人肺动脉平滑肌细胞对人肺动脉内皮细胞增殖的影响

Human pulmonary arterial smooth muscle cells regulate proliferation of human pulmonary arterial endothelial cells via survivin signaling pathway under hypoxic condition

目的探讨缺氧条件下,细胞共培养体系中人肺动脉平滑肌细胞(HPASMCs)经由Survivin信号通路调控人肺动脉内皮细胞(HPAECs)的增殖。方法建立Transwell共培养体系将HPASMCs和HPAECs分别接种于下室和上室,根据HPASMCs是否行YM155(Survivin抑制剂)预处理进行实验分组:常氧条件下HPASMCs与HAPECs共培养对照组(N组),常氧条件下YM155(终浓度100 nmol/l)预处理HPASMCs与HPAECs共培养组(NY组),缺氧条件下HPASMCs与HAPECs共培养组(H组)和缺氧条件下YM155预处理HPASMCs与HPAECs共培养组(HY组)。采用活细胞计数(CCK8)法检测各组中HPASMCs与HPAECs的增殖活性(吸光度,A值),实时定量PCR检测HPASMCs中Survivin m RNA的表达,Western blot检测HPASMCs中Survivin蛋白的表达。结果 N组HPASMCs与HPAECs增殖活性(0.561±0.007)、(0.619±0.013)与H组(0.777±0.030)、(0.875±0.021)比较,差异有统计学意义(q=16.615和21.333,P<0.05)。HY组HPASMCs与HPAECs增殖活性为(0.661±0.027)、(0.723±0.025),与H组比较差异有统计学意义(q=8.923和12.667,P<0.05)。共培养体系中N组HPASMCs未见m RNA和Survivin蛋白(0.017±0.001)表达,H组中HPASMCs可见m RNA(4 506±849)和Survivin蛋白(0.932±0.018)表达。HY组HPASMCs m RNA和Survivin蛋白含量为(677±183)和(0.426±0.022),与H组比较差异有统计学意义(q=15.25和50.6,P<0.05)。结论缺氧导致HPASMCs和HPAECs异常增殖,缺氧条件下共培养体系中HPASMCs经由Survivin信号通路调控HPAECs的增殖。

Objective To investigate the effect of hypoxic human pulmonary arterial smooth muscle cells （HPASMCs） on the proliferation of human pulmonary arterial endothelial cell （HPAECs） and the mechanism. Methods A Transwell coculture system of HPASMCs and HPAECs was constructed. HPAECs were put into the upper chamber while HPASMCs were put into the lower chamber. The Transwell coculture system included the following groups： normoxie eoculture of HPASMCs and HPAECs （N group）, normoxic coculture of HPASMCs pretreated with YM155 （inhibitor of survivin） and HPAECs （NY group）, hypoxic coculture of HPASMCs and HPAECs （H group）, and hypoxic cocuhure of HPASMCs pretreated with YM155 and HPAECs （HY group）. Cell proliferation was determined using a Cell Counting Kit-8 （CCK-8）. The mRNA and protein expressions of survivin in the HPASMCs were measured by qRT-PCR and Western blot respectively. Results The proliferation of HPASMCs and HPAECs in the H group [（0.777 ± 0.030） and （0.875 ± 0.021） respectively] was significantly increased compared with that of the N group [（0.561±0.007） and （0.619±0.013）,q = 16.615 and 21.333,P〈 0.05]. As compared with the H group, the proliferation of HPASMCs and HPAECs in the HY group [（0.661 ± 0.027） and （0.723 ± 0.025） respectively] significant decreased （q = 8.923 and 12.667, P 〈 0.05）. The mRNA and protein of survivin were expressed in HPASMCs of the H group [（4506 ± 849） and （0.932 ± 0.018）],but not in those of the N group （0.017 ± 0.001）. As compared with the H group, the survivin mRNA and protein expressions of HPASMCs in the HY group [（677 ± 183） and （0.426 ± 0.022）] significant decreased （q = 15.25 and 50.6, P 〈 0.05）. Conclusions Hypoxia leads to abnomaal proliferation of HPASMCs and HPAECs, and under hypoxic condition HPASMCs regulate the proliferation of HPAECs via survivin signaling pathway in the cocuhure system.