Inhibition Of Bcl-2 By ShRNA To Induce Apoptosis In Human Lens Epithelial Cells

Posterior capsule opacification(PCO)is the most common complication of cataract surgery that causes visual impairment.The main cause of PCO is the proliferation,migration and metaplasia of remnant lens epithelial cells(LECs)after cataract surgery.Although we can use laser capsulotomy to treat this complication,it has some severe complications,such as macular edema and retinal detachment. Approaches for prevention of PCO,such as the improvement of surgical techniques and IOL design,and the use of antiproliferative drugs,have greatly decreased the PCO rate,however,the occurrence is still significant.Therefore,the development of an alternative therapy for preventing PCO is of critical importance.Recently,gene therapy for PCO is making headway,which is focused on suicide genes,cell cycle genes and cytokine genes.Gene therapy provides a novel strategy for the treatment of PCO,however,the present methods have their own drawbacks.RNA interference(RNAi)is one of the most exciting discoveries in the past few years. It is triggered by double-stranded RNA(dsRNA)and causes sequence-specific mRNA degradation of single-stranded target RNAs homologous in response to dsRNA.Since its discovery,RNAi show great potential as gene therapy.We plan to investigate whether apoptosis of LECs can be induced by inhibition of bcl-2 with RNAi.Polyamidoamine(PAMAM)dendrimers have received much attention as a new class of gene carriers over the past few years.PAMAM is a non-virus nanometer-sized carrier with the advantages of high transfection efficiency and long-term stable expression without immunogenicity and genotoxicity.In the present study,we used the fifth generation of PAMAM(PAMAM G5)to transfect LECs and compare its efficiency with liposome,the common non-viral vector of gene therapy.In the present study,we constructed two pairs of bcl-2 shRNA and transfer them to human lens epithelial cells(HLECs)with liposome and PAMAM G5,separately. The expression of bcl-2 and apoptosis of HLECs were investigated,and the transfection efficiency of liposome and PAMAM G5 were compared. Partâ… Construction of Bcl-2 shRNA expression vectorPurpose To construct two pairs of Bcl-2 shRNA expression vectorsMethods Two pairs of Bcl-2 shRNA expression vectors were designed according to the Bcl-2 sequence in the GeneBank(No.NM₀00633).The targeting point in the Bcl-2 cDNA was 1210-1228,2133-2151 seperately.Two pairs of oligonucleotides were synthesized,annealed and inserted into plasmid PGCsi to generate shRNA eukaryotic expression vectors,named P1 and P2.Then,they were transformed into competence bacterium DH5α.the reconstructed plasmids were amplified and purified. DNA sequence were confirmed by gene sequenator.Results Gene sequenator showed the inserted DNA sequence were correct completely. The Bcl-2 shRNA expression vector were constructed successfully.Conclusion The Bcl-2 shRNA expression vector were constructed successfully.Partâ…¡Liposome-mediated inhibition of bcl-2 by shRNA to induce apoptosis in human lens epithelial cellsPurpose To investigate whether apoptosis of human lens epithelial cells(HLECs) can be induced by liposome-mediated inhibition of bcl-2 shRNA.Methods HLECs(SRA01/04)with transfected with Lipofectamine^TM2000 by bcl-2 shRNA P1 and P2.At 48h after transfection,the transfection rate was measured by flow cytometry.The whole cell protein was extracted and the bcl-2 protein level was detected by Western blotting.The bcl-2 mRNA level was detected by real-time PCR. The percentage of HLECs undergoing apoptosis was measured by Annexin V-FITC/PI staining.The nuclear morphology of HLECs was observed by staining with Hoechst 33258.The activity of caspase-3 was analyzed by Western blotting.Results At 48h after transfection,the rate of transfection of P1 and P2 was about 44.1±1.7%and 47.2±1.6%respectively.The protein and mRNA level of bcl-2 was greatly down-regulated.The percentage of HLECs undergoing apoptosis was greatly improved.Hoechst staining showed that bcl-2 shRNA transfected cells were in a bad growth status with nuclear fragmentation.The activity of caspase-3 was greatly improved(P＜0.05).Conclusion P1 and P2 can both down-regulate the expression of bcl-2,and induce the apoptosis of HLECs.It is feasible to use RNA interference mediated by liposome to induce the apoptosis of HLECs.Partâ…¢PAMAM-mediated inhibition of bcl-2 by shRNA to induce apoptosis in human lens epithelial cellsPurpose To investigate whether apoptosis of human lens epithelial cells(HLECs) can be induced by PAMAM-mediated inhibition of bcl-2 shRNA.Methods HLECs(SRA01/04)with transfected with PAMAM G5 by bcl-2 shRNA P1.At 48h after transfection,the transfection rate was measured by flow cytometry. The transfection rate mediated by PAMAM and liposome were compared.The whole cell protein was extracted and the bcl-2 protein level was detected by Western blotting. The bcl-2 mRNA level was detected by real-time PCR.The percentage of HLECs undergoing apoptosis was measured by Annexin V-FITC/PI staining.The nuclear morphology of HLECs was observed by staining with Hoechst 33258.The activity of caspase-3 was analyzed by Western blotting.Results At 48h after transfection,the rate of transfection of P1 mediated by PAMAM was about 48.5±1.5%,higher than liposome-mediated group which was about 41.1±1.8%.The protein and mRNA level of bcl-2 was greatly down-regulated. The percentage of HLECs undergoing apoptosis was greatly improved.Hoechst staining showed that bcl-2 shRNA transfected cells were in a bad growth status with nuclear fragmentation.The activity of caspase-3 was greatly improved(P＜0.05).Conclusion PAMAM-mediated bcl-2 shRNA can down-regulate the expression of bcl-2,and induce the apoptosis of HLECs.It has a higher thansfection rate than liposome.