Inhibition Of Human Fibroblasts Proliferation By RNA Interference

Objective:Glaucoma is a kind of irreversible eye disease which can cause blindness. The current clinical treatment is to decrease the intraocular pressure (IOP) by using drugs or surgery. The filtering surgery is the primary treatment of glaucoma, but the bleb scarring after the surgery is the main reason which leads to the failure of surgery. But the fibroblast proliferation, collagen deposition, filtering bleb scarring are the main reason for failure of glaucoma filtration surgery. A variety of factors play a role in wound healing. Especially TGF-β2, which is one of the most important regulatory factors involved in wound healing and fibrosis, plays an important role on the wound healing and bleb scarring of the eye. The study explores the use of specific TGF-β2 gene expressions in small hairpin RNA (shRNA) in order to inhibit human Tenon's capsule fibroblast proliferation. It can inhibit the bleb scarring and raise the success rate.Methods:Primary cultured human tenon's capsule fibroblast (human Tenon's capsule fibroblasts, HTCF), HTCF immunohistochemical identification. According to the principle of siRNA design, specific shRNA expression vector were constructed(no homologous sequence by BLAST analysis) and transfected of cultured HTCF; The TGF-β2 protein level in the culture supernatant was determined by enzyme-linked immunosorbent assay. The cell proliferation was detected with MTT.Results:The cell proliferation and TGF-β2 protein expression of the group which TGF-β2 specific shRNA was transfected in, was inhibited obviously. TGF-β2 recombinant vector on the proliferation of fibroblasts is influential, The influence obviously with time, the proliferation rate of TGF-β2 specific shRNA in 24h,48h,72h were 8.62%,14.13%,33.47%, negative control group was 4.77%,11.36%,13.57%. After 24h, the TGF-β2 protein expression of specific group was 543.58pg/ml. Compared with the control group and negative group. The specific group was lower (P<0.01). After 48h and 72h, the TGF-β2 protein expression of specific group decreased further to 400.26pg/ml and 202.72pg/ml, while negative group decreased not very obvious.Conclusion:The study shows that TGF-β2 specific shRNA was transfected into fibroblasts. It can significantly inhibited TGF-β2 expression of HTCF and the proliferation of fibroblasts.Compared to empty plasmid group and control group, the expression of TGF-β2 and the proliferation of fibroblasts was significantly reduced.1.Specific shRNA can significantly inhibit the proliferation of fibroblasts.2.It has a good time dependence that specific shRNA inhibit the expression of TGF-β2.3.The vector-mediated TGF-P2 specific shRNA has potential possibilities in the treatment of anti-scarring after anterior segment surgery...