CN108795873A

CN108795873A - A kind of fibroblastic preparation method and its kit

Info

Publication number: CN108795873A
Application number: CN201810634894.8A
Authority: CN
Inventors: 不公告发明人
Original assignee: Ningbo Minai Biotechnology Co Ltd
Current assignee: Ningbo Minai Biotechnology Co Ltd
Priority date: 2018-06-25
Filing date: 2018-06-25
Publication date: 2018-11-13

Abstract

The present invention provides a kind of fibroblastic preparation method, wherein, the method includes in being organized from people by containing vitamin E, ascorbic acid, Quercetin and fibroblast growth factor culture obtain fibroblast, and 47 gene of slow-virus transfection mutant heat shock protein, the fibroblast of preparation has good biological activity, 47 mutant of heat shock protein and the precollagen peptide of height expression can specific bonds, but inhibit collagen processing ripe and transhipment, fibrosis and cicatrization caused by inhibit excess collagen to express, fibroblast repairs fracture fibr tissue and inhibits inflammatory reaction simultaneously, restore the collagen structure of skin base layer；Fibroblast provided by the invention will have cicatrix of skin treatment and cosmetic result very well.

Description

A kind of fibroblastic preparation method and its kit

Technical field

The fibroblast obtained the present invention relates to a kind of fibroblastic method and by the method, especially may be used By the fibroblast for repairing cicatrix of skin.In particular it relates to be obtained by vitro culture from people's tissue Fibroblast, and build the recombinant viral vector of heat shock protein 47 so that fibroblast can stablize expression heat shock protein White 47,47 mutant of heat shock protein and precollagen peptide specific bond of height expression, but inhibit collagen processing ripe and turn Fortune, fibrosis and cicatrization caused by inhibit excess collagen to express, while fibroblast repair fracture fiber Tissue, restores the collagen structure of skin base layer；It will be expected to reach more preferably treatment and cosmetic result to skin scar.

Background technology

For scar granulation tissue through the ripe fibrous connective tissue formed of reconstruction, wherein fibrocyte is very rare, core it is elongated and Deep dye organizes interior blood vessel to reduce.Generally part is in contraction state, and color is pale or greyish white translucent, and matter is hard tough and lacks bullet Property.Operation excision is main means, but any modus operandi cannot all completely remove scar, only improve to greatest extent or Harm caused by correcting scar, and also preferably form new scar after performing the operation.For the keliod patients of lesser extent, laser, freezing Or radiotherapy is more commonly used means, but the possibility for pigmentation, epigenetic-surface reworking, infection, itch easily occur etc..

The fibroblast of skin disease rationality scar generates higher levels of collagen etc. than the fibroblast of normal skin Extracellular matrix, and its different phenotype, constitute fibroblastic heterogeneity.Cell technology is treated for cicatrix of skin New means are provided, such as research finds that extrasin beta 4 expresses reduction in keloid tissue, can be used as controlling for its Treat drug；Scar containing active factors repairs work of the gel with anti-proliferation of fibroblast, anti-inflammatory and softening, cicatricial tissue With and the dispel agate card scar essence of dispelling of scar essence glue and the active factors containing LEYA of agate card have damaged skin pigment prevented to produce It changes different, restores the normal skin tone and elasticity of skin.These means play good effect to cicatrix of skin treatment.But it is directed to The true cause that cicatrix of skin is formed inhibits excess collagen expression and fibrosis of skin, is to treat its crucial problem.

The present invention is that protein expression vector and the function of itself particularly control cicatrix of skin using fibroblast Treatment has good effect.Fibroblast (Fibroblast, FB) has been used to clinical treatment field, is treated in dermoplastic In achieve fine curative effect, the limitation of immunological rejection, ethics and oncogenicity for being not present etc. has and good faces Bed application prospect.Thus, the present invention obtains fibroblast using Vitro Culture Techniques, and builds 47 mutant of heat shock protein Recombinant viral vector, transfected fibroblast, height expression 47 mutant of heat shock protein specifically tied with procollagen peptide It closes, but inhibits collagen ripe and transhipment, fibrosis and cicatrization caused by excess collagen is expressed, while at fiber Cell repair is broken fibr tissue, restores the collagen structure of skin base layer, has no Tumor formation, is expected to repair cicatrix of skin Multiple and beauty generates better effect.

Invention content

Present inventor is by the study found that as the collagen specific molecular companion in cicatrix of skin forming process Companion, heat shock protein 47 play a significant role during fibroblast synthesizes collagen.Heat shock protein 47 is in glue It in the endoplasmic reticulum of former protein secretory cell, is combined with procollagen peptide specific, participates in the folding, assembly, modification of precollagen With transhipment etc. processes.Cicatrix of skin formation is synthesis and the degradation imbalance of collagen in wound, collagen over-deposit As a result；47 up-regulated expression of heat shock protein has promoted collagen overexpression, so that trauma skin fibrosis and cicatrization.

The present invention is by assisting procollagen peptide to fold heat shock protein 47, processing maturation and transhipment are closely related Active site carries out positional mutation, with procollagen specific bond, but influences the process that it is folded, processing is ripe and is transported, Inhibit Collagen of Fibroblasts albumen overexpression and fibrosis in cicatricial tissue.10 are obtained by site-directed mutation approach HSP47 mutant is screened through internal experiment in vitro, it is found that 47 mutant of heat shock protein is by the 78th site leucine mutation For proline, or the 132nd site serine sported into alanine, can play inhibition collagen overexpression, and Prevent fibrosis of skin and cicatrization.

The present invention is broken up using fibroblast proliferation and the characteristic of " going back to the nest ", by the HSP47 mutant gene pieces of structure The recombinant viral vector of section transfects FBs, can high expression HSP47 mutant, play HSP47 mutant inhibit collagen expression and The biological action of fibrosis and its function of fibroblast itself improve local skin microenvironment, repair fracture fiber group It knits, restores the collagen structure of skin base layer.

It is impaired that present inventor has been surprisingly found that fibroblast height expression HSP47 mutant can enter in cicatricial tissue In fibroblast, collagen expression and scar is inhibited to be excessively formed most close, the most representative cell factor of relationship The expression of transforming growth factor (transforming growth factor- β, TGF-β), the inhibition that pathologic scar is formed Produce significant effect.

The present invention provides a kind of fibroblastic preparation methods, which is characterized in that the method includes structure heat to stop The genetic fragment of gram protein 47 mutant in recombination to virus expression carrier, is packaged into after virus and transfected fibroblast, system The fibroblast of standby Cheng Kegao expression HSP47 improves fibroblast and plays more preferably inhibition collagen over-expression table Fracture fibr tissue reparation is reached, prevents fibroblast fibrosis and improved, can be used to treat cicatrix of skin.

Viral vectors of the present invention, can stablize in the cell proliferation and expression target gene, predominantly slow virus, Adenovirus, retrovirus etc., it is preferable that selection slow virus expression system.

People HSP47 gene orders are expanded by PCR from normal human peripheral blood's genomic DNA, by the mesh of HSP47 DNA fragmentation carry out rite-directed mutagenesis again, obtain HSP47 mutant, then be connected respectively on expression plasmid and be built into recombinant plasmid Afterwards, after positive colony PCR identifications and sequencing identification, the plasmid built can be stored in -80 DEG C of ultra low temperature freezers.Again will HSP47 mutant gene segments are cloned on slow sick carrier, and packaged recombination HSP47 mutant virus can transfect into fibre Cell is tieed up, the fibroblast of HSP47 mutant slow-virus transfections is prepared into.

72 hours fluorescence microscopy microscopic observation green fluorescent proteins after infection (Green fluorescent protein, GFP luminous situation), GFP develop the color more than 90% or more, show to carry HSP47 mutant gene virus transfection fibroblasts Success obtains the fibroblast of continuous expression HSP47 mutant.

It is detected according to operation instructions by PCR kit for fluorescence quantitative, has transfected HSP47 mutant virus carriers After FBs secondary cultures to the 10th generation, expression HSP47 mutant genes are higher than at least 70 times or more of the FBs of untransfected.

The fibroblast detects through immunofluorescence technique, marker α smooth muscle actins, I procollagen types Protein alpha 1 and 90% or more the expression of type III procollagen α 1.

The FBs for having transfected recombination HSP47 mutant virus has following performance：Inhibit cicatricial tissue fibroblast Excess collagen is expressed and fibrosis, inhibits inflammatory reaction, repairs fracture fibr tissue, restores the collagen egg of skin base layer White structure；And without tumor.

