rendered as shuttle shape form.Under 100 multiple low power lens, take pictures.

2. flow cytometer detection apoptosis result

Cell flow cytometer detection apoptosis as seen, the lens epithelial cell apoptosis cell number behind the ultraviolet radiation accounts for 30.9% of total cell number; And the apoptotic cell quantity that has added the TPCK intervention only accounts for 20.1% of total cellular score.

4, discussion and conclusion

TTase is the heat-stable protein of 11.8KD, in protokaryon and eukaryote, exist (Holmgren A.Glutathione-dependent synthesis of deoxyribo nucleotides:characterization of the enzymatic mechanism of Escherichia coli glutaredoxin[J] .Biol Chem, 1979,254:3272-3278.).It also is a kind of multi-functional enzyme, at many biochemical processes protein renaturation (Yoshitake S for example, Nanri H, Fernando MR, Minakami S.Possible differences in the regenerative roles played by thioltransferase and thioredoxin for oxidatively damaged proteins[J] .Biochem (Tokyo), 1994,116:42-46.), the reduction of ribonucleotide reductase (William CH, Arscott DL, Muller S.Thioredoxin reductase:two modes of catalysis have evovled [J] .Eur J Biochem, 2000,267:6110-6119.) and polymerization (the Wang G-M of catalytic proteins sulfydryl disulfide, Raghavachari N, Lou MF.Relationship of protein-glutathione mixed disulfide and thioltransferase in H ₂O ₂-induced cataract in cultured pig lens[J] .Exp Eye Res, 1997,64:693-700.) and the activity (Xing KY, the Lou MF.Effect of H that recover glycolysis and oxidation defence key enzyme ₂O ₂On human lens epithelial cells and the possible mechanism for oxidative damage repair by thioltransferase[J] .Exp Eye Res, 2002,74:113-122.).In recent years, TTase also finds to have DHAR activity (Wells WW, Wu D P, Yang YF, Rocque PA.Mammalian thioltransferase (glutaredoxin) and protein disulfide isomerase have dehydroascorbate reductase avtivity[J] .Biol Chem, 1990,265:15361-15364. and Fernando MR, Satake M, Monnier V, Lou MF.Thioltransferase mediated ascorbate recycling in human lens epithelial cells[J] .Invset Ophthalmol Vis Sci, 2004,45:230-237.) and regulation and control (the Stark DW that participated in signal transduction mechanism, Chock PB, Mieyal JJ.Glutathione-thiylradical scavenging and transferase properties of human glutaredoxin (thioltransferase): potential role in redox signal transduction[J] .Bio Chem, 2003,278:14607-14613.).Trx is the heat-stable protein of little 12KD, in high and unicellular lower eukaryote, all exist, its active site has the cysteine residues of two high conservatives, can reduce disulfide bond between the albumen (Holmgren A.Enzymatic reduction-oxidation of protein disulfides by thioredoxin[J] .Methods Enzymol, 1984,107:295-300.).The Trx of oxidation is by TR and reduced-NAD H reduction.TR is the selenoenzyme of homodimer, dependence NADH restoring system that can catalysis Trx, just as other oxidation cellular elements (Berken K of great majority, Gromer S, Schirmer RH, Muller S.Thioredoxin reductase as a pathophysiological factor and drug target[J] .Eur J Biochem, 2000,267:6118-6125.).Play an important role in the oxidation-reduction process of Trx/TR system in most cells, comprise that DNA is synthetic, cell proliferation, growth and differentiation, the regeneration of albumen, and can resist oxidative stress and apoptotic cytotoxin (Mustacichi D, Powis G.Thioredoxin reductase[J] .Biochem, 2000,346:1-8. and Berggren M, Gallegos A, Gasdaska JR, Gasdaska PY, Warneke J, Powis G.Thioredoxin and thioredoxin reductase gene expression in human tumors and cell lines, and the effects of serum stimulation and hypoxia[J] .Anticancer Res, 1996,16:3459-3466.).

The evidence of oxidation is owing to the activity decreased of antioxidant system main in the cataractous lens, and H in discovery cataractous lens and the aqueous humor ₂O ₂Level raises, and Spector etc. (Spector A.Oxidative stressinduced cataract:Mechanism of action[J] .FASEB, 1995,9 (12): 1173-1179.) proposing oxidative damage is the beginning factor that opens of Cataractogenesis.Oxidative stress can be induced the expression of albumen and enzyme numerous in the different cells in addition.Research has been found that the expression of TTase can suffer slight H at human lens epithelial cells ₂O ₂Increase by two to three times of (Raghavachari N under the oxidative damage, Kryzan K, Xing K-Y, Lou MF.Regulation of thioltransferase expression in human lens epithelial cells[J] .Invest Ophthalmol Vis Sci, 2001,42:1002-1008.), this rise mediates (Kryzan K by a series of signal transduction mechanism, Lou MF.Human thioltransferase (TTase) gene is controlled by AP-1 and mediated through redox signaling[J] .Invest Ophthalmol Vis Sci, 2002,43:1876-1883.).Trx also is (the Raghavachari N that causes the up-regulated in the human lens epithelial cells by identical mode under the stimulation of oxidative stress, Lou MF.Evidence for the presence ofthioltransferase in the lens[J] .Exp Eye Res, 1996,63:433-441.).There is the expression that studies show that Trx that certain rise phenomenon (Reddy PG also occurs when the crystalline lens of Emory mice suffers from photooxidation, Bhuyan DK, Bhuyan KC.Lens-specific regulation of the thioredoxin-1 gene, but not thioredoxin-2 upon in vivo photochemical oxidative stress in the Emory mouse[J] .Biochem Biophys Res Commun, 1999,265:345-349.).Lou infers that the temporary transient rise phenomenon of TTase and Trx stress (0.2Mm H in the low concentration oxidation in the pig crystalline lens of cultivating in 2005 ₂O ₂) occur in 6 hours of cultivation, but under oxidative stress Continuous Damage condition, the defense system collapse of TTase and Trx, express and reduce gradually (Moon S, Rohan Fernando M, Lou MF.Induction of thioltransferase and thioredoxin/thioredoxin reductase systems in cultured porcine lenses under oxidative stress[J] .Invest Ophthalmol Vis Sci, 2005,46:3783-3789.).The experimental result prompting of inventor this part, the expression of TTase and Trx is at H ₂O ₂Oxidative damage 3hr has certain rise phenomenon, and 6hr obviously reduces, and this is similar with before all results of study, just at H ₂O ₂The critical point that the expression of concentration and TTase and Trx is increased disagrees, and this is different relevant from used laboratory animal and experiment condition.And inventor's experiment also is provided with the TPCK intervention group, oxidative stress group at these two kinds of enzyme contents of 6hr, the TPCK intervention group shows TTase and Trx expresses the relatively high level that still is in, this may point out TPCK to have certain inductive effect for the expression of TTase and Trx, when crystalline lens suffers long-term oxidative stress situation, TTase in the body, Trx is not enough to remove the non-specific aggregation of albumen, there are not enough abilities to keep the lenticular transparency, and TPCK is by indirectly raising the expression of these two kinds of enzymes, simultaneously self is to the alkylating of protein disulfide, can reduce crosslinked between the crystallin disulfide bond of oxidated damage.

