脂肪与髓核来源间充质干细胞在转化生长因子β1诱导下向类髓核细胞分化

摘要

背景：大量研究表明多种组织来源的成体干细胞在体外均可向类髓核细胞分化。椎间盘髓核组织来源间充质干细胞在转化生长因子β1诱导下能否向类髓核细胞分化？其分化能力与脂肪间充质干细胞相比是否有差异目前未见报道。目的：比较脂肪间充质干细胞与髓核间充质干细胞在转化生长因子β1诱导下向类髓核细胞诱导分化能力的差异。方法：分别取大鼠腹股沟处脂肪组织与尾段脊柱，采用机械酶消化法分离培养脂肪间充质干细胞与髓核间充质干细胞。流式细胞仪检测两种细胞 CD105、CD90、CD29、CD45、CD44、CD34、CD24的表达。脂肪间充质干细胞与髓核间充质干细胞各自分为诱导组、无因子诱导组和对照组，诱导组以转化生长因子β1标准软骨诱导液培养，无因子诱导组以不含转化生长因子β1的软骨诱导液培养，对照组以含体积分数为10%胎牛血清的DMEM/F12培养液培养。培养14 d后用RT-PCR检测各组细胞Ⅱ型胶原、蛋白多糖、SOX-9基因的表达。结果与结论：两种细胞CD105、CD90、CD29表达阳性，CD45、CD44、CD34、CD24表达阴性。向类髓核细胞诱导培养14 d后两种细胞诱导组Ⅱ型胶原、蛋白多糖、SOX-9基因表达水平较对照组均明显升高，差异有显著性意义(P <0.05)；髓核间充质干细胞诱导组3种基因的表达水平明显高于脂肪间充质干细胞诱导组，差异有显著性意义(P <0.05)。提示脂肪间充质干细胞与髓核间充质干细胞均具有向类髓核细胞分化的能力，而髓核间充质干细胞相对于脂肪间充质干细胞其软骨相关基因表达更高，可能更适合于作为组织工程髓核研究的种子细胞。%BACKGROUND:A large number of studies have shown that adult stem cels derived from multiple tissues are available to differentiate towards nucleus pulposus-like celsin vitro. It is unclear whether mesenchymal stem cels derived from nucleus pulposus tissues have the ability to differentiate towards nucleus pulposus-like phenotypes induced by transforming growth factor-beta 1. Up to now, there are few reports on the difference between the differentiation ability of mesenchymal stem cels derived from nucleus pulposus tissues and adipose-derived mesenchymal stem cels. OBJECTIVE:To compare the ability of mesenchymal stem cels derived from nucleus pulposus tissues and adipose-derived mesenchymal stem cels differentiating into nucleus pulposus-like cels under induction of transforming growth factor-beta 1. METHODS:The groin fat tissue and the coccygeal spine of rats were taken respectively to isolate and culture mesenchymal stem cels derived from nucleus pulposus tissues and adipose-derived mesenchymal stem cels by mechanical enzyme digestion method. Flow cytometry was employed to detect the expression of CD105, CD90, CD29, CD45, CD44, CD34, and CD24 of both two kinds of stem cels. Mesenchymal stem cels derived from nucleus pulposus tissues and adipose-derived mesenchymal stem cels were divided into complete induction group (complete induction medium with transforming growth factor-beta 1), incomplete induction group (complete induction medium without transforming growth factor-beta 1) and control group(DMEM/F12 containing 10% fetal bovine serum and 100 mg/L penicilin/streptomycin), respectively. After 14 days of culture, real-time PCR was used to detect the expression of colagen type II, Aggrecan and SOX-9 in each group. RESULTS AND CONCLUSION:CD105, CD90, CD29 expressed positively and CD45, CD44, CD34, CD24 negatively in both two kinds of stem cels. After 14 days of induced differentiation, the expressions of colagen type II, Aggrecan and SOX-9 in the two kinds of cels were significantly higher in the complete induction groups than in the control groups (P < 0.05). Under the induction of transforming growth factor-beta 1, the expression of colagen type II, Aggrecan and SOX-9 in mesenchymal stem cels derived from nucleus pulposus tissues was significantly higher than that in adipose-derived mesenchymal stem cels (P < 0.05). These findings suggest that both two kinds of mesenchymal stem cels have the ability to differentiate towards nucleus pulposus-like cels induced by transforming growth factor-beta, and mesenchymal stem cels derived from nucleus pulposus tissues may be more suitable as seed cels for nucleus pulposus tissue engineering research.