SPC及EGFP共表达载体跟踪人羊水间充质干细胞体外定向分化

Differentiation of human amniotic fluid-derived mesenchymal stem cells traced by the co-expression vector of surfactant protein C and enhanced green fluorescent protein in vitro

目的构建肺泡表面活性蛋白C(SPC)及增强型绿色荧光蛋白(EGFP)共表达载体pcDNA3.1/SPC/EGFP,探讨其在体外跟踪人羊水间充质干细胞(AF-MSCs)定向分化为II型肺泡上皮细胞(AECII)的作用。方法采用PCR和DNA重组技术构建pcDNA3.1/SPC/EGFP表达载体,脂质体转染至AF-MSCs,G418稳定筛选;将AF-MSCs分为阴性对照组、未转染组和转染组,各组体外诱导培养后荧光显微镜观察SPC启动子调控下游EGFP基因表达活性,RT-PCR检测SPA和SPC mRNA表达水平,Western blot检测SPA和SPC蛋白表达以及电镜观察嗜锇性板层小体。结果成功构建pcDNA3.1/SPC/EGFP表达载体,测序结果与SPC启动子及EGFP序列一致;AF-MSCs体外诱导分化后,在阴性对照组中未见绿色荧光细胞,SPA和SPC mRNA及蛋白均为阴性表达,且未发现嗜锇性板层小体;在未转染组中亦未见绿色荧光细胞,而SPA和SPC mRNA(相对表达量为0.072±0.004和0.087±0.012)及蛋白(相对表达量为0.051±0.008和0.063±0.009)均为阳性表达,并发现嗜锇性板层小体;在转染组中可见绿色荧光细胞,SPA和SPC mRNA(相对表达量为0.109±0.011和0.126±0.017)及蛋白(相对表达量为0.075±0.012和0.081±0.006)均为显著表达,与未转染组相比差异均有统计学意义(t值分别为-5.50、-3.16、-2.90和-2.85,均P<0.05),亦可见嗜锇性板层小体。结论经pcDNA3.1/SPC/EGFP表达载体转染的AFMSCs在体外适当诱导下能定向分化为AECII,pcDNA3.1/SPC/EGFP表达载体可能成为跟踪AF-MSCs定向分化的工具,为肺组织再生的干细胞治疗提供研究基础。

Objective To construct the co-expression vector of pulmonary surfactant protein C and enhanced green fluorescent protein （pcDNA3.1/SPC/EGFP） and study the differentiation of human amniotic fluid-derived mesen- chymal stem cells （AF-MSCs） into type Ⅱ alveolar epithelial ceils （AECII） traced by pcDNA3.1/SPC/EGFP expression vector in vitro. Methods Polymerase chain reaction （PCR） and recombinant DNA technology were used to construct the pcDNA3.1/SPC/EGFP expression vector. After transfected by pcDNA3.1/SPC/EGFP expression vector, AF-MSCs were selected by G418. AF-MSCs were randomly divided into 3 groups, including control group, non-transfection group and transfection group. After AF-MSCs differentiated in vitro, EGFP gene expression regulated by SPC promoter was observed under fluorescence microscope; SPA and SPC mRNA expressions were detected by RT-PCR; SPA and SPC protein expressions were measured by Western blot, and lamellar bodies were observed under transmission electron microscopy. Results DNA sequence analyses verified that the pcDNA3.1/SPC/EGFP expression vector had been constructed successfully. After AF-MSCs differentiated in vitro, the expression of green fluorescence was observered only in transfection group. The levels of SPA and SPC mRNA and protein in control group were negative, which were positive in non-transfection group and transfection group. And the expression levels of SPA and SPC mRNA （0. 109 ± 0. 011, t = -5.50 and 0. 126 ± 0. 017, t = -3.16 ; all P 〈0.05 ） and protein （0.075 ± 0.012, t = -2.90 and 0. 081 ± 0.006, t = -2.85 ; all P 〈 0.05 ） in transfection group were significantly higher than in non-transfection group （SPA and SPC mRNA： 0. 072 ± 0. 004 and 0.087 ± 0. 012, SPA and SPC protein ： 0. 051 ±0. 008 and 0. 063 ± 0. 009）. Lamellar bodies were found in the differentiated cells of non-transfection group and transfection group. Conclusion AF-MSCs, transfected by pcDNA3.1/SPC/EGFP expression vector, can be differentiated into AECII like cells in vitro in the appropriate inducement and pcDNA3.1/ SPC/EGFP expression vector could be the tool to trace AF-MSCs differentiation. These may provide the basis of stem cell therapy for lung tissue regeneration.