人脐带间充质干细胞外泌体对高糖诱导脐静脉内皮细胞功能的影响

Effect of exosomes secreted by human umbilical mesenchymal stem cells on high glucose - induced human umbilical vascular endothelial cells

目的探讨人脐带间充质干细胞（hUMSCs）外泌体对高糖诱导人脐静脉内皮细胞（hUVECs）功能的影响。 方法酶消化法原代培养hUMSCs和hUVECs，传代扩增至第3代，采用流式细胞仪分别鉴定2种细胞表面标志物；收集培养的第3代hUMSCs无血清培养基，提取外泌体，在电镜下观察其超微结构，并用流式细胞分析仪鉴定其表面标志物；体外培养第3代hUVECs分为对照组、高糖组（20 mmol/L）、外泌体＋高糖组（20 mmol/L）、miR－22抑制剂（50 nmol/L）＋高糖组（20 mmol/L），孵育24 h后采用Annexin V/PI双染流式细胞术观察hUVECs凋亡，四甲基偶氮唑蓝（MTT）法观察各组细胞的增殖能力，Transwell系统中观察各组细胞的迁移功能，成血管实验检测各组内皮细胞功能。 结果流式细胞术检测第3代hUMSCs的表面标志物CD105、CD73、CD90呈高表达；同时检测第3代hUVECs表面标志物CD309、CD31、CD34呈高表达，提示分离培养的hUMSCs和hUVECs具有间充质干细胞（mesenchymal stem cells，MSCs）和内皮细胞（endothelial cells，ECSs）特异性表型特征。外泌体扫描电镜观察可见大小不一的球状膜性结构，且流式细胞仪检测CD63、CD105、CD73、CD90、CD44呈阳性表达。MTT法检测各组细胞增殖结果：对照组、高糖组、miR－22抑制剂＋高糖组、外泌体＋高糖组细胞存活率分别为（99.0±1.6）%、（75.0±2.2）%、（66.0±1.8）%、（95.0±2.6）%，与对照组比较，高糖组和miR－22抑制剂＋高糖组细胞增殖明显下降，差异均有统计学意义（t＝28.271，P〈0.05；t＝43.213, P〈0.01）,外泌体＋高糖组细胞增殖无明显下降，差异无统计学意义（t＝2.001，P〉0.05）。流式细胞仪检测各组细胞凋亡结果：对照组、高糖组、miR－22抑制剂＋高糖组、外泌体＋高糖组细胞凋亡率分别为16.44%、52.67%、45.92%、17.58%，与对照组比较，高糖组和miR－22抑制剂＋高糖组细胞凋亡率明显增高，差异均有统计学意义（t＝－13.756、－16.489，均P〈0.01）,外泌体＋高糖组细胞凋亡率略有增高，差异无统计学意义（t＝－2.182，P〉0.05）。Transwell系统中观察细胞的迁移功能：对照组、高糖组、miR－22抑制剂＋高糖组、外泌体＋高糖组细胞迁移数量分别为834.5个、295.8个、252.2个、779.5个，与对照组比较，高糖组和miR－22抑制剂＋高糖组细胞迁移数量明显减少，差异均有统计学意义（t＝18.280、27.266，均P〈0.01）,外泌体＋高糖组细胞迁移数量略有减少，差异无统计学意义（t＝1.985，P〉0.05）。体外成管实验结果：对照组、高糖组、miR－22抑制剂＋高糖组、外泌体＋高糖组细胞成管数量分别为29.8个、13.3个、11.3个、29.6个，与对照组比较，高糖组和miR－22抑制剂＋高糖组细胞成管数量明显减少，差异均有统计学意义（t＝9.030，P〈0.05；t＝10.671，P〈0.01）,外泌体＋高糖组细胞成管数量略有减少，差异无统计学意义（t＝0.083，P〉0.05）。 结论hUMSCs外泌体可有效抑制高糖诱导hUVECs的凋亡、促进细胞增殖、迁移和血管再生作用，为寻找糖尿病微血管病变治疗的新靶点提供实验依据。