Under the oxidative stress condition of certain hour, the expression of TTase and Trx and active increasing for Cell protection are necessary, they are by repairing some crucial enzyme or albumen in the oxidative damage, for example glutathione peroxidase, hydrogen peroxide detoxication enzyme (Xing K, Lou MF.The possible physiological function of thioltransferase in cells[J] .FASEB, 2003,17:2088-2090.) etc.; TTase can also strengthen the recycling ability of Ascorbate to reduce accumulation (the Fernando MR of hydroascorbic acid in the cell of poisoning, Nanri H, Yoshitake S, Nagata-Kuno K, Minakami S.Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells[J] .Eur J Biochem, 1992,209:917-922.).Trx can pass to H to electronics from hydrogen-peroxide reduction albumen there ₂O ₂Destroy combination (the Yoshitake S of it and protein disulfide, Nanri H, Fernando MR, Minakami S.Possible differences in the regenerative roles played by thioltransferase and thioredoxin for oxidatively damaged proteins [J] .Biochem (Tokyo), 1994,116:42-46.), remove hydrogen peroxide (the Chae H of toxicity, Robison K, Poole L, Church G, Storz G, Rhee S.Cloning and sequencing of thiol-specific antioxidant from mammalian brain:alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes[J] .Proc NatlAcad Sci USA, 1994,91:7017-7021.), thereby the albumen/enzyme of the oxidative damage of having regenerated.Inventor's experimental result also demonstrates in front 3 hours, and the active of these two kinds of enzymes obviously improves.Under oxidative stress status, be used for Cell protection.But under oxidative stress damage for a long time, the so lasting powerful infringement of can not loading of the enzyme of these repair systems, cause active reduction own and decay (the Moon S of content, Rohan Fernando M, Lou MF.Induction of thioltransferase and thioredoxin/thioredoxin reductase systems in cultured porcine lenses under oxidative stress[J] .Invest Ophthalmol Vis Sci, 2005,46:3783-3789.).The inventor has also observed identical phenomenon in the experiment of this part, time after 3 hours, the content of TTase and Trx reduces gradually, monitored significant reduction (compare difference with matched group significant difference is arranged) in 6 hours, lenticular opacity is progressively deepened in time subsequently, develops into total cataract, and the nuclear district is obviously muddy, muddy irreversibility, these reparation enzymes also can't be discharged duties again and be brought into play the antioxidative effect this moment.The experimental result of this part is coincide therewith, also is a bright spot of second portion experiment, has shown under the effect of TPCK this two kinds of enzymes ageing change when crystalline lens suffers oxidative stress of TTase, Trx.

Many apoptosis mechanism that studies show that might be that some cataract are such as Cytological Basis (Li WC total in the radiational cataract forming process in the past, Kuazak JR, Dunn K.Lens epithelial cell apoptosis appears to be a common cellular basis for noncongenital cataract development in human and animals[J] .Cell Biol, 1995,130:169-181.).Apoptosis betides lens epithelial cells at first, so that lens epithelial cells comes off from epithelial layer, its density lowers, thereby a little less than keeping the homeostatic effect slightly of crystalline lens, can also so that the structure of crystallin change, thereby the increase of the cross-linked polymeric between the albumen causes light scattering to strengthen, and the crystalline lens transparency lowers.And TPCK is as a kind of anti-apoptosis agent, verifiedly can intervene many pathophysiological processes, for example can dwindle the Infarction volume behind the transient focal cerebral ischemia, other has many research to find that TPCK by the activity of some protease in the blocking-up cell, has brought into play certain inhibitory action to antiapoptotic factors in the various kinds of cell.Carry out the TUNEL apoptosis based on this this part experiment by the crystalline lens on isolated culture and detect to observe TPCK for the impact of lens epithelial cell apoptosis; results suggest; under the oxidative stress condition, TPCK can Cell protection to a certain degree avoids suffering the apoptosis under stress situation.In the streaming apoptosis analysis of the lens epithelial cells of former culture, also can see identical result, for the lens epithelial cells that suffers at first the oxidative stress damage, the apoptosis quantity that has added the lens epithelial cells of TPCK intervention obviously is less than the quantity of oxidative stress damage group.Preliminary analysis, TPCK may suppress the apoptosis of the different cells that many factors causes, lens epithelial cells be can block and superoxide anion and some other toxic cell factors when suffering oxidative damage, discharged, these superoxide anions and the toxic cell factor have caused the disorder of crystalline lens metabolism just, for example make the ion channel function imbalance on the lens epithelial cells film, the inside and outside ion distribution level of cell is unbalance, and then imbibition has caused the preliminary generation of lenticular opacity.

In view of front two parts to the protective effect of TPCK to lenticular opacity, strongly attract the inventor to think deeply from the sulfydryl join protection, but not the passive reproducibility sulfydryl of keeping starts with, new way and mechanism of action that research health crystallin is assembled.(be called for short: UPP) impact for Cataractogenesis has had new progress to the Ubiquitin-proteasome path recently, UPP is relatively more classical signal transduction pathway, in the past more research is to concentrate on Tumorigenesis and treatment aspect, there has been research that proteasome inhibitor is used for tumor and has obtained certain effect (Orlowski RZ, Dees EC.The role of the ubiquitination-proteasome pathway in breast cancer:applying drugs that affect the ubiquitin-proteasome pathway to the therapy ofbreast cancer[J] .Breast Cancer Res, 2003,5:5-7.).Thereby up-to-date about the cataractous UPP of studies show that can by the impaired crystallin of fast degradation stoped its in crystalline lens gathering prevent muddy generation (Xinyu Zhang, Edward J.Dudek, Bingfen Liu, Linlin Ding, Alexandre F.Fernandes, Jack J.Liang, Josepb Horwitz, Allen Taylor, Fu Sbang.Degradation of C-terminal truncated α A-crystallins by the ubiquitin-proteasome pathway[J] .Invest Ophthalmol Vis Sci, 2007,48:4200-4208.).About it concrete treatment effect countries in the world of cataract be there is not yet relevant research report.In conjunction with up-to-date research trends, the inventor imagines this classical protein quality control mechanism of UPP and has exercised the effect that is similar to the DTT compounds, the impaired disulfide bond of degeneration that namely can dissociate and form, the aggregation extent of reduction protein.The intervention effect that previous experiments has been observed DTT is considerably beyond excellent such as the card woods with dosage GSH or other clinical eye drops commonly used, and this has pointed out the disulfide bond that dissociates, and to keep the reduced form sulfydryl more effective than passive.

