Adjustment Of Apoptosis Of Bcl-2 And Influence Of LN On The Expression Of Bcl-2 Of Rabbit Cornea Endothelial Cells

Objective: (1) By investigating the effect of Bcl-2 antibody on apoptosis of the rabbit's corneal endothelial cells, the correlation between Bcl-2 and apoptosis was considered; (2) The influence of laminin (LN) on the expression of bcl-2 in rabbit's cornea endothelial cells was to be researched.Methods: (1) The change of morphology and number of rabbit's corneal endothelial cells in vitro was observed by inverted microscope. The cells number was counted every day. On the basis of these numbers, cell growth curve was graphed; After being cultured for 24h in the medium with and without antibody of Bcl-2, corneal endothelial cells were induced apoptosis by being exposed to ultraviolet ray. After being cultured for another 24h close behind, apoptosis rate of the cells was detected by flow cytometry and the morphology and structure of corneal endothelial cells was observed by transmission electron microscope. (2) Cultured rabbit corneal endothelial cells were given LN of different concentrations. ELISA was exerted to measure OD value. The optimum concentrations of LN of each group were got after the OD value was compared. Then cultured rabbit corneal endothelial cells were divided into three groups as fellow: normal control group, LN group, negative control group. Immunohistochemical technique and ELISA were exerted to to assess the expression levels of bcl-2 .Results: (1) Cell growth curve showed that the cell growth accord with general rules . It was showed by flow cytometry that the apoptosis rate of the cells cultured in the medium with antibody of Bcl-2 was higher significantly than those cultured in the medium without antibody of Bcl-2. This suggests that Bcl-2 can inhibit cells' apoptosis. As shown by the microscope, the cells cultured in the medium without antibody of Bcl-2 (the control group) connected in mass tightly as petals. The density of cells was moderate. Cells were attached to petri dish. The nucleus were in the same size. Comparing with the control group, the cells of the test group grew weakly. Somecells could not be attached to the bottom of the dish. Some round cells which relied on each other drifted on the culture medium. Some vacuoles could be watched in plasm. The nucleus were different in size. (2) By ELISA it showed that within certain concentrations (20~60 mg /L), the LN could promote the expression bcl-2 gene in rabbit's corneal endothelial cells in a dose-dependent manner. And the optimal promoting effect was at the concentration of 60mg/L. Bcl-2 expression score showed that a strong positive expression happened in LN group but a weak expression in the control group. But the negative control group had a negative expression by immunohistochemical stain. The results of ELISA showed that LN could promote the gene expression of bcl-2 in rabbit's corneal endothelial cellsConclusions: Bcl-2 could protect corneal endothelial cells from apoptosis; LN could promote the gene expression of bcl-2 in rabbit's corneal endothelial cells.