CN109627341A - Hormone induction zinc finger shape polypeptide and preparation method thereof and the application in pharmacy - Google Patents
Hormone induction zinc finger shape polypeptide and preparation method thereof and the application in pharmacy Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, are related to hormone induction zinc finger shape polypeptide and preparation method thereof and pharmaceutical usage, are especially chemical synthesis hormone induction zinc finger shape polypeptide in preparation and inhibit the purposes in inflammatory reaction drug.The present invention synthesizes the hormone induction zinc finger shape polypeptide being made of 54 amino acid by chemically synthesized mode, which can significantly inhibit the ocular inflammatory response of endotaxin induction after zoopery vitreous chamber intervention.By the retina m ü ller cell and retinal microvascular endothelial cell assay of originally culture, hormone induction zinc finger shape polypeptide passes through the secretion for the inflammatory factor for inhibiting nuclear factor NF-kB p65 phosphorylation that p65 nuclear transfer is inhibited to inhibit endotaxin induction as the result is shown.The present invention confirms that the hormone induction zinc finger shape polypeptide synthesized in vitro has significant anti-inflammatory activity by internal animal experiment and cell assay in vitro.It can be used for preparing the drug for the treatment of diseases associated with inflammation.
Description
Technical field
The invention belongs to field of biotechnology, are related to hormone induction zinc finger shape polypeptide and preparation method thereof and medicinal use
On the way, it is especially chemical synthesis hormone induction zinc finger shape polypeptide and inhibits the purposes in inflammatory reaction drug in preparation.
Background technique
Prior art discloses the effects of human body internal hormone extensively, participates in the metabolism of substance, regulates and controls the multiple devices of body
Stress reaction the etc. when growth and development of official and the normal function for maintaining organ, also participation body cope with various stimulations, is to maintain
The indispensable important substance of one kind of body homeostasis；Wherein being most widely used clinically with glucocorticoid is studied
Show that its immunosupress can help inflammatory reaction caused by body fight a variety of causes, it has also become treatment disease saves life
Order essential drug.
In clinical practice, glucocorticoid is widely used in treatment multi-infection and non-infectious disease, and
Significant clinical effectiveness is obtained, but its adjoint drug side-effect also causes more serious damage, including metabolism to body
Disorder, osteoporosis etc..Such as, in eye part disease treatment, though glucocorticoid local use also result in it is serious concurrent
Disease, such as corticosteroid glaucoma, glucocorticoid induced cataract etc., these complication can lead to serious irreversible inpairment of vision.
Significant clinical effectiveness and serious complication in view of glucocorticoid, those skilled in the art are quasi- to seek a kind of novel hormonal and replaces
For drug, make the antiphlogistic effects that can play hormone and the drug side-effect without hormone.
One kind is had found in the researchers such as D'Adamio F in 1997 are studied using dexamethasone stimulation thymocyte
Albumen with zinc-finger, and it is named as hormone induction zinc finger shape albumen (GILZ).As a kind of hormone induction albumen,
The anti-inflammatory activity of GILZ was explored in several animal models, comprising: utilizes inflammatory bowel disease model, discovery is overexpressed
The significantly wilder mouse of transgenic mice colon site inflammation of GILZ is light；GILZ, which is overexpressed transgenic mice, can also mitigate notochord
Post-injury inflammatory reaction, the activation for showing as the notochord damage location T cell in the mouse that GILZ is overexpressed is suppressed, proinflammatory disease
The release of the factor such as IL-1 β and TNF-α is reduced；Equally in the mouse model of autoimmune cerebral spinal inflammation, GILZ
It can interact with NF-kB and mitigate inflammatory reaction；GILZ, which is overexpressed to pass through, in the endotoxemia of LPS induction mitigates inflammation
The release and then mitigation body inflammatory reaction of inflammation factor；In arthritis mouse model, GILZ overexpression can be sent out with mimic hormone
Antiphlogistic effects are waved, and the reaction of Gilz-knockout mouse arthritic inflammation significantly aggravates；The early-stage study of the application is lured in endotoxin
In the uveitis model led, endotoxin is significantly suppressed after being overexpressed with slow virus over-express vector induced retinal GILZ and is lured
The ocular inflammatory response led.
To sum up numerous test results confirm that GILZ inhibits the activity of inflammatory reaction, however these are tested or pass through building
GILZ-/- or GILZ overexpression transgenic mice, or the overexpression by constructing GILZ or
Person silence plasmid or viral vector transduction cell or tissue, since those technologies itself have risk and ethics
Defect limits its clinical application.
