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目的:在体外检测Nrf3基因对大肠癌LoVo的生长影响作用。方法:构建其融合蛋白真核表达载体,脂质体介导该真核表达载体转染大肠癌LoVo细胞系,FCM观察其在体外对大肠癌LoVo细胞系细胞周期和凋亡的影响;荧光显微镜观察候选基因亚细胞定位。结果:构建融合蛋白真核表达载体pEGFP-N1-Nrf3。RT-PCR方法检测Nrf3的表达,与芯片检测结果基本一致。荧光显微镜下观察Nrf3定位于细胞核内。FCM分析显示Nrf3影响下LoVo G2/M+S期细胞所占比例较对照组明显减少,G0/G1细胞所占比例明显增加。证实Nrf3可抑制大肠癌LoVo细胞的DNA合成和有丝分裂,促使细胞阻滞于G0/G1期,抑制大肠癌LoVo细胞的体外生长。FCM分析显示Nrf3在体外对大肠癌LoVo细胞的凋亡无影响。结论:Nrf3在体外具有抑制大肠癌增殖的功能,对大肠癌的细胞凋亡无影响,为肿瘤抑制基因。
Objective: To investigate the effect of Nrf3 gene on the growth of colorectal cancer LoVo in vitro. Methods: The fusion protein eukaryotic expression vector was constructed. The eukaryotic expression vector was transfected into LoVo cell line by lipofectamine. The effect of FCM on the cell cycle and apoptosis of LoVo cell line was observed by FCM. Fluorescence microscopy Observe candidate gene subcellular localization. Results: The fusion protein eukaryotic expression vector pEGFP-N1-Nrf3 was constructed. RT-PCR method to detect the expression of Nrf3, and the chip test results are basically the same. Nrf3 was located in the nucleus under fluorescence microscope. FCM analysis showed that the proportion of cells in LoVo G2 / M + S phase decreased significantly under the influence of Nrf3 compared with the control group, and the proportion of G0 / G1 cells increased significantly. Confirmed that Nrf3 can inhibit DNA synthesis and mitosis in colorectal LoVo cells, promote cell arrest in G0 / G1 phase and inhibit growth of colorectal LoVo cells in vitro. FCM analysis showed that Nrf3 had no effect on the apoptosis of colorectal cancer LoVo cells in vitro. Conclusion: Nrf3 has the function of inhibiting the proliferation of colorectal cancer in vitro, has no effect on the apoptosis of colorectal cancer, and is a tumor suppressor gene.