Scleraxis慢病毒基因感染人羊膜间充质干细胞向肌腱细胞的定向分化

Enhanced tenogenic differentiation by Scleraxis overexpression in human amniotic mesenchymal stem cells

背景:Scleraxis作为肌腱细胞特异性表达分子,不仅参与肌腱祖细胞的聚集及分化,还影响肌腱细胞外基质的形成。人羊膜间充质干细胞具有多向分化潜能,在体外不同诱导条件下可分化为骨、软骨及其他结缔组织。目的:探讨Scleraxis慢病毒基因感染人羊膜间充质干细胞能否向肌腱细胞定向分化并观察其分化效果。方法:取足月产胎盘羊膜组织,两步酶消化法分离人羊膜间充质干细胞并采用倒置相差显微镜观察和流式细胞鉴定。取第3代细胞分3组进行培养,单纯人羊膜间充质干细胞培养组为空白组,人羊膜间充质干细胞经Slclerxis基因慢病毒感染后为过表达组,人羊膜间充质干细胞经不携带Scleraxis基因慢病毒感染后为空质粒组。细胞培养7 d内CCK-8法检测各组细胞增殖能力细胞。细胞培养后3 d和7 d,分别进行实时荧光定量PCR和Western Blot检测评价各组细胞向肌腱细胞定向分化的效果。结果与结论:(1)CCK-8检测显示:培养7 d内,过表达组、空质粒组与空白组细胞在增殖能力上无明显差异(P>0.05);(2)Westen blot检测显示:过表达组Scleraxis蛋白表达水平明显高于空质粒组和空白组(P<0.05);(3)实时荧光定量PCR显示:3 d时,过表达组Ⅰ型胶原、Ⅲ型胶原、纤连蛋白及肌腱蛋白C m RNA表达水平明显高于空质粒组(P<0.05),而腱调蛋白的表达与空质粒组无明显差异(P>0.05);7 d时,Ⅰ型胶原、Ⅲ型胶原、纤连蛋白、肌腱蛋白C及腱调蛋白的表达水平明显高于空质粒组(P<0.05);(4)结果提示:人羊膜间充质干细胞经Scleraxis慢病毒基因感染后可向肌腱细胞定向分化。

BACKGROUND:Human amniotic mesenchymal stem cells(hAMSCs)are adult stem cells with multipotential differentiation,which can be induced to differentiate into bone,cartilage and other connective tissues.Meanwhile,as a highly specific marker of tenocytes,Scleraxis is involved in aggregation and differentiation of tendon progenitor cells as well as the formation of tendon extracellular matrix.OBJECTIVE:To investigate whether hAMSCs have the ability of differentiation into tenocytes by ectopic expression of Scleraxis.METHODS:Agreed by puerpera,the amniotic membrane from the full-term placenta was separated,and hAMSCs were isolated by a two-step enzyme digestion,observed under inverted phase contrast microscope,and identified by flow cytometry.Passage3cells were induced via plasmid-mediated Scleraxis overexpression in overexpression group.Untransfected cells cultured in normal medium served as blank control group,and those with empty plasmid transfection were defined as empty plasmid group.Cell proliferation was tested in each group using cell counting kit-8within7days of culture.Real-time quantitative PCR and western blot were used to assess the tenogenic differentiation of hAMSCs in each group at3and7days of culture.RESULTS AND CONCLUSION:Findings from the cell counting kit-8indicated that the cell viability had no significant differences among the groups within7days of culture(P>0.05).Western blot results showed the protein expression of Scleraxis in the treatment group was significantly higher than that in the other two groups(P<0.05).Real-time PCR results showed,at3days of culture,the expression of collagen type I,collagen type III,Fibronectin and Tenascin-C in the overexpression group was significantly higher than that in the empty plasmid group(P<0.05),but the expression of Tenomodulin had no difference(P>0.05);at7days of culture,the expressions of collagen type I,collagen type III,Fibronectin,Tenascin-C and Tenomodulin in the overexpression group were significantly higher than those in the empty plasmid group(P<0.05).In summary,hAMSCs can be differentiated into tenocytes by ectopic expression of Scleraxis.