Summary of the invention
It is an object of the invention to the hormone induction zinc finger shapes that have biological activity new by iii vitro chemical synthesis synthesis
Polypeptide and its pharmaceutical usage.
It is a protein chain by 135 Amino acid profiles based on hormone induction zinc finger shape albumen disclosed in the prior art,
Since chemical synthesis albumen must limit polypeptide chain length, in the synthesis process polypeptide cross grow to often result in peptide chain structure it is abnormal and
Influence peptide chain correct synthesis, the present invention only have chosen its proline rich structural domain (the 98th to No. 135 amino acid:
KTLASPEQLEKFQSRLSPEEPAPEPETPEAPGGSAV), terminated into cell-penetrating peptide YGRKKRRQRRR, then in the N ' of polypeptide
Hormone induction zinc finger shape polypeptide chain described in chemical synthesis: YGRKKRRQRRRKTLASPEQLEKFQ SRLSPEEPAPEP
ETPEAPGGSAV；The polypeptide chain of iii vitro chemical synthesis has water solubility well, can thoroughly be dissolved in water, can promote this
Polypeptide enters cell, and PBS either cell culture medium without assisting any organic solvent, and can guarantee the administration of polypeptide
The safety of mode and administration route.
Specifically, the chemical synthesising technology scheme that the present invention uses is:
The amino acid sequence of the end C ' the proline richness dimerization domain positioned at GILZ is chosen (from 98 amino acids to 135 ammonia
Base acid), upper cell-penetrating peptide is connected at the end N ' of the sequence, the polypeptide segment etc. is made to can smoothly enter into cell, the polypeptide sequence are as follows:
YGRKKRRQRRRKTLASPEQLEKF QSRLSPEEPAPEPETPEAPGGSAV。
The hormone induction zinc finger shape polypeptide that the further object of the present invention is to provide synthesis inhibits inflammation medicine in preparation
Application in object.
In the present invention, the intervention experiment of the hormone induction zinc finger shape polypeptide of SD rat intraocular inflammation model has been carried out,
The results show that hormone induction zinc finger shape polypeptide can successfully enter m ü ller cell under the guidance of cell-penetrating peptide；Swash
Element induction zinc finger shape polypeptide can significantly inhibit endotaxin induction anterior ocular segment inflammatory reaction；It is intraocular to significantly inhibit endotaxin induction
Inflammatory cell exudation, including in anterior chamber and vitreous chamber；And inhibit the secretion of endotaxin induction retina MCP-1, in inhibition
The secretion of toxin-induced view mTNF-a inhibits the secretion of endotaxin induction retina IL-1 β, inhibits endotaxin induction view
The secretion of film ICAM-1 inhibits endotaxin induction retina m ü ller cell colloid, inhibits endotaxin induction retina GFAP
And the amount and inhibition endotaxin induction retina m ü ller cell APQ4 abnormal expression of vimentin, inhibit endotaxin induction view
The amount of nethike embrane AQP4 and the NF-kB p65 nuclear transfer of inhibition endotaxin induction and the NF-kB p65 for inhibiting endotaxin induction
Phosphorylation.
The present invention confirms the hormone induction zinc finger shape albumen synthesized in vitro by internal animal experiment and cell assay in vitro
Polypeptide has significant anti-inflammatory activity.It can be used for preparing the drug for the treatment of diseases associated with inflammation.
