CN104873958A - Application of recombinant and syncretic Tat-Cygb protein in anti-hepatic fibrosis - Google Patents
Application of recombinant and syncretic Tat-Cygb protein in anti-hepatic fibrosis Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and relates to novel application of recombinant and syncretic Tat-Cygb protein. Experiment research testifies that the Tat-Cygb can cure inflammation, heal wound, resist liver fibrosis, and promote the reversion of the liver fibrosis, and is hopeful to provide novel treatment means of recombinant and syncretic Tat-Cygb human cell globulin for clinic.
Description
Technical field:
The invention belongs to field of biomedicine technology, relate to the application of recombination fusion protein in antiinflammatory, wound healing and anti-hepatic fibrosis.
Background technology:
Inflammation (inflammation): be the defense reaction that the biological tissue with vascular system occurs damage factor infects and the most basic pathology process of numerous disease.The generation evolution of inflammation was generally divided into for three phases: telangiectasis, permeability are hyperfunction; Platelet adhesion and leukoplania; Granulation tissue hyperplasia.Inflammation is clinical common pathological process, can betide body and respectively organize and organ.Acute inflammation has the changes such as red, swollen, hot, pain, with often with general reactions such as heating, leukocytosiss.After pro-inflammatory cytokine acts on body, trigger cell damage is downright bad on the one hand; On the other hand, stimulate body disease-resistant function to increase, make the interior bad border of body and reach new equilibrium between interior boundary and outer bad border.
Wound healing (wound healing): be after body suffers External Force Acting, the tissue such as skin occurs, from the healing recovery process of breaking or after defect, comprising regeneration and granulation tissue hyperplasia, the scar tissue formation etc. of various tissue.
The basic process of skin wound healing mainly comprises: acute inflammation stage, hyperplasia phase, cicatrization phase, epidermis and other tissue regeneration phase.Three kinds of situations are divided into: wound is only limitted to skin epidermis according to the order of severity of wound; Slightly severe one skin and subcutaneous tissue rupture and occur wound; Serious wound can have muscle, tendon, neural fracture.
Wound healing can be divided into general healing, secondary healing, subcrustal healing three types.
1) general healing (healing by first intention): the time of general healing is short, forms cicatrix less.Be more common in that as few in tissue defects such as operative incisions, edge of wound is neat, without infecting, through bonding or the involutory tight wound of wound surface after sewing up, only have a small amount of blood clot in wound, inflammatory reaction is slight, and promoting epidermization just can cover wound in 24 ~ 48 hours.Granulation tissue just can grow from edge of wound at the 3rd day and to be filled up by wound very soon, and within 5 ~ 6 days, collagen fiber are formed, and within about 2 ~ 3 weeks, heal completely.
2) secondary healing (healing by second intention): see that tissue defect is comparatively large, edge of wound is not whole, cannot be neatly involutory, or with the wound infected.Compared with primary healing, mainly contain following difference: 1. because slough is many, or owing to infecting, continue to cause local organization degeneration, necrosis, inflammatory reaction is obvious.Only have infection control, remove slough, regeneration could start.2. wound is large, and wound contraction is obvious, bottom wound and edge grow granulation tissue wound filled and led up.The time of 3. healing is longer, and the cicatrix of formation is larger.
3) subcrustal healing (healing under scab): form the hard crust of pitchy after the blood of wound surface, transudate and downright bad material drying, heal under crust.After epithelium regeneration completes, namely crust comes off.If exudate is more under crust, if there is bacteriological infection, crust hinders exudate to discharge, and is unfavorable for healing.This kind of healing required time is longer than without crust person, because first crust must dissolve by promoting epidermization in this case, then could grow.
So wound healing principle of medication should comprise and reduces wound surface, prevents from damaging and promoting tissue regeneration.The mode that the degree of damage and the regeneration capacity of tissue determine to repair, the time of healing and the size of cicatrix.The regeneration capacity of tissue mainly obtains during evolution, but still is subject to the impact of whole body and local condition.Systemic factor (as age, nutrition) and local factor (as infected and foreign body rejection, local blood circulation, innervation, ionizing radiation etc.) two aspects all can affect the progress of Regeneration and Repair.
