外源性联合应用EPCs和SDF-1α对大鼠肝移植缺血再灌注的保护作用

Exogenous SDF-1α delivery combined with EPCs transplantation produces a superior protective effect on hepatic ischemia reperfusion injuryed rats

摘要:

目的：探讨外源性注射骨髓来源的血管内皮祖细胞（endothelial progenitor cells，EPCs）或基质细胞源性因子-1α（stro－mal derived factor-1 alpha，SDF-1α）或两者联合，比较性评估3种方法对大鼠肝移植术后缺血再灌注损伤（ischemia reperfusion injury，IRI）的影响。方法：LEWIS大鼠骨髓EPCs提取培养，流式表型鉴定，激光共聚焦功能鉴定；建立大鼠原位肝移植模型，随机分为5组：A组（假手术组），B组（手术组），C组（SDF-1α组），D组（EPCs组），E组（SDF-1α+EPCs组）；再灌注12 h后处死各组大鼠，免疫组化检测肝组织再生以及肝细胞生长因子（hepatocyte growth factor，HGF）的表达情况；WB检测肝组织血管内皮生长因子（vascular endothelial growth factor，VEGF）、HGF、转化生长因子-β（transforming growth factor-beta，TGF-β）、B细胞淋巴瘤/白血病-2（B cell lymphoma/lewkmia-2，Bcl-2）、含半胱氨酸的天冬氨酸蛋白水解酶3（cysteinyl aspartate specific proteinase-3，Caspase-3）蛋白表达；ELISA检测VEGF、HGF、TGF-β表达。结果：LEWIS大鼠骨髓提取的EPCs CD34、CD133的阳性比例分别为78.32%、70.76%，同时具有结合ac-LDL和UEA-1功能的EPCs，阳性率达80%以上；SDF-1α诱导EPCs穿过的细胞数[（79.6±6.3）个]明显高于对照组[（36.2±2.9）个]（P=0.000）；各组大鼠肝组织中，C、D组肝组织HGF分泌和再生情况免疫评分明显高于A、B组（P=0.000），E组免疫评分明显高于C、D组（P=0.000）；各组VEGF、HGF、TGF-β、Bcl-2、Caspase-3蛋白表达结果，C、D组VEGF、HGF、TGF-β、Bcl-2蛋白表达明显高于A、B组（P=0.000），E组VEGF、HGF、TGF-β蛋白表达明显高于C、D组（P=0.000），而C、D、E组Bcl-2蛋白表达比较差异无统计学意义。B、C、D组Caspase-3蛋白表达明显高于A组（P=0.000），而E组Caspase-3蛋白明显低于C、D组（P=0.000）；ELISA结果与Western blot结果一致。结论：外源性联合应用EPCs和SDF-1α能有效保护肝移植缺血再灌注的损伤，诱导肝细胞再生，减少肝细胞凋亡。

Abstract:

Objective：To investigate the effects of exogenous SDF-1αdelivery or EPCs transplantation or SDF-1αcombined with EPCs transplantation on hepatic ischemia reperfusion injuryed rats. Methods：EPCs were extracted from bone marrow of LEWIS rats and cultured for days for phenotype identification with flow cytometry and functional identification with confocal laser. The orthotopic liver transplantation models were established in mice，which were divided randomly into five groups：A group，sham group；B group，surgery group；C group，SDF-1α group；D group，EPCs group；E group，SDF-1α+EPCs group. The rats were euthanased after 12 h reperfusion，and the liver tissues in each group were assayed with immunohistochemistry for liver regeneration and HGF ex－pression. The protein levels of VEGF，HGF，TGF-β，Bcl-2 and Caspase-3 in each group were assayed with Western blot，and the expression of VEGF，HGF and TGF-β in liver homogenates with ELISA. Results：The CD34 and CD133 positive rates of EPCs extracted from LEWIS rats were 78.32% and 70.76 respectively，and the binding capacity of ac-LDL and UEA-1 in EPCs accounted for over 80%. The number of EPCs（79.6±6.3） induced by SDF-1αwas significant higher than that in control group（36.2±2.9）（P=0.000）. In liver tissues of each group，the immunohistochemical scores in C and D groups of HGF secretion and liver regeneration were significantly higher than those in A and B groups（P=0.000），and the immunohistochemical scores in E group was significant higher than those in C and D groups（P=0.000）. The results of proteins ex－pression showed that the expressions of VEGF，HGF，TGF-β and Bcl-2 proteins were higher in C and D groups than in A and B groups（P=0.000）；the expressions of VEGF，HGF and TGF-β proteins were significantly higher in E group than in C and D groups（P=0.000），but there was no significant difference in Bcl-2 expression among C，D，E groups. The expressions of Caspase-3 protein were significantly higher in B，C，D groups than in A group（P=0.000），but the expression of Caspase-3 was significant lower in E group than in C and D groups（P=0.000）. The ELISA got the same results. Conclusion：Exogenous SDF-1α delivery combined with EPCs transplantation produces a superior protective effect on hepatic ischemia reperfusion injuryed rats，which could induce the re－generation of liver and reduce hepatocyte apoptosis.