HSP47siRNA’s Influence On TGF-β1to Stimulate The Proliferation And Collagen Synthesis Of The Human Tenon’s Capsule Fibroblasts

Objective:Imitating the formation of scar after glaucoma surgery by using thesynthesis and proliferation of Tenon ’s fibroblast (HTCF) through factor-β1(TGF-β1). Observing the influence of proliferation of TGF-β1and the changesof recombinant plasmid stimulated by HSP47siRNA. Discussing that whetherthe HSP47gene with RNA can become a new method to against the formationof scar after glaucoma surgery.Methods:1、HTCF`s invitroculture and identification: take HTCF from the patientwho take glaucoma surgery, use the method of tissue block culture to cultivateTenon`s fibroblasts. The cell will get merged when grow to80%-90%, then takesubculture of them. Identify the chromosome of cells further, offer themVimentin and Kevatin chromosome and identify whether the cell becomesfibroblasts.2、HSP47siRNA`s establishment and identification:aiming at the siRNAsequence of HSP47gene design, clone to psiRNA-hHl-neo G2, transformcompetent cell, pick up PCR to identify, extract plasmids and identify andanalysis.3、Test the influence stimulated by TGF-β1to HTCF by CCK8: Under thecircumstance of LipofectamineTM2000`s mediation, grouping them like this:① normal cell group.②psiRNA-hHl-neo G2-HSP47and TGF-β1（5ng/ml）group.③TGF-β1（5ng/ml）group.④Empty vector and TGF-β1（5ng/ml）.⑤comparinggroup:contains culture medium.Test the CCK8after24h,48h and72h, compareand analysis the A value at450mm absorbancy, then draw a bar chart of eachgroup.4、Collagen change detection cell with picric Sirius red staining:Test group:①the normal cells group;②psiRNA-hHl-neo G2-HSP47+TGF-β1(5ng/ml) group;③TGF-β1(5ng/ml) group;④the dead body of+TGF-β1(5ng/ml) group; The cells were seeded in6well plates, were transfected in72hcells after grouping according to fixed, further picric acid-Sirius red staining,observing the change of intracellular I type, type III collagen in under polarizedlight microscopy, analysis of cells was measured by I type, type III collagencontent changes to software, parallel statistical analysis.Results:1、It is easy to cultivate HTCF and it is active out of the body. And it isstable after subculture. The identification results are that Vimentin is positiveand Kevatin is negative. It shows that the cell cultivated out of the bodybecomes fibroblasts.2、Established HSP47-siRNA(psiRNA-hHl-neo G2-HSP47), get2500kbcarrier segment and500bp segment after Double Digestion. Analysis results andsequential design are the same which proves that recombinant plasmid succeed.3、 CCK8assay: After HSP47siRNA transiently transfected byTGF-β1-stimulated human eye Tenon’s capsule fibroblasts promote the role ofTGF-β1on cell proliferation. It will be affected by a certain impact, cellproliferation activity will decline, Fibroblasts by psiRNA-hH1-neo G2-HSP47transfection and TGF-β1(5ng/ml) in24h cells after stimulation of A valuebegan to appear the difference. In addition, the empty vector+TGF-β1(5ng/ml) cell A value group and TGF-β1(5ng/ml) group compared24h,48h and72h haveno statistically significant difference (P>0.05),besides,the other groups at thesame time cell A value contrast the differences were statistically significant(P<0.05). And psiRNA-hHl-neo G2-HSP47+TGF-β1(5ng/ml) cells A value of3time points were set lower than the normal cells compared with TGF-β1(5ng/ml) groups were more highly statistically significant (P <0.01); In eachgroup and compared with24h results showed that psiRNA-hHl-neo G2-HSP47and TGF-β1Effect of48h and72h on human Tenon ’s capsule fibroblastproliferation effect is more significant, the difference was statistically significant(P<0.05).4、Picric acid-Sirius red staining of cells in each group after staining,polarized light microscope to see after by TGF-β1-induced cell TGF-β1(5ng/ml)group of type I, III collagen density was significantly increased, Microscopicarrangement of collagen fibers becomes dense and colorful refractionsignificantly enhanced with the normal cell group and psiRNA-hHl-neoG2-HSP47+TGF-β1(5ng/ml) differences between groups were compared, andthe difference was statistically significant (P <0.05); The content of type I, typeIII collagen psiRNA-hHl-neo G2-HSP47transfected cells decreasedsignificantly, fiber loose arrangement, refraction weakened, and empty vector+TGF-β1(5ng/ml) group, TGF-β1(5ng/ml) is highly statistically significantgroup differences (P<0.01); Empty vector+TGF-β1(5ng/ml) group differenceswith TGF-β1(5ng/ml) group with no statistical significance (P>0.05).Conclusions:1、Human eye Tenon’s capsule fibroblasts in vitro method is easy and fast tocultivate and can be regarded as the ideal vector after glaucoma filtrationoperation.2、Using the TGF-β1can effectively promote the human eye Tenon’sfibroblast proliferation.Besides it also can promote cell type I, type III collagen synthesis in order to simulate the scar formation after glaucoma filteringoperation of local fibrosis mechanism and provide basis for further animalexperiment.3、With the chemical synthesis of siRNA and psiRNA-hHl-neo G2vector,we successfully constructed the recombinant plasmid targeting HSP47geneeasily. This method can be applied to clinical treatment of RNA interferencetechnology better.4、HSP47-siRNA can reduce TGF-β1on human Tenon’s capsule andpromote the proliferation of fibroblasts. After transfection, the cells of type I,type III collagen synthesis decreased, indicating that HSP47and TGF-β1expression in synchronous with using RNA interference to affect the expressionof HSP47.It also can affect the profibrotic effects of TGF-β1that in order tofurther the application of RNA interference technique in glaucoma filtrationsurgery and provide theory basis.