BAX Gene Transfer To Inhibit The Proliferation Of Human Lens Epithelial Cells

PrefacePosterior capsule opacification (PCO) is the most common complication ofcataract surgery that causes visual impairment. PCO develops in 28.4％of patientsover the first 5 years after surgery. In children the incidence is almost 100％ifposterior capsulorhexis has not been performed. The main cause of PCO is theproliferation, migration and metaplasia of remnant lens epithelial cells (LECs) aftercataract surgery.Laser capsulotomy, which is so far the main treatment for PCO, can lead tosevere complications and can't be applied to young children for they can not be safelypositioned. Approaches for prevention of PCO, such as the improvement of surgicaltechniques and IOL design, and the use of antiproliferative drugs, have greatlydecreased the PCO rate, however, they have not yet completely eradicated it.Therefore, the development of an alternative therapy for preventing PCO is of criticalimportance.Recently, gene therapy for PCO is making headway, which is focused on suicidegenes, cell cycle genes and cytokine genes. Gene therapy provides a novel strategy forthe treatment of PCO, however, the present methods have their own drawbacks.We plan to inhibit the proliferation of LECs by transduction of anapoptosis-inducing gene. BAX, as a pro-apoptotic gene, plays an important role inregulating apoptosis. Previous researches demonstrate that overexpression of BAXgene can effectively suppress the tumor growth or significantly enhance the killingeffects of radiotherapy or chemotherapy on tumor cells both in vitro and in vivo.In our study, to explore the possibility of pro-apoptotic gene therapy for PCO, we investigated the effects of BAX gene overexpression on apoptosis of human lensepithelial cells (HLECs) by observing the morphology, ultrastructure and caspase 3activation of HLECs after BAX gene transfer.PartⅠComparison of transfection efficiency by techniques ofnucleofection or lipofectionPurpose To compare the transfection efficiency by methods of lipofection ornucleofection.Methods HLECs (SRA01/04) were cultured in vitro. The plasmid C3-EGFP-BAX, containing a cDNA encoding BAX protein, was amplified on a small scale andthen sequenced. After verification, plasmid C3-EGFP-BAX, along with an emptyplasmid vector C3-EGFP that expresses green fluorescent protein (GFP), wasamplified on a middle scale and purified. HLECs were transfected with plasmidC3-EGFP using lipofection or nucleofection. The transfection efficiency wasdetermined by counting the GFP positive cells under fluorescent microscope or byflow cytometry.Results The cultured HLECs grew healthily, the character of which consistedwith primary HLECs, and a large number of cells were available to undertake thefollowing experiments. Sequence analysis of plasmid C3-EGFP-BAX showed 100％match to the published sequence for human BAX-α. After amplification andpurification the plasmid C3-EGFP-BAX and C3-EGFP, with high concentration andpurity, were eligible for gene transfer. HLECs could be successfully transfected by thetwo methods of lipofection or nucleofection. 48 hours after gene transfer, theefficiency of GFP expression was 45.3％±10.5％with lipofection and 78.3％±17.5％with nucleofection.Conclusion HLECs could be successfully transfeeted by the two techniques oflipofection or nucleofection, with nucleofection exhibiting higher transfectionefficiency.PartⅡBAX gene transfer to HLECs andits effect on BCL2 expressionPurpose To detect the expression of BAX and BCL2 gene products in HLECs after the cells were transfected with plasmid C3-EGFP-BAX.Methods The baseline expression level of BAX and BCL2 gene in HLECs wereexmined first. Then BAX gene was transferred to HLECs with nucleofector technique.48 hours after transfection, BAX gene mRNA expression was measured by fluorescentreal-time RT-PCR and BAX protein expression was determined by westernimmunoblotting and immunofluorescence. The expression level of BCL2 gene wasalso examined using the same techniques. HLECs transfected with an empty plasmidvector C3-EGFP and untransfected HLECs were used as controls.Results 48 hours after BAX gene transfection, HLECs exhibited a significantincrease in the expression level of BAX gene mRNA (2.70-fold) and BAX protein(2.30-fold), compared to cells transfected with plasmid C3-EGFP. The expressionlevel of BCL2 mRNA and protein in BAX-transfected group was 97.7％and 60.1％ofthat in control group, which didn't show significant difference. As a result, theBAX-transfected ceils showed a significant increase in the mRNA ratio (2.72-fold)and protein ratio (3.83-fold) of BAX/BCL2, compared with control cells.Conclusion BAX and BCL2 protein could be detected in normal HLECs,predominately present in the cytosol. After treatment with plasmid C3-EGFP-BAX,expression of BAX gene was greatly enhanced and so was the BAX/BCL2 ratio, whilethe BCL2 expression level was not significantly affected.PartⅢInfluence of BAX overexpression on apoptosis of HLECsPurpose To investigate the influence of BAX overexpression on the apoptosisof HLECs.Methods After BAX gene transfer, the cell death was evaluated byfluorescence-activated cell sorter analysis of Annexin V. Cell morphology wasexamined by phase contrast microscope and fluorescent microscope. Nuclearmorphology was observed by staining with Hoechst 33258. Ultrastructure of cells wasassessed by transmission electromicroscope. And the expression of active caspase 3was detected by westem immunoblotting. HLECs transfected with an empty plasmidvector C3-EGFP and untransfected HLECs were used as controls.Results After BAX gene transfer, the mortality rate in successfully transfectedcells was approximately 40％, which showed a significant difference from cellstransfected with plasmid C3-EGFP, whose mortality rate was about 30％. The mortality rate at different time points didn't differ significantly. Morphology studiesshowed that BAX-transfected cells were in a bad growth status with fuzzy profile,cellular shrinking, condensation and margination of chromatin and even nuclearfragmentation. The expression level of active caspase 3 was significantly increased by2.44-fold in BAX-transfected cells than in control cells.Conclusion Overexpression of BAX gene could trigger the death of HLECswith apoptosis as the main pathway.Summary1. HLECs could be successfully transfected by the techniques of nucleofection orlipofection, with mean transfection efficiency 78.3％and 45.3％respectively.2. BAX and BCL2 protein could be detected in normal HLECs. BAX gene transfercould induce overexpression of BAX protein while the BCL2 level was notsignificantly affected. As a result, the BAX/BCL2 ratio was upregulated.3. BAX gene transfer could trigger the death of HLECs with apoptosis as the mainpathway.