Hepatic fibrosis (Hepatic fibrosis, HF): the total pathologic stage being multiple chronic hepatic diseases, hepatic fibrosis sustainable development can change liver cirrhosis even hepatocarcinoma into, and the middle and advanced stage of hepatopathy all fatal complication can occur.China is a hepatopathy big country, and high incidence and high mortality are the maximum features of hepatic disease.Hepatic fibrosis is the self-regeneration reaction that liver reply different pathogeny (inflammation, ethanol, parasite, chemical toxicant, immunity, Nutrition and Metabolism obstacle, disturbance of blood circulation, chronic viral infection, bile duct obstruction etc.) damage stimulates; The hepatocytes secrete cytokine profiles of damage, cytokine and some chemical mediator of adjoint sinusoidal endothelial cell, secretion of platelet activate hepatic stellate cell jointly, it is made to be converted into liver myofibroblast (myofibroblasts, MFB), MFB is by paracrine and Autocrine, generation is extracellular matrix (the extra cellular matrix of main component in a large number with collagen, ECM), the transition deposition of ECM, synthetics and degradation is unbalance, causes the generation of hepatic fibrosis, development.Research proves, hepatic fibrosis is reversible, and once develop into liver cirrhosis even hepatocarcinoma, people will feel simply helpless.Hepatic fibrosis is commitment and the only stage which must be passed by of liver cirrhosis, can reverse in any case, if but cause of disease sustainable existence, hepatic fibrosis increases the weight of gradually, lobules of liver and blood vessel etc. are reconstructed gradually, the normal configuration of liver is destroyed, and interval appears in central vein district and portal area, pseudolobuli is formed, and namely develops into irreversible liver cirrhosis.
Try to explore hepatic fibrosis pathogenesis, seek effective remedy measures, to reduction pathogenesis of cirrhosis rate and mortality rate significant.At present, treating liver fibrosis research mainly concentrates on the following aspects: 1) protect hepatocyte, suppresses HSC to activate, and 2) suppress ECM synthesis, promote that it is degraded, 3) and T suppression cell factor active, conditioning signal path.
Cytoglobin (Cytoglobin, Cygb) is the newcomer in hexa-coordinate haemachrome globulin (hxHb) superfamily found in vertebrates in recent years, expresses in multiple biology and tissue.Research shows, cytoglobin Cygb may be relevant with the generation of cancer.In the malignant tumor such as the esophageal carcinoma, pulmonary carcinoma and colon cancer, the expression of Cygb is usually repressed, expresses Cygb, can suppress the normal growth of lung carcinoma cell by the method for gene therapy.
Cell-penetrating peptide, i.e. CPPs, be also called protein transduction domain or film transduction peptide, be a kind of by 30 or less aminoacid form can the polypeptide of permeates cell membranes, bioactive substance can be delivered as many materials such as peptide, albumen, antisense nucleotide and liposomees.The appearance of cell-penetrating peptide, solves the difficult problem causing drug effect to seriously undermine because medicine cannot enter cell.Tat is the more deep cell-penetrating peptide of research in recent years, by the fusion with biomacromolecule (as nucleic acid or protein), can make fusion rotein transduce eukaryotic cells film and enter in cell.
The antiinflammatory of restructuring fusion Tat-Cygb human archeocyte globulin of the present invention, wound healing and effect of anti hepatic fibrosis, up to now, there is not yet relevant report both domestic and external.
Summary of the invention:
The invention provides the application of recombinant human fusion Tat cytoglobulin in anti-inflammatory.
The invention provides the application of recombinant human fusion Tat cytoglobulin in wound healing.
The invention provides the application of recombinant human fusion Tat cytoglobulin in anti-hepatic fibrosis.
Tat ?Cygb anti-inflammatory experimental result display: can be found out by the contrast of model group and each administration group, each administration group all can alleviate the auricle edema of mice to some extent, high dose TAT ?Cygb group with Dexamethasone group compared with model group, all there is significant difference (p<0.01), wherein high dose TAT ?the antiphlogistic effects of Cygb be slightly inferior to Dexamethasone group, but there was no significant difference (p>0.05) therebetween, and low dosage TAT ?Cygb group mice auricle edema degree comparatively Dexamethasone group and high dose group high, there was no significant difference (p<0.05) compared with model group.