Fibroblast of the present invention for cicatrix of skin reparation can recombinate HSP47 with subculture in vitro separately culture (preferably its vitamin E containing 0.5ng-100ng, 0.5ng-200ng are anti-by serum-free medium by the FBs of mutant virus transfection Bad hematic acid, 10ng-500ng Quercetins, 1ng-100ng fibroblast growth factors and 1 × serum-free additive) subculture in vitro separately training It supports, when cell culture was by the 5-6 days, cell growth fusion reaches 90% or more, and it is 0.25% to need use quality volume ratio Pancreatin (preferably it contains 0.05%EDTA) had digestive transfer culture.

Take the cellular morphology observed under secondary culture to 3 generations and 10 generation FBs inverted microscopes, the FBs of untransfected and other The FBs for transfecting target gene has similar cellular morphology.

The fibroblast for cicatrix of skin reparation is overexpressed in HSP47 mutant body, in external tumor formation experiment Tumor formation phenomenon is not found, ensure that its safety.

The fibroblast for cicatrix of skin reparation is externally co-culturing in fact with fibroblasts from hyperplastic scar It inhibits Proliferation of Hypertrophic Scar Fibroblasts and collage synthesis and α-SMA and I-type collagen to express in testing, promotes Its apoptosis occurs；In rabbit scar model body in transplantation experiments, with individual Allogenic Cultured Dermal Fibroblasts Transplantation and saline control group It compares, inhibits I-type collagen and the secretion of the TGF-β 1 of 1 gene expression of TGF-β and inflammatory factor, have to cicatrix of skin Significantly repair；Internal experiment in vitro shows without tumor.

The present invention also provides a kind of reagent kit products, which is characterized in that the kit includes：

1) fibroblast serum free medium；

2) packaged HSP47 mutant height expression virus；

3) II Collagenase Types；

4) pancreatin；

5) active compound：Vitamin E, ascorbic acid, Quercetin and fibroblast growth factor

6) serum-free additive；

7) operation instructions；

Wherein, the operation instructions include above-mentioned method.

The present invention also provides the applications that the fibroblast is used for cicatrix of skin reparation, by ensure that in vitro culture Obtain sufficient amount of fibroblast, and with expression HSP47 mutant proteins are stablized, play its to cutaneous scar tissue at Fibrocyte excess collagen is expressed and fibrosis, repairs fracture fibr tissue, restores the collagen knot of skin base layer The function of structure is expected to be used for treatment cicatrix of skin.

Description of the drawings

Human fibroblasts Immunofluorescence test α-SMA, the COLI α 1 and COLIII α 1 that in Fig. 1 prepared by embodiment 1 are expressed It is horizontal

Fig. 2 is expression of the HSP47 mutant in the 10th generation FBs of transfection HSP47 mutant slow virus

Fig. 3 is HSP47 prepared by embodiment 3_muSlow-virus transfection FBs is to Proliferation of Hypertrophic Scar Fibroblasts level view

Fig. 4 is HSP47 prepared by embodiment 3_muSlow-virus transfection FBs is to fibroblasts from hyperplastic scar collage synthesis knot Fruit is schemed

Fig. 5 is HSP47 prepared by embodiment 3_muSlow-virus transfection FBs is to fibroblasts from hyperplastic scar α-SMA and I type The protein expression result figure of collagen

Fig. 6 is HSP47 prepared by embodiment 1-3 methods_muIn slow-virus transfection rabbit FBs transplanting rabbit ear scar model serum 1 secretion level figure of TGF-β

Fig. 7 is HSP47 prepared by embodiment 1-3 methods_muI type glue in slow-virus transfection rabbit FBs transplanting rabbit ear cicatricial tissues 1 gene expression dose figure of former albumen and TGF-β

Specific implementation mode

The present invention provides a kind of fibroblastic preparation methods, which is characterized in that from people organize in extract it is unicellular, It obtains fibroblast by containing vitamin E, ascorbic acid, Quercetin and fibroblast growth factor culture, and slow virus turns 47 (47kDa heat shock protein, HSP47) gene of mutant heat shock protein is contaminated, being prepared into can high expression heat shock The fibroblast of protein 47 mutant, 47 mutant of heat shock protein and precollagen peptide specific bond of height expression, but inhibit Collagen processing is ripe with transhipment, fibrosis and cicatrization caused by inhibit excess collagen to express, while at Fibrocyte reparation is broken fibr tissue, restores the collagen structure of skin base layer, can be used to treat skin spot.

74 mutant of heat shock protein of the present invention is to expand heat shock protein 47cDNA sequences by round pcr, And HSP47 mutant is obtained using site-directed mutagenesis technique and is connected to disease through restriction enzyme site by its recombination to construct to expression plasmid On malicious expression vector, gene recombined virus carrier transfected fibroblast.The HSP47 mutant is by the 78th bright ammonia in site Acid mutation is proline, or the 132nd site serine is sported alanine.

HSP47 mutant genes segment of the present invention is to be expanded and recombinated on expression plasmid by round pcr, warp Restriction enzyme site is connected to recombination to expression plasmid, then recombinates on virus expression carrier, then pack 293T cells and be prepared into The virus of height expression target gene.

Viral vectors of the present invention, can stablize in the cell proliferation and expression target gene, predominantly slow virus, Adenovirus, retrovirus etc., it is preferable that selection slow virus expression system is commercially produced product, can be purchased from commercial company It buys.

The present invention provides the preparation methods of packaging HSP47 mutant slow virus, which is characterized in that by people's HSP47 sequences After the full RNA and reverse transcription extracted from normal human peripheral blood by PCR obtains cDNA, purpose is obtained using HSP47 primer amplifications Segment, HSP47 primers are normal chain：5'-CCGCTCGAGCGG TAC GCG AGG GAG GAC GAA G-3 ', antisense strand：5'- TCCCCCGGGGGA GAT ATT GAG CAG AGC GTA G-3 ' contain XhoI and Sma I restriction enzyme sites respectively；It will amplification Obtained HSP47 target fragments distinguish above-mentioned primer and mutation specific probe carries out targeted mutagenesis, and probe sequence is respectively： 5’-CCGGAGCACGGGACCCGAG-3 ' or 5 '-CTTCGACCCGCGAGCTGAC-3 ' obtains 1 He of HSP47 mutant respectively Mutant 2.

HSP47 mutant and pGMLV-PA1 will be obtained and carry out XhoI/Sma I double digestions, in T4 DNA connections after double digestion Under enzyme effect, is prepared in 4 DEG C, connection reaction in 12 hours and clone connection liquid, after conversion competent escherichia coli cell DH5a, carried out Positive colony PCR identifications and sequencing identification；PCR product detected through gel electrophoresis and sequencing identification meet HSP47 mutant size and After sequence, correct bacterium solution switching will be sequenced in the LB liquid medium of the 10ml antibiotic of benzyl containing ammonia, 37 DEG C of overnight incubations are used Endotoxin-free plasmid is small to put forward middle amount kit progress plasmid extraction, extracts qualified recombinant plasmid and places -80 DEG C of ultra low temperature freezers Medium-term and long-term preservation；The HSP47 mutant genes clip size is 1272bp；

HSP47 mutant DNA fragments and PGMLV-PA1 carriers are subjected to XhoI/Sma I double digestions respectively, in T4 DNA It connects under enzyme effect, HSP47 segments and digestion PGMLV-PA1 carriers is prepared into clone's connection in 37 DEG C, connection reaction in 12 hours Liquid after converting competent escherichia coli cell DH5a, carries out positive colony PCR identifications and sequencing identification；

It takes cell state good, the 293T cells of exponential phase is in, after cell count, according to the culture of each 10cm Ware 6 × 10⁶A cell number is inoculated in culture dish, 37 DEG C, 5%CO₂Incubator in overnight incubation；It is removed before transfection in second day Culture solution changes 5ml Opti-MEM culture solutions；Take 9 μ g packaging mixed liquors and 3 μ g slow virus expression plasmids that 1.5ml Opti- are added In MEM, gently mixing, takes 36 μ l lipofectamine 2000 to be added in 1.5ml Opti-MEM, gently mixing, room temperature are put Set 5min；Plasmid solution and 2000 dilutions of lipofectamine are mixed, room temperature 20min is set；Mixed liquor be slowly added dropwise to In the culture solution of 293T cells, mixing, in 37 DEG C, 5%CO₂It is cultivated in cell incubator；It is discarded after culture 6h mixed containing transfection With the culture medium of object, the PBS liquid cleaning that 10ml is added is primary, is fallen after the soft transfection mixture for shaking culture dish to wash remnants It abandons；It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO₂Continue to cultivate 48-72h in incubator；

According to cell state, the 293T cell supernatants of 48h after transfection are collected；In 4 DEG C, 4000g centrifuges 10min, removes Cell fragment；With 0.45 μm of filter filtering supernatant in 40ml ultracentrifugation pipes；Trim sample respectively, will carry viral supernatants The ultracentrifugation pipe of liquid is put into ultracentrifuge one by one, and setting parameter of noncentricity is 25000rpm, centrifugation time 2h, centrifugation Temperature is controlled at 4 DEG C；It after centrifugation, discards supernatant, removal remains in the liquid on tube wall, and virus is added and preserves liquid, repeatedly Piping and druming is resuspended；After fully dissolving, high speed centrifugation 10000rpm takes supernatant fluorescence spectrometry titre, packages after centrifuging 5min HSP47 mutant slow virus according to 50 μ l 2E+8TU/ml packing, be stored in -80 DEG C of ultra low temperature freezers；

Fibroblast convenient sources of the present invention, i.e. discarded skin, blood and hair follicle；It is preferably derived from Discarded skin.Due to fibroblast convenient sources, cell culture obtains simply, and can be obtained from individuation can accomplish individuation Using.