Internal animal experiment of the invention and cell assay in vitro include: endotoxin (LPS) vitreous chamber intervention production eye
Interior inflammatory model (SD rat)；2 hours after the intervention of endotoxin vitreous chamber, hormone induction zinc finger shape polypeptide vitreous chamber is
Treatment group (GILZ-p), the control polypeptide with concentration compare group (Control-p)；Endotoxin intervention after 24,48,72 and
96 hours, assess hormone induction zinc finger shape polypeptide treatment group and control peptide processing group rat anterior ocular segment inflammatory reaction；Inside
24 hours after toxin intervention, rat eye is taken to do paraffin section and HE dyeing, assessment hormone induction zinc finger shape polypeptide treatment
Group and control peptide processing group rat intraocular inflammation cell ooze out situation；24,48,72 and 96 hours after endotoxin infusion, take big
Rat retina neuroepithelial tissue row western blot detection, detects hormone induction zinc finger shape polypeptide treatment group and control
The secretion of inflammatory factor in peptide processing group retina；24,48,72 and 96 hours after endotoxin intervention, take rat retina refreshing
Transepithelial tissue row western blot detection, detects hormone induction zinc finger shape polypeptide treatment group and control peptide processing group is big
Rat retina m ü ller cell colloid situation；24,48,72 and 96 hours after endotoxin intervention, rat eye is taken to make ice
Freeze slice, Immunofluorescence test hormone induction zinc finger shape polypeptide treatment group and control peptide processing group rat retina m ü ller
The expression and distribution of the colloidization label GFAP and m ü ller cell function albumin A QP4 of cell；The retina m ü of originally culture
The hormone induction zinc finger shape polypeptide of ller cell, various concentration stimulates 24 hours, detects its cell toxicant with CCK8 kit
Property；The hormone induction zinc finger shape polypeptide of the retina m ü ller cell of originally culture, various concentration stimulates 24 hours, streaming
Cell technology detects the cell proportion that film is successfully worn by hormone induction zinc finger shape polypeptide；The retina m ü ller of originally culture
Cell, endotoxin are added the hormone induction zinc finger shape polypeptide of various concentration, collect cell after culture after stimulating 2 hours,
Western blot detects the amount of NF-kB p65 in nucleus and cytoplasm, the amount of Intracellular phosphorylation p65；Originally culture
The hormone induction zinc finger shape polypeptide of various concentration, culture 24 is added in retina m ü ller cell, endotoxin after stimulating 2 hours
Cell, the protein expression of Immunofluorescence test NF-kB p65 etc. are collected after hour.
In the embodiment of the present invention, hormone induction zinc finger shape is detected using the retina m ü ller cell of originally culture
The cell poisons of polypeptide find the m ü ller cell normally cultivated in the hormone induction zinc finger shape polypeptide of various concentration
Cell activity can't be changed after stimulation, i.e., the polypeptide does not have cytotoxicity.With drug safety；And then utilize endotoxin
The validity of the intraocular inflammation model testing hormone induction zinc finger shape polypeptide of induction, hormone induction zinc finger shape polypeptide
(1mM, 2 μ L) intravitreal significantly inhibits the ocular inflammatory response of endotaxin induction, and it is aobvious to show as anterior ocular segment inflammatory reaction
Work mitigates, the exudation of intraocular inflammation cell substantially reduces, the amount of layer of retina,neuroepithelial energy inflammatory factor is remarkably decreased, retina
M ü ller cell colloid is suppressed significantly, and shows that the polypeptide synthesized in vitro has anti-inflammatory activity；Further utilize original
The retina m ü ller cell research hormone induction zinc finger shape polypeptide for being commissioned to train feeding inhibits the mechanism of inflammation, this is more as the result is shown
Peptide can significantly inhibit the nuclear factor NF-kB p65 phosphorylation of endotaxin induction, and then inhibit p65 nuclear transfer.
The present invention confirms that the hormone induction zinc finger shape polypeptide synthesized in vitro has significant anti-inflammatory work from multiple angles
Property, new treatment means are provided for treatment diseases associated with inflammation.
Detailed description of the invention
The m ü ller cell (A) of Fig. 1: immunofluorescence dyeing vimentin identification originally culture, Green:vimentin；
blue:DAPI；(B) hormone induction zinc finger shape polypeptide of the primary retina m ü ller cell through various concentration after identified
Stimulation 24 hours, CCK8 kit detect the activity of m ü ller cell, assess the cell toxicant of hormone induction zinc finger shape polypeptide
Property.
Fig. 2: FCM analysis successfully wears the m ü ller cell proportion of film through hormone induction zinc finger shape polypeptide, with
The increase of hormone induction zinc finger shape polypeptide concentration is also gradually increased by the m ü ller cell proportion that polypeptide wears film, is prompted
Hormone induction zinc finger shape polypeptide can successfully enter m ü ller cell under the guidance of cell-penetrating peptide.
Fig. 3: after endotoxin intravitreal, the inflammatory reaction of anterior ocular segment peaked at 24 hours, after gradually mitigate
And continue to 96 hours, compared with control peptide (Control-p), 1mM hormone induction zinc finger shape polypeptide (GILZ-p, 2 μ L)
Intravitreal significantly inhibits endotaxin induction anterior ocular segment inflammatory reaction.
