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目的:利用顺铂处理肺癌、宫颈癌细胞和人胚肾细胞,检测人类端粒酶逆转录酶催化亚单位(hTERT)mRNA的选择性剪接是否发生变化。方法:将培养的肺癌A549和H1299细胞,宫颈癌C33A、SiHa和Ca Ski细胞及人胚肾293FT细胞分为实验组和对照组,用顺铂处理实验组细胞,DMSO处理对照组细胞;设计hTERTα+β+、α+β-、α-β+、INS3及DEL[e2]引物,提取两组细胞的总RNA,利用RT-PCR检测hTERT mRNA剪接异构体表达。结果:与对照组细胞相比,随着顺铂浓度的增加,肺癌A549和H1299细胞,宫颈癌C33A、SiHa和Ca Ski细胞以及人胚肾293FT细胞中hTERTα+β-和INS3异构体比例升高,而DEL[e2]异构体变化不明显。结论:在顺铂引起的DNA损伤情况下,细胞可利用hTERT mRNA选择性剪接调控机制进行调节,通过改变不同异构体的量和所占比例,从而调节端粒酶功能活性。
OBJECTIVE: To detect whether there is any change in the alternative splicing of human telomerase reverse transcriptase catalytic subunit (hTERT) mRNA using cisplatin treatment of lung cancer, cervical cancer cells and human embryonic kidney cells. Methods: The cultured lung cancer A549 and H1299 cells, cervical cancer C33A, SiHa and Ca Ski cells and human embryonic kidney 293FT cells were divided into experimental group and control group. The cells in experimental group and DMSO were treated with cisplatin. The hTERTα The total RNA of the two groups of cells was extracted and the expression of hTERT mRNA splicing is detected by RT-PCR. Results: Compared with control cells, the ratio of hTERTα + β-and INS3 isoforms increased in A549 and H1299 cells, C33A, SiHa and Ca Ski cells in cervical cancer and 293FT cells in human embryonic kidney with the increase of cisplatin concentrations High, while DEL [e2] isomers did not change significantly. CONCLUSION: In the case of DNA damage caused by cisplatin, cells can be regulated by the alternative splicing regulation of hTERT mRNA. The functional activity of telomerase can be regulated by changing the amount and proportion of different isoforms.