Fibroblast preparation method of the present invention, i.e.,：Discarded skin is passed through containing 1 dual anti-× PBS of 2 times of mycillins (pH7.4) washing uses the II Collagenase Type 5-20mL that 1: 1 mass volume ratio is 0.1% afterwards three times, disappears in 4 DEG C of refrigerator casees Change overnight；It is that 0.25% pancreatin digests 4 hours in 37 DEG C of incubators that dermis layer tissue mass volume ratio is taken after digestion, after digestion 1500rpm is centrifuged 10 minutes, and physiological saline resuspension is added after abandoning supernatant in suction, is filtered with 100 μm of cell sieves；Cell suspension 1000rpm is centrifuged 10 minutes, and cell precipitation is added complete serum-free medium after being washed 2 times with physiological saline resuspension and (contains 0.5ng-100ng vitamin Es, 0.5ng-200ng ascorbic acid, 10ng-500ng Quercetins, 1ng-100ng at fiber growth because Son and 1 × serum-free additive) it is resuspended, 1-10 × 10 are pressed after tongue disk orchid dyeing counting⁶Cell/ml is added to recombined human fibre and connects egg It is cultivated in white coated tissue culture plate or culture dish, in 37 DEG C, 5%CO₂It is cultivated 24-72 hours in incubator；

Culture, which is inhaled after 24-72 hours with 5ml pipettes, abandons culture solution, and fresh serum free culture solution is added and carries out changing liquid, carefully Born of the same parents' culture plate or culture dish continue to place 37 DEG C, 5%CO₂Continue to cultivate in incubator；

Digestion is carried out when fibroblastic growth fusion reaches 80% or more and expands bottle, and culture solution is absorbed, takes the pH value to be 7.4 1 × PBS is added after washed once in 0.25% pancreatin to culture bottle of 0.5ml, and 200 μ l tires are added after 2-3 minutes in digestion Cow's serum terminates digestion, adds physiological saline, is blown and beaten, is drawn in 15ml centrifuge tubes, then washed with physiology salt with 5mL pipettes It washs once, is added in the same 15mL centrifuge tubes, 1000 rpms of centrifugations collect fibroblast in 10 minutes；It is used after centrifugation 1mL fresh mediums are resuspended, and count, and the fresh serum-free mediums of 10mL are added with 5mL pipettes, are added to 1 bottle of fine even egg Continue in white coated culture bottle at 37 DEG C, 5%CO₂It is cultivated in incubator, a fresh medium was changed every 2 days；Wait for into fibre Dimension cell growth fusion is when reaching 80% or more, by above-mentioned through collected by trypsinisation fibroblast, continuous passage 3 be commissioned to train it is foster, The fibroblast for obtaining 109 or more, is resuspended with 4ml physiological saline, tongue disk orchid dyeing counting obtains largely into fiber finer Born of the same parents save backup under the conditions of placing 4 DEG C；

Part fibroblast forms in 40% Fibroblast culture solution, 10% dimethyl sulfoxide (DMSO) and 50% fetal calf serum Frozen stock solution be stored in -196 DEG C of liquid nitrogen containers, in case from now on recover after use.

Fibroblast detects through immunofluorescence technique, marker α smooth muscle actins, I procollagen types protein alpha 1 It is almost 100% with the expression of type III procollagen α 1.

Fibroblast of the present invention for cicatrix of skin reparation, preparation method are as follows：It will recombination HSP47 mutation Body slow virus is added in fibroblastic six well culture plate with 10 μ l 1E+7TU/ml transfects respectively, system 2ml, is mixed It is even, 37 DEG C, 5%CO₂Incubator in be incubated 8-12 hour after, replacement serum-free medium (vitamin containing 0.5ng-100ng E, 0.5ng-200ng ascorbic acid, 10ng-500ng Quercetins, 1ng-100ng fibroblast growth factors and the addition of 1 × serum-free Object)；When cell growth fusion reaches 90%, is digested with 0.25% pancreatin, be transferred to 25cm²It is grown in culture bottle, 1 hole corresponds to 1 A culture bottle, in 25cm²Continue had digestive transfer culture culture when fusion reaches 90% in culture bottle；Growth transfection is divided into 3 groups：Untransfected Control group, blank slow-virus transfection group and HSP47 slow-virus transfection groups are transfected；

72 hours fluorescence microscopy microscopic observation green fluorescent proteins after infection (Green fluorescent protein, GFP luminous situation), GFP develop the color more than 90% or more；

It is detected according to operation instructions by PCR kit for fluorescence quantitative, the use of HSP47 primers is positive-sense strand 5 '- GCGAACGATTCGACTCAAGC-3 ', antisense strand：5'-CATCCCGACAGAAAGGCACT-3'；HSP47 slow virus has been transfected to turn After dying fibrocyte secondary culture to the 10th generation, 1 gene of expression HSP47 mutant is above untransfected and blank slow virus 73 times and 51.45 times of the 1st generation FBs of transfection, 2 gene of HSP47 mutant are above the of untransfected and blank slow-virus transfection 1 73.5 times of generation FBs and 31.82 times.

Can be with subculture in vitro separately culture, that is, the FBs for recombinating HSP47 mutant slow-virus transfections passes through complete serum-free medium (preferably its vitamin E containing 0.5ng-100ng, 0.5ng-200ng ascorbic acid, 10ng-500ng Quercetins, 1ng-100ng at Fibroblast growth factor and 1 × serum-free additive) subculture in vitro separately culture, when cell culture was by the 5-6 days, cell growth fusion reaches To 90% or more, pancreatin (preferably it contains 0.05%EDTA) had digestive transfer culture that use quality volume ratio is 0.25% is needed；

Take the cellular morphology observed under secondary culture to 3 generations and 10 generation FBs inverted microscopes, the FBs of untransfected and other There is the FBs for transfecting target gene similar cellular morphology, cell to be grown at fusiformis.

Serum-free medium of the present invention is commercially available, including BIOWIT serum free mediums, OriCell^TMSerum free medium, StemXVivo serum free mediums andSerum free medium etc., more preferably Use OriCell^TMSerum free medium.

The present invention externally co-cultures with fibroblasts from hyperplastic scar real for the fibroblast of cicatrix of skin reparation It inhibits Proliferation of Hypertrophic Scar Fibroblasts and collage synthesis and α-SMA and I-type collagen to express in testing, promotes Its apoptosis occurs.

The present invention for cicatrix of skin reparation fibroblast in rabbit scar model body in transplantation experiments, and it is individual Allogenic Cultured Dermal Fibroblasts Transplantation is compared with saline control group, inhibit I-type collagen and 1 gene expression of TGF-β and inflammation because The TGF-β 1 of son is secreted, and is had to cicatrix of skin and is significantly repaired；Internal experiment in vitro shows without tumor.

1) 500ml fibroblasts serum free medium；

2) the packaged HSP47 mutant height expression viruses of 500 μ L 1E+8TU/ml；

3)10⁴IU/mL II Collagenase Types；

4) 0.25% pancreatin of 100ml；

5) 50 times of active compounds of 10mL：25ng-5 μ g vitamin Es, 25ng-10 μ g ascorbic acid, 500ng-25 μ g Mongolian oaks Pi Su, 50ng-5 μ g fibroblast growth factors

6) 50 times of serum-free additives of 10mL；

7) operation instructions；

Wherein, the operation instructions include above-mentioned method.

The present invention also provides fibroblastic applications for cicatrix of skin reparation, by ensure that in vitro culture Sufficient amount of fibroblast is obtained, and with expression HSP47 mutant proteins are stablized, plays the heat shock protein of its height expression White 47 mutant and precollagen peptide specific bond, but inhibit collagen processing ripe and transhipment, to inhibit collagen mistake Fibrosis and cicatrization caused by degree expression, while fibroblast repairs fracture fibr tissue and inhibits inflammatory reaction, it is extensive The function of the collagen structure of multiple skin base layer is expected to be used for treatment skin spot.