Tat ?Cygb Wound Healing Experiments result display: TAT ?Cygb and Cygb all can promote wound healing, both suppositions all have anti-inflammatory effect because having peroxidase activity, thus promotion wound healing.In addition, due to TAT ?Cygb to wear the function of film peptide with TAT, can transdermal cell, act in cell, promote dermal cell growth, therefore comparatively Cygb is more obvious for effect.
Tat ?Cygb anti-hepatic fibrosis experimental result display:
Utilize CCI
4cause hepatic fibrosis in mice model, by observe mice growth conditions regulating liver-QI tissue appearance, measure serum and tissue in every liver function index content and pathological section analysis verification, treatment group liver has recovered color and gloss, and in liver, collagen contents obviously reduces; In Liver function grade, treatment group Serum ALT and AST content reduce all to some extent, and close to normal group, there were significant differences compared with model group; During fibrosis blood serum designated object detects, the change of HA, LN, C-III, C-IV equal size proves that TAT-Cygb can reduce the content of hepatocyte epimatrix (ECM); Hepatic tissue index changes of contents shows, TAT-Cygb treatment group can alleviate the consumption of superoxide dismutase, SOD content is made to recover normal level, the content of lipid peroxidation product MDA and the imino group composition Hyp of collagen fiber can be reduced simultaneously, in summary, by tail vein injection TAT-Cygb fusion rotein, CCl can be promoted
4the hepatic fibrosis in mice caused reverses.
The application of pharmaceutical composition in anti-inflammatory that the fusion Tat-Cygb albumen that the invention provides to recombinate forms for active component.
The application of pharmaceutical composition in wound healing that the fusion Tat-Cygb albumen that the invention provides to recombinate forms for active component.
The application of pharmaceutical composition in anti-hepatic fibrosis that the fusion Tat-Cygb albumen that present invention also offers to recombinate forms for active component.
Accompanying drawing illustrates:
Fig. 1 recombinates TAT-Cygb to the acutely inflamed impact of mice auricle swelling
Wherein: 1--model group; 2--Dexamethasone group; 3--low dose group; 4--high dose group
Fig. 2 rat trauma model builds
Fig. 3 skin wound healing situation
Fig. 4 rat skin wound tissue section HE dyeing (the 16th day, × 100)
Wherein: (a)-normal skin; (b)-matched group; (c)--mercurochrome group; (d)-low dose group; (e)-high dose Cygb group; (f)-low dosage TAT-Cygb group; (g)-high dose TAT-Cygb group
Fig. 5 respectively organizes mouse liver outward appearance
Wherein: (a)-normal group; (b)-just modeling complete primary school Mus; (c)-model group; (d)-low dosage TAT-Cygb group; (e)-high dose TAT-Cygb group
Fig. 6 respectively organizes mouse liver HE and dyes (40 times)
Wherein: (a)-normal group; (b)-just modeling complete primary school Mus; (c)-model group; (d)-low dosage TAT-Cygb group; (e)-high dose TAT-Cygb group
Fig. 7 respectively organizes mouse liver sirius red dyeing (40 times)
Wherein: (a) ?normal group; (b) ?firm modeling complete primary school Mus; (c) ?model group; (d) ?low dosage TAT ?Cygb group; (e) ?high dose TAT ?Cygb group
Fig. 8 respectively organizes mouse liver Masson trichrome stain (40 times)
Wherein: (a)-normal group; (b)-just modeling complete primary school Mus; (c)-model group; (d)-low dosage TAT-Cygb group; (e) high dose TAT-Cygb group
Fig. 9 TAT-Cygb fusion rotein is on the impact of each group of mice serum ALT and AST
Wherein: the mice that the firm modeling of 1-is complete; 2-normal group; 3-model group; 4-high dose TAT-Cygb group; 5-low dosage TAT-Cygb group; * p<0.05vs model group; * p<0.01vs model group
Figure 10 TAT-Cygb fusion rotein is on the impact of each group of mice serum HA, LN, C-III, C-IV
Wherein: the mice that the firm modeling of 1-is complete; 2-normal group; 3-model group; 4-high dose TAT-Cygb group; 5-low dosage TAT-Cygb group; * p<0.05vs model group; * p<0.01vs model group
Figure 11 TAT-Cygb fusion rotein is on the impact of each group of murine liver tissue SOD, MDA, Hyp
Wherein: 1 ?the complete mice of firm modeling; 2 ?normal group; 3 ?model group; 4 ?high dose TAT ?Cygb group; 5 ?low dosage TAT ?Cygb group; * p<0.05vs model group; * p<0.01vs model group
embodiment:
Following examples are only and help those skilled in the art to understand the present invention better, but do not limit the present invention in any way.
