脐带间充质干细胞对妊娠期糖尿病大鼠血脂代谢及免疫功能的影响

Effects of umbilical cord-derived mesenchymal stem cells on blood lipid metabolism and immune function in pregnant diabetes rats

目的探讨脐带间充质干细胞(UC-MSCs)对妊娠期糖尿病(GDM)大鼠血脂代谢及免疫功能的影响。方法分离人UC-MSCs,分别在2D及3D条件下进行培养,观察细胞形态并进行细胞表面标志物检测。加入γ干扰素(INF-γ)进行预处理,分为IFN-γ组、IFN-γ+2D UC-MSC组、IFN-γ+3D UC-MSC组。采用酶联免疫吸附试验检测上清中白细胞介素(IL)-6、IL-8、IL-10、转化生长因子-β1(TGF-β1)和前列腺素E2(PGE-2)水平。采用随机数字表法将50只SD雌性大鼠分为:对照组(n=10)、妊娠糖尿病组(GDM组,n=10)、妊娠糖尿病+0.9%氯化钠溶液组(GDM+NS组,n=10)、2D培养UC-MSC移植组(2D UC-MSCs移植组,n=10)、3D培养UC-MSC移植组(3D UC-MSCs移植组,n=10),除对照组普通饲料喂养外,其余4组均采用高脂饮食喂养4周后交配,建立GDM大鼠模型。GDM模型成功后2D UC-MSCs移植组及3D UC-MSCs移植组进行尾静脉注射UC-MSCs,GDM+NS组注射同等量的0.9%氯化钠溶液。移植14 d后检测各组大鼠甘油三酯、低密度脂蛋白(LDL-C)及总胆固醇及IL-6、IL-8、IL-10、TGF-β1和PGE-2水平。结果(1)2D UC-MSCs细胞贴壁生长,呈成纤维样或梭形,3D UC-MSCs细胞巢状支架上黏附生长良好,分布均匀。(2)2D培养的UC-MSCs阳性表达CD44、CD90、CD105,阴性表达CD34、CD45、CD133。3D培养下的UC-MSCs阳性表达CD44、CD90、CD105,阴性表达CD34、CD45、CD103。(3)与IFN-γ组相比,IFN-γ+2D UC-MSC组与IFN-γ+3D UC-MSC组IL-6、IL-8水平均明显降低,IL-10、TGF-β1及PGE-2水平均明显升高,差异均有统计学意义(P<0.05)。与IFN-γ+2D UC-MSC组相比,IFN-γ+3D UC-MSC组IL-6、IL-8水平均明显降低,IL-10、TGF-β1及PGE-2水平均明显升高,差异均有统计学意义(P<0.05)。(4)与GDM组相比,2D UC-MSCs移植组及3D UC-MSCs移植组血糖水平明显下降,胰岛素水平明显升高,差异均有统计学意义(P<0.05)。与2D UC-MSCs移植组相比,3D UC-MSCs移植组血糖水平明显下降,胰岛素水平明显升高,差异均有统计学意义(P<0.05)。(5)与GDM组相比,GDM+NS组甘油三酯、LDL-C、总胆固醇水平均无明显变化,差异均无统计学意义(P>0.05);与GDM组相比,2D UC-MSCs移植组及3D UC-MSCs移植组甘油三酯、LDL-C、总胆固醇水平均明显下降,差异均有统计学意义(P<0.05);与2D UC-MSCs移植组相比,3D UC-MSCs移植组的甘油三酯、LDL-C、总胆固醇水平均明显下降,差异均有统计学意义(P<0.05)。(6)与CDM组相比,2D UC-MSCs移植组及3D UC-MSCs移植组IL-6、IL-8水平明显均降低,IL-10、TGF-β1及PGE-2水平均明显升高,差异均有统计学意义(P<0.05);与2D UC-MSCs移植组比较,3D UC-MSCs移植组IL-6、IL-8水平均明显降低,IL-10、TGF-β1及PGE-2水平均明显升高,差异均有统计学意义(P<0.05)。结论构建IFN-γ预处理3D UC-MSCs可增强免疫调控能力,治疗GDM大鼠模型,改善临床症状,降低炎症发应,为MSC治疗GDM提供可行性。

