课题背景：作者前期实验采用10%的二甲亚砜联合成纤维细胞生长因子成功的将大鼠骨髓间充质干细胞诱导为神经元样细胞，但7 h后细胞即开始死亡，24 h后大部分细胞已经脱离培养瓶瓶底，说明高浓度化学诱导剂对细胞有毒性作用。膀胱匀浆上清不是化学制剂，而1%二甲亚砜为低浓度化学制剂，对细胞均无明显毒性作用。

相关链接：近年来的研究表明，环境因素在干细胞分化过程中起关键作用。Jang等应用受损伤的肝脏组织匀浆与造血干细胞进行非接触性嵌合培养，在体外诱导造血干细胞向肝脏上皮分化过程中细胞表面出现肝脏上皮细胞特异标记的表达。国内杨景全等报道，大鼠脊髓匀浆上清液可在体外有效地诱导骨髓间充质干细胞分化为神经样细胞。张婷等也报道，在缺血再灌注损伤大鼠肾脏匀浆诱导下，骨髓干细胞可向肾小管上皮样细胞分化。骨髓间充质干细胞在体内分化为何种细胞，可能受很多因素的影响，其所处微环境的改变、宿主对其的诱导趋化作用及其可能分泌的某些趋化因子、生长因子等可能是很重要的诱导因素。

应用要点：骨髓干细胞在体外经诱导可分化为平滑肌细胞,文献报道的诱导剂多为血小板衍生生长因子、转录生长因子以及平滑肌细胞共培养等。本实验采用首先应用低浓度的二甲亚砜（1%DMSO）对大鼠骨髓间充质干细胞进行初诱导8 h，然后应用膀胱匀浆上清液再诱导7d，通过改变干细胞生存的微环境，成功地将骨髓间充质干细胞诱导分化为平滑肌样细胞。

摘要
目的：环境因素对干细胞的分化起到重要的作用。实验拟观察经低浓度二甲亚枫进行预诱导后，大鼠骨髓间充质干细胞在体外经膀胱组织匀浆上清液诱导定向分化为平滑肌样细胞的可行性。
方法：实验于2007-04/2007-09在河北医科大学解剖教研室下属细胞培养室完成。①实验材料：4～6周龄100～150 g健康清洁级SD大鼠1只，雌雄不限，8周龄220~250 gSD大鼠4只，由河北医科大学实验动物中心提供，实验过程中对动物处置符合动物伦理学标准。②实验方法：采用骨髓干细胞培养基和贴壁法从SD大鼠骨髓中分离骨髓间充质干细胞，并在体外扩增、传代。选择8周龄SD大鼠进行膀胱组织上清液的制备。取第4代骨髓间充质干细胞用1%二甲亚砜预诱导8 h后，应用膀胱匀浆上清液诱导7 d。以未进行诱导的骨髓间充质干细胞为阴性对照组。③实验评估：观察细胞形态学的变化，并运用免疫细胞化学检测特异性α-平滑肌肌动蛋白（α-SMA）的表达。
结果：骨髓间充质干细胞经诱导分化后，细胞呈梭形平滑肌样，融合后形成峰谷状排列，并表达平滑肌特异性蛋白标志物α－平滑肌肌动蛋白，诱导分化率为（45.6±3.5）%，与阴性对照组相比差异具有显著性意义（P < 0.05）。
结论：采用此方法成功诱导骨髓间充质干细胞分化为平滑肌样细胞。
关键词：干细胞；平滑肌；细胞培养；分化

王振显，蔡文清，宋永周，瞿长宝，史正亮，郭威.膀胱匀浆上清液在大鼠骨髓间充质干细胞诱导分化为平滑肌样细胞中的作用[J].中国组织工程研究与临床康复，2008，12(8):1422-1425(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1422(ps).pdf]

Rat bone marrow mesenchymal stem cells differentiate into smooth muscle cells in vitro induced by the supernatant of homogenized bladders

Abstract

AIM：Environmental factors play important roles in the differentiation of stem cells. This study explored the potential of rat bone marrow mesenchymal stem cells (MSCs) to differentiate into smooth muscle cells by the supernatant of homogenized bladder.

METHODS: The experiment was conducted in the cell culture laboratory affiliated to the Department of Anatomy, Hebei Medical University from April to September 2007. ①One 4-week-old healthy SD rat of 100-150 g, either male or female, and four 8-week-old SD rats of 220-250 g were provided by the experimental animal center of Hebei Medical University. All procedures employed in this study were consistent with the animal ethical standards. ②MSCs were isolated from SD rat bone marrow by total marrow culture associated with attachment method, purified and cultured in vitro. The supernatant of homogenized bladder was made with 8-week-old SD rats. The MSCs of the 4th generation were cultured in DMEM with 1% dimethyl sulfoxide (DMSO) for 8 hours before changing the supernatant of homogenized bladders to induce differentiation for 7 days. MSCs not treated served as negative control. ③Morphology of cells was observed and immunocytochemistry was performed to detect the expression of specific α-smooth muscle actin (SMA).

RESULTS: After induction, MSCs showed smooth muscle-like cells and spindle-shaped. The cells fused as a hill-and-valley growth pattern, and expressed the specific α-SMA. The differentiation rate was (45.6±3.5)%, which was statistically significant compared with negative control group (P < 0.05).

CONCLUSION: MSCs are induced into smooth muscle cells in vitro by this method.

Wang ZX, Cai WQ, Song YZ, Qu CB, Shi ZL, Guo W.Rat bone marrow mesenchymal stem cells differentiate into smooth muscle cells in vitro induced by the supernatant of homogenized bladders.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(8):1422-1425(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-8/8k-1422(ps).pdf]