In cataractous generating process, the gathering of protein and precipitation are reasons of lenticular opacity.In crystallin, α crystallin and β crystallin are all responsive to the degraded of protein lyase, the C end that the α crystallin is hydrophilic, the most N of β crystallin end can be by the protein cleavage enzymatic degradation, and these hydrophilic end play an important role to the water solublity of crystallin.The γ crystallin is relatively stable, and it is considered to a kind of structural protein in the crystalline lens at first, relatively is not easy to be subject to the degraded of protein lyase.Cysteine (the Cysteine that all contains some in three kinds of crystallins, Cys), free sulfhydryl groups among the Cys (SH) suffers oxidative damage (Lou MF.Redox regulation in the lens [J] .Progress in Retinal and Eye Research extremely easily, 2003,22 (5): 657-682.) further assemble the formation disulfide bond, cause the degeneration precipitation of albumen, the generation of lenticular opacity.The newest research results of Regius professor's ophthalmology shows alpha-crystal albumen and contains the Reducing agent combined effect of sulfydryl, can make in the human cataracts crystalline lens the active enzyme of having lost (the Schey KL that " comes back to life ", Little M, Fowler JG.Characterization of human lens major intrinsic protein structure[J] .Invest Ophthalmol Vis Sci, 2000,41 (1): 175-183.).α crystallin and Tensiomin make glutathione reductase activity " rise " 79%, and α crystallin and dithiothreitol, DTT (DTT) can make the active increase by 200% of thioltransferase.This has further disclosed the importance of chaperone activity in keeping the crystalline lens metabolism of alpha-crystal albumen, molecular chaperones can " be saved " class because protein Misfolding is accumulated the conformation disease that causes, and clinical practice Drug companion treats the conformation disease and obtained gratifying result first.The α crystallin is being brought into play multi-functional effect in the transparency, and it is required that the oligomeric property of the characteristic of α crystallin and protein is kept the crystalline lens pellucidity.Horwitz(Horwitz J.Alpha crystallin can function as a molecular chaperone[J in 1992] .Natl Acad Sci, .1992,89:10449-10453.) the cataractous relation of α crystallin age-dependent proposed: the effect of α crystallin by molecular chaperones is corrected in false folding in the crowded fibrocyte of aging or the albumen of impaired degeneration, the aggregate and precipitate that suppresses successively Denatured protein is kept lenticular transparent.Given this degraded of α crystallin has participated in the generation of lenticular opacity, the α crystallin fragment aggregate and precipitate in crystalline lens that sustains damage and degraded by protease, finally can cause the crystalline lens insoluble protein to increase, transmittance lowers, and muddiness produces gradually.α A _1-162Crystallin is the main degradation products of m-protein lyase, and studies show that out that its content in general normal crystalline lens is few, (Thampi P appears in the crystalline lens of diabetic cataract, Hassan A, Smith JB, Abraham EC.Enhanced c-terminal truncation of α A-and α B-crystallins in diabetic lenses[J] .Invest Ophthalmol Vis Sci, 2002,43:3265-3237.), this has illustrated that also it only is being subjected to oxidative stress, ionizing radiation, when medicine or toxicity stimulate the damage of high sugared state for example, just can occur and assemble easily and then cause lenticular muddiness.

Therefore inventor's imagination when the new way that the Effect of Anti crystallin is assembled: the gathering of crystallin whether depend primarily on whether form in the molecule or intermolecular disulphide crosslinked, and keep dissociating-SH just stops above-mentioned crosslinked possible condition, and nonessential condition.The experimental result of this part shows, under the stimulation of ultraviolet radiation, can detect α A in the lens epithelial cells _1-162Crystallin, this is the sheet segment molecule that the α A crystallin in the lens epithelial cells is produced by protease cracking under the damage of oxidative stress, this fragment since expose cysteine in this body structure-SH, so that easy coupling polymerization between the fragment, the albuminous degeneration aggregate and precipitate causes lenticular muddiness.There are some researches show that the chemical proteasome inhibitor of using the UPP system in lens epithelial cells can make the level rise (the Dudek EJ of impaired protein, Shang F, Valverde P, Liu Q, Hobbs M, Taylor A.Selectivity of the ubiquitin pathway for oxidatively modified proteins:relevance to protein precipitation diseases[J] .Faseb J, 2005,19:1707-1709.).And designed a kind of siRNA Ubc4(ubiquitin polymerase E2 of the UPP of rise activity the inventor) and be transfected into lens epithelial cells after, increased the activity of UPP, the result shows and can remove impaired and by the small fragment α A of protease cracking _1-162Crystalline lens is than the cell α A wherein of untransfected _1-162The content of crystallin obviously reduces.Ubc4 is the ubiquitin polymerase in a kind of UPP system, because its content in lens cell is limited, the inventor constructs the gene take liposome as carrier and passes through the RNA transfer techniques, these speed limit compositions of overexpression in crystalline lens, increased the protein degradation efficient of UPP, so that even the micromolecule degradation fragment of crystallin obtains removing, stop the formation of disulfide bond between these impaired crystallins, non-specific crosslinked, gathering enhancing formation high molecular weight material (abbreviation: HMW), keep the lenticular transparency.

In front two parts experiment, the inventor has observed the generation that TPCK can to a certain degree delay crystalline lens muddy degree under the oxidative stress damage, and can stablize crystallin under oxidative stress damage condition.And crucial antioxidase TTase in the lens epithelial cells and the expression of Trx have been improved.

In a word, by whole experimental result, the inventor learns that TPCK can regulate and control the protein in the crystalline lens, has brought into play certain function in cataractous forming process.Cataractous pathogenesis is extremely complicated, the result that the inside and outside multiple factors of body produces the crystalline lens comprehensive function, crystalline lens all is in dynamic differentiation process in whole life route, its normal differentiation state is prerequisite (the Nakamura T that keeps transparency, Pichel JG, Williams-Simons L.An apoptotic defect in lens differentiation caused by human p53 is rescued by a mutant allele[J] .Pro Natl Acad Sci, 1995,92 (13): 6142-6146.), break up the not normal cataract that then forms.The albumen that contains free sulfhydryl groups in the crystalline lens is more, if by the control concentration of alkylating reagent and activity, partially enclosed sulfydryl, crystalline protein is no longer occured or that disulphide occurs less is crosslinked, should stop or reduce protein aggregation, maintenance crystal light transmission.According to above-mentioned hypothesis, inventor's design is carried out alkylation with the sulfydryl of crystalline protein.By the enclosure portion sulfydryl, both can suppress the formation of a large amount of disulphide; The combination that also can be at war with to the disulphide polymer that has formed impels to reopen disulfide bond.Can carry out alkylating reagent to the sulfydryl of protein has a lot, such as iodoacetamide, iodomethane, DNF, benzoyl halide, chloromethyl ketone and derivant thereof etc.And the methyl ketone structure among the TPCK has been born such effect just; can be to the coordinating protection effect of GSH associated metabolic regulatory enzyme or albumen (TTase, Trx/TR etc.) when the inventor also detects TPCK and delays lenticular opacity, thus can long oxidation stress stimulation under raise these two kinds of enzymes activity reach the antioxidative effect.