Fig. 4: Western blot detection discovery, 1mM hormone induction zinc finger shape polypeptide (2 μ L) intravitreal are aobvious
It writes and inhibits the exudation of endotaxin induction intraocular inflammation cell, including in anterior chamber and vitreous chamber.
Fig. 5: Western blot detection discovery, 1mM hormone induction zinc finger shape polypeptide (2 μ L) intravitreal are aobvious
Write the secretion for inhibiting endotaxin induction retina MCP-1.
Fig. 6: Western blot detection discovery, 1mM hormone induction zinc finger shape polypeptide (2 μ L) intravitreal are aobvious
Write the secretion for inhibiting endotaxin induction view mTNF-a.
Fig. 7: Western blot detection discovery, 1mM hormone induction zinc finger shape polypeptide (2 μ L) intravitreal are aobvious
Write the secretion for inhibiting endotaxin induction retina IL-1 β.
Fig. 8: Western blot detection discovery, 1mM hormone induction zinc finger shape polypeptide (2 μ L) intravitreal are aobvious
Write the secretion for inhibiting endotaxin induction retina ICAM-1.
Fig. 9: Immunofluorescence test discovery 1mM hormone induction zinc finger shape polypeptide (2 μ L) intravitreal significantly presses down
Endotaxin induction retina m ü ller cell colloid processed.Red:GFAP,blue:DAPI.
Figure 10: Western blot detection discovery, 1mM hormone induction zinc finger shape polypeptide (2 μ L) intravitreal
Significantly inhibit the amount of endotaxin induction retina GFAP and vimentin.
Figure 11: Immunofluorescence test scientific discovery, 1mM hormone induction zinc finger shape polypeptide (2 μ L) intravitreal
Significantly inhibit endotaxin induction retina m ü ller cell APQ4 abnormal expression.Green:AQP4,blue:DAPI.
Figure 12: Western blot detection discovery 1mM hormone induction zinc finger shape polypeptide (2 μ L) intravitreal is aobvious
Write the amount for inhibiting endotaxin induction retina AQP4.
Figure 13: the retina m ü ller cell of originally culture, western blot detection discovery hormone induction zinc finger shape egg
White polypeptide can significantly inhibit the NF-kB p65 nuclear transfer of endotaxin induction.
Figure 14: the retina m ü ller cell of originally culture, Immunofluorescence test find that hormone induction zinc finger shape albumen is more
Peptide can significantly inhibit the NF-kB p65 nuclear transfer of endotaxin induction.Red:p65；blue:DAPI
Figure 15: the retina m ü ller cell of originally culture, hormone induction zinc finger shape polypeptide can significantly inhibit interior
The NF-kB p65 phosphorylation of toxin-induced.
It elaborates with reference to the accompanying drawing to specific embodiment provided by the invention
Specific embodiment
Embodiment 1
Main agents: hormone induction zinc finger shape polypeptide chain 1:
YGRKKRRQRRRKTLASPEQLEKFQSRLSPEEPAPEPETPEAPGGSAV polypeptide chain 2:
YGRKKRRQRRRKTLASPEQLEKFQSRLSP EEPAPEPETPEAPGGSAKFITC (C-terminal band FITC label,
For doing FCM analysis), control polypeptide is YGRKKRRQRRR, and the synthesis of all polypeptide chains entrusts Suzhou to shine by force biology
Science and Technology Ltd., purity are higher than 95%.GILZ, the purchase of TNF-α antibody are in proteintech company, NF-kB p65, phosphoric acid
Change the purchase of p65 antibody in Cell Signaling company, IL-1 β, the purchase of MCP-1, ICAM-1, Vimentin and GFAP antibody in
Abcam company.
