骨髓间充质干细胞对脂多糖诱导人肺泡上皮细胞凋亡的影响及旁分泌作用

Influence of bone marrow mesenchymal stem cells on apoptosis of lipopolysaccharide-induced human alveolar epithelial cells and its paracrine

目的探讨骨髓间充质干细胞(mesenchymal stem cells,MSCs)对脂多糖(lipopolysaccharide,LPS)诱导人肺泡上皮细胞株A549细胞凋亡的影响和旁分泌作用。方法培养人骨髓MSCs和A549细胞,将A549细胞分为PBS组、PBS+MSCs组、LPS组和LPS+MSCs组,培养12h后采用Annexin V/PI双染色流式细胞仪测定A549细胞凋亡情况,采用MTT法测定细胞增殖情况,采用Western blot法检测A549细胞bcl-2和caspase-3蛋白含量,采用ELISA法测定A549细胞培养液中肝细胞生长因子和角质细胞生长因子水平。结果 LPS组、LPS+MSCs组A549细胞凋亡率[(28.65±4.75)%、(12.73±2.98)%]和培养液中肝细胞生长因子水平[(211.57±21.37)、(702.15±39.53)ng/L]高于PBS组[(2.92±0.38)%、(94.14±8.42)ng/L]和PBS+MSCs组[(2.27±0.43)%、(139.35±18.62)ng/L](P<0.05),LPS+MSCs组高于LPS组(P<0.05);LPS+MSCs组A549细胞的吸光度(optical density,OD)490值(0.579±0.063)和培养液中角质细胞生长因子水平[(0.594±0.045)ng/L]高于PBS组[0.261±0.027、(0.249±0.023)ng/L]、PBS+MSCs组[0.283±0.042、(0.301±0.032)ng/L]和LPS组[0.298±0.046、(0.310±0.037)ng/L](P<0.05);LPS+MSCs组bcl-2蛋白表达(0.97±0.21)高于LPS组(0.54±0.08)与PBS组(0.46±0.09)及PBS+MSCs组(0.63±0.11)(P<0.05);LPS组A549细胞caspase-3蛋白表达及(1.26±0.31)高于LPS+MSCs组(0.53±0.14)与PBS组(0.58±0.15)和PBS+MSCs组(0.08±0.00)(P<0.05)。结论人骨髓MSCs具有抗LPS诱导的A549细胞凋亡和旁分泌的作用。

Objective To investigate the influence of bone marrow mesenchymal stem cells （MSCs） on apoptosis of lipopolysaecharide （LPS）-induced human alveolar epithelial cell line A549 and its paraerine action. Methods The human bone marrow MSCs and A549 cells were cultured, and the A549 cells were divided into PBS group, PBS＋ MSCs group, LPS group, and LPS＋MSCs group. The cells apoptosis was measured by Annexin V/PI double staining flow cytometry. MTT method was used to assay cell proliferation. Western blot method was used to detect the contents of hcl-2 and caspase-3 proteins of A549 cell. ELISA method was used to determine the levels of hepatocyte growth factor and keratinocyte growth factor of A549 cells broth. Results The A549 cells apoptosis rates （（28.65±4.75）%, （12.73±6 2.98） %） and hepatocyte growth factor levels （（211.57±21.37）, （702.15± 39.53） ng/L） were significantly higher in LPS group and LPS＋MSCs group than those in PBS group （（2.92±0.38）%, （94.14±8.42）ng/L） and PBS＋ MSCs group （（2.27±0.43）%, （139.35±18.62） ng/L） （P〈0.05）, and were significantly higher in LPS＋MSCs group than those in LPS group （P〈0.05）. The optical density 49o value of A549 cell （0. 579±0. 063） and keratinocyte growth factor level （（0. 594±0. 045） ng/L） in LPS＋MSCs group were significantly higher than those in PBS group （0. 261±0. 027, （0.249±0.023） ng/L）, PBS-MSCs group （0.283±0.042, （0.301±0.032） ng/L） and LPS group （0.298±0.046, （0. 310±0. 037） ng/L） （P〈0.05）. The expression level of bcl-2 protein was significantly higher in LPS＋MSCs group （0.97±0.21） than that in LPS group （0.54±0.08） , PBS group （0.46±0.09） and PBS＋ MSCS group （0.63±0.11） （P〈0.05）. The expression level of caspase-3 protein was significantly higher in LPS group （1.26±0.31） than that in LPS＋MSCs group （0.53±0. 14）, PBS group （0. 58±0. 15） and PBS＋ MSCs group （0. 08±0. 00） （P〈0.05）. Conclusion Human bone marrow MSCs have anti LPS-induced A549 apoptosis and paracrine role.