The preparation method of the common drug preparation of TPCK and compositions thereof

The present invention prepares injectable powder and generally adopts conventional freeze-drying, as solvent, the steps include: to get TPCK with water, adds excipient, is dissolved in water, and regulates pH, adds active carbon, filtration sterilization, and plug is partly rolled in fill, and lyophilization, tamponade are rolled lid and are got final product.Used excipient is selected from one or more in mannitol, gelatin hydrolysate, glucose, lactose, dextran, albumin, the pH adjusting agent etc.Every bottle contains TPCK0.1 ~ 4mg.

The present invention prepares injectable powder also can adopt spray drying method, as solvent, the steps include: to get TPCK with water, adds or do not add excipient (excipient is the same), is dissolved in water, and adds active carbon, filtration sterilization, and spray drying, aseptic subpackaged, tamponade is rolled lid and is got final product.Every bottle contains TPCK0.1 ~ 4mg.

When the present invention prepares small-volume injection, preparation gets final product as solvent with water for injection, also can add appropriate amount of auxiliary materials, adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzoic acid, dimethyl acetylamide, pH adjusting agent, surfactant, cyclodextrin, antioxidant, complexing of metal ion agent, the antibacterial.Injection can be mixed with solution, microemulsion, emulsion, liposome, microsphere, microcapsule or other is suitable for the ordered structure of high drug level, wherein can comprise the medicament that postpones absorption, such as Monostearate, gelatin, ethylene-ethyl acetate, polyanhydride, polyglycolic acid, collagen protein, polyorthoesters or polylactic acid etc. absorbs with the prolongation that reaches injectable composition.Every contains TPCK0.1 ~ 4mg.

The present invention prepares glucose infusion liquid or sodium chloride transfusion, with water for injection as solvent, adding the preparation of an amount of glucose or sodium chloride gets final product, also can add appropriate amount of auxiliary materials, adjuvant is selected from one or more in ethanol, propylene glycol, glycerol, Polyethylene Glycol, benzoic acid, dimethyl acetylamide, pH adjusting agent, surfactant, antioxidant, cyclodextrin, complexing of metal ion agent, the antibacterial.Every bottle contains TPCK0.1 ~ 4mg.

The present invention prepares the oral formulations such as tablet, capsule, granule, oral liquid, and adjuvant can be lactose, starch, dextrin, stearate etc., routinely technology preparation.Can comprise the high molecular polymer carrier, such as hydroxypropyl methylcellulose or polyoxyethylene etc., discharge with the prolongation that reaches Orally administered composition.

In the present invention, the embodiment of the above-described specific embodiment and the following stated all is in order to set forth better the present invention, is not to limit scope of invention.

Below by embodiment the present invention is described in detail.

Used animal, instrument and equipment, reagent and preparation thereof etc. all are from above-mentioned description or meet above-mentioned requirement in the experiment of following examples.

Embodiment 1, TPCK are on the impact of the muddy degree of crystalline lens oxidative stress and heat stability

1, materials and methods

(1) laboratory animal

10 of healthy SD rats, male, 6W age is about body weight 180g.Provided by the The 2nd Army Medical College Experimental Animal Center.The cleaning level is also raised in animal center.

(2) key instrument and consumptive material

(3) main agents

(4) preparation of main agents and solution

1. TC-199 culture fluid

Get one bag of TC-199(9.5g/ bag), add 2.2gNaHCO according to explanation ₃, add distilled water and mend to 1L, regulate pH value to 5.5.Deposit in 4 ℃.Use front Entkeimung.

2. the TPCK solution of 0.25mM

TPCK molecule formula weight is 351.84.Get the TPCK powder of a 100mg, be dissolved in the distilled water of 100ml, add again the TWEEN80(of 0.1ml as a kind of solubilizing agent, can promote to dissolve the effect of TPCK), be the solution of 2.8mmol/L(1/351.84 * 1000=2.8mmol/L).Deposit for 4 ℃.Use after the Entkeimung in super-clean bench with 10 times of front dilutions.

3. glucose oxidase thing (glucose oxidase, GO, 0.025U/ μ l)

Get GO(and contain 10KU) add the dd H of 1ml ₂O is mixed with 10U/ μ l.Take out the distilled water that 10 μ l add 90 μ l, this moment, concentration was 1U/ μ l.With 40 times of front dilutions, making its final concentration is 0.025U/ μ l.-20 ℃ of preservations.With Entkeimung in the front super-clean bench.Add GO in order to keep stable H ₂O ₂Concentration.

④H ₂O ₂（0.24mM）

H ₂O ₂Density be 1.122g/ml, the molecule formula weight is 34, so (1.122 * 30% * 100/34)/0.1=9.9mol/L is the 10mol/L that thinks generally that the chances are.Be mixed with 0.24mM concentration, getting about 40 times of 1 μ l dilutions is the H of 0.24mM ₂O ₂, (1 * 10 ^-3* 10/0.24)=42ml.Deposit under the room temperature.Use front Entkeimung.

5. (Phosphate-Bufer-Solution is called for short: PBS) 10 * phosphate buffer

With the distilled water dissolving of 800ml, regulate pH value to 7.2～7.4, be settled to 1000ml, 4 ℃ of preservations are with front 10 times of dilutions.

6. crystallin lysate

A liquid is Tris (MW 121.14) 2.428g of 0.2mol/L, adds 100ml distilled water (ddH ₂O); B liquid is that HCL (37%, the 1.19) 0.84ml of 0.1mol/L adds 100ml distilled water (ddH ₂O).Then A liquid 25ml is evenly mixed with B liquid 41.4ml, regulating acid-base value is 7.4 to PH, and this solution is the crystallin lysate.

(5) experimental technique

1. crystalline lens isolated culture

A, animal crystalline lens are prepared

10 of healthy SD rats, male, 6W age is about body weight 180g.(the cervical vertebra dislocation method is put to death after 0.6～0.7/100g) intraperitoneal anesthesia to adopt 10% chloral hydrate, around the 70% alcohol wipe Dual-eyelid, take out eyeball, put into PBS and clean blood, rinsing be placed on for twice under the super-clean bench aseptic condition with micro-shears corneoscleral junction partially afterwards about 0.2mm cut off at the place, carefully extend to both sides in the breach place, again with two microforcepses tractive breach both sides crystalline lens of carefully giving birth to gently.