The functional verification of " embodiment 1 " restructuring Tat-Cygb anti-inflammatory
According to method described in the patent No. 201010202986.2, preparation restructuring fusion Tat-Cygb albumen.
1) mice caused by dimethylbenzene xylene ear swelling test: by 40 25g male and female KM white mice half-and-half, by often organizing 5 male Mus and 5 female Mus, being divided into 5 groups at random, being respectively high dose TAT-Cygb group, low dosage TAT-Cygb group, positive controls, model group;
2) in every left auricle of white mice, be evenly coated with the dimethylbenzene of 30 μ L, cause acute auricle edema inflammation, the left ear redness of visible white mice swells;
3) after 30min, to the even coating of the left auricle of white mice, matched group is that auris dextra is wide.High dose TAT ?Cygb group (500 μ g/ only), low dosage TAT ?Cygb (100 μ g/ only), positive controls (Dexamethasone ointment), model group (normal saline);
4), after 1h, de-cervical vertebra puts to death mice, dabs medicine remaining on the left auricle of white mice, cut left and right ear with eye scissors with cotton swab;
5) lay the identical position of two ears with card punch (diameter 8mm), obtain two circular auricles, be placed in respectively on analytical balance and weigh, record.
Result shows, the auricle edema rate (Fig. 1) of each experimental mice, can be found out by the contrast of model group and each administration group, each administration group all can alleviate the auricle edema of mice to some extent, high dose TAT ?Cygb group with Dexamethasone group compared with model group, all there is significant difference (p<0.01), wherein high dose TAT ?the antiphlogistic effects of Cygb be slightly inferior to Dexamethasone group, but there was no significant difference (p>0.05) therebetween, and low dosage TAT ?Cygb group mice auricle edema degree comparatively Dexamethasone group and high dose group high, there was no significant difference (p<0.05) compared with model group.Experimental result shows, the TAT of low dosage ?Cygb antiphlogistic effects more weak, and the TAT of high dose ?Cygb there is good antiphlogistic effects.
" embodiment 2 " skin wound healing animal model is verified
1) normal raising 10 (200g ± 20g) male KM rat 7d, start modeling;
2) lumbar injection 3% pentobarbital sodium (30mg/kg) anesthetized rat, this dosage can make rat maintain comatose state 2h ~ 3h;
3) cut off most of hair of rat back with ophthalmology curved scissors, coat appropriate depilatory cream;
4) after 10min ~ 15min, wipe remaining depilatory cream and hair by Medical cotton, then wipe clean with warm water, note not allowing rat suffer from cold;
5) with 75% alcohol disinfecting rat dorsum skin, then evenly cut 6 identical circular wound surface with the circular card punch of diameter 12mm at rat back, Medical cotton is stopped blooding, and modeling completes;
Promote wound healing treatment:
1) lumbar injection 3% pentobarbital sodium (15mg/kg) anesthetized rat before each administration;
2) right front portion wound surface be high dose TAT ?Cygb group, smear every day aseptic TAT ?Cygb (80 μ g/ time), twice on the one;
3) left front portion wound surface be low dosage TAT ?Cygb group, smear every day aseptic TAT ?Cygb (30 μ g/ time), twice on the one;
4) right middle wound surface is high dose Cygb group, smear every day aseptic TAT ?Cygb (80 μ g/ time), twice on the one;
5) left portion wound surface is low dosage Cygb group, smear every day aseptic TAT ?Cygb (30 μ g/ time), twice on the one;
6) right back portion wound surface is positive controls, smears mercurochrome (100 μ L/ time) every day, twice on the one;
7) left back portion wound surface is negative control group, smears physiological saline solution (100 μ L/ time) every day, twice on the one.
