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目的构建过表达和干扰RN181基因的肝癌MHCC97L重组细胞系,并研究RN181对该细胞体外增殖的影响。方法基于基因克隆技术,构建plncx2-GFP-IRES/RN181重组载体,常规鉴定后,与pVSV-G共转染GP2-293细胞包装逆转录病毒。该病毒与RN181干扰慢病毒分别感染MHCC97L细胞,流式分选表达绿色荧光的细胞,RT-PCR、Western blotting鉴定后,CCK-8实验和软琼脂克隆形成实验检测细胞增殖情况。结果重组载体构建成功,重组细胞荧光良好,RT-PCR、Western blotting分别证实重组细胞中RN181的mRNA和蛋白表达水平在重组细胞系间均出现差异有统计学意义(P<0.05)。增殖实验显示RN181上调后细胞生长减慢,克隆数也显著少于对照组(P<0.05),而RN181干扰后上述现象可得到逆转。结论成功构建了过表达和干扰RN181的MHCC97L重组细胞系,初步证实了RN181在体外对MHCC97L细胞增殖的抑制作用。
Objective To construct a recombinant human hepatocellular carcinoma cell line MHCC97L that overexpresses and interferes with RN181 gene and to study the effect of RN181 on the proliferation of this cell line in vitro. Methods Recombinant plncx2-GFP-IRES / RN181 vector was constructed based on gene cloning technology. After routine identification, GP2-293 cells were co-transfected with pVSV-G to package retroviruses. The virus and RN181 interference lentivirus were infected with MHCC97L cells, flow cytometry expression of green fluorescent cells, RT-PCR, Western blotting identification, CCK-8 experiments and soft agar colony formation assay to detect cell proliferation. Results The recombinant vector was successfully constructed and the fluorescence of recombinant cells was good. The mRNA and protein expression levels of RN181 in recombinant cells were significantly different between recombinant cell lines (P <0.05) by RT-PCR and Western blotting respectively. Proliferation experiments showed that after RN181 upregulation, cell growth slowed down and the number of clones was also significantly less than that of the control group (P <0.05). However, the above phenomenon was reversed after RN181 interference. Conclusion The MHCC97L recombinant cell line that overexpresses and interferes with RN181 has been successfully constructed and the inhibitory effect of RN181 on the proliferation of MHCC97L cells in vitro has been demonstrated.