B, method

Take out complete crystalline lens and put into PBS, remaining on every side vitreous body is carefully peeled off, again crystalline lens careful rolling back and forth on filter paper of rinsing blotted surface moisture and the glutinous unnecessary impurity that connects several times, afterwards crystalline lens is put into the TC-199 culture fluid through Entkeimung, move into 24 well culture plates after soaking the several seconds, add 600 μ l TC-199 culture fluid, put into 37 ℃, 5%CO ₂Then incubator 2 hours adds the H of 0.04mM ₂O ₂GO with 0.025U/ μ l makes H ₂O ₂Final concentration is about 0.02mM, and experimental group adds the TPCK of 100 μ l again, and matched group only adds culture fluid.

C, grouping

H ₂O ₂Oxidative stress is respectively matched group and experimental group, every group of 10 SD rat lens with the other TPCK treatment group that adds.

2. external evoked crystallin thermal denaturation

Prepare before A, the mensuration

Get and only add H ₂O ₂Each 2 in the SD rat lens of oxidative stress and adding TPCK treatment group crystalline lens, move into respectively in the 2ml homogenate tube, add the 1.8ml protein lysate, place and use repeatedly homogenate of electric homogenizer (to note every stopping homogenate about 30s and after leaving standstill 30s on the ice chest, continuing homogenate on the ice chest, be interrupted homogenate), after grinding evenly, 12,000rpm, 4 ℃ are centrifugal 20 minutes, measure protein concentration with BCA method, microplate reader 540nm wavelength after getting the supernatant dilution, and two groups of rat lens albumen supernatant are transferred to same concentration (7.5 μ g/ μ l).

B, method

The equal serosity of the crystallin that concentration level is consistent moves into 1cm optical path cuvette, volume 2ml; Constant water bath box is preheated to 50 ℃, and sample is at first measured initial absorbance in 320nm wavelength place, and distilled water is done reference, after the heating, measures the absorbance at a 320nm wavelength place, and does reference with distilled water in per 5 minutes.

(6) statistics and analysis

All data mean ± standard deviation Expression is used the medical statistical software of SPSS18.0 and is carried out the t check, p＜0.01 expression significant difference highly significant, p＜0.05 expression significant difference is remarkable, p〉0.05 expression no difference of science of statistics.

2, experimental result

(1) crystalline lens isolated culture

First three hour of crystalline lens isolated culture in TC-199, observe through H ₂O ₂The crystalline lens of induced oxidation damage and TPCK matched group crystalline lens are all without obviously changing; Afterwards, along with the prolongation of time, H ₂O ₂Induce damage group crystalline lens the muddiness that increases the weight of gradually to occur, develop into whole bark matter even full lenticular opacity from the front or rear cortex muddiness that begins, the muddy degree of rat lens that adds TPCK in the same time all is lower than in not dosing group rat (Fig. 1).

(2) external evoked crystallin thermal denaturation

1. the BCA method is measured protein concentration

According to 7 protein standard substances (concentration is respectively 0.025,0.05,0.1,0.2,0.3,0.4,0.5 μ g/ μ l), measures 540nm place absorbance and be respectively 0.096,0.138,0.163,0.251,0.302,0.377,0.500.The drawing standard curve sees that Fig. 2 (annotates: R in the formula ²Value is more better near 1).

2. thermal stability determination

In one hour experiment, H ₂O ₂The muddy degree of the supernatant of the crystalline lens mixed protein of induced oxidation damage increases very fast, and the absorbance at the 320nm place rises very fast, and curve is precipitous, and the anti-oxidative damage ability descends; H ₂O ₂The crystalline lens mixed protein supernatant that has added TPCK in the time of the induced oxidation damage becomes muddy gradually after water-bath, degree slowly increases the weight of, and the absorbance at the 320nm place rises slower, the slope of curve low (Fig. 3).

3, conclusion

Oxidative stress and thermal induction degeneration are the risk factors of pathogenesis of cataract.

Embodiment 2, TPCK are on the impact of crystalline lens antioxidase and apoptosis situation

1, materials and methods

(1) laboratory animal

30 of healthy SD rats, male, 6W age is about body weight 180g.Provided by the The 2nd Army Medical College Experimental Animal Center.The cleaning level is raised in animal center.

(2) key instrument and consumptive material

30 of healthy SD rats, male, 6W age is about body weight 180g.Provided by the The 2nd Army Medical College Experimental Animal Center.

The cleaning level is raised in animal center.

(3) main agents

β-actin, TTase, Trx primer synthesize by the prompt base in the English Weihe River, Shanghai (Invitrogen Trading Co., Ltd) trade Co., Ltd.

(4) preparation of main agents and solution

1. (Phosphate-Bufer-Solution is called for short: PBS) 10 * phosphate buffer

Ditto.

2. (Tris HCL-Buffer-Solution is called for short: TBS) the TBS-T buffer

NaCl 8g

KCL 2g

Tris 3g

Water distilled water with 800ml dissolves fully, regulates pH value to 7.4, is settled to 1000ml, high pressure-temperature sterilization, this is the TBS buffer, TBS-T is that the TWEEN(TWEEN that adds again 1ml is a kind of non-ionic detergent, the effect of renaturation antigen is arranged, can improve specific identification ability).Preserve under the room temperature.

3. 10mg/ml ethidium bromide (Ethidium bromide, EB)

The 100mg ethidium bromide is dissolved in the distilled water of 10ml fully and get final product, and keeps in Dark Place in 4 ℃.

4. 2 * sds gel sample loading buffer

Be made into first the 2 * SDS sample loading buffer room temperature preservation that does not contain DTT, existing with now being added on the above-mentioned buffer from the DTT storage liquid (20 ℃ of preservations) of 1mol/L before use.

5. crystallin lysate

Same first.

6. 30% propylene phthalein amine

Take by weighing propylene phthalein amine 29g, N, the two ammonium acrylate 1g of N '-methylene add distilled water 60ml, are heated to add distilled water after 37 ℃ of dissolvings and be settled to 100ml, filtration sterilization.Put 4 ℃ of preservations in the brown bottle.

7. 10% sodium lauryl sulphate (is called for short: SDS)

Claiming the 10g sodium lauryl sulphate to add distilled water 90ml is heated to 68 ℃ and adds and add distilled water to 1000ml after concentrated hydrochloric acid is regulated PH to 7.2.

8. concentrate glue buffer (1mol/L Tris-HCL pH=6.8)

12.12g Tris is dissolved in the 80ml distilled water, with adding water to 100ml, 4 ℃ of preservations behind the concentrated hydrochloric acid adjusting pH to 6.8.

