论文部分内容阅读
目的探讨丝氨酸/苏氨酸激酶15(serine/threoninekinase15,STK15)基因在胃癌细胞有丝分裂中的作用。方法应用RNA干扰技术(RNAi)抑制胃癌细胞株MKN45细胞STK15基因表达;realtime定量PCR及Westernblot检测干扰前后STK15mRNA及蛋白质表达的变化;倒置显微镜观察MKN45细胞形态的变化;流式细胞仪检测细胞周期的变化;MTT法检测细胞增殖速度的变化;免疫荧光结合激光共聚焦显微镜观察MKN45细胞微管及有丝分裂表型的改变。结果STK15基因沉默后,其mRNA及蛋白质表达明显下降,realtimePCR结果显示,转染后48h,STK15-组STK15mRNA水平较对照siRNA组的下降了89.54%;Westernblot灰度比半定量检测显示,STK15蛋白水平较对照siRNA组降低了57.18%。较多MKN45细胞呈圆形改变并呈现G2期细胞的DNA含量,STK15-组3个时间点的圆形细胞占18.95%,而对照siRNA组为8.34%,差异有统计学意义(P<0.05);流式细胞仪检测结果显示,STK15-组G2期DNA含量细胞平均值为26.13%,而对照siRNA组G2期DNA含量细胞平均值12.46%(P<0.05)。细胞增殖速度减慢(P<0.05)。细胞有丝分裂表型发生改变(P<0.05)。结论STK15基因在MKN45细胞有丝分裂过程中可能起着关键作用,阻断其表达可导致MKN45细胞有丝分裂停滞。
Objective To investigate the role of serine / threonine kinase 15 (STK15) gene in the mitosis of gastric cancer cells. Methods RNA interference (RNAi) was used to inhibit the expression of STK15 gene in gastric cancer cell line MKN45. The changes of STK15 mRNA and protein expression were detected by realtime quantitative PCR and Western blot. The morphology of MKN45 cells was observed by inverted microscope. The cell cycle was detected by flow cytometry The changes of cell proliferation were detected by MTT assay. The changes of microtubules and mitosis phenotype of MKN45 cells were observed by immunofluorescence combined with laser confocal microscopy. Results STK15 mRNA and protein expression was significantly decreased after STK15 gene silencing. Realtime PCR results showed that the STK15 mRNA level in STK15-treated cells decreased 89.54% 48 h after transfection compared with control siRNA group. Western blot showed that STK15 protein level Compared with the control siRNA group decreased 57.18%. More MKN45 cells showed a round shape and showed the DNA content of G2 phase cells. The number of round cells in STK15-group at 3 time points was 18.95%, while it was 8.34% in control siRNA group, the difference was statistically significant (P <0.05) The results of flow cytometry showed that the average DNA content of G2 phase in STK15- group was 26.13%, while that in control siRNA group was 12.46% (P <0.05). Cell proliferation slowed down (P <0.05). Cell mitosis phenotype changed (P <0.05). Conclusion The STK15 gene may play a key role in the mitosis of MKN45 cells. Blocking the expression of STK15 may result in mitotic arrest of MKN45 cells.