骨髓间充质干细胞条件培养基对小鼠肺泡上皮细胞增殖的影响及机制

Effects of bone marrow mesenchymal stem cells conditioned medium on proliferation of mouse alveolar epithelial cells and its mechanism

目的探讨骨髓间充质干细胞条件培养基(BMSCs-CM)对小鼠肺泡上皮细胞增殖的影响及机制。方法分离与培养小鼠骨髓间充质干细胞(BMSCs),流式细胞仪检测细胞表面标记物示CD44阳性表达率为92.5%、CD34阴性表达率为97.3%,提示成功分离与培养BMSCs,且纯度较高。当第2代细胞融合70%~80%时,提取BMSCs-CM。将人肺泡上皮细胞A549随机分为对照组、无血清组、BMSCs组、BMSCs-CM组。对照组以含10%血清的DMEM/F12培养基培养,无血清组以无血清的DMEM/F12培养基培养,BMSCs组加入已接种小鼠BMSCs的Transwell用无血清的DMEM/F12培养基共培养,BMSCs-CM组以获取的小鼠BMSCs-CM培养。培养24 h时,采用CCK-8法检测各组细胞相对存活率。将体外常规培养的肺泡Ⅱ型细胞(ATⅡ)随机分为对照组、BMSCs-CM组,对照组以无血清的DMEM/F12培养基培养24 h,BMSCs-CM组以获取的小鼠BMSCs-CM培养24 h,采用RT-PCR法检测钠通道蛋白α(α-ENa C)相对表达量。结果无血清组细胞相对存活率为(80.70±0.70)%,BMSCs组为(85.83±1.98)%,BMSCs-CM组为(91.28±1.91)%,BMSCs组与BMSCs-CM组均高于无血清组(P<0.05或<0.01)。对照组α-ENa C相对表达量为1.00±0.00,BMSCs-CM组为3.16±1.50,两组比较P<0.05。结论 BMSCs-CM可促进肺泡上皮细胞增殖,其作用机制可能与促进肺泡Ⅱ型细胞α-ENa C表达有关。

Objective To investigate the effects of bone marrow mesenchymal stem cells conditioned medium（BMSCs-CM） on the proliferation of mouse alveolar epithelial cells and its mechanism. Methods Mouse bone marrow mesenchymal stem cells（BMSCs） were isolated and cultured in vitro,and the cell surface markers were identified by flow cytometry. The results showed that the positive rate of CD44 expression was 92. 5%,and the negative expression rate of CD34 was 97. 3%,suggesting the isolated and cultured cells were BMSCs with high purity. When the second generation cell fusion reached 70% to 80%,BMSCs-CM was extracted. Human alveolar epithelial cells A549 were randomly divided into four groups. The control group was cultured in DMEM/F12 medium containing 10% serum,the serum-free group was cultured in serum-free DMEM/F12 medium,the BMSCs group was co-cultured with serum-free DMEM/F12 medium in Transwells that had been inoculated with mouse BMSCs,and the BMSCs-CM group was cultured with acquired mouse BMSCsCM. After 24 hours of culture,the relative viability of the cells in each group was measured by using the CCK-8. The in vitro cultured alveolar type Ⅱ cells AT Ⅱ were randomly divided into the control group and BMSCs-CM group. The cells in the control group were cultured in serum-free DMEM/F12 medium for 24 h,and the cells in the BMSCs-CM group were cultured for 24 h in mouse BMSCs-CM. RT-PCR was used to detect the relative expression of sodium channel protein α（α-ENa C）. Results The relative survival rates of the serum-free group,the BMSCs group,and the BMSCs-CM group were（80. 70 ± 0. 70） %,（85. 83 ± 1. 98） %,and（91. 28 ± 1. 91） %,respectively. Both the BMSCs group and the BMSCsCM group were higher than the serum-free group（P〈0. 05 or P〈0. 01）. The relative expression of α-Ena C in the control group and the BMSCs-CM group was 1. 00 ± 0. 00 and 3. 16 ± 1. 50,respectively; significant difference was found between the two groups（P〈0. 05）. Conclusion BMSCs-CM can promote the proliferation of alveolar epithelial cells by promoting the expression of α-ENa C in alveolar type Ⅱ cells.