大鼠羊水间充质干细胞向Ⅱ型肺泡上皮细胞定向分化的体外实验

In vitro differentiation of rat amniotic fluid-derived mesenchymal stem cells into type Ⅱ alveolar epithelial cells

目的：观察体外诱导大鼠羊水间充质干细胞（ AF-MSCs ）向Ⅱ型肺泡上皮细胞的定向分化。方法选取10只清洁级妊娠SD大鼠，收集羊水标本，分离、培养AF-MSCs，以流式细胞术分析其表型标记、实时荧光定量聚合酶链反应（ qRT-PCR）检测Oct-4 mRNA表达（以大鼠胚胎干细胞作为阳性对照）；根据不同体外诱导培养方法，将AF-MSCs分为A（空白对照组）、B、C、D、E共5组，各组体外不同诱导方法处理后qRT-PCR检测肺表面活性蛋白（ SP） A、SPB、SPC、SPD和TTF1 mRNA表达水平，免疫荧光法检测SPA、SPC蛋白水平以及电镜观察嗜锇性板层小体。结果大鼠AF-MSCs在含20％胎牛血清、4μg/L碱性成纤维细胞生长因子的L-DMEM培养基中呈旋涡状生长，第3代AF-MSCs表型标记中 CD29、CD44、CD73、CD90、CD105和 CD166阳性表达率分别为（99.1±7.9）％、（99.2±7.4）％、（75.6±5.2）％、（98.9±8.1）％、（92.9±7.3）％和（89.3±6.7）％，而CD34和CD45表达阴性；Oct-4 mRNA相对表达量（0.690±0.059）显著低于大鼠胚胎干细胞阳性对照（1.000±0.002）（P＜0.01）；体外诱导分化后，A组SPA、SPB、SPC、SPD和TTF1 mRNA及SPA、SPC蛋白均为阴性表达，而B组SPA、SPB、SPC、SPD和TTF1 mRNA相对表达量分别为0.426±0.043、0.368±0.028、0.492±0.058、0.327±0.024和0.183±0.018，均显著高于其他各组（均P＜0.01），SPA、SPC蛋白绿色荧光呈强阳性表达；同时，在B组中观察到嗜锇性板层小体。结论 AF-MSCs在体外能通过特定的诱导定向分化为Ⅱ型肺泡上皮细胞。

Objective To explore the in vitro differentiation of rat amniotic fluid-derived mesenchymal stem cells （ AF-MSCs ） into type Ⅱ alveolar epithelial cells （ AECⅡ）.Methods Flow cytometry was used to analyze the phenotypes of AF-MSCs from 10 pregnant Sprague-Dawley rats.And the Oct-4 mRNA expression level was detected by quantitative real-time polymerase chain reaction （ qRT-PCR）.Rat embryonic stem cell was used as a positive control.According to different culturing methods , AF-MSCs were randomly divided into 5 groups of A （control group）, B, C, D and E.After in vitro differentiation, SPA, SPB, SPC, SPD and TTF1 mRNA expressions were detected by qRT-PCR, SPA and SPC protein expressions measured by immunofluorescence and lamellar bodies observed by transmission electron microscopy.Results AF-MSCs could grow spirally in L-DMEM medium containing 20%fetal bovine serum and 4 μg/L basic fibroblast growth factor.The expressions of such surface antigens of AF-MSCs （ third passage） as CD29 （99.1 ±7.9）%, CD44 （99.2 ±7.4）%, CD73 （75.6 ±5.2）% , CD90 （98.9 ± 8.1）%, CD105 （92.9 ±7.3）% and CD166 （89.3 ±6.7）% were positive while CD34 and CD45 were negative.And the expression of Oct-4 mRNA （relative quantity：0.690 ±0.059） was significantly lower than rat embryonic stem cells （ relative quantity：1.000 ±0.002 ） positive control group （ P〈0.01 ）.After in vitro differentiation, the expressions of SPA , SPB, SPC, SPD and TTF1 mRNA and SPA and SPC proteinwere negative in group A and positive in group B.The expressions of SPA , SPB, SPC, SPD and TTF1 mRNA （relative quantity：0.426 ±0.043, 0.368 ±0.028, 0.492 ±0.058, 0.327 ±0.024 and 0.183 ± 0.018） and SPA and SPC protein in group B were significantly higher than other groups （ all P〈0.01）.Lamellar bodies could be found in the differentiated cells of group B.Conclusion Rat AF-MSCs from amniotic fluid may differentiated into AECⅡlike cells in vitro.