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:
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. -
-
[1] DAZZI F, KRAMPERA M. Mesenchymal stem cells and autoimmune diseases[J]. Best Pract Res Clin Haematol, 2011, 24(1): 49-57. DOI: 10.1016/j.beha.2011.01.002. [2] CONTRERAS-KALLENS P, TERRAZA C, OYARCE K, et al. Mesenchymal stem cells and their immunosuppressive role in transplantation tolerance[J]. Ann N Y Acad Sci, 2018, 1417(1): 35-56. DOI: 10.1111/nyas.13364. [3] CHAMBERLAIN G, FOX J, ASHTON B, et al. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing[J]. Stem Cells, 2007, 25(11): 2739-2749. [4] LOEBINGER MR, JANES SM. Stem cells as vectors for antitumour therapy[J]. Thorax, 2010, 65(4): 362-369. DOI: 10.1136/thx.2009.128025. [5] HASHEMI SM, GHODS S, KOLODGIE FD, et al. A placebo controlled, dose-ranging, safety study of allogenic mesenchymal stem cells injected by endomyocardial delivery after an acute myocardial infarction[J]. Eur Heart J, 2008, 29(2): 251-259. [6] IKEGUCHI R, KAKINOKI R, OHTA S, et al. Recipient bone marrow-derived stromal cells prolong graft survival in a rat hind limb allotransplantation model[J]. Microsurgery, 2017, 37(6): 632-640. DOI: 10.1002/micr.30128. [7] PURROY C, FAIRCHILD RL, TANAKA T, et al. Erythropoietin receptor-mediated molecular crosstalk promotes T cell immunoregulation and transplant survival[J]. J Am Soc Nephrol, 2017, 28(8): 2377-2392. DOI: 10.1681/ASN.2016101100. [8] GALLATIN WM, WEISSMAN IL, BUTCHER EC. A cell-surface molecule involved in organ-specific homing of lymphocytes. 1983[J]. J Immunol, 2006, 177(1): 5-9. [9] SHEN L, GAO Y, QIAN J, et al. A novel mechanism for endothelial progenitor cells homing: the SDF-1/CXCR4-Rac pathway may regulate endothelial progenitor cells homing through cellular polarization[J]. Med Hypotheses, 2011, 76(2): 256-258. DOI: 10.1016/j.mehy.2010.10.014. [10] GHADGE SK, MÜHLSTEDT S, OZCELIK C, et al. SDF-1α as a therapeutic stem cell homing factor in myocardial infarction[J]. Pharmacol Ther, 2011, 129(1): 97-108. DOI: 10.1016/j.pharmthera.2010.09.011. [11] TARI K, ATASHI A, KAVIANI S, et al. Erythropoietin induces production of hepatocyte growth factor from bone marrow mesenchymal stem cells in vitro[J]. Biologicals, 2017, 45: 15-19. DOI: 10.1016/j.biologicals.2016.10.010. [12] LIU N, TIAN J, CHENG J, et al. Effect of erythropoietin on the migration of bone marrow-derived mesenchymal stem cells to the acute kidney injury microenvironment[J]. Exp Cell Res, 2013, 319(13): 2019-2027. DOI: 10.1016/j.yexcr.2013.04.008. [13] LIAO W, ZENG F, KANG K, et al. Lipoxin A4 attenuates acute rejection via shifting TH1/TH2 cytokine balance in rat liver transplantation[J]. Transplant Proc, 2013, 45(6): 2451-2414. DOI: 10.1016/j.transproceed.2013.01.069. [14] LI B, TIAN L, DIAO Y, et al. Exogenous IL-10 induces corneal transplantation immune tolerance by a mechanism associated with the altered Th1/Th2 cytokine ratio and the increased expression of TGF-β[J]. Mol Med Rep, 2014, 9(6):2245-2250. DOI: 10.3892/mmr.2014.2073. [15] MOTA AP, VILAÇA SS, DAS MERCÊS FL JR, et al. Cytokines signatures in short and long-term stable renal transplanted patients[J]. Cytokine, 2013, 62(2): 302-309. DOI: 10.1016/j.cyto.2013.03.001. [16] 金潇, 周松, 胡建敏, 等.间充质干细胞在肾脏移植中的应用研究进展[J].实用医学杂志, 2018, 34(3): 499-502. DOI: 10.3969/j.issn.1006-5725.2018.03.040.JIN X, ZHOU S, HU JM, et al. Advances in the application of mesenchymal stem cells in renal transplantation[J]. J Pract Med, 2018, 34(3): 499-502. DOI: 10.3969/j.issn.1006-5725.2018.03.040. [17] XIE J, WANG Y, BAO J, et al. Immune tolerance induced by RelB short-hairpin RNA interference dendritic cells in liver transplantation[J]. J Surg Res, 2013, 180(1): 169-175. DOI: 10.1016/j.jss.2012.10.021.