Wu Guizhu, Zheng Xiu, Jiang Zhongqing, Wang Jinhua, Song Yanfeng
Department of Obstetrics and Gynecology, First Affiliated Hospital of Fujian Medical University, Fuzhou, 350005, China.
J Huazhong Univ Sci Technolog Med Sci. 2010 Jun;30(3):285-90. doi: 10.1007/s11596-010-0344-5. Epub 2010 Jun 17.
This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells (ADSCs) into myoblasts, which may provide a new strategy for tissue engineering in patients with stress urinary incontinence (SUI). ADSCs, isolated and cultured ex vivo, were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine (5-aza), 5% FBS, and 5% horse serum. Cellular morphology was observed under an inverted microscope. Ultrastructural changes occurring during the differentiation were observed by transmission electron microscopy and confocal laser scanning microscopy. Cellular immunohistochemical staining was applied to determine the expression of desmin protein in cells with and without induced differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect mRNA and protein expression, respectively, of sarcomeric and desmin smooth muscle proteins. The results showed that ADSCs were mainly of a spindle or polygon shape. Flow cytometry analysis revealed that ADSCs did not express CD34, CD45, and CD106 but high levels of CD44 and CD90, which confirmed that the cultured cells were indeed ADSCs. After induction with a 5-aza-containing solution, morphological changes in ADSCs, including irregular cell size, were observed. Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface. Many organelles were observed and the cytoplasm was found to contain many mitochondria, rough endoplasmic reticulum (rER), and myofilament-like structures. Cell immunohistochemical staining revealed different levels of desmin expression in each phase of the induction process, with the highest expression level found on day 28 of induction. RT-PCR and Western blot results confirmed significantly higher desmin gene expression in induced cells compared with control cells, but no significant difference between the two groups of cells in sarcomeric protein expression. It was concluded that under specific induction setting, ADSCs can be induced to differentiate into myoblasts, providing a potential new option in stem cell transplantation therapy for SUI.
本研究旨在诱导分离纯化的脂肪来源基质细胞（ADSCs）分化为成肌细胞，这可能为压力性尿失禁（SUI）患者的组织工程提供一种新策略。通过流式细胞术鉴定离体分离培养的ADSCs，并在含有添加了5-氮杂胞苷（5-aza）的DMEM、5%胎牛血清（FBS）和5%马血清的诱导培养液存在的情况下诱导其分化为成肌细胞。在倒置显微镜下观察细胞形态。通过透射电子显微镜和共聚焦激光扫描显微镜观察分化过程中发生的超微结构变化。应用细胞免疫组织化学染色来确定诱导分化和未诱导分化的细胞中结蛋白的表达。分别使用逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹法检测肌节和平滑肌结蛋白的mRNA和蛋白表达。结果显示，ADSCs主要呈纺锤形或多边形。流式细胞术分析显示，ADSCs不表达CD34、CD45和CD106，但高表达CD44和CD90，这证实培养的细胞确实是ADSCs。在用含5-aza的溶液诱导后，观察到ADSCs的形态变化，包括细胞大小不规则。细胞逐渐从长纺锤形变为多边形和具有微绒毛的星形细胞。观察到许多细胞器，并且发现细胞质中含有许多线粒体、粗面内质网（rER）和肌丝样结构。细胞免疫组织化学染色显示在诱导过程的每个阶段结蛋白表达水平不同，在诱导第28天表达水平最高。RT-PCR和蛋白质免疫印迹结果证实，与对照细胞相比，诱导细胞中的结蛋白基因表达显著更高，但两组细胞在肌节蛋白表达上无显著差异。得出的结论是在特定诱导条件下，ADSCs可被诱导分化为成肌细胞，为SUI的干细胞移植治疗提供了一种潜在的新选择。
