抗坏血酸作用下兔骨髓间充质干细胞外基质分泌与结构改变

Ascorbic acid influences on extracellular matrix and structure of rabbit bone marrow mesenchymal stem cells

背景:细胞外基质对干细胞的影响是目前组织工程学的研究热点,而关于自身细胞外基质的合成、分泌及对细胞的影响却少有研究报道。目的:体外培养兔骨髓间充质干细胞,观察抗坏血酸对其细胞外基质与整体结构改变的影响。方法:通过差速贴壁法分离获取兔骨髓间充质干细胞,流式细胞技术检测CD44、CD45、CD31的表达。将兔骨髓间充质干细胞用含抗坏血酸(20 mg/L)的培养基进行干预,观察细胞形态、大体结构、超微结构及组织学改变,RT-PCR检测细胞外基质相关基因的表达。结果与结论:(1)经流式细胞学分析,第2代骨髓间充质干细胞中95%以上的细胞表达CD44,而CD45与CD31的表达极低;(2)抗坏血酸干预后,骨髓间充质干细胞增殖良好,细胞间界限难以分辨,干预10-14 d后,形成了"膜片状"结构;(3)苏木精-伊红染色后可见细胞分层排列,Masson染色后可见大量细胞外基质成分,透射电镜下可见胞内含大量内质网与"囊泡样"结构;(4)与未经抗坏血酸干预的对照组相比较,经抗坏血酸干预的实验组纤维连接蛋白的mRNA表达显著升高(P<0.05),而Ⅰ型胶原的mRNA表达虽稍提高,但两组间差异无显著性意义;(5)结果表明,抗坏血酸促进了兔骨髓间充质干细胞的增殖转化、细胞外基质的合成分泌、膜片状结构的形成,在组织工程应用中具有巨大潜能。

BACKGROUND:The effect of extracellular matrix on stem cells is the focus of tissue engineering.However,there are few reports about the synthesis and secretion of extracellular matrix as well as its effects on cells.OBJECTIVE:To isolate,culture and identify rabbit bone marrow mesenchymal stem cells(BMSCs),and to explore the changes of extracellular matrix and whole structure under the intervention of ascorbic acid.METHODS:Rabbit BMSCs were isolated by differential adherent method of the bone marrow,and the expression of CD44,CD45 and CD31 was identified by flow cytometry.The BMSCs were cultured in the culture medium containing 20 mg/L ascorbic acid.Then the cell morphology,gross structure,ultrastructure,and histological changes of BMSCs were observed.The expression of extracellular matrix related genes was detected by RT-PCR.RESULTS AND CONCLUSION:Over 95%passage 2 BMSCs could express CD44,but the expression levels of CD45 and CD31 were extremely low.Intervention with ascorbic acid enhanced the proliferation of BMMSCs with unclear cell boundaries.A cell-sheet structure formed at 10-14 days after intervention.Hematoxylin-eosin staining results showed a layered cell arrangement,and Masson staining findings showed a large amount of extracellular matrix composition.Abundant endoplasmic reticula and vesicle-like structure were observed under the transmission electron microscope.RT-PCR findings showed that ascorbic acid significantly increased the expression of fibronectin mRNA in the BMSCs(P<0.05),but slightly increased the mRNA expression of collagen type I.All these findings indicate that ascorbic acid not only increases the proliferation and transformation of rabbit BMSCs,but also promotes the synthesis and secretion of extracellular matrix,which has great potential in tissue engineering applications