An Experimental Investigation Of Tetrandrine On The Isolated Rabbit Corneal Stroma Cells In Vitro

Objective We plan to explore the antiproliferative and apoptosis effect about tetrandrine and clear out the extent of the effect , which one is more important, antiproliferation or apoptosis;study the mechanism of gene.Methods The first part 1. The primary cultured and subcultured keratocytes from NewZealand white rabbits and cells identification ;2,The clells were exposed to diferent concentrations of tetrandrine (2μg/ml,3μg/ml和4μg/ml) in experimental groups and the culture medium containing 20ul PBS in contrast group. We detect the expression of PCNA by immunocytochemistry,and determine proliferation index ;3,We observe the apoptosis index by FCM.4,To compare proliferation index and the apoptosis index.The second part To detect the expression of P53mRNA and c-myc mRNA by reverse transcription-polymerase chain reaction(RT-PCR), experiment include tetrandrine 2μg/ml group and contrast group. Results The first part 1. After the treatment of tetrandrine,48 hours later,the expression of PCNA depressed with the concentrations of tetrandrine's increasing, and the index of proliferation also depressed;G1 phase cells increased with increased concentrations of Tet.2,The index of apoptosis multed as the concentrations of tetrandrine's increased;3,The slope rate of the curve about index of proliferation larger than slope rate of the curve about index of apoptosis .The second part Normal corneal stromal cells and the group with tetrandrine both have P53mRNA expressing , compared with the control group, P53mRNA expression was significantly increased, and the difference was statistically significant (P <0.05); Normal corneal stromal cells and the group with tetrandrine both have c-myc mRNA expressing, compared with the control group, c-myc mRNA expression was significantly reduced, and the difference was statistically significant (P <0.05).Conclusion 1. The expression of PCNA was inhibited as the result of tet on corneal stromal cells, with the concentration increasing, the rate of cell proliferation declined; Tet blocked cell in the G1 phase 2,Tet induced cells apoptosis, with the concentration increasing , the apoptosis index also increased; 3,Among the concentration of 0 ~ 4μg/ml, Tet has both antiproliferation and induce apoptosis on the corneal stromal cells, but the main effect was antiproliferation;4,Normal corneal stromal cells and the group with tetrandrine both have P53mRNA and c-myc mRNA expressing, Tet-induced corneal stromal cells to increase expression of P53mRNA (P <0.05),and inhibit expression of c-myc mRNA (P <0.01).