中国修复重建外科杂志

中国修复重建外科杂志

TGF-β1 联合 VEGF 对人羊膜间充质干细胞向韧带成纤维细胞体外分化作用的实验研究

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目的 探讨人羊膜间充质干细胞(human amniotic mesenchymal stem cells,hAMSCs)是否具有 MSCs 特性,以及经 TGF-β1 和 VEGF 联合诱导后是否具有向韧带成纤维细胞分化的能力。 方法 取自愿捐赠的足月产妇胎盘,采用胰蛋白酶胶原酶联合消化法分离培养 hAMSCs,流式细胞术检测 hAMSCs 表型分子,免疫荧光染色检测 hAMSCs 其角蛋白-19(cytokeratin-19,CK-19)和波形蛋白表达情况。取第 3 代 hAMSCs,分别使用含 TGF-β1 和 VEGF 的 L-DMEM/F12 成骨诱导培养基(实验组)和普通 L-DMEM/F12 培养基(对照组)培养,细胞增殖-毒性检测试剂盒(cell counting kit-8,CCK-8)检测两组细胞增殖能力;培养 5、10、15 d 分别采用免疫荧光染色及实时荧光定量 PCR 检测韧带及血管生成相关特异性蛋白和基因表达。 结果 倒置相差显微镜观察示 hAMSCs 呈单层贴壁生长;流式细胞术结果示 hAMSCs 表达 MSCs 表型分子;免疫荧光染色示 hAMSCs 高表达波形蛋白、低表达 CK-19;hAMSCs 具有向成骨、成软骨及成脂细胞分化的能力。CCK-8 法检测示,7 d 时两组细胞均达增殖高峰,实验组细胞增殖能力于 7 d 后显著高于对照组(P<0.05)。免疫荧光染色示,培养 5、10、15 d 时实验组 Ⅰ 型胶原、Ⅲ 型胶原、纤维连接蛋白(Fibronectin)、细胞连接素(Tenascin-C)表达染色均较对照组增强。实时荧光定量 PCR 结果示,随时间延长实验组 Ⅰ 型胶原、Ⅲ 型胶原、Fibronectin、α-肌动蛋白(α-smooth muscle actin,α-SMA)、VEGF mRNA 相对表达量均逐渐上调(P<0.05)。除培养 5 d 两组 Ⅰ 型胶原、Ⅲ 型胶原、VEGF mRNA 相对表达量比较差异无统计学意义(P>0.05)外,其余各时间点实验组 Ⅰ 型胶原、Ⅲ 型胶原、Fibronectin、α-SMA 和 VEGF mRNA 相对表达量均显著高于对照组(P<0.05)。 结论 hAMSCs 具有 MSCs 特征,且体外增殖能力良好,可作为组织工程种子细胞来源;体外诱导后韧带成纤维细胞及血管生成相关特异性基因表达上调,韧带成纤维细胞特异性蛋白分泌增加,TGF-β1 联合 VEGF 可作为构建组织工程韧带的生长因子选择。

Objective To investigate whether human amniotic mesenchymal stem cells (hAMSCs) have the characteristics of mesenchymal stem cells (MSCs) and the differentiation capacity into ligament fibroblastsin vitro. Methods The hAMSCs were separated through trypsin and collagenase digestion from placenta, the phenotypic characteristics of hAMSCs were detected by flow cytometry, the cytokeratin-19 (CK-19) and vimentin of hAMSCs were tested through immunofluorescence staining. The hAMSCs at the 3rd passage were cultured with L-DMEM/F12 medium containing transforming growth factor β1 (TGF-β1) and vascular endothelial growth factor (VEGF) as the experimental group and with single L-DMEM/F12 medium as the control group. The morphology of hAMSCs were observed by inverted phase contrast microscope; the cellular activities and ability of proliferation were examined by cell counting kit-8 (CCK-8) method; the ligament fibroblasts related protein expressions including collagen type I, collagen type III, Fibronectin, and Tenascin-C were detected by immunofluorescence staining; specific mRNA expressions of ligament fibroblast s and angiogenesis including collagen type I, collagen type III, Fibronectin, α-smooth muscle actin (α-SMA), and VEGF were measured by real-time fluorescence quantitative PCR. Results The hAMSCs presented monolayer and adherent growth under inverted phase contrast microscope; the flow cytometry results demonstrated that hAMSCs expressed the MSCs phenotypes; the immunofluorescence staining results indicated the hAMSCs had high expression of the vimentin and low expression of CK-19; the hAMSCs possessed the differentiation ability into the osteoblasts, chondroblasts, and lipoblasts. The CCK-8 results displayed that cells reached the peak of growth curve at 7 days in each group, and the proliferation ability in the experimental group was significantly higher than that in the control group at 7 days (P<0.05). The immunofluorescence staining results showed that the expressions of collagen type I, collagen type III, Fibronectin, Tenascin-C in the experimental group were significantly higher than those in the control group at 5, 10, and15 days after culture (P<0.05). The real-time fluorescence quantitative PCR results revealed that the mRNA relative expressions had an increasing tendency at varying degrees with time in the experimental group (P<0.05). The relative mRNA expressions of collagen type I, collagen type III, Fibronectin, α-SMA, and VEGF in the experimental group were significantly higher than those in the control group at the other time points (P<0.05), but no significant difference was found in the relative mRNA expressions of collagen type I, collagen type III, and VEGF between 2 groups at 5 days (P>0.05). Conclusion The hAMSCs possesses the characteristics of MSCs and good proliferation ability which could be chosen as seed cell source in tissue engineering. The expressions of ligament fibroblasts and angiogenesis related genes could be up-regulated, after inductionin vitro, and the synthesis of ligament fibroblasts related proteins could be strengthened. In addition, the application of TGF-β1 and VEGF could be used as growth factors sources in constructing tissue engineered ligament.

关键词: 人羊膜间充质干细胞; 韧带成纤维细胞; TGF-β1; VEGF; 组织工程韧带

Key words: Human amniotic mesenchymal stem cells; ligament fibroblasts; transforming growth factor β1; vascular endothelial growth factor; tissue engineered ligament

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