间接共培养环境下脂肪干细胞诱导分化的研究

Induced Differentiation of Adipose-Derived Stem Cells in Indirect Co-culture

目的肌腱病的治疗是临床的难点,脂肪干细胞(ADSCs)具有多向分化潜能,为肌腱病的治疗提供了新思路。此课题在体外构建ADSCs与肌腱细胞的间接共培养模式以探索其机制,为其临床应用提供参考。方法分别采集培养兔肌腱细胞和ADSCs及富血小板血浆(PRP)。选用孔径为0.4μm的共培养嵌盒和6孔板来构建间接共培养模式。分别将P3代肌腱细胞、ADSCs接种于内、外室,另外设置PRP干预组。分别在第3,7,14天时,收集各组内、外室细胞并进行观察及检测,分析间接共培养环境对ADSCs诱导分化与胞外基质表达产生的影响。结果间接共培养14 d,免疫组化结果显示单纯干细胞培养组ADSCs的Ⅰ型胶原与腱调蛋白(TNMD)的表达程度显著高于阴性的干细胞对照组,低于阳性肌腱细胞对照组,而PRP干预组的表达优于单纯干细胞实验组。结论间接共培养的环境下,可以诱导ADSCs上调Ⅰ型胶原和腱调蛋白的表达。PRP的干预对诱导ADSCs细胞外基质的分泌有促进作用。

Objective To build an indirect co-culture system for adipose-derived stem cells（ADSCs） and tenocytes in vitro, explore the differentiation mechanism of ADSCs, and provide a reference for the clinical applications in treatment of tendinopathy. Methods The isolated tenocytes of rabbits was cultured. ADSCs were collected in vitro and be expanded. Rabbit blood was prepared and had plasma activated to get the plasma extraction. An indirect co-culture system was established by using co-culture kit of 0.4 um to ensure circulation of culture fluid and avoid direct contact of cells. An additional PRP was added and indirect coculture group was set up. The corresponding protein levels of tenomodulin（TNMD） and type I collagen by immunohistochemistry methods at various time points（ 3,7,14 d） to study the secretion of extra cellular matrix and differentiation of ADSCs induced by the co-culture environment. Results On the 14th day,the results of immunohistochemical staining were measured for statistical analysis. The statistical data showed that experimental groups had a notable higher type I collagen and TNMD expression than the negative control but lower than the positive control. The expression in the PRP group was higher than that in the stem cells group. Conclusion The type I Collagen and TNMD expression of ADSCs could be induced by the environment of being co-cultured with tenocytes. There is some significant effect of PRP on the secretion of extra cellular matrix of ADSCs in the environment of co-culture.