8) growing state of wound surface epidermis granulation tissue is observed every day, and the growth healing state of epithelial tissue, and Taking Pictures recording.Wrap wound dressing upon administration, avoid infection.
The preparation of Skin slice and dyeing:
After administration 15d, get wound healing place skin histology and fix, do skin histology paraffin section, the size of scar tissue after observation wound healing.
TAT ?Cygb on the impact of rat skin wound healing:
KM rat 10 is raised, modeling after 7d under normal condition.The rat back of the skin puncher of 12mm after depilation is utilized evenly to make a call to 6 holes in order.Represent respectively high dose TAT ?Cygb group (160 μ g/d), low dosage TAT ?Cygb group (60 μ g/d), high dose Cygb group (160 μ g/d), low dosage Cygb group (60 μ g/d), positive controls (mercurochrome, 200 μ L/d), negative control group (physiological saline solution, 200 μ L/d) (Fig. 2).Before medication every day, the growth situation of perusal rat wound surface epidermis granulation tissue, and the growth healing rate of epithelial tissue, Taking Pictures recording.
Can be found by the tracing observation after medication every day, the TAT of each dosage ?Cygb and Cygb group and mercurochrome group, matched group wound surface repair degree and mode of healing has comparatively marked difference (Fig. 3).Modeling first day, respectively form face difference little, wound area size is identical, and surface ratio is more moistening, medication the 4th day, the wound surface of the TAT ?Cygb group of visible high low dosage, Cygb group and mercurochrome group grows granulation tissue, be with a small amount of transudate, local has grown thin layer or some columnar epithelium, and matched group wound has formed brown incrustation, this is formed afterwards by the blood of wound surface, downright bad material and transudate drying, after medication the 7th day, wound area reduces, TAT ?Cygb group and the high dose Cygb group wound skin edge of wound of high low dosage obviously shrink, granulation tissue growth is intact, wound is ruddy, and low dosage Cygb group and mercurochrome group wound form crust skin, similar to matched group, this mode in subcrustal healing is generally than slow without crust wound healing, although be at edge of wound hyperplastic epidermis basal cell equally, but it carries out under crust, must after dissolving part crust, constantly could move regeneration epidermis by periphery to wound surface center, until promoting epidermization completes, crust just can come off, therefore this mode of healing is consuming time longer, after medication the tenth day, wound area continues to reduce, and high low dosage TAT ?Cygb group wound is tending towards healing, and high dose Cygb group wound granulation tissue growth is intact, low dosage Cygb group, mercurochrome group and matched group still have crust, the supracutaneous decrustation of partial regeneration.After medication the 13 day, high low dosage TAT ?Cygb group and high dose Cygb formed face and heal, but still needed time and treat the complete epithelization of epidermis, low dosage Cygb group decrustation, wound granulation tissue growth is intact, and mercurochrome group and matched group are still shown in fritter crust, and healing is not good enough.
Experimental result shows, TAT ?Cygb and Cygb all can promote wound healing, both suppositions all have anti-inflammatory effect because having peroxidase activity, thus promote wound healing.In addition, due to TAT ?Cygb to wear the function of film peptide with TAT, can transdermal cell, act in cell, promote dermal cell growth, therefore comparatively Cygb is more obvious for effect.
Normal skin is made up of epidermis, corium and subcutaneous tissue, and visible a large amount of knot hoof tissue and skin appendages in skin corium, as sweat gland, sebaceous gland and hair follicle etc.And the rat of full thickness skin excision, its skin appendages is totally disrupted, and in agglutination, granulation tissue hyperplasia, after reconstructing maturation, forms the fibrous connective tissue be made up of collagen fiber and elastic fiber, i.e. scar tissue.
