体外诱导人脐带华通胶间充质干细胞向内皮细胞分化过程中APJ表达变化

Expression and variance of APJ in umbilical cord-derived human mesenchymal stem cells induced into endothelial cell in vitro

目的研究人脐带华通胶间充质干细胞(human umbilical cord Wharton's Jelly-mesenchymal stem cells,HUW-MSCs)体外诱导向内皮细胞分化过程中表面APJ受体表达的变化,探讨APJ是否可作为鉴定内皮细胞的早期细胞表面标志物。方法剖宫产取脐带后分别用酶消化和组织贴壁法培养获得脐带华通胶间充质干细胞,用酶消化法获得人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)。取2×10~7第2代HUW-MSCs用单克隆APJ-APC荧光抗体标志后行流式分选,测定HUW-MSCs实验前细胞表面表达APJ的水平。实验分3组:诱导组MSCs用含血管内皮细胞生长因子5μg/L、碱性成纤维细胞生长因子10μg/L、表皮生长因子10μg/L和10%胎牛血清(fetal bovine serum,FBS)M199培养基培养;共同培养组MSCs先与HUVECs在Transwell 6孔培养板中,用含10%FBS的M199培养5-7 d后转入25 cm^2培养瓶中继续培养,培养液为含10%FBS的M199和培养1 d内皮细胞的M199(2:1)混合培养基:单纯培养组细胞培养基为含10%FBS的M199。培养周期为14 d。分别于第7、10、14天流式检测各组细胞APJ表达水平,同时检测内皮细胞表面APJ水平作为阳性对照。另外于第7、14天检测3个实验组、内皮细胞组、HUW-MSCs组内皮细胞早期标志物CD309表达变化情况,比较各实验组之间APJ与CD309变化趋势。结果HUW-MSCs流式分选出5×10~3APJ^+-HUW-MSCs,表面表达APJ基本呈阴性。诱导组、共同培养组和单纯培养组细胞形态均较HUWMSCs有显著改变,诱导组呈圆形、线性、网状,共同培养、单纯培养组细胞呈卵圆形、长梭形。另外,诱导组生长最快,增殖能力最强;单纯培养组次之;共同培养组细胞生长缓慢,增殖能力最弱。第7、10、14天,3组细胞CD309表达分别为诱导组1.8%、2.6%和8.8%,共培养组0.5%、2.5%和4.7%,单纯培养组0.3%、2.1%和2.7%。第7、10、14天各组APJ阳性表达率分别为诱导组0.5%、0.9%和5.2%,共培养组0.4%、0.8%和3.0%,单纯培养组0.2%、0.6%和2.1%。结论HUW-MSCs表面APJ表达基本呈阴性,HUW-MSCs向内皮细胞诱导分化过程中APJ表达逐渐上调,与内皮细胞早期标志物CD309变化趋势基本一致,可作为早期内皮细胞鉴定的标志物。

Objective To explone the expression and variation, induced by cytokines in vitro, of APJ in mesenchymal stem cells from human umbilical cord Wharton＇s Jelly. To identify APJ is whether a surface mark of vascular endothelial cell or not in prophase of differentiation. Methods The umbilical cord was obtained from cesarean section.We isolated the Wharton＇s Jelly and umbilical veins endothelial cells and cultured the adherent cells. Cells labeled with monoclonal fluorescent an- tibody reaching up to 2× 10^7 of P2 generation were undergone sorting through flow cytometer. Cells were divided into 3 subgroups, induction, cocuhure and haplo-cuhivation. The concentration ofVEGF, β-FGF and EGF in culture media,M199,for induction subgroup was 5 μg/L, 10 μg/L, 10 μg/L respectively. MSCs were first cocuhured with HUVEC in non-contact system by Transwell for 5- 7 days, then we fed them with mixed M199 making up with fresh media and suction from endothelio- cyte in volume ratio of 1：2 in common culture flask. All of the M199 contained 10% FBS and 1% mycillin.The whole cultivation cycle was fortnight, and we checked out the APJ in 3 subgroups in d7, d10 and d14 and CD309 in d7, d10 and d14 respectively. Results About five thousands of APJ＋-HUW-MSCs was obtained after sorting. Morphous of cells in 3 subgroups had changed dramati- cally compared to MSCs cultured by EMEM/F12,which presented shapes as follow round,filate and reticulum cells in induction group, oval and elongated fusiform cells were cocuhure and haplo-cuhi- vation. As for reproductive activity, the most was induction, the weakest was cocuhure. Expression of CD309 in d7,dl0 and d14 of the induction, cocuhure and haplo-cuhivation were（ 1.8% vs. 2.6% vs. 8.8%）, （0. 5% vs. 2. 5% vs. 4.7%） and（0.3% vs. 2. 1% vs. 2.7%） respectively. The per- centage of APJ positive cells in induction were （0. 5% vs. 0.9% vs. 5.2%） in d7,dl0 and d14, （0.4% vs. 0.8% vs. 3.0% ） respectively with cocuhure. In haplo-cuhivation, they were （0. 2% vs. 0.6% vs. 2. 1%）. Conclusion Expression of receptor APJ in the surface of MSCs is approaching to negative. In the induction of HUW-MSCs into vascular endothelial cells, APJ expression enhance gradually in accordance with CD309, a pristine mark of vascular endothelial cells, and APJ may be a new mark of identification of vascular endothelial cells in prophase of differentiation.