Hereinafter, specific embodiments of the present invention are illustrated, but the technical scope of the present invention is not limited to these examples.

Embodiment 1

Recombinate the structure and packaging of HSP47 mutant slow virus carriers

It is extracted from normal human peripheral blood by blood total RNA extraction reagent box (purchase of Beijing Tiangeng biology) total RNA, and use one-step method Reverse Transcriptase kit (purchase of Promega companies of the U.S.) by acquisition mRNA reverse transcriptions for cDNA,

According to international ncbi database, HSP47 primers, as normal chain are designed：5'-CCGCTCGAGCGG TAC GCG AGG GAG GAC GAA G-3 ', antisense strand：5'-TCCCCCGGGGGA GAT ATT GAG CAG AGC GTA G-3'；Contain respectively XhoI and Sma I restriction enzyme sites are synthesized through Beijing Nuo Sai companies.The HSP47 target fragments for being obtained amplification by round pcr, Reaction system is：Primer concentration is 20 μm of ol, and each 2 μ L, cDNA templates that are added respectively add 3 μ L, 10 × PCR buffer solution of people, 5 μ L, 10mM dNTP mixtures 0.5 μ L, ddH₂29.5 μ L, Hot Start Taq enzymes of O 0.5 μ L, 50 μ L of total volume.Reaction condition is： 95 DEG C of 5min, 95 DEG C of 60sec, 49 DEG C, 50sec, 72 DEG C 60sec, 5 cycle after carry out 95 DEG C of 5min, 95 DEG C of 60sec again, 67 DEG C, 50sec, 72 DEG C 60sec, 25 cycle after re-extend 10min.PCR product cuts glue after agarose electrophoresis, is given birth to Tiangeng Change scientific and technological (Beijing) Co., Ltd gel reclaims kit and recycles DNA fragmentation.

Using above-mentioned HSP47 primers and point mutant probe, i.e.,：5 '-CCGGAGCACGGGACCCGAG-3 ' or 5 '- CTTCGACCCGCGAGCTGAC-3 ', carries out positional mutation PCR, and reaction system is：Primer concentration is 20 μm of ol, each that 2 μ are added L；Concentration and probe concentration is 20 μm of ol, 2 μ L；HSP47 fragment templates add 3 μ L, 10 × PCR buffer solution of people, 5 μ L, 10mM dNTP mixing Object 0.5 μ L, ddH₂29.5 μ L, Hot Start Taq enzymes of O 0.5 μ L, 50 μ L of total volume；Reaction condition and acquisition HSP47 mutation Body segment is for example above-mentioned, that is, obtains 1 (HSP47 of HSP47 mutant_mu- 1) and 2 (HSP47 of HSP47 mutant_mu-2)。

HSP47 mutant and pGMLV-PA1 (the lucky triumphant purchase in Shanghai) use XhoI/Sma I (U.S. Thermo respectively Fisher companies) carry out double digestion 2 hours, segment is acted in T4 DNA ligases (Japanese TAKARA companies) both after double digestion Under, prepare clone's connection liquid, (U.S. conversion competent escherichia coli cell DH5a in 4 DEG C, connection reaction in 12 hours Invitrogen companies) positive colony PCR identifications and sequencing identification are carried out afterwards；Identify HSP47 mutant genes clip size about For 1272bp, meet HSP47 mutant size and sequence.Correct bacterium solution switching will be sequenced in the LB of the 10ml antibiotic of benzyl containing ammonia In fluid nutrient medium, 37 DEG C of overnight incubations are put forward middle amount kit and are carried out plasmid pumping with Beijing Tiangeng biology endotoxin-free plasmid is small It carries, extracts and preserved for a long time in qualified recombinant plasmid -80 DEG C of ultra low temperature freezers of placement.

It takes cell state good, is in the 293T cells (Invitrogen companies of the U.S.) of exponential phase, cell count Afterwards, according to the culture dish 6 × 10 of each 10cm⁶A cell number is inoculated in culture dish, 37 DEG C, 5%CO₂Incubator in cultivate Overnight；Culture solution is removed before transfection in second day, changes 5ml Opti-MEM culture solutions；Take 9 μ g packaging mixed liquors and 3 μ g slow virus tables It is added in 1.5ml Opti-MEM up to plasmid, gently mixing, takes 36 μ l lipofectamine, 2000 (U.S. Invitrogen Company) it is added in 1.5ml Opti-MEM, gently mixing, is placed at room temperature for 5min；Mix plasmid solution and lipofectamine 2000 dilutions set room temperature 20min；Mixed liquor is slowly added dropwise into the culture solution of 293T cells, mixing, in 37 DEG C, 5%CO₂ It is cultivated in cell incubator；The culture medium containing transfection mixture is discarded after culture 6h, the PBS liquid cleaning that 10ml is added is primary, It is abandoned after the soft transfection mixture for shaking culture dish to wash remnants；It is slowly added to the cell culture medium containing 10% serum 20ml, in 37 DEG C, containing 5%CO₂Continue to cultivate 48-72h in incubator；

According to cell state, the 293T cell supernatants of 48h after transfection are collected；In 4 DEG C, 4000g centrifuges 10min, removes Cell fragment；With 0.45 μm of filter filtering supernatant in 40ml ultracentrifugation pipes；Trim sample respectively, will carry viral supernatants The ultracentrifugation pipe of liquid is put into ultracentrifuge one by one, and setting parameter of noncentricity is 25000rpm, centrifugation time 2h, centrifugation Temperature is controlled at 4 DEG C；After centrifugation, discarding supernatant, removal as possible remains in the liquid on tube wall, and virus is added and preserves liquid, Gently piping and druming is resuspended repeatedly；After fully dissolving, high speed centrifugation 10000rpm after centrifuging 5min, takes supernatant fluorescence spectrometry to drip Degree, packaged HSP47 mutant slow virus dispense according to 50 μ l 2E+8TU/ml, are stored in -80 DEG C of ultra low temperature freezers.

Same method is mutated for HSP47 gene critical sites, and it is prominent that we have also prepared other 8 HSP47 Variant slow virus, i.e. HSP47_mu- 3, the 70th site serine sports proline；HSP47_mu- 4, the 82nd site serine Sport proline；HSP47_mu- 5, the 124th site arginine sports proline；HSP47_mu- 6, the 228th site essence ammonia Acid mutation is alanine；HSP47_mu- 7, the 243rd site tyrosine sports proline；HSP47_mu- 8, the 265th site silk Histidine mutations are alanine；HSP47_mu- 9, the 326th site leucine sports alanine；HSP47_mu- 10, the 378th position Point mutant serine is proline.

Embodiment 2

The preparation of human fibroblasts

Discarded skin uses 1: 1 quality volume after 1 × PBS (pH7.4) washings three times dual anti-containing 2 times of mycillins Than II Collagenase Types (Sigma Co., USA's purchase) 5-20mL for 0.1%, digested overnight in 4 DEG C of refrigerator casees；It is taken after digestion Dermis layer tissue mass volume ratio is that (U.S. Thermo Fisher are public for 0.25% pancreatin (containing mass volume ratio 0.02%EDTA) Department's purchase) it is digested 4 hours in 37 DEG C of incubators, 1500rpm is centrifuged 10 minutes after digestion, and physiological saline is added after abandoning supernatant in suction It is resuspended, is filtered with 100 μm of cell sieves；Cell suspension 1000rpm is centrifuged 10 minutes, and washing 2 is resuspended with physiological saline in cell precipitation Fibroblast serum-free medium (vitamin E containing 0.5ng-100ng, 0.5ng-200ng ascorbic acid, 10ng- are added after secondary 500ng Quercetins, Sigma Co., USA's purchase；1ng-100ng fibroblast growth factors, the purchase of R&D companies of the U.S.；1 × without blood Clear additive, the purchase of Thermo Fisher companies of the U.S.) it is resuspended, 1-10 × 10 are pressed after tongue disk orchid dyeing counting⁶Cell/ml adds Enter into the coated tissue culture plate of recombinant human fibronectin polypeptide or culture dish and cultivate, in 37 DEG C, 5%CO₂It is trained in cell incubator It supports 48-72 hours.The fibroblast culture medium for replacing fresh factor-containing is continuously cultivated again, until cell adherent growth goes out Come.

When cell growth is closeer, digested using 0.25% (mass volume ratio) pancreatin, with 10ml containing cell because The fibroblast culture medium of son is transferred to the coated 75cm of new recombinant human fibronectin polypeptide after being resuspended²In culture bottle, in 37 DEG C, 5%CO₂Culture is merged until growth in cell incubator reaches 90% or so, and 0.25% pancreatin is used to carry out had digestive transfer culture training It supports, i.e., 1 bottle passes on 2 bottles, and the fibroblast culture medium for supplementing fresh factor-containing continues to cultivate, when cultivating to 5 generation, at Fibrocyte is digested using 0.25% pancreatin, that is, is obtained fibroblast, can be resuspended to the tire ox containing 10% dimethyl sulfoxide (DMSO) It is frozen in liquid nitrogen in serum.

