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构建人同源盒基因NKX3·1cDNA真核表达载体,研究其在前列腺癌细胞PC-3、LNCaP中的表达及对细胞的促凋亡作用.以人前列腺癌细胞LNCaP细胞中的总RNA为模板,RT-PCR扩增NKX3·1基因全长编码片段,将NKX3·1cDNA重组到真核表达载体pcDNA3·1(+)中;将pcDNA3·1-NKX3·1表达载体瞬时转染前列腺癌细胞PC-3和LNCaP细胞,用RT-PCR和Western印迹检测NKX3·1cDNA在转录水平和蛋白水平的表达;绘制细胞生长曲线,观察NKX3·1对前列腺癌细胞增殖的抑制作用;用DNA/ladder和流式细胞术检测NKX3·1对前列腺癌细胞凋亡的影响,进一步用RT-PCR检测凋亡相关基因caspase3、caspase8、caspase9、Apaf1、survivin和Bcl2表达的变化.人同源盒基因NKX3·1cDNA真核表达载体pcDNA3·1-NKX3·1经酶切及测序鉴定正确.pcDNA3·1-NKX3·1转染PC-3和LNCaP细胞后,经RT-PCR和Western印迹证明能有效表达NKX3·1.生长曲线显示,前列腺癌细胞转染NKX3·1cDNA后细胞增殖受到抑制;前列腺癌细胞转染NKX3·1cDNA48h后,DNA电泳呈现具有凋亡特征的DNAladder;流式细胞术检测出现明显凋亡峰;RT-PCR检测凋亡相关基因.结果显示,caspase3、caspase8、caspase9基因表达明显增加,Bcl2基因表达明显减少.本研究成功构建了真核表达载体pcDNA3·1-NKX3·1,转染PC-3和LNCaP细胞后能有效表达,并对细胞具有诱导凋亡作用.
To construct eukaryotic expression vector of human homeobox gene NKX3.1cDNA and study its expression in prostate cancer cells PC-3, LNCaP and its effect on cell apoptosis.With the total RNA in human prostate cancer LNCaP cells as a template The full-length coding fragment of NKX3.1 gene was amplified by RT-PCR and the NKX3.1 cDNA was recombined into the eukaryotic expression vector pcDNA3.1 (+). The pcDNA3.1-NKX3.1 expression vector was transiently transfected into prostate cancer PC -3 and LNCaP cells. The expression of NKX3.1cDNA at transcription and protein level was detected by RT-PCR and Western blotting. The cell growth curve was drawn to observe the inhibitory effect of NKX3.1 on the proliferation of prostate cancer cells. The effect of NKX3 · 1 on the apoptosis of prostate cancer cells was detected by flow cytometry and the expression of caspase3, caspase8, caspase9, Apaf1, survivin and Bcl2 were further detected by RT-PCR. The expression vector pcDNA3.1-NKX3.1 was verified by restriction enzyme digestion and sequencing.PCR-3 and LNCaP cells transfected with pcDNA3.1-NKX3.1 were proved to express NKX3.1 efficiently by RT-PCR and Western blot. Growth curves show that the prostate cancer cells transfected NKX3 · 1cDNA proliferation After 48 hours of transfection of NKX3.1cDNA into prostate cancer cells, DNA electrophoresis showed a DNAladder with apoptotic features. The apoptotic peak was detected by flow cytometry and apoptosis-related genes were detected by RT-PCR.The results showed that caspase3, caspase8, The expression of caspase9 gene was significantly increased and the expression of Bcl2 gene was significantly reduced.In this study, the eukaryotic expression vector pcDNA3.1-NKX3 · 1 was successfully constructed and transfected into PC-3 and LNCaP cells, .