Zoopery:
The adult rat normally raised is taken, (100g/2ml) induced rat general anesthesia is injected intraperitoneally in 10% chloraldurate,
After compound tropicamide eye drops eye droppings expands pupil, the induction local anaesthesia of Oxybuprocaine hydrochloride eye drops eye droppings, adjustment microscope is extremely
Clearest state；33G micro syringe is taken, injection (the hormone induction zinc finger of 125ug/ml endotoxin or 1mM is extracted
The control polypeptide chain of shape polypeptide chain or isoconcentration, 2ul) in the oblique 45 ° of inserting needles of corneoscleral junction 1mm, needle is seen through pupil
Start to push pin after point to vitreous chamber, after needle point rest on the withdraw of the needle after vitreous chamber 50 seconds, keep warm after applying Ofloxacin salve
Send animal house raising after to revival back to；
Polypeptide chain lysate is injected after intravitreal endotoxin after 2 hours again, 24,48,72 after endotoxin injection
And 96 hours, it scores the prosthomere inflammation of rat, it is complete that (100g/2ml) induction is injected intraperitoneally in 10% chloraldurate of rat
Body anesthesia, is subsequently placed under slit-lamp and rat prosthomere inflammatory reaction is assessed and photographed to record according to the method for following table；
Paraffin section and HE dyeing: it injects polypeptide chain lysate after intravitreal endotoxin after 2 hours again, is injecting
24 hours after endotoxin, eyeball is taken to carry out HE dyeing counting exudation inflammatory cell, rat is injected intraperitoneally with 10% chloraldurate
It is lethal after (100g/2ml) induced rat general anesthesia and cervical dislocation, it takes eyeball to be placed in eyeball fixer under microscope, takes
Material is carefully, to keep the integrality of eyeball；Take the eyeball fixed, alcohol serial dehydration (by low concentration to high concentration: 70%,
80% and 90% is dense, and each concentration is dehydrated 30 minutes) after, then with absolute alcohol and the immersion of dimethylbenzene equivalent mixed liquor 15 minutes, two
Toluene 115 minutes, dimethylbenzene II 15 minutes, until the complete bleach of eyeball tissue；Transparent eyeball block is placed in solubilized
In the paraffin of change, it is put into wax-dissolving box heat preservation；It is embedded after paraffin is completely immersed in tissue block.Embedded wax stone is fixed on
It on slicer, is cut into a thickness of 5 μm of thin slices, is attached on glass slide, put in 45 DEG C of insulating boxs and dry；Piece of drying is put into Soviet Union
It is dyed in another name for aqueous solution several minutes；Color separation in sour water and ammonium hydroxide, each several seconds；Flowing water enters distilled water a moment after rinsing 1 hour；
Enter to be dehydrated in 70% and 90% alcohol each 10 minutes；Enter alcohol eosin stains liquid to dye 2~3 minutes；Slice after dyeing is through pure
Dehydration of alcohol, then through dimethylbenzene make to be sliced transparent, observation is stored at room temperature after marking mounting.Piece dyed through HE, under microscope
Anterior chamber and vitreous chamber emigrated cell number are counted, emigrated cell, which is counted, carries out blind counting with statistics using the third party；
Retina frozen section and Immunofluorescence test: polypeptide chain is injected again after 2 hours after intravitreal endotoxin
Lysate takes eyeball to carry out frozen section 72 hours after endotoxin injection.By rat, excessively anesthesia causes 10% chloraldurate
Extremely, the eyeball of rat is taken to be immersed in eyeball fixer under the microscope 2 hours, successively with 20% sucrose, 30% saccharose gradient
Dehydration 2 hours；Eyeball after serial dehydration is placed under microscope, removes cornea, crystal with microscissors, optic cup is made.Eyeball is set
In embedded box, -80 ° of refrigerators being placed on the embedding of OCT embedding medium and freeze 30 minutes, latter 10 μm of frozen section machine-cut thickness are cut
Piece is simultaneously adhered to anticreep glass slide, and slice is placed 2 hours at room temperature, and 4% paraformaldehyde fixes 2 hours at room temperature, PBS punching
It washes 30 minutes, 0.1%triton-X100 (preparation of 0.1% sodium citrate) wears film 30 minutes, lowlenthal serum confining liquid closing slice
30 minutes, 4 ° of refrigerators were incubated for primary antibody (Rabbit anti GILZ Polyclonal antibody, 1:500) overnight, and PBS is rinsed
3 times, five minutes every time, 37 ° of (488 Affini Pure Goat Anti-rabbit of Alexa Fluor of incubating secondary antibody 2 hours
IgG；555 Affini Pure Goat Anti-rabbit IgG of Alexa Fluor), PBS flushing 3 times, five minutes every time,
DAPI dyes 3 minutes (1:10000), and PBS is rinsed 3 times, and mounting after five minutes, is placed in fluorescence microscopy under the microscope and claps every time
According to；
