摘要
目的:探讨转染外源性BAK基因及其过表达对胃癌细胞的诱导凋亡作用和分子机制. 方法:构建携带有BAK基因的真核表达载体并转染胃癌MKN-45细胞1-5 d后,RT-PCR和Western Blotting法检测BAK基因表达;细胞计数、MTT比色法检测癌细胞生长活性,流式细胞仪分析细胞周期时相改变,透射电镜、末端TdT酶标记技术检测癌细胞凋亡;比色法检测癌细胞内Caspase-3活性改变. 结果:转染1-5 d后,癌细胞BAK mRNA和蛋白表达水平显著增强(P<0.0 1),癌细胞体外生长抑制11.6-35.3% (P<0.01),增生活性抑制10.2-32.4%(P<0.01),细胞周期出现G0/G1期阻滞,部分癌细胞出现胞体缩小、核固缩等凋亡形态学改变,凋亡率为21.4%(P<0.01);癌细胞Caspase-3活性增强4.45倍(P<0.01). 结论:转染外源性BAK基因使其过表达能激活Caspase- 3,显著诱导胃癌MKN-45细胞凋亡,有望成为胃癌治疗的新途径.
AIM: To explore the apoptosis-inducing effects of extrinsic BAK gene transfer and its over-expression on gastric cancer cells and its molecular mechanisms. METHODS: The eukaryotic expression for BAK gene was constructed and transferred into gastric cancer MKN-45 cell line. After being transferred for 1 to 5 days, cellular BAK gene expression was detected by RT-PCR and Western blotting methods. The growth activities of cancer cells were detected by cell count and MTT colorimetry. Cell cycle changes were assayed by flow cytometry. Cellular apoptosis was assayed by electronic microscopy and in situ terminally labeled transferase technique (TUNEL). Cellular caspase-3 activities were observed by colorimetric method. RESULTS: After being transferred for 1 to 5 days, cellular BAK mRNA and protein expression levels were significantly increased (P<0.01). In vitro growth of gastric cancer cells was inhibited by 11.6-35.3% (P<0.01). The cellular proliferation activities were decreased by 10.2-32.4% (P<0.01), with cell cycle being blocked at G0/G1 phase. Partial cancer cells presented the characteristic morphological changes of apoptosis, with the apoptotic rates being 21.4% (P<0.01). The cellular caspase-3 activities were enhanced by 4.45 times(P<0.01). CONCLUSION: Transfection of extrinsic BAK gene, resulting in its over-expression, can significantly induce apoptosis of gastric cancer MKN-45 cells through activating caspase-3, which is a potential strategy for gene therapy of gastric cancer.
出处
《世界华人消化杂志》
CAS
2004年第5期1025-1029,共5页
World Chinese Journal of Digestology
