| Effect on the proliferation and survival of human Lens epithelial cells transferred by TGF-β1 geneIntroductionCataracta is one of the most frequently growing blind ophthalmocace, The pathogenesy is still mis-classification. The research discovers that lens epithelium cells take place a series of retrogression and proliferation Changes in the course. At the same time, The changes of morphous correlact with the categories of cataracta. In the course, many kinds of cytokines become the hot spots. Many researchs indicated that Transforming growth factor-β1 correlated with cataracta very much, especially subcapsulal cataracta. But there is few student on the mechanism of it.Transforming growth factor-β(TGF-β), a multifunctional cytokine, has been implicated in many biological responses, including cellular growth and differentiation, intercellular adhesion, epithelial mesenchymal transformation of many cell types, promoting the production and deposition of extracellular matrix, wound healing and immunological regulation etc.Gene transfer, which means to induce aim gene into eukaryotic cells with special method, has following advantages: 1,The transfected cells are able to express aim gene continuously and effectively that can modulate the growth of themselves and other effective cells and to obtain specific functions; 2,The endogenous aim proteins produced by transfected cells have undergone authentic post-translational modification and therefore have improved potencies to combine with receptors on cells' surface as well as more biological activities; 3,The price of gene transfer is lower than that of purified recombinant proteins. Therefore, it is of great value to induce aim gene express on the HLEC, in order to research the mechanism of eataraeta. ObjectionThe purpose of the present study is to investigate the effect of EGFP on human lens epithelial cell by transferred by pEGFP-TGF-β1 plasmid., to investigate the future of EGFP on the filed of ophthalmology, to investigate the effects of TGF-β1 on proliferation and survival of human lens epithelial cell, to investigate the mechanism of TGF-β/Smad4 pathway on HLEC by transfected with pEGFP-TGF-β1 plasmidMethods1,HLEC-B3 Cultured and Treatmented: Human lens epithelial cell line HLE B-3 were cultured with DMEM (including 15% FCS, 100mg/l penicillin, 125rag/l streptomycin), in 5%Co2, 37℃saturated atmosphere.2,To construct pEGFP-TGF-β1 plasmid with Gene transfer technique: pSNAV-TGF-β1 and pEGFP-TGF-β1 were cleavaged by EcoR I and Sal I, The fragments were reclaimed (1.8kb,4.7kb), reclaiming linear carrying agent, linking the two DNA, transforming, screening and cloning.3,Immortalized human lens epithelial cellline(HLEC-B3) was transfered by TGF-β1 gene: The cells were cultured in 5%Co2, 37℃saturated atmosphere in order to 90%. It were transferred according to Lipofectamine2000 direction for use.4,The morphologic change of HLEC transferred by TGF-β1 gene was observed with inverted microscope.5,The expression of EGFP in HLEC transferred by pEGFP-TGF-β1 plasmid was detected by fluorescent inverted microscope, to transfection efficency.6,The effect of proliferation on HLEC was detected through cell growth eurveand MTT colorimetric assay.7,Cell apoptosis and cell cycle of HLEC-B3 transferred by TGF-β1 gene were examined by flow cytometry.8,RT-PCR for TGF-β1,p21,CDK2 and Smad4mRNA: Using Trizol reagent of total Mrna was extracted from LECs. The first chain of cDNA was reverse transcription, and PCR products were amplified. After agarose gel electrophoresis, images were made and PCR products were scanned by KODAK image analysis system.9,Western-blot analysis detected TGF-β1,p21,CDK2 and Smad4 protein: Cell sediment was resolved, dissociated, centrifugalized and extracted the supernatant. The first antibody and the second antibody were added in, room temperature incubating and washing the membrane, ECL for coloration.10,Statistical analysis: SPSS software was used, with a P＜0.05 considered statistically significant.Results1,Contructed pEGFP-TGF-β1 plamid successfully.2,HLEC-B3 cells were transfered successfully by TGF-β1gene, EGFP can expressed in HLEC, The fluorescence expressed in cell is uniform and stable.3,TGF-β1 suppressed the growth rate of HLEC very much.4,Flow cytometric analyzed that TGF-β1 enhanced the cell apoptosis (20.98±1.73 % after 24 hours and 43.61±1.39% after 48hours transfered). The proportions of G1 phase cells were increased to (72.01±1.88)% after 24hours and (74.73±2.22)% after 48hours transfered, while the proportions of S phase cells were decreased to(20.45±2.24)% after 24hours and(19.37±1.42)% after 48hours transfered.5,RT-PCR detected that TGF-β1mRNA began to raised after 24h, culminated after 48h, decreased after 72h, returned to normal after 96h. P21Mma companied with it. but CDK2 decreased with it.6,Western-blot detected that the consequence consisted with RT-PCR.. The effect of TGF-β1 gene was demonstrated from the lever of transcription and translation.7,Smad4 protein raised with the lever of TGF-β1 gene at various time point, but disappeared very quickly.Conclusions1,TGF-β1 genes can successfully transfer HLEC-B3.2,EGFP can express successly in HLEC without cytotoxicity.3,TGF-β1 can inhibits proliferation and induce apoptosis of HLEC-B3.4,TGF-β1 can increase p21, but decreas CDK2, to impact the cell cycle of HLEC.5,TGF-β/Smad4 pathway take part in the course.
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