Tongue is taken to expect 1.0 × 10 after blue dyeing counting⁵For FBs cells kind in the burnt special culture dish of copolymerization, ice PBS washes three Time, 5 minutes every time；When cell is half-dried, covering fixes 15 minutes with 4% cold paraformaldehyde, is protected from light；After sucking paraformaldehyde, It is washed three times, every time 5 minutes with 1 × PBS of ice；0.5%Triton X-100 covering cell 10 minutes, 1 × PBS of ice wash three times, often Secondary 5 minutes；Import fetal calf serum (Fetus bovine serum, FBS) room temperature of host identical as secondary antibody is closed 30 minutes；Match Primary antibody processed dilutes 200 times, i.e. rabbit-anti people α smooth muscle actins (α-Smooth muscle actin, α-SMA), I types with FBS Procollagen α 1 (type I Collagen alpha-1, COL1 α 1) and 1 (type III of type III procollagen α Collagen alpha-1, COLIII α 1) it is mostly anti-；It is separately added into primary antibody covering cell, tinfoil wraps up 4 DEG C and is protected from light overnight.It takes Go out cell and warms to room temperature about 1h again；1 ‰ Tween of ice is washed twice, 5 minutes every time, in shaking table.1 × PBS of ice is washed once, 5 minutes, In shaking table；Prepare fluorescent marker secondary antibody：Ab50598 Goat anti-rabbit IgG (H&L) TRITC is prepared with 1 × PBS, Concentration 1: 200；Secondary antibody is added, is incubated at room temperature 1 hour (being protected from light).1 ‰ Tween of ice is washed twice, 5 minutes every time, in shaking table.Ice 1 × PBS is washed once, 5 minutes, in shaking table.DAPI contaminates core, is dripped per ware 1, covers all cell.1 ‰ Tween of ice washes two It is secondary, 5 minutes every time, in shaking table.1 × PBS of ice is washed once, 5 minutes, in shaking table.Anti- fluorescent quenching mountant is added, is protected from light.Carefully Born of the same parents' machine testing in confocal fluorescent microscopic (German Lycra company).

For Fig. 1 the results show that human fibroblasts are through above-mentioned Immunofluorescence test, A is α-SMA in confocal fluorescent microscopic Under the green fluorescence observed, be almost 100% with its fluorescence signal is compared under light field,；B is COLI α 1 aobvious in confocal fluorescent The fluorescent red-orange observed under micro mirror is almost 100% with its fluorescence signal is compared under light field；C is that COLIII α 1 are being copolymerized The green fluorescence observed under burnt fluorescence microscope shows fibroblast express alpha-SMA, COLI α 1 and 1 positive rates of COLIII α Also it is almost 100%.

Embodiment 3

The preparation of HSP47 mutant slow-virus transfection human fibroblasts

Recombination each 10 μ l 1E+7TU/ml of HSP47 mutant slow virus are added to 50 μ g/ml's of human fibroblasts In six well culture plates, system 2ml, mixing, 37 DEG C, 5%CO₂Incubator in be incubated 8-12 hour after, be replaced with fiber Cell non-serum culture solution (vitamin E containing 0.5ng-100ng, 0.5ng-200ng ascorbic acid, 10ng-500ng Quercetins, 1ng-100ng fibroblast growth factors, 1 × serum-free additive)；When cell growth fusion reaches 90%, with 0.25% pancreas Enzymic digestion is transferred to 25cm²It is grown in culture bottle, 1 hole corresponds to 1 culture bottle, in 25cm²When fusion reaches 90% in culture bottle after Continuous had digestive transfer culture culture；Growth transfection is divided into 3 groups：Untransfected control group (Con-FB), blank slow-virus transfection group (Blank- FB), HSP47 mutant slow-virus transfection groups (HSP47 has been transfected_mu-FB)。

The transfected fibroblast of acquisition by PCR kit for fluorescence quantitative according to the operation instructions (U.S. Invitrogen companies) detection HSP47 mutant gene expressions are normal chain using HSP47 primers：5'- CCGCTCGAGCGG TAC GCG AGG GAG GAC GAA G-3 ', antisense strand：5'-TCCCCCGGGGGA GAT ATT GAG CAG AGC GTA G-3'；PCR system and reaction condition are carried out by method in embodiment 1, and Fig. 2 is that HSP47 mutant is transfecting Expression in the 10th generation FBs of HSP47 mutant slow virus.The FBs of recombination HSP47 mutant slow virus carriers is transfected After secondary culture to the 10th generation, 1 gene of expression HSP47 mutant is above the 1st generation of untransfected and blank slow-virus transfection FBs73 times and 51.45 times, 2 gene of HSP47 mutant is above the 1st generation FBs 73.5 of untransfected and blank slow-virus transfection Times and 31.82 times.

Embodiment 4

The fibroblastic reagent kit product for cicatrix of skin reparation prepared

Fibroblastic kit for cicatrix of skin reparation includes：

1) 500ml fibroblasts serum free medium；

2) the packaged HSP47 mutant height expression viruses of 500 μ L 1E+8TU/ml；

3)10⁴IU/mL II Collagenase Types；

4) 0.25% pancreatin of 100ml；

6) 50 times of serum-free additives of 10mL；

7) operation instructions；

Wherein, the operation instructions include the method for embodiment 1-3 descriptions.

Embodiment 5

HSP47 mutant slow-virus transfection human fibroblasts co culture system in vitro is tested

The fibroblasts from hyperplastic scar (being bought from U.S. ScienCell) of logarithmic growth phase is with 3x10⁵Cells/well Concentration is inoculated in 6 hole Transwell culture plates (0.3 μm, Corning companies of the U.S.), UltraCULTURE^TMFree serum culture, 1.5mL is inoculated with per hole, human fibroblasts, 3x10 is added in cell⁵Cell/room, 1.5ml set 37 DEG C, 5%CO₂Incubator is trained altogether It supports, is grouped.Blank control A groups：It is added without fibroblast in cell and co-cultures (Control), multiple holes 6；At fiber Cell controls B groups：Normal fibroblast (FB) is added in cell to co-culture, multiple holes 6；Test C groups：It is added and implements in cell Blank slow-virus transfection FBs (Blank-FB) prepared by example 3 is co-cultured, multiple holes 6；Test D groups：Embodiment 3 is added in cell The HSP47 of preparation_mu- 1 slow-virus transfection FBs (HSP47_mu1- FB) it co-cultures, multiple holes 6；Test E groups：It is added and implements in cell HSP47 prepared by example 3_mu- 2 slow-virus transfection FBs (HSP47_mu2- FB) it co-cultures, multiple holes 6；In vitro culture 1-7 days, in inversion Microscopically observation, photograph.

The cytochrome oxidase isozymes of blank control are good, directional, radial.And normal fibroblast control group, blank Slow-virus transfection FBs experiment C groups, HSP47_muSlow-virus transfection FBs tests D and E groups, and fibroblasts from hyperplastic scar number subtracts Few, protrusion shortens, and shape becomes irregular, arranges aobvious confusion, and refractivity is weaker.

By U.S.'s Bio-Rad cell counters, according to operation instructions respectively on day 1, the 2nd day, the 3rd day, the 4th day With the 5th day detection Proliferation of Hypertrophic Scar Fibroblasts activity.It is recognised that blank control A groups and other each groups from Fig. 3 It compares, Proliferation of Hypertrophic Scar Fibroblasts speed faster, between the two and has significant difference (* * P=0.011)；And FB compares B groups and Blank-FB experiment C groups and HSP47_muSlow-virus transfection FBs experiments D with E groups are compared, and hyperplastic scar is at fibre It ties up cell proliferation rate faster, between the two and there is significant difference (* P=0.036), show HSP47 prepared by the present invention_mu Slow-virus transfection FBs inhibits Proliferation of Hypertrophic Scar Fibroblasts, its apoptosis is promoted to occur.

Each group takes per hole co-cultures the 1st day 50 μ L of supernatant, and 100 DEG C are dried, and adds 4mol/L NaOH, 120 DEG C, 10min sequentially adds oronain, perchloric acid solution, and develop the color about 15min at 65-70 DEG C, with Beckman DU-600 spectrophotometers Colorimetric surveys absorbance value, wavelength 550nm；Its hydroxyproline in the supernatant of hydroxyproline colorimetric determination each group cell Content, analyzes fibroblasts from hypertrophic scars collage synthesis, and Fig. 4 results are shown：HSP47 prepared by embodiment 3_muSlow-virus transfection FBs There are inhibiting effect, HSP47 to fibroblasts from hyperplastic scar collage synthesis_muThe cell of slow-virus transfection FBs experiment D and E groups Collage synthesis tests C groups, * P=0.038 significantly lower than FB control B groups and Blank-FB.And FB control B groups and Blank-FB are real C groups are tested compared with blank control A groups, collage synthesis is significantly reduced, * P=0.042；Two groups of experimental data exists through examining Statistical significance.

