目的研究N-(6-氨乙基)-5-氯-1-萘磺酰胺（W7）在体外对人脂肪间充质干细胞（hADSCs）转分化为内皮细胞（EC）的影响。方法"以含40-ng/ml血管内皮生长因子（VEGF）和10-ng/ml碱性成纤维细胞生长因子（bFGF）的干细胞无血清分化培养基进行细胞培养，分为空白对照组（不含W7的分化培养基）和高（30-μmol/L）、中（20-μmol/L）、低（10-μmol/L）剂量W7组。hADSCs加药8-d后，采用流式细胞术（FCM）检测各组EC von Willebrand因子(vWF)和血管选择性钙黏素（VE-Cadherin）表型变化，激光共聚焦显微镜检测钙荧光探针（Fluo-3）标记的细胞内［Ca2+］i变化；同时将W7处理后的hADSCs种植在Matrigel胶上，观察细胞成血管能力；采用Western blot技术分析不同浓度药物处理8-d后细胞外调节激酶（ERK）和磷酸化ERK(p-ERK)变化。结果"分化至8-d时，与空白对照组相比，hADSCs转分化后的细胞VE-Cadherin 和vWF表达量随W7浓度降低而显著上升（P＜0.01），胞质［Ca2+］i显著升高（P＜0.01）。W7干预组的培养细胞均有管腔样血管结构形成，空白对照组的细胞无血管管型形成。与空白对照组相比，不同剂量W7干预组间细胞的ERK表达水平差异无统计学意义（P＞0.05）；随着W7浓度的降低，p-ERK表达水平明显升高（P＜0.05）。结论"适当浓度的W7可促进hADSC向EC诱导分化，其机制可能与促进细胞内［Ca2+］i增加，激活细胞分化过程中的ERK/MAPK信号通路有关。
Objective To explore the effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide（W7） on the differentiation from human adipose-derived mesenchymal stem cells (hADSCs) to endothelial cells. MethodshADSCs were cultured with serum-free differential medium containing 40 ng/ml vascular〖HT〗〖HJ〗〖HK〗〖CD10〗〖HT6，7.5〗基金项目：科技部社会公益专项（2005DIBIJ086）Supported by the Special Social Commonweal Research Programs of the Ministry of Science and Technology（2005DIBIJ086）〖HT〗〖HJ〗〖GK2!2〗endothelial growth factor (VEGF) and 10ng/ml basic fibroblast growth factor (bFGF). Cells were divided into control group (differential medium without W7), high-dose group (containing 30 μmol/L W7), medium-dose group (containing 20 μmol/L W7), and low-dose group ( containing 10 μmol/L W7). The hADSCs were cultured for 8 days, and then the changes in the phenotypes of von Willebrand factor (vWF) and vessel-selective cadherin (VE-Cadherin) were detected by flow cytometry (FCM). The intracellular Ca2+ labeled with Fluo-3 was detected by laser confocal microscopy. After hADSCs planting on Matrigel, their angiogenic potentials were observed under the inverted phase contrast microscope, and the expression of extracellular regulated kinase (ERK) and phosphorylated extracellular regulated kinase (p-ERK) were evaluated by Western blot. ResultsAfter the hADSCs were cultured for 8 days, compared with the control group, the expressions of vWF and VE-Cadherin significantly increased along with the decrease of W7 level and the intracellular Ca2+ also significantly increased (P＜0.01). Lumina-like vascular structure was formed in W7 treatment groups, but not in the blank control group. Compared with the blank control group, the expression of ERK showed no significant in W7 treatment groups (high-, medium-, and low-dose groups)（P>0.05）; however, along with the decrease of W7 levels, the expression of p-ERK significantly increased（P<0.05). ConclusionW7 in proper levels can effectively induce the differentiation from hADSCs to endothelium by increasing the intracellular Ca2+ level and thus activating the ERK/MAPK pathway."
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