体外构建人羊膜间充质干细胞膜片及成骨分化潜能

In vitro construction of human amniotic mesenchymal stem cell sheet and its osteogenic differentiation

背景:人羊膜间充质干细胞属于成体干细胞,其来自于废弃的胎盘,来源广泛,可以无创获取,具有免疫原性低、生长周期短等特点,是组织工程种子细胞的重要来源,目前人羊膜间充质干细胞已经用于临床糖尿病的治疗。目的:探索一种简便的方法构建人羊膜间充质干细胞膜片,并研究其成骨分化潜能。方法:将第3代人羊膜间充质干细胞高密度接种于普通培养皿中,加入成膜片诱导培养基以构建人羊膜间充质干细胞膜片,通过组织学染色以及扫描电镜观察细胞膜片的特性。取第3代人羊膜间充质干细胞高密度接种于培养皿中,加入成膜片诱导培养基培养7 d,再换用成骨诱导培养基培养14 d以构建成骨诱导的人羊膜间充质干细胞膜片。通过茜素红染色、免疫组化染色、碱性磷酸酶活性、RT-PCR以及Western blot检测人羊膜间充质干细胞膜片的成骨分化潜能。结果与结论:①苏木精-伊红染色可见人羊膜间充质干细胞膜片由多层细胞累积而成,细胞分布均匀;②扫描电镜观察可见人羊膜间充质干细胞膜片呈复层结构,胞外有大量的胞外基质产生,细胞包埋于胞外基质中;③人羊膜间充质干细胞膜片成骨诱导14 d,茜素红染色后可见橘红色沉淀,免疫组化染色后细胞周围有大量Ⅰ型胶原产生;④与未诱导的人羊膜间充质干细胞膜片相比,成骨诱导14 d后人羊膜间充质干细胞膜片碱性磷酸酶活性显著升高(P<0.01),Ⅰ型胶原、骨钙蛋白、Runt相关转录子2的mRNA和蛋白表达量显著升高(P<0.05);⑤该实验应用一种简便、经济的方法在普通培养皿上成功构建了人羊膜间充质干细胞膜片,体外研究证实人羊膜间充质干细胞膜片具有良好的成骨分化潜能。

BACKGROUND:Human amniotic mesenchymal stem cells(hAMSCs)are a kind of adult stem cells that can be extracted from discarded placenta.Compared with other mesenchymal stem cells,hAMSCs have many advantages such as noninvasive isolation,low immunogenicity and short growth cycle,and thus hAMSCs are an important source of tissue engineering seed cells.Currently,hAMSCs have been applied in the clinical treatment of diabetes.OBJECTIVE:To explore a simple method to fabricate hAMSC sheets and to study their potential to differentiate into osteocytes.METHODS:Passage 3 hAMSCs were seeded into culture plates at a high density,and the sheet-forming medium was added to fabricate hAMSC sheet.The structural characteristics of hAMSC sheets were evaluated by histological staining and scanning electron microscopy.The sheet-forming induction medium was added to the passage 3 hAMSCs for 7 continuous days,and then replaced by the osteogenic induction medium for 14 days to construct osteogenic-induced hAMSC sheets.The potential for osteogenic differentiation of hAMSC sheets was assessed by alizarin red staining,immunohistochemical staining,alkaline phosphatase activity,RT-PCR,and western blot assay.RESULTS AND CONCLUSION:Hematoxylin-eosin staining results indicated that hAMSC sheets had a multi-layered structure with the cells stacked layer-by-layer and evenly distributed.Under the scanning electron microscope,the hAMSC sheets had a multi-layered structure,and a large amount of extracellular matrices that enveloped the fusiform cells were produced.After 14 days of osteogenic induction,orange-red precipitation was observed by alizarin red staining.Immunohistochemical staining results showed a large amount of type II collagen.Compared with non-induced hAMSC sheet,alkaline phosphatase activity was significantly increased in the osteogenic induced sheet(P<0.01).The expression levels of type I collagen,osteocalcin,and Runt-related transcript factor 2 mRNA and protein were significantly higher in the osteogenic-induced hAMSC sheet group than the non-induced hAMSC sheet group(P<0.05).Our findings indicate that this is a simple and economic method to construct the hAMSC sheets in the normal culture medium.Moreover,hAMSC sheets have a good osteogenic differentiation potential that has been confirmed in vitro.