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目的:应用特异性小干扰RNA(siRNA)下调97-H细胞中脑源性神经生长因子(BDNF)表达,观察对细胞凋亡和侵袭的影响并探讨相关分子机制。方法:在人肝细胞癌(HCC)细胞系97-H中,采用蛋白质印迹法检测BDNF的表达,采用ELISA方法检测培养上清上BDNF的分泌水平。特异性BDNF-siRNA转染细胞,采用FITC-phalloidin染色方法检测actin细胞骨架的变化,采用western blot方法检测细胞内RhoA、Rac1、Cdc42的活化情况。同时,流式细胞术检测细胞凋亡,Transwell小室测定细胞侵袭能力的变化。结果:97-H细胞培养上清中BDNF含量为(119.08±6.21)ρg/mL。在97-H细胞中,特异性BDNF-siRNA显著抑制BDNF的表达,干扰细胞内actin细胞骨架聚合,RhoA或Rac1活性受到抑制,同时与对照组相比,凋亡细胞百分比增加至(27.00±1.71)%,P=0.000,侵袭细胞数减少至(26.9±1.6)%,P=0.000。结论:干扰BDNF的表达能显著降低HCC细胞侵袭能力,其机制可能与阻断actin细胞骨架聚合、以及RhoA或Rac1活化相关。BDNF信号通路可能作为阻断HCC发展演进的新靶点,有待于进一步深入研究。
OBJECTIVE: To investigate the effect of BDNF on the apoptosis and invasion of 97-H cells by using specific small interfering RNA (siRNA) and to explore the related molecular mechanisms. Methods: The expression of BDNF was detected by Western blotting in human hepatocellular carcinoma (HCC) cell line 97-H. The secretion of BDNF on culture supernatant was detected by ELISA. Specific BDNF-siRNA transfected cells were used to detect the changes of actin cytoskeleton by FITC-phalloidin staining. The activation of RhoA, Rac1 and Cdc42 were detected by western blot. At the same time, apoptosis was detected by flow cytometry. Transwell chamber was used to determine the change of cell invasiveness. Results: The content of BDNF in 97-H cell culture supernatant was (119.08 ± 6.21) ρg / mL. In 97-H cells, specific BDNF-siRNA significantly inhibited the expression of BDNF and interfered with the actin cytoskeleton polymerization, while the activity of RhoA or Rac1 was inhibited. Compared with the control group, the percentage of apoptotic cells increased to (27.00 ± 1.71 )%, P = 0.000, the number of invasive cells decreased to (26.9 ± 1.6)%, P = 0.000. CONCLUSION: Interference of BDNF expression can significantly reduce the invasiveness of HCC cells. The mechanism may be related to blocking actin cytoskeleton polymerization and RhoA or Rac1 activation. BDNF signal pathway may be used as a new target to block the evolution of HCC development needs further study.