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[目的]研究RIN1过表达对人胃癌细胞MKN28增殖和细胞周期的影响。[方法]采用PCR法扩增人RIN1基因,并构建重组真核表达质粒pcDNA3.1(+)-RIN1。采用脂质体转染法将pcDNA3.1(+)-RIN1及空载体pcDNA3.1(+)转染至人胃癌MKN28细胞,G418法筛选阳性克隆;Western blot法和免疫荧光法鉴定RIN1的高表达。CCK-8法和平板克隆实验检测RIN1高表达对MKN28细胞增殖的影响。流式细胞术检测RIN1高表达对MKN28细胞周期的影响。[结果]成功构建RIN1真核表达载体,RIN1基因全长为2 353bp。G418法筛选获得高表达RIN1的MKN28克隆细胞株MKN28/RIN1-1和MKN28/RIN1-2,Western blot结果显示这两株细胞分别为MKN28/3.1的3.21倍和3.17倍。过表达RIN1基因后,MKN28细胞增殖显著性加快,克隆数显著性增加;细胞周期改变,G0/G1期细胞减少而S期和G2/M期细胞增加。[结论]RIN1基因过表达加速MKN28细胞从G1期向S期的转化促进细胞增殖;RIN1可能成为胃癌治疗的一个潜在靶点。
[Objective] To investigate the effect of RIN1 overexpression on the proliferation and cell cycle of human gastric cancer cell line MKN28. [Method] The human RIN1 gene was amplified by PCR and the recombinant eukaryotic expression plasmid pcDNA3.1 (+) - RIN1 was constructed. The recombinant plasmid pcDNA3.1 (+) - RIN1 and empty vector pcDNA3.1 (+) were transfected into human gastric cancer MKN28 cells by lipofection method. The positive clones were screened by G418 method. The expression of RIN1 was identified by Western blot and immunofluorescence expression. The effects of RIN1 overexpression on the proliferation of MKN28 cells were detected by CCK-8 assay and plate cloning assay. Flow cytometry was used to detect the effect of RIN1 overexpression on the cell cycle of MKN28 cells. [Result] The RIN1 eukaryotic expression vector was successfully constructed. The full length of RIN1 gene was 2 353 bp. The MKN28 / RIN1-1 and MKN28 / RIN1-2 cells were screened by G418. The results of Western blot showed that the two cells were 3.21-fold and 3.17-fold higher than that of MKN28 / 3.1 respectively. After overexpression of RIN1 gene, the proliferation of MKN28 cells was significantly accelerated, and the number of clones was significantly increased. The cell cycle was changed, the cells in G0 / G1 phase decreased and the cells in S phase and G2 / M phase increased. [Conclusion] RIN1 gene overexpression accelerates the transformation of MKN28 cells from G1 phase to S phase and promotes cell proliferation. RIN1 may be a potential therapeutic target for gastric cancer.