Objective To investigate the effect of umbilical cord-derived mesenchymal stem cells on blood lipid metabolism and immune function in gestational diabetes mellitus(GDM)rats.Methods Human umbilical cord mesenchymal stem cells(UC MSCs)were isolated and cultured in 2D and 3D conditions,respectively.Cell morphology was observed and cell surface markers were detected.Joinγ-interferon(INF-γ)retreatment,divided into IFN-γgroup,IFN-γ+2D UC-MSC group,IFN-γ+3D UC-MSC group,use ELISA to detect interleukin-6(IL-6),interleukin-8(IL-8),interleukin-10(IL-10)and transforming growth factor-β1(TGF-β1)and prostaglandin E2(PGE-2)levels in the supernatant.Fifty SD female rats were divided into the control group(n=10),pregnant diabetes group(GDM group,n=10),pregnant diabetes+normal saline group(GDM+NS group,n=10),2D cultured UC-MSC transplantation group(2D UC-MSCs transplantation group,n=10),3D cultured UC-MSC transplantation group(3D UC-MSCs transplantation group,n=10)according to the random number table method.In addition to the control group,the other four groups were fed a high-fat diet for four weeks and then mated to establish the rat model of GDM.After the success of the GDM model,the 2D UC-MSCs transplantation group and 3D UC-MSCs transplantation group were injected with UC MSCs via the tail vein,and the GDM+NS group was injected with the same amount of normal saline.Triglycerides,low-density lipoprotein(LDL-C),total cholesterol,IL-6,IL-8,IL-10,TGF-β1 and PGE-2 levels were detected 14 days.Results(1)2D UC-MSCs cells adhered to the wall and grew in a fibrous or spindle shape,while 3D UC-MSCs cells adhered to the nest-like scaffold well and distributed evenly.(2)UC-MSCs in 2D culture expressed CD44,CD90,CD105 positively,and CD34,CD45,CD133 negatively.UC MSCs in 3D culture expressed CD44,CD90,CD105 positively,and CD34,CD45,CD103 negatively.(3)Compared with IFN-γgroup,the levels of IL-6 and IL-8 in IFN-γ+2D UC-MSC group and IFN-γ+3D UC-MSC group decreased significantly,the levels of IL-10 and TGF-β1 and PGE-2 in IFN-γ+2D UC-MSC group and IFN-γ+3D UC-MSC group increased significantly,the differences were statistically significant(P<0.05).And compared with IFN-γ+2D UC-MSC group,the levels of IL-6 and IL-8 decreased significantly in IFN-γ+3D UC-MSC group,the levels of IL-10,TGF-β1 and PGE-2 in IFN-γ+3D UC-MSC group were significantly increased,the differences were statistically significant(P<0.05).(4)Compared with the GDM group,the level of blood glucose in the 2D UC-MSCs transplantation group and 3D UC-MSCs transplantation group decreased significantly,the level of insulin increased significantly,the differences were statistically significant(P<0.05).Compared with the 2D UC-MSCs transplantation group,the level of blood glucose in the 3D UC-MSCs transplantation group decreased significantly,the level of insulin increased significantly,the differences were statistically significant(P<0.05).(5)Compared with the GDM group,there were no significant changes in triglycerides,LDL-C,and total cholesterol levels in the GDM+NS group,and the differences were not statistically significant(P>0.05).Compared with the GDM group,the levels of triglycerides,LDL-C,and total cholesterol in the 2D UC-MSCs transplantation group and the 3D UC-MSCs transplantation group decreased significantly,and the differences were statistically significant(P<0.05).Compared with the 2D UC-MSCs transplantation group,the levels of triglycerides,LDL-C,and total cholesterol in the 3D UC-MSCs transplantation group decreased significantly,and the differences were statistically significant(P<0.05).(6)Compared with the CDM group,the levels of IL-6 and IL-8 in the 2D UC-MSCs transplantation group and the 3D UC-MSCs transplantation group decreased significantly,and the levels of IL-10 and TGF-β1 and PGE-2 increased significantly,the differences were statistically significant(P<0.05).Compared with the 2D UC-MSCs transplantation group,the levels of IL-6 and IL-8 in the 3D UC-MSCs transplantation group decreased significantly,and IL-10 and TGF-β1 and PGE-2 increased significantly,the differences were statistically significant(P<0.05).Conclusion Construction of IFN-γpretreatment 3D UC-MSCs can enhance the immune regulation ability,treat GDM rat models,improve clinical symptoms,reduce inflammatory response,and provide feasibility for MSC to treat gestational diabetes.