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目的探讨抑制人肝癌细胞HepG2中Rac1的活性对其生长及侵袭能力的影响。方法应用逆转录病毒载体,将Rac1基因的阴性突变体Rac1N17导入HepG2细胞,经嘌呤霉素筛选后获得表达Rac1N17的阳性细胞克隆Rac1N17-HepG2,荧光显微镜下观察绿色荧光蛋白的表达,四甲基偶氮唑(MTT)法检测细胞增殖抑制并绘制细胞生长曲线,细胞基质黏附实验检测对细胞的黏附能力的影响,Transwell法检测细胞侵袭能力的改变。结果荧光镜下可见绿色荧光蛋白主要分布于HepG2细胞核中,在细胞质中也有少量表达,证实了病毒载体质粒转染成功。MTT法表明Rac1的活性抑制后,HepG2细胞的增殖能力无明显变化(P>0.05);细胞基质黏附实验显示HepG2细胞与基质的黏附能力随着时间的延长而逐渐减弱(P<0.05);Transwell侵袭实验结果显示Rac1的活性抑制后,Rac1抑制组穿过PET滤膜的HepG2细胞数明显少于对照组(P<0.05),表明其侵袭能力明显受抑制。结论Rac1基因在肝癌细胞的黏附、侵袭及过程中发挥重要的作用,有望成为肝细胞癌基因治疗新的靶点。
Objective To investigate the effect of inhibiting Rac1 activity on the growth and invasion of HepG2 cells. Methods Rac1N17, a negative mutant of Rac1, was introduced into HepG2 cells by retroviral vector. The positive cell clone Rac1N17-HepG2 expressing Rac1N17 was obtained by puromycin screening. The expression of green fluorescent protein (GFP) was observed under fluorescence microscope. The inhibition of cell proliferation was measured by MTT assay and the cell growth curve was drawn. Cell adhesion assay was used to detect the effect of cell adhesion. Transwell assay was used to detect the change of cell invasiveness. Results Fluorescence microscopy showed that the green fluorescent protein was mainly distributed in the nucleus of HepG2 cells and also expressed in the cytoplasm. It was confirmed that the plasmid vector was transfected successfully. MTT assay showed that the proliferation of HepG2 cells did not change significantly (P> 0.05). The adhesion of HepG2 cells to matrix decreased gradually with time (P <0.05). Transwell The invasion assay showed that after Rac1 was inhibited, the number of HepG2 cells passing through the PET filter in Rac1 group was significantly less than that in control group (P <0.05), indicating that the invasion ability was significantly inhibited. Conclusion Rac1 gene plays an important role in the adhesion, invasion and progression of hepatocellular carcinoma cells and may be a new target of hepatocellular carcinoma gene therapy.