Ma Xiang-Dong, Ma Xing, Sui Yan-Fang, Wang Weng-Liang, Wang Chun-Mei
Department of Obstetrics and Gynecology, Xijing Hospital, Fourth Military Medical University, 17 Changle Xilu, Xi'an 710033, Shaanxi Province, China.
World J Gastroenterol. 2003 May;9(5):946-50. doi: 10.3748/wjg.v9.i5.946.
To investigate gap junctional intercellular communication (GJIC) in hepatocellular carcinoma cell lines, and signal transduction mechanism of gap junction genes connexin32(cx32),connexin43(cx43) in human hepatocarcinogenesis.
Scarped loading and dye transfer (SLDT) was employed with Lucifer Yellow (LY) to detect GJIC function in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal control liver cell line QZG. After Fluo-3AM loading, laser scanning confocal microscope (LSCM) was used to measure concentrations of intracellular calcium (Ca(2+))i in the cells. The phosphorylation on tyrosine of connexin proteins was examined by immunoblot.
SLDT showed that ability of GJIC function was higher in QZG cell than that in HHCC and SMMC-7721 cell lines. By laser scanning confocal microscopy, concentrations of intracellular free calcium (Ca(2+))i was much higher in QZG cell line (108.37 nmol/L) than those in HHCC (35.13 nmol/L) and SMMC-7721 (47.08 nmol/L) cells. Western blot suggested that only QZG cells had unphosphorylated tyrosine in Cx32 protein of 32 ku and Cx43 protein of 43 ku; SMMC-7721 cells showed phosphorylated tyrosine Cx43 protein.
The results indicated that carcinogenesis and development of human hepatocellular carcinoma related with the abnormal expression of cx genes and disorder of its signal transduction pathway, such as decrease of (Ca(2+))i, post-translation phosphorylation on tyrosine of Cx proteins which led to a dramatic disruption of GJIC.
研究肝癌细胞系中的缝隙连接细胞间通讯（GJIC），以及缝隙连接基因连接蛋白32（cx32）、连接蛋白43（cx43）在人类肝癌发生中的信号转导机制。
采用刮除负载及染料传递（SLDT）技术，用荧光黄（LY）检测肝癌细胞系HHCC、SMMC - 7721及正常对照肝细胞系QZG中的GJIC功能。Fluo - 3AM负载后，用激光扫描共聚焦显微镜（LSCM）测量细胞内钙离子（Ca(2+)）i浓度。通过免疫印迹检测连接蛋白酪氨酸磷酸化情况。
SLDT显示，QZG细胞的GJIC功能能力高于HHCC和SMMC - 7721细胞系。通过激光扫描共聚焦显微镜观察，QZG细胞系（108.37 nmol/L）细胞内游离钙离子（Ca(2+)）i浓度远高于HHCC（35.13 nmol/L）和SMMC - 7721（47.08 nmol/L）细胞。蛋白质印迹法表明，只有QZG细胞中32 ku的Cx32蛋白和43 ku的Cx43蛋白存在未磷酸化的酪氨酸；SMMC - 7721细胞显示Cx43蛋白酪氨酸磷酸化。
结果表明，人类肝癌的发生发展与cx基因的异常表达及其信号转导途径紊乱有关，如细胞内钙离子（Ca(2+)）i浓度降低、连接蛋白酪氨酸翻译后磷酸化，导致GJIC严重破坏。
