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目的探讨在双层共培养体系中缝隙连接在血管内皮细胞(endothelial cell,EC)调节血管平滑肌细胞(smooth muscle cell,SMC)增殖能力中的作用。方法采用植块法分别培养大鼠主动脉内皮及平滑肌细胞,并将2种细胞分别接种至Transwell insert膜的两侧以建立内皮细胞与平滑肌细胞双层共培养模型,并用透射电镜观察2种细胞在Tran-swell insert膜两侧生长情况。用3H-TdR掺入法观察双层共培养模型中平滑肌细胞增殖情况,并观察缝隙连接阻断剂18α-甘草次酸(18α-GA)对平滑肌细胞增殖能力的影响。结果在共培养第1天,EC/SMC组与SMC/SMC组的平滑肌细胞3H-TdR掺入率无统计学差异[(10900±1320)cpm/wellvs(10430±1200)cpm/well,P>0.05]。第2天,EC/SMC组中平滑肌细胞3H-TdR的掺入率有低于SMC/SMC组[(17200±1734)cpm/wellvs(20900±1659)cpm/well]的趋势,但两者差异未达到统计学标准。共培养第3天时,EC/SMC组平滑肌细胞3H-TdR掺入率显著低于SMC/SMC组,两者相比有显著性差异[(25800±1984)cpm/wellvs(33070±3569)cpm/well,P<0.05]。在用18α-GA预处理24h后,EC/SMC共培养3d后平滑肌细胞3H-TdR的掺入率明显高于同期未处理EC/SMC组,两者相比有显著性差异[(30500±2650)cpm/wellvs(25800±1984)cpm/well,P<0.05]。结论双层共培养模型中内皮细胞通过缝隙连接可抑制血管平滑肌细胞的增殖能力。
Objective To investigate the role of gap junctions in the proliferation of vascular smooth muscle cells (SMC) in endothelial cells (ECs) in a double co-culture system. Methods The aorta endothelial cells and smooth muscle cells of rats were cultured by explant method. The two kinds of cells were inoculated on both sides of Transwell insert to establish a double co-culture model of endothelial cells and smooth muscle cells. Transmission electron microscope Growth on both sides of Tran-swell insert. The 3H-TdR incorporation method was used to observe the proliferation of smooth muscle cells in double-layer co-culture model. The effect of 18α-glycyrrhetinic acid (18α-GA), a gap junction blocker, on the proliferation of smooth muscle cells was observed. Results There was no significant difference in 3H-TdR incorporation between the EC / SMC group and the SMC / SMC group on day 1 of co-culture [(10900 ± 1320) cpm / well vs (10430 ± 1200) cpm / well, P> 0.05]. On the second day, the 3H-TdR incorporation rate of smooth muscle cells in EC / SMC group was lower than that in SMC / SMC group [(17200 ± 1734) cpm / wellvs (20900 ± 1659) cpm / well] Did not meet the statistical standards. On day 3 of co-culture, the 3H-TdR incorporation rate of smooth muscle cells in EC / SMC group was significantly lower than that in SMC / SMC group [(25800 ± 1984) cpm / well vs 33070 ± 3569 cpm / well, P <0.05]. After pretreatment with 18α-GA for 24h, the 3H-TdR incorporation rate of smooth muscle cells in EC / SMC co-cultured for 3 days was significantly higher than that in untreated EC / SMC group [(30500 ± 2650 ) cpm / wellvs (25800 ± 1984) cpm / well, P <0.05]. Conclusions Endothelial cells can inhibit the proliferation of vascular smooth muscle cells through gap junction in double co-culture model.