By the rat trauma skin tissue slice after medication 16d, observable often organizes the size in rat site of injury scar repairing region.As shown in (Fig. 4), only can see the scar tissue of vitreous degeneration in the visual field of matched group B rat skin site of injury under 100 times of mirrors, the skin appendages such as hair follicle around, sweat gland and sebaceous gland are outside the visual field; Equally, mercurochrome group C rat skin wound due to subcrustal healing comparatively slow, can't see normal skin architecture in a visual field; Low dosage Cygb group D, can see partial skin appendages in a visual field of 100 times of mirrors; High dose Cygb group E, low dosage TAT ?Cygb group F and high dose TAT ?the visible skin appendages around of Cygb group G draw close to center, scar repairing region reduces successively.Infer TAT ?Cygb and Cygb because of it can improve cellular oxidation stress, prevent fibroblast hyper-proliferative from moving and extracellular matrix excessive buildup, make little than matched group and mercurochrome group of scar tissue, another reverse side, TAT ?Cygb and Cygb all by anti-inflammatory effect promote wound comparatively fast heal, epithelium is formed comparatively fast, wherein TAT ?Cygb group more obvious than Cygb group effect, and matched group and mercurochrome group due to subcrustal healing slower, in agglutination, the motion of rat exacerbates the repeated infection of wound simultaneously, make granulation tissue hyperplasia, cause scar tissue larger.
" embodiment 3 ": Tat-Cygb fusion rotein treatment hepatic fibrosis in mice
The structure of hepatic fibrosis mouse model and treatment:
1) experiment grouping: model group, TAT ?Cygb high dose group, TAT ?Cygb low dose group, normal group, every 7 white mice one group, totally 28 white mice.
2) hepatic fibrosis mouse model builds: the KM white mice of normal raising 25g one week, starts modeling.Model group, high dose TAT ?Cygb group and low dosage TAT ?the white mice of Cygb group with 1mL/100g body weight lumbar injection CCl
4solution, lumbar injection once every three days, and inject 16 times altogether, modeling lasts 46 days.3 times use the CCl of 15%
4solution, the CCl of middle 10 uses 25%
4solution, the CCl of last 3 uses 30%
4solution.The normal saline of normal group mice by intraperitoneal injection same volume.All white mice are all raised with enough Mus grain and water.
3) TAT ?Cygb treatment: treat that modeling in 46 days completes, start immediately treatment.High dose TAT ?Cygb group with 120 μ g/ (secondary) tail vein injection TAT ?Cygb fusion rotein, low dosage TAT ?Cygb group with 60 μ g/ (secondary) tail vein injection TAT ?Cygb fusion rotein, model group and normal group white mice are with 200 μ L/ (secondary) tail vein injection saline.Every other day tail vein injection once, injects 15 times altogether, last surrounding.
In modeling 46d, mice lethargy is depressed, and fur is clean, and belly swells, and movable minimizing is drowsiness, and food-intake and drinking-water reduce, and urine is yellow, illustrates that mouse liver is because of CCl
4impaired, and then have influence on the healthy growth of mice.And normal group fur is smooth, body weight is constant, more active, and food-intake is all normal with drinking-water.
In one month of administration, high dose TAT ?Cygb group mice active gradually, fur becomes smooth, and food-intake and drinking-water are tending towards normal.Low dosage TAT ?Cygb group mice is slightly better than model group, and activity is more active, but fur is not smooth.And model group mice still has that fur is unclean, drowsiness, food-intake and the less symptom of drinking-water.Mouse liver outward appearance:
In administration after 1 month, dissect mice, perusal liver outward appearance, and Taking Pictures recording.
As (Fig. 5), difference and the change of each group of mouse liver outward appearance obviously can be found out.Normal group mouse liver quality is soft, and glossiness is high, smooth surface, and clear-cut margin is neat, and whole liver is kermesinus, sees (Fig. 5 a); The hard glossiness of model group mouse liver quality is low, and rough surface has graininess, and edge is more blunt, and the jaundice of whole liver, trends towards liver cirrhosis, and indivedual hepatic portion and diaphragm and peripheral organs adhesion, as (Fig. 5 b); Model group mouse liver quality after one month is still comparatively hard, and glossiness is low, and rough surface has graininess, and the edge liver completeer than firm modeling takes a turn for the better to some extent, and whole liver is still more yellow, sees (Fig. 5 c); Low dosage TAT ?Cygb group mouse liver quality softer than model group, but still harder than normal group, glossiness is low, and there is fuzzy granule on surface, and edge is sharper keen, and liver color is slightly yellow, sees (Fig. 5 d); High dose TAT ?Cygb group mouse liver quality soft, glossiness obviously increases, and surface is more smooth, and clear-cut margin is neat, and liver is kermesinus, sees Fig. 5 e.