Objective To explore the effect of exosomes secreted by human umbilical mesenchymal stem cells （hUMSCs） on high glucose -induced human umbilical vascular endothelial cells （hUVECs）. Methods hUMSCs and hUVECs were primary cultured by enzyme digestion method and the surface markers of P3 generation was detected by using flow cytometry （FCM）. The serum - free medium of hUMSCs was collected to extract exosomes and then observed their uhrastructure was observed under an electron microscope and the surface markers were detected by using FCM again. The P3 generation hUVECs were divided into 4 groups in vitro：high glucose （20 mmol/L） group, exosomes with high glucose （20 mmol/L） group, miR -22 inhibitors （50 nmol/L） with high glucose （20 mmol/L） group,and a con- trol group. After 24 hours＇ incubation, Annexin V/PI staining method and FCM assay was used to evaluate cell apoptosis, 3 - （g,5 - dimethyl - 2 - thiazolyl） - 2,5 - diphenyl - 2 - H - tetrazolium bromide （MTr） and Transwell assay were used to detect the ability of cell proliferation as well as migration and tube formation assay to determine endothelial cell functions. Results Surface markers like CD105 , CD73 , CD90 had a high expression rate on hUMSCs while others like CD3o9 ,CD31, CD34 had a relatively high expression rate on hUVECs. Besides, CD63, CD105, CD73, CDgo and CD44 were positive in the exosomes, which suggested that separately - cultured hUMSCs and hUVECs had specificity phenotypic characteristics of mesen- chymal stem cells（ MSCs ） and endothelial cells （ECSs）. Ball -like membranous structures with uneven sizes were seen in the exosomes under the scanning electron microscope. Cell viability in the con- trol group,high glucose group, miR - 22 inhibitors with high glucose group, exosomes with high glucose group were （99.0 ± 1.6） %, （75.0 ±2.2） %, （66.0 ± 1.8 ） %, （95.0 ± 2.6） %, respectively, by MTT method. Compared with the control group, high glucose group and miR - 22 inhibitors with high glucose group cell proliferation were significant- ly decreased, the difference was statistically significant（ t = 28. 271, P 〈 0.05 ; t = 43. 213, P 〈 0.01 ）, exosomes with high glucose group cell proliferation was not significantly decreased, the difference was not statistically significant（t = 2.001 ,P 〉 0.05 ）. Cell apoptosis rate in the control group, high glucose group, miR- 22 inhibitom with high glucose group, exosomes with high glucose group were 16.44% ,52.67% ,45.92% , 17.58% , respectively, by FCM. Com- pared with the control group, high glucose group and miR - 22 inhibitors with high glucose group cell apoptosis rate were significantly higher, the difference was statistically significant（ t = - 13. 756, - 16. 489, all P 〈 0.01 ）, exosomes with high glucose group cell apoptosis rate increased slightly, the difference was not statistically significant （t = -2. 182 ,P 〉 0.05 ）. Cell migration in the control group, high glucose group, miR -22 inhibitors with high glucose group, exosomes with high glucose group were 834.5 number,295.8 number,252.2 number,779.5 number, respec- tively, by Transwell assay. Compared with the control group, high glucose group and miR - 22 inhibitors with high glu- cose group cell migration significantly reduced, the difference was statistically significant（t = 18. 280,27. 266, all P 〈 0.01 ）, exosomes with high glucose group cell migration decreased slightly, the difference was not statistically signifi- cant（ t = 1. 985 ,P 〉 0.05 ）. Tube formation number in the control group, high glucose group, miR -22 inhibitors with high glucose group, exosomes with high glucose group were 29.8 number, 13.3 number, 11.3 number,29.6 number, respectively, by tube formation assay. Compared with the control group, high glucose group and miR - 22 inhibitors with high glucose group tube formation number significantly reduced, the difference was statistically significant （t = 9.030 ,P 〈 0.05 ;t = 10.671 ,P 〈 0.01 ）, exosomes with high glucose group tube formation number decreased slightly, the difference was not statistically significant（ t = 0. 083, P 〉 0.05 ）. Conclusions Exosomes of hUMSCs do have a protective effect on the injured hUVECs induced by high glucose, which can inhibit the apoptosis of hUVECs, promote their proliferation as well as keep their migration and tube formation functions. This study has provided an experimental basis for a novel therapeutic target of diabetic microvascular lesions.