【摘要】 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.更多还原

【Abstract】 The eukaryotic expression vector of human homeobox gene NKX3.1 cDNA was constructed to study its expression and function of inducing apoptosis in human prostate cancer cells. The NKX3.1 complete decoding sequence was amplified by RT-PCR using RNA from LNCaP cell and cloned into pcDNA3.1（+） vector to generate pcDNA3.1-NKX3.1 which was transiently transfected into prostate cancer PC-3 and LNCaP cells. The expression of NKX3.1 mRNA and protein was verified by RT-PCR and Western blotting.The Cell growth curve was drew to show the proliferation inhibition, and the cell apoptosis was examined by DNA electrophoresis and flow cytometry （FCM）. The expressions of some apoptosis-related genes, caspase3,caspase8, caspase9, Apaf1, survivin and Bcl₂ in the transfected cells were further detected by RT-PCR. The sequence of cloned NKX3.1 cDNA was identical to that documented in GenBank. RT-PCR and Western blotting indicated that NKX3.1 cDNA could be effectively expressed in the transfected PC-3 and LNCaP cells. The proliferation inhibition of the transfected cells was showed by the cell growth curve. The DNA ladder in DNA electrophoresis and the apoptosis peak in FCM were found in the transfected cells. The results of RT-PCR showed that the expressions of caspase 3, caspase8 and caspase9 genes were markedly increased, while the expression of Bcl₂ gene was decreased after transfection of pcDNA3.1-NKX3.1 for 48 hours. The eukaryotic expression vector pcDNA3.1-NKX3.1 was successfully constructed and could be effectively expressed in PC-3 and LNCaP cells that could be induced to apoptosis after transfection of pcDNA3.1-NKX3.1 plasmid.更多还原