Take above-mentioned co-cultivation 1 day 2 × 10⁶Fibroblasts from hyperplastic scar, be detected using western blot analysis, Protein Extraction：1000rpm is centrifuged 10 minutes after cell suspension, abandons culture solution, and PBS is cleaned 3 times.It is added according to cell concentration corresponding Extraction buffer, on ice jog 15min.It collects in lysate to EP pipes, 14,000rpm centrifugation 15min.Take supernatant to new EP Guan Zhong.Protein sample concentration mensuration：It is carried out with reference to BCA protein quantifications kit (KGPBCA) specification of the triumphant base in Nanjing.It will carry The protein solution and 5 × sample-loading buffer for having taken sample are mixed by 5: 1, boil 5min.Prepare 5% deposition glue, 8% separation gel, It is about 10ng per hole applied sample amount.Electrophoresis：80V constant pressures 50 minutes, 120V constant pressures electrophoresis to the rigid plastic emitting bottom of bromophenol blue stop, pvdf membrane Methanol pre-processes 3~5 seconds, puts to transfer liquid and infiltrates half an hour.Gel is taken out, is put to filter paper, gel is formed and transfers heap Lamination simultaneously removes bubble removing.Under cryogenic conditions (4 DEG C), 100V constant pressures 60~120 minutes.Hybond membrane is taken out, TBST is rinsed 5 minutes, Three times；5% skimmed milk power solution room temperature is closed 1 hour；TBST washes film 5min, three times.Suitable primary antibody diluted concentration (mouse is added Anti-human α-SMA antibody (1: 1000), anti-human 1 antibody of Col I α (1: 1000) of mouse), β-actin are used as with reference to egg In vain, it stays overnight for 4 DEG C.TBST washes film 5min, three times；37 DEG C of incubation 1h of rabbit-anti mouse secondary antibody diluent；TBST washes film 5min, three times；It steams Distilled water rinses film 2min, discards liquid.It washes altogether three times.Hybond membrane is placed on a transparent plastic sheet, it will with clean pipettor Chemiluminescence luminous substrate is equably added to the surface of film, and reaction is made to continue 5min.The extra ECL of film surface is sucked with filter paper Luminescent solution uses film development, fixing in darkroom, and takes pictures.Western blot, which is analyzed, by software I mage J carries out gray analysis.

It is the protein expression gray analysis knot for passing through Western blotting and detecting α-SMA and I-type collagen in Fig. 5 Fruit shows compared with blank control A groups that FB compares B groups, Blank-FB using β-actin albumen idiosyncrasy bands as standard Test C groups, HSP47 prepared by embodiment 3_muSlow-virus transfection FBs co-culture experiments D and E groups, albumen reaction band obviously subtract It is weak；HSP47 in Fig. 5 A and B_muα-the SMA of slow-virus transfection FBs experiment D and E groups and I-type collagen expression are significantly lower than FB pairs C groups, * P=0.024 are tested according to B groups and Blank-FB.And FB control B groups and Blank-FB experiment C groups and blank control A group phases Than α-SMA and I-type collagen expression are significantly reduced, * P=0.027.HSP47_muSlow-virus transfection FBs experiment D and E groups and Control group compares, and a-SMA albumen lowers 83.89% and 84.73% respectively, and I-type collagen lowers 87.36% He respectively 87.77%, show HSP47 prepared by the present invention_muSlow-virus transfection FBs expresses fibroblasts from hypertrophic scars α-SMA and Col I α 1 There is inhibiting effect.

Ill vitro test method is co-cultured according to 5 cell of embodiment, the HSP47 mutant of other 8 positional mutation is carried out Screening, experimental result is shown in the following table 1.

As can be seen that HSP47 in table 3_mu1And HSP47_mu2Slow-virus transfection FBs and other each HSP47 mutant slow virus The FBs of transfection is compared, and the Proliferation of Hypertrophic Scar Fibroblasts of co-cultivation obviously inhibits, between the two and with significant Difference (* * P=0.009).HSP47_mu1And HSP47_mu2The fibroblasts from hyperplastic scar that slow-virus transfection FBs is co-cultured, glue Original synthesis is significantly lower than the FBs, * P=0.034 with other each HSP47 mutant slow-virus transfections.HSP47_mu1And HSP47_mu2Slowly α-SMA and the I-type collagen expression for the fibroblasts from hyperplastic scar that virus transfection FBs is co-cultured obviously are respectively lower than it The FBs of his each HSP47 mutant slow-virus transfection,^#P=0.011 and^&P=0.008.

It co-cultures in experiment in vitro and finds with fibroblasts from hyperplastic scar, HSP47_mu1And HSP47_mu2Slow-virus transfection FBs has preferably Inhibiting proliferation scar fibroblast proliferation, collage synthesis, α-SMA and l than other HSP47 mutant The expression of collagen type；Show the 78th site mutation of heat shock protein 74 or the 132nd site mutation, to cicatrix of skin Reparation may have good effect.

Embodiment 6

Rabbit ear cicatrix of skin model skin experiment in vivo

24 adult rabbits purchased from Department Of Medicine, Peking University's animal center, half male and half female, 2.0~2.5kg of weight, family Rabbit auricular vein injects 3% yellow Jackets (30mg/kg), and performing the operation with the outside of belly of picking up the ears after anesthesia cuts off a diameter of 10~15mm Full thickness skin, strike off perichondrium.At ear two, each surface of a wound interval 15mm or more, surface of a wound exposure, the 20d after surface of a wound epithelialization, Cicatrix of skin hyperplasia is formed, and rabbit ear cicatrix of skin model is prepared into.This model continues no less than 3 weeks.

After modeling success, it is randomly divided into three groups of A, B, C and D group, every group 6, A groups are to be prepared using embodiment 1-3 methods HSP47_mu- 1 slow-virus transfection rabbit FBs transplantation groups (HSP47_mu1- FB), B groups are to be prepared using embodiment 1-3 methods HSP47_mu- 2 slow-virus transfection rabbit FBs transplantation groups (HSP47_mu2- FB), skin carries out skin corium skin at rabbit ear cicatrix of skin two Lower injection 5 × 10⁵Cell, 50 μ l of volume；Rabbit fibroblast transplantation group (FB) prepared by C groups embodiment 2, with the same sample prescription of A groups Case injects fibroblast；D groups are saline control group (Control), same to inject 50 μ l physiological saline.

Three groups of rabbit modeling ear modeling positions and normal auris dextra are visually observed after the last administration, and take pathological biopsy, It is fixed with 10% formalin, paraffin embedding, slice, Hematoxylin-eosin (HE) dyeing, using the green skies production in purchase Beijing Hematoxylin-eosin staining kit is carried out according to operation instructions, and tissues observed changes under light microscope.

Rabbit ear color is pink, poor, soft before modeling, and pathological analysis thereon arrange by visible clearly capillary, follicular orifice Row are neat, and the 30th day after modeling, the variation of cicatrix of skin animal model local skin pathology is the positive evidence of model success or not, After model is successfully prepared, skin histology at modulus type, the visible local skin fibroblast proliferation of microscopy, collagenous fibres increase increasing Slightly, fine and close and disorganized, during which there is a small amount of inflammatory cell.Specific rabbit ear cicatricial tissue variation see the table below 1.

Table 1

Note：Cicatrix of skin local skin pathology variation classification reference standard is "-"：Skin histology is normal, and institutional framework is complete It is whole；"+"：Fibroblast proliferation, collagenous fibres increase thickening, fine and close and disorganized, during which there is a small amount of inflammatory cell；"+ +"：Fibroblast had significant proliferation, collagenous fibres showed increased thickening is fine and close and disorganized, during which there is inflammatory cell；"++ +"：Fibroblast is largely proliferated, collagenous fibres showed increased thickening, very fine and close and disorganized, during which there is more inflammatory Cell.

The HSP47 that in 1 result of table prepared by the present invention_muSlow-virus transfection rabbit FBs transplantation treatment rabbit ear scar models, are being transplanted Repairing well occurs in the 14th Lepus ear scar afterwards, i.e., Proliferation of Hypertrophic Scar Fibroblasts is inhibited well, collagen Fiber, which substantially reduces, to attenuate, and inflammatory cell liquid significantly reduces in tissue.Do not find that tumor formation is existing in each group animal model body simultaneously As.

