潜在抑癌基因TXNIP对胃癌MKN-45细胞生物学特性的影响

Influence of potential tumor suppressor gene TXNIP in biological characteristics of gastric cancer MKN-45 cells

目的:探讨硫氧还蛋白相互作用蛋白(TXNIP)基因对胃癌MKN-45细胞生物学特性的影响,阐明TXNIP与胃癌发生发展的关系。方法:将TXNIP基因插入载体pcDNA3.1构建PC-TXNIP真核表达载体,并将其转染胃癌MKN-45细胞建立稳定转染细胞系(MKN-TXNIP),利用RT-RCR法、免疫细胞化学法检测转染细胞中的TXNIP基因及细胞原位蛋白表达。每种检测实验均分为MKN-TXNIP组、MKN-PC组和MKN-45组(空白对照组)。采用生长曲线法、平板克隆形成实验法、流式细胞术和Transwell小室实验法等检测稳定表达TXNIP胃癌细胞株生长、增殖状态、细胞周期和侵袭能力等生物学特性。结果:与MKN-PC组和MKN-45组比较,MKN-TXNIP组中TXNIP基因可稳定表达。与MKN-PC组和MKN-45组比较,MKN-TXNIP组稳定表达的细胞株生长速度明显减慢(P<0.05)。平板克隆形成实验,MKN-TXNIP组细胞平均克隆形成率明显低于其他2组(P<0.05)。细胞周期检测,与MKN-45组比较,MKN-TXNIP组处于G0/G1期的细胞百分比明显升高(P<0.05),而处于G2/M期的细胞百分比明显降低(P<0.05)。流式细胞术,各组细胞的凋亡率均约为2.0%,各组间比较差异均无统计学意义(P>0.05)。Transwell小室实验,MKN-TXNIP组MKN-45细胞穿膜率低于其他2组(P<0.05)。结论:TXNIP基因对胃癌细胞增殖、分裂及侵袭转移能力可能具有抑制作用,并可同时影响胃癌细胞的细胞周期,但其对细胞凋亡的影响作用较小。

Objective To investigate the influence of TXNIP in the biological characteristics of gastric cancer MKN-45 cells,and to clarify the relationships between TXNIP and the carcinogenesis and progression of gastric cancer.Methods The cDNA of TXNIP was subcloned into a constitutive vector pcDNA3.1followed by transfection in MKN-45 cells by using liposome.RT-PCR and immunocytochemistry were used to detect the expressions of TXNIP gene and protein in MKN-TXNIP cells.Each detection experiment was divided into MKN-TXNIP group,MKN-PC group and MKN-45group（blank control group）.Flow cytometry was used to analyze the apoptosis and cell cycles of gastric carcinoma cells.The cell growth curves and the colony assay were used to detect the growth and proliferation of MKN-45 cells.Transwell chamber was used to detect the invasion.Results Compared with MKNPC group and MKN-45 group,TXNIP gene could express stably in MKN-TXNIP group.The growth speed of the MKN-45 cells in MKN-TXNIP group was slower than those in MKN-45 group and MKN-PC group（P〈0.05）.The results of colony formation assay showed that the colony formation rate in MKN-TXNIP group was lower than those in MKN-45 group and MKN-PC group（P〈0.05）.The percentage of the cells in G0/G1 phase in MKNTXNIP group was higher significantly（P〈0.05）,and the percentage of the cells in G2/M plase was slower than those in MKN-45 group and MKN-PC group.The flow cytometry results showed that the apoptotic rates of the cells in each group were about 2.0%,and there were no significant differences between various groups（P〉0.05）.The results of migration assay suggested that the cell migration rate of the MKN-45 cells in MKN-TXNIP group was significantly lower than those in MKN-45 group and MKN-PC group（P〈0.05）.Conclusion TXNIP gene can inhibit the growth,proliferation,division and invasion of gastric cancer cells.It can also influence the cell cycle,but it only has little influence in the apoptosis of MKN-45 cells.