The primary muller cell culture of rat retina: newborn SD rat (2-3 days) is sterilized with ANER DIAN after sacrificed by decapitation
It impregnates 5 minutes, takes eyeball under super bacterium platform microscope and separate retinal tissue, with culture after 0.25% pancreatin digestion minute
Base terminates digestion, and 1200rpm is centrifuged 5 minutes, abandons supernatant, with packing after fresh culture resuspension cell in T75 culture bottle (10
Newborn rat dispenses 5 bottles), it is placed in routine culture in cell incubator, with there are also the DMEM F12 culture medium culture of 20%FPS,
Liquid is changed every other day, and long to being passed on after 80% fusion after muller cell, reaching can be used for subsequent examination after identifying after three generations
It tests；
Muller cellular identification: well-grown Muller cell is passaged on cell climbing sheet simultaneously overnight incubation, more than 4%
Polyformaldehyde fixes 30 minutes, and 0.1%triton-X100 (preparation of 0.1% sodium citrate) is added after PBS cleaning and wears film 30 minutes,
The closing of lowlenthal serum confining liquid slice 30 minutes, 4 ° of refrigerators were incubated for primary antibody (Vimentin, 1:500) overnight, PBS flushing 3 times, often
Secondary five minutes, 37 ° of incubate secondary antibody 2 hours (Alexa Fluor 488Affini Pure Goat Anti-rabbit IgG), PBS
It rinses 3 times, five minutes every time, DAPI dyed 3 minutes (1:10000), and PBS is rinsed 3 times, and mounting after five minutes, is placed in glimmering every time
Viewed under light microscopy is simultaneously taken pictures.The cell of the Vimentin positive is muller cell, and the muller cell of originally culture passes
Purity is up to 95% or more after to 3 generations；
CCK8 experiment: cytotoxicity of the hormone induction zinc finger shape polypeptide to muller cell of various concentration is detected, with every
20000, hole cell inoculation is in 96 orifice plates, respectively with the hormone induction zinc finger shape egg of final concentration of 0.01,0.1,1 and 10mM
White polypeptide handles cell.After 24 hours, 0.1mg CCK8 is added into every hole, 37 DEG C are incubated for 4 hours, in spectrophotometer
Sample absorbance value is detected at 490nm.Each drug concentration sets 6 multiple holes, and 3 repeated experiments；
Flow cytometer inspection: the muller cell normally cultivated in six orifice plates, the also FITC that various concentration is added are marked
Hormone induction zinc finger shape polypeptide (0.01,0.1,1 and 10mM), continue culture 24 hours after collect cell, fluidic cell
There are also the muller cells of FITC signal for instrument detection；
Western blot immunoblotting assay: by the layer of retina,neuroepithelial of collection or the retina of originally culture
Muller cell is added appropriate single Detergent Lysis liquid according to the amount of cell and splits (containing PMSF), and BSA kits albumen is dense
Degree.By the albumen supernatant of extraction and 5 X albumen sample-loading buffer (0.25M Tris-HCL, pH6.8,50% glycerol, 20%- mercaptos
Base ethyl alcohol, 10%SDS, 0.0012% bromophenol blue), it is put into progress boiling water bath 10min in boiling water.Take 40ug albumen sample containing SDS
5%-12% polyacrylamide gel in electrophoresis, be then transferred on pvdf membrane；Pvdf membrane TBS (20mM Tris-Hcl
PH7.4,150mM NaCl), pvdf membrane is impregnated with the TBST containing 5% skimmed milk power, room temperature shaker is closed 2 hours；Use confining liquid
Dilute corresponding primary antibody and 4 DEG C of overnight incubations；TBST is sufficiently washed pvdf membrane 5-6 times, 5min/ times；37 DEG C of shaking tables of secondary antibody are incubated for 2
Hour.It is mixed, is added dropwise in 1:1 ratio with enhancement solution in ECL reagent and stable Peroxidase Solution after washing away extra secondary antibody
Working solution sucks extra substrate solution with filter paper, is covered with preservative film, X on pvdf membrane, reacting several minutes after fluorescent belt is obvious
Developing liquid developing, fixing solution fixing are sequentially placed into after ray film tabletting；It develops photographic film and analyzes result；Film is dried, film is scanned,
Film gray value is analyzed with BandScan, analyzes result；
Experimental result is examined poor using the average and standard deviation of independent experiment is indicated three times and more than three times with t
Different, P value, which is less than, thinks there is statistical difference, and all statistics use SPSS16.0 software；
Test result is shown:
Hormone induction zinc finger shape polypeptide no cytotoxicity: it is dyed through immunofluorescence vimentin, is accredited as primary training
Feeding rat retina m ü ller cell (Figure 1A), is passaged to 96 orifice plates, more with the hormone induction zinc finger shape albumen of various concentration
Peptide stimulates 24 hours, whether has cytotoxicity with CCK8 cytotoxic agent box detection hormone induction zinc finger shape polypeptide itself.