Vancouver cicatrix of skin marking scales scoring is carried out to rabbit ear scar after each group cell transplantation, statistical result see the table below 2。

Table 2

Explanation：It scores skin colour, blood vessel, flexibility, thickness, pain and itch.0 grade of lock evaluation project is both needed to It compares and carries out with the skin at other positions of body.And it is required that special slide pressing scar must be used to be observed after 2 seconds.Best result 18 points, minimum point 0 point, score is higher to illustrate that scar situation is more serious, conversely, then influence degree is smaller.

The HSP47 that in 2 result of table prepared by the present invention_muSlow-virus transfection rabbit FBs transplantation treatment rabbit ear scar models, are being transplanted Start within the 7th day rabbit ear scar color and luster, blood vessel, flexibility, thickness, pain and itch afterwards to occur improving well, shows to pass through this Invent the HSP47 prepared_muSlow-virus transfection rabbit FBs transplantation treatments, which have cicatrix of skin, significantly repairs curative effect.

Embodiment 7

Rabbit ear scar model collagen and inflammatory Cytokines Expression analysis

Rabbit ear scar model uses recombination HSP47 prepared by embodiment 1-3 the methods in embodiment 6_muSlow-virus transfection Rabbit FBs experiment in vivo acquires rabbit peripheral blood by ear central artery after transplanting 7 days, 14 days, 30 days and 60 days, passes through 1000rpm Centrifugation obtains serum after ten minutes.Water is secreted using 1 enzyme linked immunosorbent assay (ELISA) of TGF-β (ELISA) kit detection TGF-β 1 Flat (being purchased from R&D companies of the U.S.), is detected according to 1 ELISA kit operation instructions of U.S. R&D company's T GF- β.In Fig. 6 HSP47_muSlow-virus transfection rabbit FBs transplanting A and B groups TGF-β 1, which is secreted, is substantially less than FBs transplanting C groups, * * P=0.012；And FBs The TGF-β 1 that transplanting component C is secreted is horizontal also significantly lower than saline control D groups, * * P=0.042；Show HSP47_muSlow virus Transfection FBs inhibits the TGF-β 1 of rabbit ear scar model inflammatory factor to secrete, and rabbit ear scar inflammatory reaction is contributed to improve and promote Into scar repairing.

Rabbit ear scar model uses recombination HSP47 prepared by embodiment 1-3 the methods in embodiment 6_muSlow-virus transfection In rabbit FBs experiment in vivo, 7 days after transplanting rabbit ear cicatricial tissue 0.5g or so are taken, use total tissue RNA extracts kit (north Capital Tiangeng biology purchase) extraction total serum IgE, and will be obtained using one-step method Reverse Transcriptase kit (purchase of Promega companies of the U.S.) It is cDNA to obtain mRNA reverse transcriptions.The primer sequence of Real-time PCR is as follows, type I collagen：Positive-sense strand：5'-CCT GGAAAG AAT GGAGAT GAT G-3 ', antisense strand：5'-ATCCAAACC ACT GAAACC TCT G-3'；TGF-β1：Just Adopted chain：5 '-GCCAGAGTG GTT ATC 1_IT TGA TG-3 ', antisense strand：5'-AGT GTG TTA TCC CTG CTG TCA C-3'；GAPDH：Positive-sense strand：5 '-GGTGAAGGTCGG TGT GAA CG-3 ', antisense strand：5'-CTC GCT CCT GGAAGATGGTG-3’。

Obtained cDNA templates are subjected to RT-PCR and use ABl7500 real-time quantitative instrument detection I-type collagen and TGF-β The expression of 1 target gene.Real time-PCR detection architectures are as follows：(U.S. Invitrogen is public by 10 μ L SYBR Premix Department), each 1 μ L (10pmol/L) of upstream and downstream primer, 2 μ L cDNA and 6ul ddH₂O amounts to 20ul；Reaction condition：96℃ 5min；96 DEG C of 1min, 56 DEG C of 45sec；45 cycles；96 DEG C of 3min, 60 DEG C of 1min.HSP47 in Fig. 7_muSlow-virus transfection FBs transplants A and B groups I-type collagen and 1 gene expression of TGF-β is substantially less than FBs transplanting C groups, * * P=0.008；And FBs is moved It is horizontal also significantly lower than saline control D groups, * P=0.033 to plant the TGF-β 1 that component C is secreted；Show HSP47_muSlow-virus transfection FBs inhibits the expression of the collagen over-expressed in rabbit ear scar model, while also inhibiting 1 base of inflammatory factor TGF-β Because of expression, help to prevent rabbit ear scar caused by collagen overexpression fibrosis due to the scar that generates, while also pressing down Inflammatory reaction is made.

Claims

1. a kind of fibroblastic preparation method, which is characterized in that it is unicellular the method includes being extracted in being organized from people, Fibroblast, and slow-virus transfection are obtained by containing vitamin E, ascorbic acid, Quercetin and fibroblast growth factor culture Mutant heat shock protein 47 (47kDa heat shock protein, HSP47) gene, being prepared into can high expression heat shock protein The fibroblast of white 47 mutant；

Wherein, heat shock protein is expanded by archaeal dna polymerase chain reaction (polymerase chain reaction, PCR) technology White 47 cDNA sequence, and HSP47 mutant is obtained using site-directed mutagenesis technique, by its recombination to construct to expression plasmid, through enzyme Enzyme site is connected on virus expression carrier, gene recombined virus carrier transfected fibroblast；

47 mutant of the heat shock protein is that the 78th leucine is sported proline, or by the 132nd mutant serine For alanine.

2. according to the method described in claim 1, it is characterized in that, wherein, 72 hours fluorescence microscopies are green under the microscope after infection The luminous situation of color fluorescin (Green fluorescent protein, GFP), GFP develop the color more than 90% or more；

It is preferred that the operation instructions according to PCR kit for fluorescence quantitative are detected, it is slow 47 mutant of heat shock protein has been transfected After the fibroblast secondary culture to the 10th generation of viral vectors, expression 47 mutant gene of heat shock protein is higher than untransfected At least 70 times or more of fibroblast；

It is further preferred that the fibroblast detects through immunofluorescence technique, glue before marker α smooth muscle actins, I types Former protein alpha 1 and 90% or more the expression of type III procollagen α 1.

3. according to claim 1-2 any one of them methods, which is characterized in that described to have transfected 47 mutant of heat shock protein The fibroblast of slow virus has following performance：47 mutant of heat shock protein and precollagen peptide specific bond of height expression, But inhibit collagen processing ripe with transhipment, fibrosis and cicatrization caused by inhibit excess collagen to express, Fibroblast repairs fracture fibr tissue and inhibits inflammatory reaction simultaneously, restores the collagen structure of skin base layer；And Without tumor.

4. according to claim 1-3 any one of them methods, which is characterized in that by people HSP47 sequences by PCR from normal person After the full RNA and reverse transcription of peripheral blood extraction obtain cDNA, target fragment is obtained using HSP47 primer amplifications, HSP47 primers are Normal chain：5'-CCGCTCGAGCGG TAC GCG AGG GAG GAC GAA G-3 ', antisense strand：5'-TCCCCCGGGGGA GAT ATT GAG CAG AGC GTA G-3 ' contain XhoI and Sma I restriction enzyme sites respectively；The piece for the HSP47 mesh that amplification is obtained Above-mentioned primer and mutation specific probe carry out targeted mutagenesis to section respectively, and probe sequence is respectively：5'- CCGGAGCACGGGACCCGAG-3 ' or 5 '-CTTCGACCCGCGAGCTGAC-3 ' obtains HSP47 mutant 1 and dashes forward respectively Variant 2.