9. separation gel buffer (1.5mol/L Tris-HCL pH=8.8)

18.16g Tris is dissolved in the 80ml distilled water, with adding water to 100ml, 4 ℃ of preservations behind the concentrated hydrochloric acid adjusting pH to 8.8.

10. 10% Ammonium Persulfate 98.5 (is called for short: AP)

Be made into 10% a small amount of storage liquid and 4 ℃ of preservations with distilled water, because slowly decomposition of Ammonium Persulfate 98.5, preferably matching while using.

⑾ confining liquid

5% defatted milk powder is dissolved among the TBST.

⑿ 5 * Tris-glycine electrophoretic buffer

Tris 125mmol/L 15.1g

Glycine 1.25mol/L 94g

SDS 10%50ml

Add distilled water to 1000ml, dilution is 5 times during use, pH=8.3.

⒀ 5 * transfering buffering liquid

Add distilled water to 1000ml, dilution is 5 times during use.

⒁ lens epithelial cells culture fluid

Comprise in every 50ml culture fluid in the DMEM F12 culture medium of 45ml, and the 5ml hyclone (Fetal bovine serum, be called for short: FBS), three anti-(they being 10%FBS, 1% 3 kind of antibiotics mixture) of 0.9ml.

(5) experimental technique

1. the cultivation of crystalline lens isolated culture and lens epithelial cells

A, animal crystalline lens are prepared (same first)

30 of healthy SD rats, male, 6W age is about body weight 180g.(the cervical vertebra dislocation method is put to death after 0.6～0.7/100g) intraperitoneal anesthesia to adopt 10% chloral hydrate, around the 70% alcohol wipe Dual-eyelid, take out eyeball, put into PBS and clean blood, rinsing be placed on for twice under the super-clean bench aseptic condition with micro-shears corneoscleral junction partially afterwards about 0.2mm cut off at the place, carefully extend to both sides in the breach place, again with two microforcepses tractive breach both sides crystalline lens of carefully giving birth to gently.

B, crystalline lens isolated culture method

Take out complete crystalline lens and put into PBS, remaining on every side vitreous body is carefully peeled off, again crystalline lens careful rolling back and forth on filter paper of rinsing blotted surface moisture and the glutinous unnecessary impurity that connects several times, at last crystalline lens is placed the TC-199 culture fluid through Entkeimung, move into 24 well culture plates after soaking the several seconds, add 600 μ l TC-199 culture fluid, put into 37 ℃, 5%CO ₂Incubator 2 hours is rejected and is cultivated the crystalline lens (may be because operation is damaged to crystalline peplos) that becomes muddy after 2 hours, then transfers in the 24 new orifice plates, adds the H of 0.24mM ₂O ₂GO with 0.025U/ μ l makes H ₂O ₂Final concentration is roughly 0.02mM, and experimental group adds the TPCK of 100 μ l again, and matched group only adds culture fluid.

C, lens epithelial cells cultural method

Take out freeze-stored cell from liquid nitrogen, constantly rock in 37 ℃ of water baths until dissolve, taking the Cell sap of dissolving in the super-clean bench wherein, Cell sap (1ml) carefully is drawn onto in the 15ml centrifuge tube with pasteur pipet, the culture fluid mixing that adds again 4ml, low-speed centrifugal, 1000rpm, 5min.Discard supernatant after centrifugal, add immediately the 2ml culture fluid, pasteur pipet piping and druming is mixed into single cell suspension (as far as possible avoiding producing bubble).Add the culture fluid about 3ml in the preprepared culture bottle, the single cell suspension that piping and druming is good is added in the culture bottle.Whether microscopically observation of cell state is blown and beaten evenly.The cell that recovery is good is put into 5% CO ₂Cultivate in the incubator.The next day observation of cell growth conditions.Continue behind the cell attachment to cultivate, changed a Cell sap, went down to posterity once in 6～7 days in 2～3 days.The third generation cell state that goes down to posterity is best, namely can be used for experiment.

D, lens epithelial cells oxidative stress damage method

Lens epithelial cells is without H ₂O ₂The oxidative stress damage method, because lens epithelial cells is relatively more fragile, H ₂O ₂Oxidative damage may cause irreversible damage, even also can't reverse with interfering effects of drug.So the inventor adopts ultraviolet irradiation to cause the oxidative stress damage.With cultured cells uncap in super-clean bench, about the distance irradiation 30min apart from the about 10cm of Burdick lamp.

E, grouping

H ₂O ₂Oxidative stress is respectively matched group and experimental group with adding in addition the TPCK treatment group, and the crystalline lens that simple culture of ex vivo crystalline lens is not done any processing is blank group, every group of 10 SD rat lens.Immunohistochemistry detects with the experiment of TUNEL apoptosis same as described above.Lens epithelial cells is cultivated and is divided into H ₂O ₂Oxidative stress and TPCK intervene, and are respectively matched group and experimental group.

2. RT-PCR detects the mrna expression of TTase and Trx

A, the total RNA of crystalline lens extract

The crystalline lens of getting isolated culture 3hr, 6hr, 12hr places the homogenate tube of 2ml, and the Trizol electric homogenizer that every pipe adds the 1ml pre-cooling carries out homogenate.

B, Trizol one step extraction process

Operating process is seen Figure 11.

The evaluation of C, nucleic acid samples

The purity testing of nucleic acid samples

The purity spectrophotometric determination of nucleic acid samples.The reading of observation nucleic acid samples under 260nm and two wavelength of 280nm (is called for short: OD) value, judge the purity of nucleic acid samples according to the ratio of OD 260/OD 280, ideal ratio is 1.5～1.8, shows that RNA solution purity is high, contain protein, DNA impurity is less, is fit to the RT-PCR experiment.Calculate the concentration (μ g/ μ l) of RNA sample according to formula:

Concentration=OD 260 * nucleic acid extension rate of RNA sample * 40/1000

The detection of RNA sample integrity

Adopt RNA denaturing formaldehyde gel electrophoresis, operating process is seen Figure 12.

Points for attention

Whole process is worn disposable glove, avoids RNA enzymatic degradation RNA and causes the pollution of RNA, uses disposable plastic vessel and the automatic pipet(te) extracting RNA of sterilization, avoids using public instrument to cause the cross-contamination of RNA enzyme.

D, design of primers

Utilize on the internet Pubmed to search the genes of interest total length, Jie Ji trade Co., Ltd designs and synthesizes by the English Weihe River, Shanghai.Design result compares with Blast, guarantees its specificity.Total amount 2ODU ₂₆₀, adopt PAGE mode purification, effectively removed non-full length fragment, primer efficient is high.The employing distilled water redissolves, then forward primer 10 μ l and downstream primer 10 μ l mix homogeneously, and (this is that 20 ℃ of PCR Primer Mix) ， – deposit to add the DEPC water of 30 μ l again.