As shown in Figure 1B, the hormone induction zinc finger shape polypeptide stimulation m ü ller cell of various concentration is after 24 hours, m ü ller cell
It is active and unaffected, that is to say, that hormone induction zinc finger shape polypeptide itself does not have cell poisons；
Hormone induction zinc finger shape polypeptide can succeed with m ü ller cell combination: in order to promote hormone induction zinc
Finger-like polypeptide successfully breaks through cell membrane and enters cell, we terminate the preceding paragraph cell-penetrating peptide in the N ' of the polypeptide, while in C '
The FITC that can be fluoresced in the connection of end facilitates us successfully to be contacted with each other with Flow Cytometry detection with m ü ller and enters m
The hormone induction zinc finger shape polypeptide of ü ller.As shown in Fig. 2, the hormone induction zinc finger shape polypeptide of various concentration handles m
ü ller cell is after 2 hours, it can a degree of and m ü ller cell combination；
Hormone induction zinc finger shape polypeptide inhibits the anterior ocular segment inflammatory reaction of endogenous toxic material induction: as shown in figure 3, endotoxin glass
Glass body cavity injects significant induced rat anterior ocular segment inflammatory reaction, shows as iris hyperemia, exudation of anterior chamber, hypopyon and pupil contracting
Small etc., inflammatory reaction peaked at 24 hours and continues to 96 hours.Compared with control peptide, inflammatory reaction is in hormone induction zinc
Finger-like protein polypeptide processing group significantly mitigates, and at 24,48,72 and 96 hours, there were significant differences for inflammatory score.
Hormone induction zinc finger shape polypeptide inhibits the intraocular inflammation cell exudation of endotaxin induction: as shown in figure 4, inside
24 hours after toxin intravitreal, a large amount of inflammatory cell exudation was widely distributed in corpus ciliare choroideae and vitreous chamber
It is interior.By the counting to inflammatory cell is oozed out in eyeball, the intervention of discovery hormone induction zinc finger shape polypeptide is significantly suppressed
The quantity of inflammatory cell in eyeball；
Hormone induction zinc finger shape polypeptide inhibits the secretion of the retinitis inflammation factor of endotaxin induction: monocyte becomes
Change factor M CP-1, tumor necrosis factor TNF-alpha, interleukin I L-1 β and Intercellular Adhesion Molecule ICAM-1, is that mediation is intraocular non-
Often important medium, participates in the Cascaded amplification of intraocular inflammation.As viewed in figures 5-8, after endotoxin intravitreal in retina
MCP-1, TNF-α, the amount of IL-1 β and ICAM-1 gradually rise, and hormone induction zinc finger shape polypeptide can be to a certain degree
The upper secretion for inhibiting these inflammatory factors.In the above result shows that hormone induction zinc finger shape polypeptide is able to suppress
The inflammatory reaction of poison induction.
