The Experimental Study On Apoptosis Of Human Lens Epithelial Cells By10-23Deoxyribozyme Silencing Survivin Gene

Objective: The experimental design specific10-23DNAzymefor Survivin mRNA sequences，The liposome (Lipofectamine2000) mediatedspecific10-23DNAzyme of Survivin gene was transfected into human lensepithelial cells (HLECs) of SRA01/04cell line, To study and explore the effectof inhibition of Survivin gene expression on apoptosis of HLECs and SurvivinmRNA, to provide the experimental basis and new method for prevention andtreatment of after cataract（posterior capsule opacification,PCO）. Methods:HLECs immortal line SRA01/04cultured in vitro, the cells were divided intothree groups, Blank control group: HLECs+complete medium, Negativecontrol group: HLECs+liposome+non specific10-23DNAzyme, Theexperimental group: HLECs+liposome+specific10-23DNAzyme. The use ofantisense technology, liposome as carrier respectively, specific and non specific10-23DNAzyme was transfected into the HLECs.Transfection of24h and48h,we use inverted phase contrast microscope to Observate the growth conditionand morphologic change of HLECs. After transfection of48h, we use RT-PCRmethod to test the expression of Survivin mRNA in HLECs, Flow cytometry(FCM) combined with annexin V/PI (Annexin V/PI) double staining wasadopted to detect the apoptosis of HLECs. Results: Using inverted phasecontrast microscope, After24h,the HLECs cells of blank control group and negative control group were oval or round, dispersed affixed to the bottom ofthe culture bottle, the cell body colorless translucent cells, abundant cytoplasm,the cells containing coarse alignment and compact particles are not consistent, afew cells projecting, combined with other cells heave. After48h, cell gap isreduced, convex cell bodies out less, round in shape. The experimental groupHLECs at24h after transfection, the cells became round, chromatincondensation, cytoplasm condensed. After transfection of48h, part of the cellshave cracked into shape is not a circle of apoptotic bodies, some cells have beenseparated from the bottom of the culture flask, floating in the culture medium.After transfection of48h,The RT-PCR method and the Gray scale scanningsemi quantitative results are shown: The HLECs Survivin mRNA relativeexpression levels of blank control group, negative control group andexperimental group were:0.726±0.148,0.700±0.115and0.314±0.130.Therelative content of Survivin mRNA in experimental group compared with theblank control group and negative control group respectively, were reduced by56.75%and55.14%, The difference was statistically significant(P=0.000<0.05).When compared with the relative content of Survivin mRNAof the blank control group and negative control group,The difference was notstatistically significant (P=0.055>0.05). Flow cytometry (FCM) combined withannexin V/PI (Annexin V/PI) double staining showed: After transfection of48h,apoptosis cells in the blank control group was2.33±0.23%, negative control ofcell apoptosis rate was3.57±0.20%,The blank control group and negative control group in comparison, the two groups had no statistically significantdifference in the rate of apoptosis in HLECs(P=0.072>0.05). Apoptosis cells inthe experimental group was19.52±1.16%, compared with the blank controlgroup and negative control group, the apoptosis rate of HLECs increasedsignificantly, the difference was statistically significant (P=0.000<0.05).Conclusion: Specific expression of10-23DNAzyme can effectively inhibitSurivivn mRNA of HLECs; After the decline of Survivin gene expression inHLECs could promote apoptosis of HLECs.