β-actin detects primer:

Forward primer 5 '-AGCCTTCCTTCTTGGGTA-3 '

Downstream primer 5 '-TCAGTAACAGTAAGCCTA-3 ', product are 338bp;

TTase detects primer:

Forward primer 5 '-CAAATTTCCGGCAGTGTGTGTC-3 '

Downstream primer 5 '-GAGCCATGCAATGGAGTCTGAGTA-3 ', product are 87bp;

Trx detects primer:

Forward primer 5 '-TACAACAGCTCACCGGAGCAAG-3 '

Downstream primer 5 '-GGCCATTAGCATGGCTGGAC-3 ', product are 194bp.

(be called for short: RT-PCR) E, reverse transcription-polymerase chain reaction

At first carry out reverse transcription reaction, number is prepared the reverse transcription reaction pipe per sample, adds first the following reagent of total system 20 μ l, the reverse transcription reaction condition be (37 ℃, 15min * 3, this is reverse transcription reaction; 85 ℃, 5sec, this is the inactivation reaction of reverse transcription):

F, polymerase chain reaction

The following reaction reagent that adds cumulative volume 30 μ l:

Amplification condition is as follows:

Arrange 62 ℃ up and down about 2 ℃ of scopes interior 4 (namely 59 ℃, 60 ℃, 62 ℃, 63 ℃ four annealing temperatures) in annealing temperature before the upper simple PCR machine, carry out simple PCR, in the hope of groping optimum annealing temperature.

G, agarose gel electrophoresis

Glue: 50 * TAE electrophoretic buffer 1ml that packs in conical flask, use dd H ₂O 49ml and 0.5g agar Icing Sugar.Heating makes the agarose dissolving in microwave oven, makes solution be cooled to 60 ℃, and adding EB is 0.5 μ g/ μ l to final concentration, fully mixing.Comb is placed in position at distance base plate 0.5～1.0mm, and warm agarose solution is poured in the rubber moulding, and gel thicknesses and checks under the tooth of comb or whether between cog has bubble between 3～5mm.(place 30～45min) in room temperature, carefully remove comb, gel is put into electrophoresis tank, adding there was not the dark capacity electrophoretic buffer of the about 1mm of glue face just after gel solidifies fully.

Electrophoresis: after getting 10 μ l PCR products and sample loading buffer mixing, slowly mixture is added in the sample cell with micropipettor, wherein the first swimming lane adds DNA ladder.The voltage of will switching on transfers to 110mv, makes DNA anode (red line) mobile.Electrophoresis to bromophenol blue and the blue or green FF of dimethylbenzene moved out of suitable distance in gel.Cut off the electricity supply, take out glue observed result and taking pictures under uviol lamp.

The result judges: present white fluorescent under the ultraviolet light, can see the position of migrating of band, the gel images photograph determines that the mRNA of TTase/Trx and confidential reference items β-actin has the rear upper RT-PCR instrument of expression.

H、Real-time?PCR

Use SYBR Green fluorescent dye imbedding method to carry out the Real time pcr amplification of above-mentioned purpose gene.Reaction system is as follows:

RT-PCR is according to the optimal temperature conditions of above-mentioned electrophoresis: 95 ℃, and 2min → (95 ℃, 15s → 62 ℃, 30s → 72 ℃, 15s) circulation is 40 times → 72 ℃, 5s; Simultaneously with 95 ℃, 15s → 62 ℃, 30s → 72 ℃, the chain melt curve analysis of the condition monitoring amplified production of 15s, the result shows and all becomes overlapping unimodal, confirms that the specificity of primer is better.Analysis software is obtained mean CT-number, carries out quantitative analysis with the ratio of the mRNA relative expression quantity of purpose and reference gene.

3. Western Blot detects the expression of TTase and Trx

The extraction of A, sample total protein

The crystalline lens of getting isolated culture 3hr, 6hr, 12hr places the homogenate tube of 2ml, every pipe add 900 μ l pre-coolings the crystallin lysate, sample places on ice and carries out homogenate with Potter-Elvehjem Tissue Grinders.Homogenate leaves standstill 20min after the homogenate on ice fully, and rear 4 ℃, 12000rpm, centrifugal 20min.Getting its supernatant transfers in another new 1.5ml EP pipe.

B, protein quantification

Adopt BCA determination of protein concentration test kit detection by quantitative protein content: according to 0,1,2,4,8,12,16,18,20 μ l are added to 96 orifice plates, add the deionization distilled water and supply 20 μ l with the protein standard substance that configures in the test kit.Add successively 20 μ l samples in 96 orifice plates, last every hole all adds BCA working solution 200 μ l(A liquid: B liquid is 50:1, mixing), place 30min for 37 ℃.540nm wavelength place microplate reader is measured the OD value.The standard curve of making according to standard substance albumen calculates the sample protein concentration.Each sample adds the albumen sample-loading buffer of 1/4 volume, and (be sample: albumen sample-loading buffer=4:1), place the EP pipe, boiling water bath 10min makes albuminous degeneration.-80 ℃ save backup.

C, SDS-PAGE gel electrophoresis

Separation gel according to following formulated 12%:

Encapsulating: in the gap, pour into rapidly separation gel, reserve concentrated glue space (the long 1cm that adds again of tooth), with pipettor careful cover one deck isopropyl alcohol at separation gel solution, gel vertically is put in room temperature, 30min.Inclining the cover layer isopropyl alcohol, and the deionized water wash gel is for several times to take out unpolymerized separation gel, with napkin sucking-off residual liquid.

And then according to the concentrated glue of following formulated 5%:

Add concentrated glue, the loading comb is installed.Gel vertically is put in room temperature, carefully shifts out comb behind the 30min.With micro sample adding appliance application of sample in order, the first hole adds molecular weight of albumen standard (Maker).Order adds sample specimen, and every sample adds albumen 30 μ g and adds to the bottom, whenever adds one and should wash sample injector in the groove buffer.Positive pole connects lower groove, voltage 80V, beginning electrophoresis.Behind about 1hr, observe bromophenol blue and reach separation gel bottom, powered-down.

Transferring film: after transferring film is finished nitrocellulose filter put into the plate that fills 5% defatted milk powder solution, hatch 2hr in 37 ℃ of shaking tables, the sealing heterogenetic antigen.Nitrocellulose membrane is put into microscler glass container, add the antibody (being the primary antibodie that the defatted milk powder of 2ml adds 2 μ l) of 1:1000,4 ℃ of shaking tables spend the night.Put into and wash 20min * 3 time under the TBST room temperature of glass dish with about 200ml, remove excessive primary antibodie.Film is changed in the microscler glass drying oven, the goat-anti rabbit two that adds 1:2000 is anti-again.The room temperature shaking table is hatched 2hr.Take out film and wash film 3 times with TBST again.The BCIP/NBT colour developing, and preserve image.When experiment repeated, conditionally complete was consistent.

