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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 pcDNA3.1 / SPC / EGFP co-expression vector of alveolar surfactant protein C (SPC) and enhanced green fluorescent protein (EGFP), and to investigate the possible mechanism of directional differentiation of human amniotic mesenchymal stem cells (AF-MSCs) Alveolar epithelial cells (AECII). Methods The pcDNA3.1 / SPC / EGFP expression vector was constructed by PCR and DNA recombination technology. The liposome was transfected into AF-MSCs and G418 was stably selected. The AF-MSCs were divided into negative control group, untransfected group and transfected group The expression of SPA and SPC mRNA was detected by RT-PCR. The expression of SPA and SPC protein was detected by Western blot and the osmiophilic lamellar bodies were observed by electron microscopy. RESULTS: The pcDNA3.1 / SPC / EGFP expression vector was constructed successfully. The sequencing results were consistent with those of SPC promoter and EGFP. After the differentiation of AF-MSCs in vitro, no green fluorescent cells were found in the negative control group. The SPA and SPC mRNA and protein (P <0.05), and no osmiophilic lamellar bodies were observed. No green fluorescent cells were observed in the untransfected cells. The expressions of SPA and SPC mRNA (relative expression amount was 0.072 ± 0.004 and 0.087 ± 0.012) and protein The expression levels were 0.051 ± 0.008 and 0.063 ± 0.009 respectively), and the osmiophilic lamellar bodies were found. In the transfection group, the green fluorescent cells, SPA and SPC mRNA (relative expression amount was 0.109 ± 0.011 and 0.126 ± 0.017) and protein (relative expression 0.075 ± 0.012 and 0.081 ± 0.006) were significantly expressed, compared with the untransfected group differences were statistically significant (t values were -5.50, -3.16, -2.90 and -2.85, both P <0.05), also see the osmiophilic lamellar bodies. Conclusions AFMSCs transfected by pcDNA3.1 / SPC / EGFP expression vector can be differentiated into AECII under proper induction in vitro. The pcDNA3.1 / SPC / EGFP expression vector may be a tool to track the directional differentiation of AF-MSCs. Provide the basis for stem cell therapy research.