表达反义细胞周期蛋白cyclin D1载体对胰腺癌细胞生长和凋亡的影响

Effects of antisense cyclin D1 expressing vector on the cell growth and apoptosis of pancreatic carcinoma

目的观察反义细胞周期蛋白ｃｙｃｌｉｎＤ１对胰腺癌细胞生长的影响。方法采用Ｓｏｕｔｈｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ方法检测了５株人胰腺癌细胞系中ｃｙｃｌｉｎＤ１的扩增及表达情况，发现ＰＣ７细胞中基因有扩增及过度表达。我们构建了反义（ＡＳ）ｃｙｃｌｉｎＤ１的表达载体，采用ｌｉｐｏｆｅｃｔｉｎ转染方法转染ＰＣ７细胞，获得转化细胞系ＰＣ７／ＡＳｃｙｃｌｉｎＤ１。经Ｎｏｒｔｈｅｒｎｂｌｏｔ、Ｗｅｓｔｅｒｎｂｌｏｔ检测转化细胞系，表明有外源反义ｃｙｃｌｉｎＤ１的表达，而内源性ｃｙｃｌｉｎＤ１ｍＲＮＡ表达及蛋白合成下调，并进行了细胞凋亡分析。结果转化细胞系细胞恶性生物学行为及表型部分逆转，细胞生长速度、ＤＮＡ合成及细胞增殖和代谢能力均明显下降，软琼脂集落形成能力减低。流式细胞术分析发现细胞被阻滞于Ｇ１期，ＤＮＡ凝胶电泳、原位凋亡检测发现凋亡细胞增多。结论通过反义ＲＮＡ技术，下调在细胞周期调控中起重要作用的ｃｙｃｌｉｎＤ１的表达，可使胰腺癌细胞恶性表型部分逆转。

Objective To observe the effects of antisense (AS) cyclin D1 expressing vector on the cell growth and apoptosis of pancreatic carcinoma. Methods Examination of the amplification and expression of cyclin D1 in 5 human pancreatic carcinoma cell lines. Our study found the gene amplification and overexpression of cyclin D1 in PC 7 cell line cells. We then constructed the antisense cyclin D1 vector and transfected the PC 7 cell line with lipofectin. The resultant transformant cell line, PC 7/AS cyclin D1, showed the expression of exogenous antisense cyclin D1 mRNA and down regulation of endogenous cyclin D1 mRNA expression and inhibition of its protein synthesis detected by Northern blot and Western blot respectively. Results The transformant cells showed retardation of cell growth and partial reversion of the malignant phenotype, including decrease of the rates of cell growth, DNA synthesis, cell proliferation and metabolism, and also the ability of soft agar colony formation. The tumorogenesis of the transformant cells in nude mice was suppressed. G 1 arrest was revealed by flow cytometry. Apoptosis was identified by DNA fragmentation and in situ TUNEL detection. Conclusion Down scaling of the expression of cyclin D1, which plays an important role in the regulation of the cell cycle, can effectively inhibit the proliferation of carcinoma cells and increase cell apoptosis.