Objective: To observe the senescent effect of human pulmonary arterial endothelial cells (HPAEC) stimulated by cigarette smoke extract (CSE) and the effect of secretion of senescent cells on human pulmonary arterial smooth muscles cell (HPASMC) proliferation and migration. Methods: HPAEC was treated with different concentrations of CSE in vitro and cell proliferation was determined by CCK8, senescence cells analyzed by detecting the β-gal activity, and the senescent proteins of cells measured by Western blot. The concentration of senescence-associated secretory phenotype (SASP) was detected by ELISA and the expression of MCP-1 and TGF-β1 was measured by Real-time PCR. The number of the proliferated cells was measured by Transwell assay and immunoflurescence. Results: The HPAEC was aging with the stimulation concentration of CSE increasing and the stimulation time prolonging (P<0.05). Western blot indicated that the senescent associated protein p53 or p21 increased markedly after 48 h and 72 h CSE-exposure (n=3, P<0.05). The SA-β-Gal staining showed that the number of senescent cells increased as the exposure time prolonged. Compared with the control group, cell viability of 48 h group(1.8±0.1) and 72 h group (1.8±0.1) decreased significantly. The flow cytometry showed a significant difference between the CSE group(14.1±1.2) and the control group(28.5±1.8) in S phase(P<0.01), indicating cell cycle arrest. The SASP was increasing as the CSE-exposure prolonged. Compared with the control group(177±39), the 48 h group(460±43) and the 72 h group(609±64) showed a marked increase in MCP-1(P<0.05). For TGF-β1, it had a same tendency and a significant difference between the control group(121±18) and the 48 h group(413±32) or 72 h group(606±67, both P<0.05). In the meantime, the bFGF increased after 48 h stimulation(291±13, P<0.05). Besides MCP-1, TGF-β1 showed a significant difference between the control group and the 72 h CSE-exposure group (P<0.01). Premature cells could secrete SASP which induced HPASMC proliferation. After different times of conditioned medium stimulation, HPASMC proliferated especially at 72 h(P<0.05) . The immnoflorescence and Transwell assay confirmed this finding. Conclusion: CSE could induce senescence of HPAEC and SASP production which improved HPASMC proliferation and migration.
目的： 观察烟草烟雾提取物(CSE)对肺动脉内皮细胞(HPAEC)衰老的影响以及细胞衰老相关分泌表型(SASP)对肺动脉平滑肌细胞(HPASMC)的增殖和迁移效应。 方法： 对HPAEC给予不同浓度的CSE，分别于0、6、12、24、48和72 h观察并使用CCK8法检测细胞活性，流式细胞术检测细胞的增殖情况，利用β-半乳糖苷酶(SA-β-gal)衰老染色对衰老细胞进行拍照染色计数，Western blot法检测衰老相关蛋白的表达情况，免疫荧光检测细胞中蛋白的表达量，实时PCR检测衰老相关基因的表达，酶联免疫吸附试验(ELISA)检测分泌的细胞因子浓度，Transwell检测HPASMC的迁移情况。 结果： 与0 h时比较，48 h时细胞活性明显降低，吸光度值为(1.8±0.1)，差异有统计学意义(P<0.05)；72 h时细胞活性(1.8±0.1)下降更加明显(P<0.01)。流式细胞术检测细胞周期发现CSE刺激后S期细胞(14.1±1.2)数量较前(28.5±1.8)明显下降，细胞周期阻滞较为明显且具有统计学意义(P<0.01)。细胞衰老相关分泌表型(SASP)的含量不断升高，其中MCP-1含量0 h时为(177±39)，48 h时为(460±43)，72 h时为(609±64)；TGF-β1含量0 h时为(121±18)，48 h时为(413±32)，72 h时为(606±67)；bFGF含量0 h时为(123±17)，48 h时为(291±13)，72 h时为(471±43)，差异均有统计学意义(均P<0.05)。Western blot法可见HPAEC中衰老相关蛋白p53或p21随着CSE刺激浓度和刺激时间的增加表达也逐渐增高(P<0.05)，SA-β-gal衰老染色可见随着时间延长视野内衰老细胞的数量明显增加。免疫荧光和Transwell小室实验图像均可验证细胞增殖情况。 结论： CSE可诱导HPAEC发生衰老并能够使平滑肌细胞发生迁移。.
Keywords: Cigarette smoke; Human pulmonary artery endothelial cells; Myocytes, smooth muscle.