摘要
目的探讨NHE-1反义基因表达存肿瘤细胞内pH(pHi)调节及凋亡调控中的作用。方法以NHE-基因的反义基因转染人耐药小细胞肺癌H446/CDDP细胞,采用荧光标记法研究反义基因对肺癌细胞的酸化效应,使用细胞计数的方法进行细胞生长检测,采用免疫组织化学方法研究细胞凋亡基因Caspase3的表达,使片流式细胞仪双染法研究NHE-1反义基因对细胞凋亡的影响,采用透射电镜的方法检测NHE-1反义基因转染的肺癌细胞是否存在细胞凋亡。结果细胞酸化效应显示,NHE-1反义基闪转染的人耐药小细胞肺癌H446/CDDP细胞可使pHi下降至6.86±0.01,与空载体转染的人耐药小细胞肺癌细胞H446/CDDP(7.25±0.02)相比明显降低(P<0.01)。反义基因转染的人耐药小细胞肺癌H446/CDDP细胞在48 h后生长明显受到抑制。免疫组织化学方法显示转染NHE-1反义基因的人耐药小细胞肺癌H446/CDDP细胞凋亡基冈Caspase3有表达,而空载体转染的人耐药小细胞肺癌细胞H446/CDDP Caspase3没有表达。流式细胞仪双染法研究显示NH-1反义基因转染组比空载体组的人耐药小细胞肺癌H446/CDDP细胞凋亡率明显增加(P<0.01)。透射电镜发现NHE-1反义基因转染的人耐药小细胞肺癌H446/CDDP细胞存在细胞凋亡,细胞脱落大量小块胞质,细胞体积明显缩小。结论细胞酸化能使人耐药小细胞肺癌H446/CDDP细胞生长受到抑制,凋亡基因表达增强,产生细胞凋亡。人耐药小细胞肺癌H446/CDDP细胞NHE-1基因表达能通过阻止其胞内酸化,对维持其抗凋亡生长性质具有重要作用。
Objective To evaluate the effects of cell acidosis and apoptosis of Na＋/H＋ exchanger- 1 （ NHE-1 ） antisense gene on drug-resistant human small cell lung cancer（ SCLC ） H446/DDP cells. Methods An expression recombinant of antisense gene was constructed with the NHE-1 gene and a retroviral vector. It was transfected to drug-resistant human small cell lung cancer（SCLC） cell line H446/CDDP cells. Intracellular pH （pH） values were measured with fluorescence spectrophotometer. The proliferation of cancer cells was assayed by cytometry. The expression of apoptosis gene of cancer cells was assayed by immune histochemical method. The cell apoptosis of Na＋/H＋ exchanger-1（NHE-1） antisense gene on drug-resistant human small cell lung cancer（SCLC） H446/DDP cells was observed by flow cytometry. The apoptosis of cancer cells was observed by transmission electron microscopy. Results An expected recombinant was successfully constructed. Compared with the control group, transfected with void vector（7.25 ± 0. 02）, pHi value decreased significantly to 6.86 ± 0.01 in recombinant-transfected H446/CDDP cells （ P 〈 0.01 ）. The cell proliferation decreased at 48 h after antisense gene transfection. There was expression of apoptosis gene Caspase3 in recombinant-transfected H446/CDDP cells, but not in the control group. Both flow cytometry and transmission electron microscopy revealed apoptosis in the recombinant-transfected H446/CDDP cells. Conclusion The NHE-1 antisense gene can induce acidification of drug-resistant human SCLC H446/CDDP cells, and may play an important role in apoptosis of drug-resistant human SCLC H446/CDDP cells.
出处
《中华肺部疾病杂志（电子版）》
CAS
2007年第1期19-23,共5页
Chinese Journal of Lung Diseases(Electronic Edition)
基金
国家自然科学基金资助项目(No.30270591)
中国博士后科学基金资助项目(No.2004035528)
关键词
细胞内
pH
肺癌
NHE-1
基因表达
Intracellular pH （pHi）
Lung cancer
NHE-1
Gene expression