According to standard protein molecular weight Maker(as shown in Figure 4), downcut gel piece corresponding to desired molecule amount, place transfering buffering liquid.Cut nitrocellulose filter and 6 3mm filter paper, size fits like a glove with the gel size.Nitrocellulose filter is soaked in 5min in the transfering buffering liquid, and the expulsion bubble.Keep flat 3 filter paper of electrode (electrode also places transfering buffering liquid) stack, extrude bubble, nitrocellulose filter is placed on the filter paper, the lower left corner is cut off one jiao and is made marks Accurate align.Gel lies against on the film, 3 filter paper that superpose again, the side's of putting electrode.Energized, 200mA, electrotransfer 55min.

4. the TUNEL method detects the apoptosis of lens epithelial cells

The preparation of A, tissue specimen

Get the rat lens of isolated culture, PBS rinsing twice is rolled gently back and forth on filter paper and is sucked unnecessary moisture, then places 4% paraformaldehyde that is placed with 1ml, and it is fixing to spend the night.

B, paraffin section TUNEL method operating process

See Figure 13.

5. flow cytometry detects lens epithelial cell apoptosis

Transfer in six orifice plates when lens epithelial cells of cultivating is grown to 90% left and right sides, cultivate and be divided into two groups after one day, add TPCK in one group, another group does not add, and then shines 30min under uviol lamp, and is apoptosis-induced.Cultivated one day, the post-stimulatory cell of trypsinization is collected cell and is done streaming again, and operating process is seen Figure 14.

6. statistics and analysis

All data mean ± standard deviation

Expression is used the medical statistical software of SPSS18.0 and is carried out the t check, p＜0.01 expression significant difference highly significant, p＜0.05 expression significant difference is remarkable, p〉0.05 expression no difference of science of statistics.

2, result

(1) RT-PCR interpretation of result

The PCR electrophoresis result shows that the band brightness of respectively organizing crystalline lens Trx exists, and the significant change (see figure 5) is arranged.RT-PCR result carries out quantitative analysis with Image analyst software, and the mRNA of Trx has high expressed in the Normal group crystalline lens; 3hr H ₂O ₂The mrna expression of the crystalline lens Trx of oxidative damage is also higher, but 6hr is H ₂O ₂The mrna expression of the crystalline lens Trx of oxidative damage obviously reduces.RT-PCR result shows that the ratio of each time point TTase and Trx/ β-actin specifically sees Table 2-1, and not medication group is H only ₂O ₂Oxidative damage group and TPCK group is compared with the blank group respectively, and TTase and the Trx expression of each time point after with TPCK all has statistical significance (see figure 6) (P＜0.05 or P＜0.01).

The first swimming lane is DNA Maker, is followed successively by matched group, 3hr oxidative damage group, and 3hr adds the TPCK group, 6hr oxidative damage group, 6hr adds TPCK group and 12hr oxidative damage group, and 12hr adds the expression of the Trx gene of TPCK group.Results suggest 3hr expression increases, and descends gradually behind the 6hr, and is very low to the 12hr expression.Electrophoresis result also analyzed best PCR condition as above 3, the amplification condition of (2).

The ratio of each time point and β-actin behind the table 2-1 rat lens oxidative damage

Annotate: ﹡ and compare (default value is 1), p＜0.05 with matched group; ﹡ ﹡ compares (default value is 1), p＜0.01 with matched group.

Show among the figure that the relative expression quantity of the mRNA of TTase and Trx is compared with matched group at 3 hours and obviously raised, 6 as a child expression reduce gradually.(the first two block diagram be respectively TTase at 3hr and 6hr, Trx, black is H ₂O ₂Group, blueness are the TPCK groups, and latter two is Trx) ﹡ p＜0.05 ， ﹡ ﹡ p＜0.01

(2) Western-Blot interpretation of result

Western Blot marking results of hybridization shows organizes respectively that cornea β-brightness of actin band is similar, and the band brightness of TTase and Trx has the significant change (see figure 7).As seen among the figure, process that the protein expression level of TTase and Trx all is increased significantly than the crystalline lens group behind the 3hr, TPCK medication group is higher than not medication group; Begin behind the 6hr to go down, but the TPCK group still is higher than not medication group but slightly is lower than Normal group.

(3) TUNEL apoptosis interpretation of result

The crystalline lens epithelial cell only has the positive of trace and dye.H ₂O ₂Oxidative damage 3hr, the visible positive expression of lens epithelial cells, apoptotic cell and dye the brown granular shape, compare with the oxidative damage group, and the apoptotic cell of TPCK processed group is less but still have a little positive to dye (among Fig. 8-1 shown in the white arrow).The negative contrast of Fig. 8-2.

(4) fluidic cell apoptosis interpretation of result

1. lens epithelial cells cultivation results

After former generation rat lens epithelial cell line (RAT-CELL-0067) recovery that Wuhan biological company limited of primary former generation is bought, the 3rd day cellular morphology, cell is almost all adherent, without the suspension dead cell, the iuntercellular free from admixture, have no pollution condition, lens epithelial cells is rendered as shuttle shape form.Under 100 multiple low power lens, take pictures, be illustrated in fig. 9 shown below:

2. flow cytometer detection apoptosis result

Cell flow cytometer detection apoptosis as seen, the lens epithelial cell apoptosis cell number behind the ultraviolet radiation 30min accounts for the C among 30.9%(Figure 10 of total cell number); Only account for D among 20.1%(Figure 10 of total cellular score and added apoptotic cell quantity that TPCK intervenes).A among Figure 10 is unmarked blank group, and the B among Figure 10 is single mark AV matched group.

3, conclusion

Under the effect of TPCK, these two kinds of enzymes of TTase, Trx show ageing change when crystalline lens suffers oxidative stress; TPCK can Cell protection to a certain degree avoids suffering the apoptosis under stress situation.

Claims (5)

1.TPCK the purposes in the anti-lenticular opacity product of preparation.

2.TPCK compositions in the purposes of preparation in the anti-lenticular opacity product.

3. purposes according to claim 1 and 2; it is characterized in that; the muddy product of described anti-crystalline lens refers in medicine, food, beverage and the reagent technical field, a kind ofly directly or indirectly is used for prevention, diagnosis, detects, protection, treatment and the research crystalline lens is muddy and the product of directly related disease.

4. purposes according to claim 3 is characterized in that, the muddy product of described anti-crystalline lens is the medicine that is directly used in prevention, diagnosis, detection, protection, treatment and research crystalline lens muddiness and directly related disease thereof.

5. purposes according to claim 1 and 2 is characterized in that, described lenticular opacity disease comprises one or more in all kinds of lenticular opacity diseases.

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