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目的:建立稳定表达RI基因的卵巢癌SKOV3细胞系,并观察外源基因对细胞体外增殖的作用。方法:以脂质体为介导,将携有RI基因的重组载体pLNCX-ri转染SKOV3细胞,实验分3组,RI基因转染组(SKOV3/RI组)、空质粒转染组(SKOV3/Neo组)和空白对照组(SKOV3组)。采用G-418筛选获得稳定转染的细胞系后,通过RT-PCR技术和免疫细胞化学染色方法鉴定各组细胞中RI基因的表达;通过MTT、细胞周期分析、细胞凋亡检测方法观察该基因对细胞生长的影响。结果:外源性RI基因已整合于细胞基因组中,并获得稳定表达。转染RI基因后的细胞生长速度减慢,明显低于对照组,差异有统计学意义。细胞周期分析显示,实验组G1期细胞增至69.23%,S期细胞降至24.34%,与另外两组相比差异有统计学意义(P<0.05)。细胞凋亡检测表明SKOV3/RI组细胞凋亡率为58.25%,与SKOV3组(17.97%)及SKOV3/Neo组(21.42%)比较,差异有统计学意义(P<0.05)。结论:通过脂质体介导,可以建立RI基因稳定表达的卵巢癌细胞系,且RI基因对SKOV3细胞的体外增殖有抑制作用。
OBJECTIVE: To establish ovarian cancer SKOV3 cell line stably expressing RI gene and to observe the effect of exogenous gene on cell proliferation in vitro. Methods: The recombinant plasmid pLNCX-ri carrying RI gene was transfected into SKOV3 cells by lipofectamine. The experiment was divided into 3 groups: SKOV3 / RI group, SKOV3 / RI group, SKOV3 / / Neo group) and blank control group (SKOV3 group). After stable transfected cell lines were screened by G-418, the expression of RI gene in each group was identified by RT-PCR and immunocytochemical staining. The gene expression was observed by MTT, cell cycle analysis and apoptosis assay Effect on cell growth. Results: The exogenous RI gene has been integrated into the cell genome and obtained stable expression. After transfected with RI gene, the cell growth rate slowed down significantly lower than the control group, the difference was statistically significant. Cell cycle analysis showed that cells in G1 phase increased to 69.23% and cells in S phase decreased to 24.34%, which was significantly different from the other two groups (P <0.05). The apoptosis rate of SKOV3 / RI group was 58.25%, which was significantly different from SKOV3 group (21.92%) and SKOV3 group (17.97%) (P <0.05). CONCLUSION: Liposome-mediated ovarian cancer cell lines with stable expression of RI gene can be established and RI gene can inhibit the proliferation of SKOV3 cells in vitro.