The Effects Of The Extraction Of Human Amniotic Membrane On Proliferation And The Expression Of TGF-β1mRNA In Cultured Corneal Fibroblasts Of Rabbit

Objective: To study the effects of the different concentrations extraction of human amniotic membrane on cultured corneal fibroblasts of rabbit proliferation and the expression of TGF-β1mRNA so as to investigate the mechanism of the inhibition of corneal scar by the extraction of human amniotic membrane.Methods: After the primary culture of the rabbit' corneal fibroblasts gained by digestion and serial subcultivation, identification with vimentin and keratin immunocytochemistry. We observed the effects of the different concentrations(40mg/L, 80mg/L, 160mg/L) extraction of human amniotic membrane on proliferation by tetrazotium (MTT) colorimetric assay and detected the expression of TGF-β1mRNA by RT-PCR on cultured corneal fibroblasts of rabbit.Results: (1) Positive reaction of cellular vimentin and negative reaction of cellular keratin in cultured fibroblasts of rabbit cornea confirmed that the cultured cells were fibroblasts of rabbit cornea by immunocytochemistry. (2) Four groups of OD values gained by MTT were significant different among the experiment groups(0.335±0.013, 0.220±0.018, 0.094±0.005) and control ones (0.415±0.026) (P<0.01). (3) The expression of TGF-β1mRNA was more significantly depressed in different concentrations of experimental groups than in control ones, there was significant difference of TGFB1/GAPDH ratio obtained by RT-PCR among the different concentrations of experimental groups (1.519±0.669, 0.617±0.014, 0.622±0.031)and control ones(1.768±0.040) (P<0.01). But there was no significant difference between the midrange concentrations and high ones (80mg/L and 160mg/L) . Conclusion: (1) The extraction of human amniotic membrane significantly inhibited fibroblastic proliferation on cultured corneal fibroblasts of rabbit in vitro. The effect of inhibition increased with the increasing protein concentrations of extraction of human amniotic membrane. (2) The extraction of human amniotic membrane also significantly inhibited the expression of TGF-β1mRNA on cultured comeal fibroblasts of rabbit and the inhibition was significantly related to the protein concentrations in the extraction of human amniotic membrane. There was no significant difference when the protein concentrations of the extraction of human amniotic membrane added to 80mg/L. The study can partially explain the mechanism of the inhibition of corneal scar proliferation by the extraction of human amniotic membrane.