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目的:采用重组慢病毒系统,在过表达和抑制NDRG2(N-myc downstream regulated gene 2)基因后,检测人结肠癌细胞系Caco-2的增殖情况。方法:分别采用NDRG2过表达慢病毒载体pLenti6-NDRG2和NDRG2干涉慢病毒载体pLKO.1-shNDRG2制备病毒并感染Caco-2细胞,经14天耐药的筛选,获得稳定感染的细胞株;采用Real time RT-PCR和Western blot方法明确NDRG2在稳转细胞株中的表达情况,同时采用MTT方法检测细胞的增殖能力,通过流式细胞仪检测细胞周期变化情况。结果:Real time RT-PCR和Western blot结果表明,在慢病毒pLenti6-NDRG2稳定感染的Caco-2细胞中,NDRG2表达上调;pLKO.1-shNDRG2稳定感染的Caco-2细胞中,NDRG2的表达下调。MTT实验结果表明,NDRG2过表达细胞株中细胞的增殖能力下降,而pLKO.1-shNDRG2慢病毒感染Caco-2后细胞的增殖能力明显增加;流式细胞术结果显示,过表达NDRG2使Caco-2细胞在G0/G1期细胞增多而S期的细胞减少,抑制细胞内源性NDRG2后G0/G1期细胞减少而S期细胞增多。结论:通过过表达和抑制NDRG2基因,验证了NDRG2作为抑癌候选基因能够有效抑制结肠癌细胞Caco-2的增殖。
OBJECTIVE: To detect the proliferation of human colon cancer cell line Caco-2 after overexpression and inhibition of NDRG2 gene by recombinant lentivirus system. Methods: The NDRG2 overexpression lentiviral vector pLenti6-NDRG2 and NDRG2 interference lentiviral vector pLKO.1-shNDRG2 were used to infect Caco-2 cells and infected with Caco-2 cells respectively. Stably infected cell lines were obtained after 14 days of drug resistance. Real- The expression of NDRG2 in stable cell lines was determined by RT-PCR and Western blot. MTT assay was used to detect the proliferation of cells. The cell cycle was detected by flow cytometry. Results: Real-time RT-PCR and Western blot showed that NDRG2 expression was up-regulated in Caco-2 cells stably infected with lentivirus pLenti6-NDRG2 and down-regulation of NDRG2 expression was found in Caco-2 cells stably infected with pLKO.1-shNDRG2 . The results of MTT assay showed that the cell proliferation ability of NDRG2 overexpression cell line was decreased, while the proliferation ability of Caco-2 lentiviral vector pLKO.1-shNDRG2 was significantly increased. Flow cytometry showed that NDRG2 overexpression caused Caco- 2 cells increased in G0 / G1 phase and decreased in S phase, which inhibited G0 / G1 phase cells and S phase cells after inhibiting endogenous NDRG2. CONCLUSION: NDRG2 as a candidate tumor suppressor gene is demonstrated to be able to effectively inhibit the proliferation of colon cancer cells Caco-2 by overexpressing and inhibiting NDRG2.