HSP47 mutant will be obtained and pGMLV-PA1 carries out XhoI/Sma I double digestions, made in T4 DNA ligases after double digestion Under, is prepared in 4 DEG C, connection reaction in 12 hours and clone connection liquid, after conversion competent escherichia coli cell DH5a, carried out positive Clone PCR is identified and sequencing identification；PCR product detected through gel electrophoresis and sequencing identification meet HSP47 mutant size and sequence Afterwards, correct bacterium solution switching will be sequenced in the LB liquid medium of the 10ml antibiotic of benzyl containing ammonia, 37 DEG C of overnight incubations, with without in Toxin plasmid is small to put forward middle amount kit progress plasmid extraction, extracts qualified recombinant plasmid and places length in -80 DEG C of ultra low temperature freezers Phase preserves；The HSP47 mutant genes clip size is 1272bp；

HSP47 mutant DNA fragments and PGMLV-PA1 carriers are subjected to XhoI/Sma I double digestions respectively, in T4 DNA connections Under enzyme effect, HSP47 segments and digestion PGMLV-PA1 carriers are prepared into clone's connection liquid in 37 DEG C, connection reaction in 12 hours, turned After changing competent escherichia coli cell DH5a, positive colony PCR identifications and sequencing identification are carried out；

It takes cell state good, the 293T cells of exponential phase is in, after cell count, according to the culture dish 6 of each 10cm ×10⁶A cell number is inoculated in culture dish, 37 DEG C, 5%CO₂Incubator in overnight incubation；Culture is removed before transfection in second day Liquid changes 5ml Opti-MEM culture solutions；Take 9 μ g packaging mixed liquors and 3 μ g slow virus expression plasmids that 1.5ml Opti-MEM are added In, gently mixing, takes 36 μ l lipofectamine 2000 to be added in 1.5ml Opti-MEM, gently mixing, is placed at room temperature for 5min；Plasmid solution and 2000 dilutions of lipofectamine are mixed, room temperature 20min is set；Mixed liquor is slowly added dropwise to 293T In the culture solution of cell, mixing, in 37 DEG C, 5%CO₂It is cultivated in cell incubator；It is discarded after culture 6h containing transfection mixture Culture medium, the PBS liquid cleaning that 10ml is added is primary, soft to shake culture dish to abandon after washing remaining transfection mixture； It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, containing 5%CO₂Continue to cultivate 48-72h in incubator；

According to cell state, the 293T cell supernatants of 48h after transfection are collected；In 4 DEG C, 4000g centrifuges 10min, removes cell Fragment；With 0.45 μm of filter filtering supernatant in 40ml ultracentrifugation pipes；Trim sample respectively, will be with vial supernatant Ultracentrifugation pipe is put into ultracentrifuge one by one, and setting parameter of noncentricity is 25000rpm, centrifugation time 2h, centrifuging temperature Control is at 4 DEG C；It after centrifugation, discards supernatant, removal remains in the liquid on tube wall, and virus is added and preserves liquid, blows and beats repeatedly It is resuspended；After fully dissolving, high speed centrifugation 10000rpm takes supernatant fluorescence spectrometry titre after centrifuging 5min, packaged HSP47 mutant slow virus is stored in -80 DEG C of ultra low temperature freezers according to 50 μ l 2E+8TU/ml packing.

5. according to claim 1-4 any one of them methods, which is characterized in that the fibroblast source discard skin, Blood and hair follicle；It is preferably derived from discarded skin.

6. according to claim 1-5 any one of them methods, which is characterized in that discarded skin passes through dual anti-containing 2 times of mycillins 1 × PBS (pH7.4) washing three times afterwards use 1: 1 mass volume ratio be 0.1% II Collagenase Type 5-20mL, in 4 DEG C of ice It is digested overnight in case case；It is that 4 are digested in 37 DEG C of incubators is small for 0.25% pancreatin that dermis layer tissue mass volume ratio is taken after digestion When, 1500rpm is centrifuged 10 minutes after digestion, and physiological saline resuspension is added after abandoning supernatant in suction, is filtered with 100 μm of cell sieves；Cell Suspension 1000rpm is centrifuged 10 minutes, and the resuspension of cell precipitation physiological saline is added serum-free medium after washing 2 times and (contains 0.5ng-100ng vitamin Es, 0.5ng-200ng ascorbic acid, 10ng-500ng Quercetins, 1ng-100ng at fiber growth because Son and 1 × serum-free additive) it is resuspended, 1-10 × 10 are pressed after tongue disk orchid dyeing counting⁶Cell/ml is added to recombined human fibre and connects egg It is cultivated in white coated tissue culture plate or culture dish, in 37 DEG C, 5%CO₂It is cultivated 24-72 hours in incubator；

Culture, which is inhaled after 24-72 hours with 5ml pipettes, abandons culture solution, and fresh serum free culture solution is added and carries out changing liquid, cell training It supports plate or culture dish continues to place 37 DEG C, 5%CO₂Continue to cultivate in incubator；

Digestion is carried out when fibroblastic growth fusion reaches 80% or more and expands bottle, culture solution is absorbed, and it is 7.4 to take pH value 1 × PBS is added after washed once in 0.25% pancreatin to culture bottle of 0.5ml, and 200 μ l fetal calf serums are added after 2-3 minutes in digestion Digestion is terminated, physiological saline is added, is blown and beaten, is drawn in 15ml centrifuge tubes with 5mL pipettes, then with brine one It is secondary, it is added in the same 15mL centrifuge tubes, 1000 rpms of centrifugations collect fibroblast in 10 minutes；1mL is used after centrifugation Fresh medium is resuspended, and counts, and the fresh serum-free mediums of 10mL are added with 5mL pipettes, are added to 1 bottle of fibronectin packet Continue in the culture bottle of quilt at 37 DEG C, 5%CO₂It is cultivated in incubator, a fresh medium was changed every 2 days；Wait for into fiber finer Intracellular growth fusion is when reaching 80% or more, and by above-mentioned through collected by trypsinisation fibroblast, continuous passage 3 is commissioned to train foster, acquisition 10⁹Above fibroblast, is resuspended with 4ml physiological saline, tongue disk orchid dyeing counting obtains a large amount of fibroblasts, puts It is saved backup under the conditions of setting 4 DEG C；

The jelly that part fibroblast forms in 40% Fibroblast culture solution, 10% dimethyl sulfoxide (DMSO) and 50% fetal calf serum Liquid storage is stored in -196 DEG C of liquid nitrogen containers, in case being used after recovering from now on.

7. according to claim 1-6 any one of them methods, which is characterized in that

The HSP47 mutant slow virus prepared is added to fibroblastic six hole with 10 μ l 1E+7TU/ml respectively to cultivate It is transfected in plate, system 2ml, mixing, 37 DEG C, 5%CO₂Incubator in be incubated 8-12 hour after, replace complete serum-free Culture medium；The serum-free medium；It is preferred that the serum-free medium vitamin E containing 0.5ng-100ng, 0.5ng-200ng Ascorbic acid, 10ng-500ng Quercetins, 1ng-100ng fibroblast growth factors and 1 × serum-free additive；Work as cell growth When fusion reaches 90%, is digested with 0.25% pancreatin, be transferred to 25cm²It is grown in culture bottle, 1 hole corresponds to 1 culture bottle, in 25cm² Continue had digestive transfer culture culture when fusion reaches 90% in culture bottle；Growth transfection is divided into 3 groups：Untransfected control group, blank are sick slowly Malicious transfection group and HSP47 mutant slow-virus transfection groups are transfected；

72 hours fluorescence microscopy microscopic observation green fluorescent proteins (Green fluorescent protein, GFP) after infection Shine situation, and GFP develops the color more than 90% or more；

It is detected according to operation instructions by PCR kit for fluorescence quantitative, the use of HSP47 primers is positive-sense strand 5 '- GCGAACGATTCGACTCAAGC-3 ', antisense strand：5'-CATCCCGACAGAAAGGCACT-3'；It is slow HSP47 mutant has been transfected After virus transfection fibroblast secondary culture to the 10th generation, express HSP47 mutant 1 and 2 genes higher than untransfected at Fibrocyte is respectively 73 times and 73.5 times.

The fibroblast of HSP47 slow-virus transfections is recombinated by complete culture solution α-MEM subculture in vitro separately cultures, works as cell culture By the 5-6 days, cell growth fusion reached 90% or more, and it is 0.25% pancreatin had digestive transfer culture to need use quality volume ratio；

Take the cellular morphology observed under secondary culture to 1 generation and 10 generation fibroblast inverted microscopes, untransfected at fiber There is the fibroblast of cell and other transfection target gene similar cellular morphology, cell to be grown at fusiformis.

8. according to claim 1-7 any one of them methods, which is characterized in that the fibroblast, in vitro with hyperplasia Proliferation of Hypertrophic Scar Fibroblasts and collage synthesis and α-are inhibited in property fibroblasts from hypertrophic scars co-culture experiments SMA and I-type collagen expression, promote its apoptosis to occur；In rabbit scar model experiment in vivo, with individual fibroblast Transplanting is compared with saline control group, inhibits I-type collagen and the TGF-β 1 of 1 gene expression of TGF-β and inflammatory factor Secretion；Have to cicatrix of skin and significantly repairs；Internal experiment in vitro shows without tumor.

9. a kind of kit, which is characterized in that the kit includes：

1) fibroblast serum free medium；

2) packaged HSP47 mutant height expression virus；

3) II Collagenase Types；

4) pancreatin；

6) serum-free additive；

7) operation instructions；

Wherein, the operation instructions include claim 1-8 any one of them methods.

10. a kind of transfection prepared such as any one of claim 1-8 the method fibroblast of recombination HSP47 viruses and Its application in repairing cicatrix of skin.