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

发布时间：2018-05-13 12:53

  本文选题：细胞分化 + 羊膜　； 参考：《中国组织工程研究》2017年33期

【摘要】：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(P0.05);(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(P0.05);(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(P0.05),而腱调蛋白的表达与空质粒组无明显差异(P0.05);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(P0.05);(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。
[Abstract]:Background as a specific expression molecule of tendon cells, Scleraxis not only participates in the aggregation and differentiation of tendon progenitor cells, but also affects the formation of extracellular matrix of tendon cells. Human amniotic mesenchymal stem cells have the potential to differentiate into bone, cartilage and other connective tissues under different induction conditions in vitro. Aim: to investigate whether human amniotic mesenchymal stem cells (MMSCs) infected with Scleraxis lentivirus gene can differentiate into tendon cells and observe its differentiation effect. Methods: human amniotic mesenchymal stem cells were isolated by two-step enzyme digestion and observed by inverted phase contrast microscope and identified by flow cytometry. The third passage of human amniotic mesenchymal stem cells were divided into three groups. The human amniotic mesenchymal stem cells were cultured as blank group and human amniotic membrane mesenchymal stem cells infected by Slclerxis gene lentivirus as overexpression group. Human amniotic mesenchymal stem cells were infected without Scleraxis gene lentivirus. The proliferative ability of cells in each group was detected by CCK-8 assay within 7 days of cell culture. After 3 days and 7 days of culture, real-time fluorescence quantitative PCR and Western Blot were used to evaluate the differentiation of the cells into tendon cells in each group. Results and conclusion the results of CCK-8 test showed that: within 7 days of culture, the expression of CCK-8 in the overexpressed group was higher than that in the control group. There was no significant difference in proliferative ability between empty plasmid group and blank group. The expression level of Scleraxis protein in overexpression group was significantly higher than that in blank plasmid group and blank group. The expression of Scleraxis protein in overexpression group was significantly higher than that in blank plasmid group and blank group. The C m RNA expression level of type 鈪，

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