Liver section HE dyeing (Fig. 6) display:
The lobules of liver structure of normal group murine liver tissue is normal, has complete reticular fiber support, the regularity of distribution, and hepatic cords is towards surrounding radiation arrangement centered by central vein, and liver funicular cell arranges compact, and form is normal, sees (Fig. 6 a); Model group murine liver tissue lobules of liver structure disturbance, portal area fibrosis also expands, and collagen deposition is around it, have fibrous septum to be formed between header, hepatocyte volume increases, with large stretch of inflammatory cell infiltration, show serious damaging pathological change, see (Fig. 6 b); Model group murine liver tissue after one month has recovery a little, but still has serious damaging pathological change, as (Fig. 6 c); Low dosage TAT ?Cygb group murine liver tissue lobules of liver structure normal, still have collagen deposition around central vein, the fibrous septum between accidental portal area fibrosis and header, liver funicular cell arranges compact, and form is normal, sees (Fig. 6 d); High dose TAT ?the lobules of liver structure of Cygb group murine liver tissue normal, hepatic cords in radiation arrangement, without inflammatory cell, indivedual accidental portal areas fibrosis, with low dosage TAT ?Cygb group and model group contrast and be clearly better, as (Fig. 6 e).HE coloration result can tentatively illustrate TAT ?Cygb to CCI
4the hepatic fibrosis caused has dose-dependent therapeutical effect.
Liver section picrosirius staining
Adopt picrosirius staining method to identify collagen fiber better.Coloration result visible (Fig. 7), normal group murine liver tissue lobules of liver is complete, and structure is normal, only has a small amount of collagen painted at central vein, portal area, hepatic portal vein regulating liver-QI aorta edges, without fibrosis, as (Fig. 7 a); A large amount of collagen deposition, fibrosis around model group murine liver tissue portal area, with inflammatory cell infiltration, form fibrous septum between header and between header and central vein, make lobules of liver structure disturbance, and form pseudolobuli, as (Fig. 7 b); Low dosage TAT ?Cygb group murine liver tissue leaflet structure complete, fibrosis is comparatively light, portal area fibrosis, and fibrous septum is reduced, and collagen is painted more shallow than model group, as (Fig. 7 c); High dose TAT ?Cygb group murine liver tissue lobule complete, structure is normal, and fubril cicatrix in accidental lobule, portal area collagen is painted shallow, and fibrosis is low, as (Fig. 7 d).Picrosirius staining show TAT ?Cygb significantly can reduce collagen content in mouse liver, alleviate degree of hepatic fibrosis.
Liver section Masson trichrome stain:
Adopt Masson trichrome stain (Fig. 8) with the collagen fiber in dividing tissue and muscle fiber.As Fig. 8 a, normal group murine liver tissue lobules of liver structure is normal, has complete reticular fiber support, and hepatic cords arranges towards surrounding radiation centered by central vein, only Hepatic artery, hepatic portal vein and a small amount of a small amount of collagen of central vein marginal existence, exist without fibrosis; Model group murine liver tissue lobules of liver is disorderly, and there is a large amount of collagen deposition portal area, forms collagen fiber interval between header and between header and central vein, and pseudolobuli is formed, with inflammatory cell infiltration, as (Fig. 8 b); Low dosage TAT ?Cygb group murine liver tissue fibrosis reduce, lobules of liver structural integrity, portal area fibrosis is comparatively light, but still has more collagen to exist around portal area, as (Fig. 8 c); High dose TAT ?Cygb group murine liver tissue lobules of liver structural integrity, in order, portal area and portal area collagen content obviously reduce in hepatic cords arrangement, and fibrosis is light, comparatively close to normal group, as (Fig. 8 d).Masson trichrome stain result show equally TAT ?Cygb significantly can reduce collagen content in mouse liver, alleviate degree of hepatic fibrosis.Liver function grade (ALT, AST):
Glutamate pyruvate transaminase (ALT) and glutamic oxaloacetic transaminase, GOT (AST) are mainly present in hepatocyte, are to detect index sensitiveer in hepatic injury, by detect its content can reflect TAT ?Cygb to hepatocellular therapeutical effect.
