间隙连接蛋白43对胰腺癌细胞株BxPC3凋亡的作用及其机制研究

目的研究间隙连接蛋白43（Cx43）在胰腺癌细胞凋亡中的作用，并探讨其机制。方法采用脂质体2000法将pcDNA—Cx43、pcDNA—Cx43N、空质粒pcDNA3．0、siRNA—Cx43和对照siRNA-NC分别转染BxPC3细胞。Western blotting检测细胞Cx43蛋白表达、细胞色素C（Cyt C）含量；流式细胞仪检测细胞凋亡、线粒体膜电位；荧光分光光度计检测细胞Caspase-9和Caspase-3活性；染料传递法测定细胞间间隙连接（GJ）。结果Cx43转染BxPC3细胞后，Cx43蛋白表达明显上调，细胞凋亡从空质粒转染组的（6．35±0．43）％增加到（14．29±1．24）％，经H2O2处理后，从（20．34±2．47）％增加到（31．27±2．56）％（P〈0．05），同时线粒体膜电位下降，CytC从线粒体释放增多，Caspase蛋白酶活性增加；而siRNA43干扰细胞后，细胞凋亡从（7．42±0．47）％减少到（5．19±1．37）％，经H2O2处理后，从（19．43±1．71）％减少到（11．67±1．97）％（P〈0．05），线粒体膜电位去极化，CytC从线粒体释放减少，Caspase酶活性下调。细胞间间隙连接指数在无GJ抑制剂B—GA情况下从对照组的14．52±0．57增加到pcDNA—Cx43转染组的23．05±3．84，在存在β—GA情况下从1．70±0．24增加到3．84±0．45（P〈0．05），但细胞凋亡率改变无显著差异。结论Cx43可通过线粒体凋亡途径促进BxPC3细胞凋亡，Cx43调节细胞凋亡存在间隙连接以外的机制。

Role of connexin 43 in apoptosis of pancreatic cancer cell line BxPC3

Objective To investigate the role of connexin 43 （Cx43） in the apoptosis of pancreatic cancer cell line BxPC and its possible mechanism. Methods pcDNA-Cx43, pcDNA-Cx43N, pcDNA3.0, siRNA-Cx43 and siRNA-NC were transfected into BxPC3 cells via liposome method. Cx43 protein and Cytochrome C （Cyt C ） concentration was determined by Western blot, and the apoptosis was analyzed by Annexin V/PI binding assay. The mitochondria apoptosis pathway involved in Cx43 associated apoptosis was examined which contains the depolarization of mitochondrial membrane potential （ MMP ） ; fluorospectrophotometer was used to measure the activities of caspase-3 and easpase-9. Gap junction intercellular communication（ GJIC ） was determined by dye-transfer method. Results Cx43 protein expression increased after BxPC3 transfection, apoptosis rate increased from （6.35± 0.43 ） % in empty vector transfection group to （ 14.29 ± 1.24） % ; after H2O2 treatment, apoptosis rate increased from （ 20.34 ± 2.47 ） % to （ 31.27 ±2.56） % （P 〈 0.05 ）. Meanwhile, mitochondrial membrane potential was decreased, Cyt C was increasingly released from mitochondria, caspases activities were increased; after siRNA43 interference, apoptosis rate decreased from （7.42±0.47）% to （5.19 ± 1.37）% , after H2O2 treatment, apoptosis rate decreased from （ 19.43± 1.71 ） % to （ 11.67 ± 1.97 ） % （ P 〈 0.05 ）. Decreased mitochondrial membrane potential and Cyt Crelease were observed, caspases activities were decreased. GJIC of pcDNA-Cx 43 transfection group increased from 14.52 ±0.57 to 23.05 ± 3.84, and it increased from 1.70 ± 0.24 to 3.84 ± 0.45 in the presence of β-GA （P 〈 0.05 ）. But the apoptosis rate was not significantly different. Conclusions Cx43 could promote BxPC3 apoptosis via mitochondrial apoptotic signal pathway, and the possible mechanism included signal pathway other than GJIC.