EPO预处理骨髓间充质干细胞预防大鼠肾移植术后急性排斥反应的初步研究

Preliminary study of the effect of EPO pretreatment of bone marrow mesenchymal stem cells on preventing acute rejection after renal transplantation in rats

    摘要
  • 摘要:
      目的  探讨促红细胞生成素（EPO）预处理骨髓间充质干细胞（BMSC）用于预防大鼠肾移植术后急性排斥反应的作用。
      方法  将BMSC分为5组，分别为对照组（不含EPO）、A组（EPO终浓度10 IU/mL）、B组（EPO终浓度100 IU/mL）、C组（EPO终浓度500 IU/mL）和D组（EPO终浓度1 000 IU/mL），每组细胞培养24 h和48 h，采用3-（4，5-二甲基噻唑-2）-2，5-二苯基四氮唑溴盐（MTT）法测定BMSC的细胞增殖率。将BMSC分为2组，分别为BMSC组（不含EPO）和EPO-BMSC组（EPO终浓度500 IU/mL），每组细胞培养48 h后，采用蛋白印迹（Western blot）法检测各组BMSC表面趋化因子受体（CXCR）4的蛋白表达水平。以Wistar大鼠为供体，SD大鼠为受体，建立肾移植急性排斥反应模型后，将受体鼠随机分为4组，每组6只，分别为对照组（未进行任何干预）、EPO组（术后立即经尾静脉注射1 mL含有500 IU的EPO溶液）、BMSC组（术后立即经尾静脉注射1 mL的1×106/mL BMSC细胞液）、EPO-BMSC组（经尾静脉注射1 mL500 IU/mL EPO体外培养的含有1×106/mL BMSC细胞液），采用血清肌酐（Scr）试剂盒测定血标本中Scr水平，采用Western blot法检测移植肾组织中干扰素（IFN）-γ和白细胞介素（IL）-4的蛋白表达水平。
      结果  培养24 h，各组BMSC增殖率比较，差异均无统计学意义（均为P > 0.05）。培养48 h，与对照组比较，C组（EPO终浓度500 IU/mL）的BMSC增殖率较高，差异有统计学意义（P < 0.05）。与BMSC组比较，EPO-BMSC组BMSC表面CXCR4的蛋白表达水平更高（P < 0.05)。肾移植术后1 d，各组受体鼠的Scr水平比较，差异均无统计学意义（均为P > 0.05）；术后5 d，与对照组比较，EPO组、BMSC组和EPO-BMSC组受体鼠的Scr水平均明显降低，差异均有统计学意义（均为P < 0.05），且EPO-BMSC组受体鼠的Scr水平低于EPO组、BMSC组，差异均有统计学意义（均为P < 0.05）。术后1 d和术后5 d，各组受体鼠移植肾组织中IL-4的蛋白表达水平比较，差异均无统计学意义（均为P > 0.05）。术后1 d，与对照组比较，EPO组、BMSC组及EPO-BMSC组受体鼠移植肾组织中IFN-γ蛋白表达水平和IFN-γ/IL-4均有不同程度降低，差异均有统计学意义（均为P < 0.05）。术后5 d，与对照组比较，EPO组、BMSC组和EPO-BMSC组中IFN-γ蛋白表达水平和IFN-γ/IL-4均有不同程度降低，差异均有统计学意义（均为P < 0.05），其中EPO-BMSC组中IFN-γ蛋白表达水平和IFN-γ/IL-4均低于EPO组和BMSC组，差异均有统计学意义（均为P < 0.05）。
      结论  EPO预处理BMSC可预防肾移植术后急性排斥反应，保护移植肾功能。

     

    Abstract:
      Objective  To investigate the role of bone marrow mesenchymal stem cells (BMSCs) pretreated with erythropoietin (EPO) in the prevention of acute rejection after renal transplantation in rats.
      Methods  BMSCs were divided into five groups: control group (without EPO), group A (pretreated with EPO at a final concentration of 10 IU/mL), group B (pretreated with EPO at a final concentration of 100 IU/mL), group C (pretreated with EPO at a final concentration of 500 IU/mL) and group D (pretreated with EPO at a final concentration of 1 000 IU/mL). In each group, the BMSCs were cultured for 24 h and 48 h. The proliferation rate of the BMSCs was determined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The BMSCs were divided into two groups: BMSC group (without EPO) and EPO-BMSC group (pretreated with EPO at a final concentration of 500 IU/mL). After 48 h culture, Western blot was adopted to measure the expression level of CXC chemokine receptor (CXCR) 4 protein in BMSCs. Wistar rats were used as the donors, and SD rats were utilized as the recipients to establish the rat models with acute rejection after renal transplantation. The recipient rats were randomly divided into four groups (n=6 in each group) including the control group (without any intervention), EPO group (injection of 1 mL of solution containing 500 IU EPO via tail vein immediately after surgery), BMSC group (injection of 1 mL of solution containing 1×106/mL BMSCs via tail vein immediately after surgery) and EPO-BMSC group (injection of 1 mL of solution containing 1×106/mL BMSCs cultured in vitro with 500 IU/mL EPO via tail vein). The level of serum creatinine (Scr) level was determined by Scr detection kit. Western blot was used to detect the expression levels of interferon (IFN)-γ and interleukin (IL)-4 proteins.
      Results  After 24 h culture, the proliferation rate of BMSCs did not significantly differ among all groups (all P > 0.05). After 48 h culture, the proliferation rate of BMSCs in group C (pretreated with EPO at a final concentration of 500 IU/mL) was significantly higher than that in the control group (P < 0.05). Compared with the BMSC group, the expression level of CXCR4 protein on the surface of BMSCs was higher in the EPO-BMSC group (P < 0.05). At 1 d after renal transplantation, the levels of Scr did not significantly differ among all groups (all P > 0.05). At 5 d after operation, the levels of Scr in the EPO, BMSC and EPO-BMSC groups were significantly lower than that in the control group (all P < 0.05). The level of Scr in the EPO-BMSC group was markedly lower than those in the EPO and BMSC groups (both P < 0.05). At postoperative 1 d and 5 d, the expression levels of IL-4 protein in the kidney tissues did not significantly differ among all groups (all P > 0.05). At 1 d after surgery, compared with control group, the expression levels of IFN-γ protein and IFN-γ/IL-4 ratio in the renal tissues in the EPO, BMSC and EPO-BMSC groups were significantly decreased to varying extents (all P < 0.05), and similar results were obtained at 5 d after surgery (all P < 0.05). The expression levels of IFN-γ protein and IFN-γ/IL-4 ratio in the EPO-BMSC group were significantly lower than those in the EPO group and BMSC group (both P < 0.05).
      Conclusions  BMSCs pretreated with EPO can prevent the incidence of acute rejection after renal transplantation and protect the renal graft function.

     

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