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目的 :检测M2-型丙酮酸激酶(pyruvate kinase M2,PKM2)在胆管癌(cholangiocarcinoma,CCA)组织中的表达情况,探讨PKM2下调对胆管癌细胞迁移、侵袭及增殖的影响。方法:实时定量逆转录聚合酶链反应(q RT-PCR)和免疫组织化学(immunohistochemistry,IHC)染色分别检测胆管癌及对应癌旁组织标本中PKM2的m RNA和蛋白表达水平。利用慢病毒表达载体系统在胆管癌细胞株Hu CCT-1、HCCC-9810中下调PKM2,分别用划痕实验、Transwell细胞侵袭实验和CCK-8比色法检测细胞迁移、侵袭及增殖能力。结果:胆管癌组织中PKM2的m RNA和蛋白表达水平明显高于对应癌旁组织。经q RT-PCR和Western blot方法证实稳定转染PKM2 sh RNA的胆管癌细胞中PKM2的m RNA和蛋白水平较对照组均明显下降(P<0.05),与空载对照组(PKM2-NC)和正常对照组(PKM2)相比,实验组细胞(PKM2-sh RNA)的迁移、侵袭及增殖能力明显减弱,差异有统计学意义(P<0.05)。结论:胆管癌组织中PKM2表达明显高于癌旁组织,PKM2 sh RNA能有效地降低胆管癌细胞中PKM2基因的表达,PKM2基因沉默可以抑制Hu CCT-1、HCCC-9810细胞的迁移、侵袭及增殖。
OBJECTIVE: To detect the expression of pyruvate kinase M2 (PKM2) in cholangiocarcinoma (CCA) tissues and to investigate the effect of down-regulation of PKM2 on the migration, invasion and proliferation of cholangiocarcinoma cells. Methods: The mRNA and protein levels of PKM2 in cholangiocarcinoma and corresponding paracancerous tissues were detected by real-time RT-PCR and immunohistochemistry (IHC). PKM2 was down-regulated in the cholangiocarcinoma cell lines Hu CCT-1 and HCCC-9810 by lentivirus expression vector system. The migration, invasion and proliferation of cells were detected by scratch assay, Transwell cell invasion assay and CCK-8 colorimetric assay respectively. Results: The expression of m RNA and protein of PKM2 in cholangiocarcinoma was significantly higher than that in paracancerous tissues. The results of q RT-PCR and Western blot showed that PKM2 mRNA and protein levels in PKC2 stably transfected cholangiocarcinoma cells were significantly lower than those in control group (P <0.05) Compared with normal control group (PKM2), the migration, invasion and proliferation of PKM2-sh RNA in experimental group were significantly decreased (P <0.05). Conclusion: The expression of PKM2 in cholangiocarcinoma tissues is significantly higher than that in paracancerous tissues. PKM2 sh RNA can effectively reduce the expression of PKM2 gene in cholangiocarcinoma cells. PKM2 gene silencing can inhibit the migration and invasion of HuCCT-1 and HCCC-9810 cells. proliferation.