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目的:分析顺铂处理后多种肿瘤细胞中CASP9基因mRNA选择性剪接异构体的表达及其变化规律。方法:用梯度浓度的顺铂处理肺癌细胞(A549、H1299)、人胚肾细胞(293FT)、宫颈癌细胞(SiHa、CaSki、C33A)6种细胞,提取各细胞的总RNA,用半定量RT-PCR和琼脂糖凝胶电泳及胶图像分析软件检测分析CASP9基因mRNA异构体的组成及其比例和相对表达水平的变化。结果:6种细胞中,CASP9f∶r1引物检测到的CASP9a和c(NR-102732)两种异构体,但随着顺铂浓度的增加,两种异构体的比例在6种细胞中均无变化;而CASP9f∶r2引物可检测到的异构体CASP9a和b、a/b比例在A549、H1299、SiHa、C33A、Ca Ski中呈逐渐增加趋势,而在293FT细胞中则呈相反趋势;在A549顺铂抗性株中,顺铂处理前后CASP9a/b的比例显著差异变化明显减弱。结论:在顺铂处理下,检测的5种细胞中的Casp9是通过增加casp9a/b的比例来应对,仅293FT例外;顺铂抗性A549细胞株在顺铂处理后casp9a/b的比例比例与细胞凋亡和抗性有关,提示调节casp9a/b的比例是治疗癌症的可能的有效途径之一。
OBJECTIVE: To analyze the expression of caspase 9 alternative splicing isoform and its variation in various tumor cells after cisplatin treatment. Methods: Six kinds of cells of lung cancer cells (A549, H1299), human embryonic kidney cells (293FT) and cervical cancer cells (SiHa, CaSki, C33A) were treated with a gradient concentration of cisplatin. Total RNA of each cell was extracted. -PCR and agarose gel electrophoresis and gel image analysis software to detect and analyze the CASP9 gene mRNA isoforms of the composition and its proportion and relative expression level changes. Results: Two isoforms of CASP9a and c (NR-102732) were detected by CASP9f: r1 primer in six kinds of cells. However, with the increase of cisplatin concentrations, the ratio of the two isoforms was The CASP9f: r2 primers detectable isoform CASP9a and b, a / b ratio in A549, H1299, SiHa, C33A, Ca Ski gradually increased, while in 293FT cells showed an opposite trend; In A549 cisplatin resistant strains, significant differences in the ratio of CASP9a / b before and after cisplatin treatment significantly decreased. Conclusions: Under cisplatin treatment, Casp9 in the five kinds of cells detected by increasing the proportion of casp9a / b to deal with only the 293FT exception; cisplatin-resistant A549 cell line in cisplatin casp9a / b after the proportion of the ratio and Apoptosis and resistance, suggesting that the regulation of casp9a / b ratio is one of the possible effective ways to treat cancer.