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目的观察核干细胞因子(nucleostemin,NS)、细胞周期蛋白B1(Cyclin B1)及抑癌基因PTEN mRNA和蛋白的表达情况。方法正常食管上皮细胞(shantou human embryonic esophageal epithelial cell line,SHEE)和永生化食管上皮恶变细胞(shantou human embryonic esophageal carcinoma epithelial cell line,SHEEC)连续传代培养,取对数生长期SHEE和SHEEC细胞,采用RT-PCR及Western blot方法检测细胞中NS、Cyclin B1及PTEN mRNA和蛋白的表达并做相关性分析,MTT法检测抑制NS表达后的细胞增殖活性。结果以SHEE细胞中NS、Cyclin B1和PTEN mRNA的表达量为1,SHEEC细胞中对应的相对表达量分别为(2.047±0.507)、(2.214±0.213)及(0.668±0.188),两组比较差异均有统计学意义(P<0.05);SHEE及SHEEC细胞中NS、Cyclin B1及PTEN蛋白表达量分别为(1.142±0.113),(3.161±0.108)、(1.585±0.056),(2.173±0.058)及(1.894±0.341),(1.162±0.524),两组比较差异均有统计学意义(P<0.05);NS mRNA和蛋白与Cyclin B1 mRNA和蛋白的表达呈正相关(r=0.832,P<0.01;r=0.991,P<0.05),与PTEN mRNA和蛋白的表达呈负相关(r=-0.924,P<0.01;r=-0.769,P<0.05);MTT检测结果显示,NS RNA干扰(RNA interference,RNAi)组与空白对照组和阴性对照组在干扰后12 h细胞增殖能力差异无统计学意义(P>0.05),而在干扰后24、36、48 h差异有统计学意义(P<0.05)。结论 SHEEC细胞中NS、Cyclin B1 mRNA和蛋白的表达量均高于SHEE,而PTEN的表达量低于SHEE,NS可能对细胞恶变有影响。
Objective To observe the expression of nucleostemin (NS), cyclin B1 and PTEN mRNA and protein. Methods SHEE and SHEEC cells were serially subcultured with SHEE and SHEEC cells in logarithmic growth phase, The expression of NS, Cyclin B1 and PTEN mRNA and protein were detected by RT-PCR and Western blot. The correlation analysis was made between the expression of NS, Cyclin B1 and PTEN mRNA and protein. The cell proliferation activity was detected by MTT assay. Results The relative expression levels of NS, Cyclin B1 and PTEN mRNA in SHEE cells were 1 (2.047 ± 0.507), (2.214 ± 0.213) and (0.668 ± 0.188), respectively (1.142 ± 0.113), (3.161 ± 0.108), (1.585 ± 0.056) and (2.173 ± 0.058) respectively in SHEE and SHEEC cells (P <0.05) (1.894 ± 0.341) and (1.162 ± 0.524), respectively, with significant difference between the two groups (P <0.05). The expressions of NS mRNA and protein were positively correlated with the expression of Cyclin B1 mRNA and protein (r = 0.832, (r = -0.924, P <0.01; r = -0.769, P <0.05). The results of MTT showed that the expression of NS RNA interference (P> 0.05). However, the difference was statistically significant at 24, 36 and 48 h after interference (P <0.05). There was no significant difference in cell proliferation between RNAi group and blank control group and negative control group at 12 h 0.05). Conclusion The expression of NS and Cyclin B1 mRNA and protein in SHEEC cells are higher than those in SHEE, while the expression of PTEN is lower than that in SHEE. NS may affect the cell malignant transformation.