As (Fig. 9), in the mice serum that firm modeling is complete, ALT content is more than 3 times of normal group, AST content is more than 2 times of normal group, illustrate that liver receives very large damage, although model group after 1 month recovers a lot, but its ALT content is still significantly higher than normal group mice, illustrate that modeling is more successful, and high dose TAT ?Cygb group mice serum ALT and AST content all significantly reduce, have significant difference compared with model group, wherein low dosage TAT ?Cygb group mice serum ALT content also remarkable in model group.
Fibrosis blood serum designated object detects (HA, LN, C ?III, C ?IV):
Hyaluronic acid (HA), laminin，LN (LN), C ?III collagen, C ?IV are all main components of hepatocyte epimatrix (ECM), by surveying its changes of contents, the degree of hepatic fibrosis can be indicated.As (Figure 10), in the mice serum that firm modeling is complete HA, LN, C ?III and C ?IV content all apparently higher than normal group, model group content after 1 month reduces, but still compared with normal group, there is significant difference, wherein high dose TAT ?LN, C in Cygb group mice serum ?III, C ?IV content significantly lower than model group, low dosage TAT ?in Cygb group serum C ?IV significantly lower than model group.
Hepatic tissue Indexs measure (SOD, MDA, Hyp):
In hepatic tissue, the change of superoxide dismutase (SOD) and malonaldehyde (MDA) can indicate the height of active oxygen and its derivative content in body.As the imino acid composition of collagen fiber, Hyp can indicate the collagen fiber level in liver indirectly.As (Figure 11), the mice that firm modeling is complete and model group increase because the oxidative stress in liver causes SOD to consume, superoxide dismutase content is significantly lower than normal group (p<0.05), the end-product MDA content of lipid peroxidation is significantly higher than normal group (p<0.01), can promote to activate hepatic stellate cell, and synthesize collagen; Hyp content is significantly higher than normal group (p<0.01), the increase of instruction collagen contents.Under high dose TAT-Cygb effect, SOD content significantly increases, MDA and Hyp content significantly reduces, and under low dosage TAT-Cygb effect, MDA content also has significant difference (p<0.05) compared with model group.
Utilize CCI
4cause hepatic fibrosis in mice model, by observe mice growth conditions regulating liver-QI tissue appearance, measure serum and tissue in every liver function index content and pathological section analysis, have studied the effect of TAT-Cygb fusion rotein to hepatic fibrosis.Observe from liver outward appearance and pathological section, treatment group liver has recovered color and gloss, and in liver, collagen contents obviously reduces, and shows that the hepatic fibrosis in mice that TAT-Cygb can promote CCI4 to induce reverses; In Liver function grade, treatment group serum alt and AST content reduce all to some extent, and close to normal group, there were significant differences compared with model group; During fibrosis blood serum designated object detects, the change of HA, LN, C-III, C-IV equal size proves that TAT-Cygb can reduce the content of hepatocyte epimatrix (ECM); Hepatic tissue index changes of contents shows, and TAT-Cygb treatment group can alleviate the consumption of superoxide dismutase, makes SOD content recover normal level, can reduce the content of lipid peroxidation product MDA and the imino group composition Hyp of collagen fiber simultaneously.In summary, by tail vein injection TAT-Cygb fusion rotein, CCl can be promoted
4the hepatic fibrosis in mice caused reverses.
Claims (2)
1. restructuring merge Tat ?Cygb human archeocyte globulin preparing the application in anti-hepatic fibrosis medicines.
2. restructuring merge Tat ?Cygb human archeocyte globulin be active component composition compositions preparing the application in anti-fibrosis medicine.
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| 吕颖慧等: "细胞珠蛋白对肝星状细胞氧化损伤的保护作用", 《生物工程学报》 * |
| 张如婧等: "TAT-cytoglobin融合蛋白的元和表达、纯化及生物活性分析", 《生物工程学报》 * |
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