目的 构建小鼠黑色素瘤细胞（B16）过表达野生型及点突变型缝隙连接蛋白43（Connexin43，Cx43）细胞模型，为以缝隙连接（Gap Junction，GJ）为靶点的中药复方、中药单药及药物单体的研究提供可靠阳性对照和阴性对照。方法 构建野生型Cx43、突变型Cx43G21R、突变型Cx43G138R重组荧光蛋白融合慢病毒表达质粒，用定点突变技术获得Cx43G21R和Cx43G138R突变体，对上述3种质粒进行双酶切和测序鉴定，并分别包装病毒感染B16细胞，使B16细胞过表达野生型Cx43、突变型Cx43G21R、突变型Cx43G138R。Western blot检测Cx43蛋白表达水平变化，荧光示踪法观察缝隙连接通讯（Gap Junction Intercellular Communication，GJIC）功能变化。结果 ①酶切及测序证明，成功构建pLVCx43-mCherry、pLVCx43-mCherryG21R、pLVCx43- mCherryG138R重组荧光蛋白融合慢病毒表达质粒。②成功感染B16细胞并筛选稳定过表达Cx43、Cx43G21R、Cx43G138R细胞株，Western blot检测显示上述细胞株Cx43蛋白表达均高于对照组。③过表达野生型Cx43后B16细胞GJIC功能较对照组增强；过表达突变型Cx43后B16细胞GJIC功能较对照组减弱。结论 过表达野生型Cx43可增强B16细胞GJIC功能，过表达突变型Cx43可抑制B16细胞GJIC功能。

Objective To construct mouse B16 melanoma cell models of wild-type overexpression Cx43 and site- specific mutant Cx43，thus to provide reliable positive and negative control for the study of gap junction targeted Chinese herbal formula，Chinese medicine ingredient，and drug monomer. Methods We constructed the recombined fluorescent protein（mCherry） infused with Lentivirus expression plasmid of wild-type Cx43（pLVCx43-mCherry），mutant R（pLVCx43G21R-mCherry） and mutant Cx43G138R（pLVCx43 G138R-mCherry）. The Cx43G21R and Cx43G138R mutants were obtained by site-specific mutagenesis. The three kinds of plasmid were performed double restriction enzyme digestion and sequencing，and then were packaged to infect B16 cells. The B16 cells overexpressed the protein of wild-type Cx43 and mutants of Cx43G21R and Cx43G138R，and then western blotting was applied for the detection of protein level. The functional changes of gap junction intercellular communication（GJIC） were assayed by fluorescent tracer under fluorescence microscope. Results The results of digestion and sequencing demonstrated that the recombined Lentivirus-fluorescent protein expression plasmids of pLVCx43-mCherry，pLVCx43-mCherryG21R and pLVCx43-mCherryG138R had been constructed successfully. We also successfully transfected the mouse B16 cells and selected transfected cell lines with stable overexpression of wild-type Cx43，site-specific mutant Cx43G21R and site-specific mutant Cx43G138R. The results of Western blotting showed that Cx43 expression level of the three kinds of cell lines was higher than that in control group. The GJIC function of B16 cells with wild-type Cx43 overexpression was dramatically higher，while that of B16 cells with site-specific Cx43 mutants overexpression was lower than the control group. Conclusion Wild-type overexpression Cx43 can promote the GJIC function in mouse B16 melanoma cells，and conversely the GJIC function can be inhibited by site-specific mutants of overexpression Cx43G21R and Cx43G138R.

R34-33

国家自然科学基金资助项目（81072906，30973811）。