Hormone induction zinc finger shape polypeptide inhibits the colloid of the retina m ü ller cell of endotaxin induction: pathologic ring
Border can induced retinal m ü ller cell colloid, the mark of colloid is that glial fibrillary acid protein GFAP expression increases,
The colloid of retina m ü ller cell is the mark of retina homeostasis, and the m ü ller cell of colloid is not only under function
Drop, while a large amount of inflammatory factor is secreted, lead to retinal inflammation cascade reaction.As shown in figure 9, endotoxin intravitreal
The intracellular GFAP of retina m ü ller is dramatically increased and is extended to view outer nuclear layer after 72 hours afterwards, and compared with control peptide, hormone is lured
The expression that zinc finger shape polypeptide significantly inhibits m ü ller cell GFAP is led, prompts hormone induction zinc finger shape polypeptide that can press down
The colloid of retina m ü ller cell processed, i.e., maintain the stable state of environment in retina to a certain extent.With immunofluorescence
As a result it matches, western blot as shown in Figure 10 also confirms that GFAP, vimentin in endotaxin induction retina
Expression quantity increase, and hormone induction zinc finger shape polypeptide after endotoxin infusion 24,48,72 and 96 hours can be one
Determine the expression for inhibiting GFAP in degree.Normal retina m ü ller cell expresses a large amount of aquaporin egg (AQP4), main to be distributed
In the m ü ller cell podocytic process soleplate contacted with vitreous chamber and the m ü ller cell body contacted with retinal vessel, view
Water in membrane tissue is shifted by the channel AQP4 into vitreous chamber and retinal vessel, and the AQP4 of m ü ller cell is to maintain view
The key of water balance in nethike embrane.When AQP4 channel abnormal, a large amount of water, which is gathered in retinal tissue, leads to retina
Oedema.As shown in figure 11, the expression decline of retina m ü ller cell AQP4 and distribution disorders after endotoxin intravitreal
It is abnormal, and hormone induction zinc finger shape polypeptide can restore the arrangement of AQP4 to a certain extent.Wesstern blot also into
One step confirms, causes the expression quantity of retina AQP4 to be gradually reduced after endotoxin intravitreal, and hormone induction zinc finger shape
The intravitreal of polypeptide restores the amount (as shown in figure 12) of retina AQP4 to a certain extent:
The NF-kB p65 nuclear transfer of hormone induction zinc finger shape polypeptide inhibition endotaxin induction: further using primary
The retina m ü ller cell of culture explores the anti-inflammatory mechanism of hormone induction zinc finger shape polypeptide.The m ü ller of endotoxin stimulation
Cell, the significant nuclear transfer of NF-kB p65, the amount cashed as p65 in p65 amount decline in endochylema simultaneously karyon increases, and swashs
Element induction zinc finger shape polypeptide can significantly inhibit NF-kB p65 nuclear transfer, this conclusion simultaneously by immunofluorescence and
Western blot confirms (as shown in Figure 13,14)；
The NF-kB p65 phosphorylation of hormone induction zinc finger shape polypeptide inhibition endotaxin induction: NF-kB p65 phosphorylation
It is the power of p65 nuclear transfer, therefore we further explore hormone induction zinc finger shape egg using the m ü ller cell of originally culture
Whether white polypeptide can inhibit the phosphorylation of p65, and as shown in figure 15, hormone induction zinc finger shape polypeptide successfully inhibits endogenous toxic material
The p65 phosphorylation that element is induced.
Claims (7)
1. hormone induction zinc finger shape polypeptide inhibits the purposes in inflammatory reaction drug in preparation；The hormone induction zinc finger
Its polypeptide of shape polypeptide is from N-terminal to C-terminal are as follows:
YGRKKRRQRRRKTLASPEQLEKFQSRLSPEEPAPEAPETPEAPGGSAVK。
2. purposes according to claim 1, which is characterized in that the hormone induction zinc finger shape polypeptide by changing in vitro
Synthesis is learned to be made, including terminating in the N ' of the polypeptide into cell-penetrating peptide YGRKKRRQRRR, hormone induction zinc described in chemical synthesis
Finger-like polypeptide chain: YGRKKRRQRRRKTLASPEQLEKFQ SRLSPEEPAPEP ETPEAPGGSAV.
3. purposes according to claim 1, which is characterized in that the hormone induction zinc finger shape polypeptide is in cell-penetrating peptide
It can enter m ü ller cell under guidance, inhibit the inflammatory reaction of endotaxin induction anterior ocular segment.
4. purposes according to claim 1, which is characterized in that the hormone induction zinc finger shape polypeptide inhibits endotoxin
The exudation of intraocular inflammation cell is induced, and inhibits the secretion of endotaxin induction retina MCP-1, inhibits endotaxin induction retina
The secretion of TNF-α inhibits the secretion of endotaxin induction retina IL-1 β, inhibits the secretion of endotaxin induction retina ICAM-1.
5. purposes according to claim 1, which is characterized in that the hormone induction zinc finger shape polypeptide inhibits endotoxin
Induced retinal m ü ller cell colloid.
6. purposes according to claim 1, which is characterized in that the hormone induction zinc finger shape polypeptide inhibits endotoxin
The amount and inhibition endotaxin induction retina m ü ller cell APQ4 abnormal expression of induced retinal GFAP and vimentin, suppression
The amount of endotaxin induction retina AQP4 processed.
7. purposes according to claim 1, which is characterized in that the hormone induction zinc finger shape polypeptide inhibits endotoxin
The NF-kB p65 nuclear transfer of induction and the NF-kB p65 phosphorylation for inhibiting endotaxin induction.
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