骨髓间充质干细胞移植修复大鼠肝缺血再灌注损伤最佳剂量的研究

The Optimal Dosage of Bone Marrow Mesenchymal Stem Cells Transplantation for Treatment of Hepatic Ischemia-Reperfusion Injury in Rats

目的探讨骨髓间充质干细胞(BMSCs)移植修复大鼠肝缺血再灌注损伤的最佳剂量,为细胞移植修复肝缺血再灌注损伤提供前期的实验依据。方法采用全骨髓直接贴壁法分离培养BMSCs,取第4代细胞进行移植。将30只健康雄性Wistar大鼠建立肝缺血再灌注损伤模型后,随机分成空白对照组、5×105个组、1×106个组、2×106个组及3×106个组5组,每组6只。后4组大鼠均在建立肝缺血再灌注损伤模型后,立即抽取200μL细胞悬液(数量分别为5×105、1×106、2×106及3×106个),通过门静脉注射;空白对照组大鼠注射等体积的PBS溶液。于移植术后24 h采血检测血清天门冬氨酸氨基转移酶(AST)和丙氨酸氨基转移酶(ALT)水平;取肝脏组织检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性及核因子-κB(NF-κB)p65蛋白的表达,并行HE染色。结果1×106个组和2×106个组与空白对照组、5×105个组及3×106个组比较,其AST、ALT及MDA水平均降低(P<0.05),而SOD活性均增高(P<0.05);NF-κB p65蛋白的表达均明显下调;肝组织的病理学损伤均改善。但该2组的AST、ALT、MDA及SOD水平比较差异均无统计学意义(P>0.05),且肝组织的病理学改变也无明显差别。结论大鼠BMSCs移植治疗肝缺血再灌注损伤的最佳剂量为1×106个细胞。

Objective To investigate the optimal dosage of bone marrow mesenchymal stem cells （BMSCs） transplantations for treatment of hepatic ischemia-reperfusion injury in rats, and to provide prophase experimental basis for it. Methods BMSCs of Wistar rats were isolated and cultivated by bone marrow adherent culture method. BMSCs of the fourth generation were prepared for cell transplantation. Thrity hepatic ischemia-reperfusion injury models of male Wistar rats were successfully established, and then were randomly divided into blank control group, 5 ×10^5 group, 1 ×10^6 group, 2 ×10^6 group, and 3 ×10^6 group, each group enrolled 6 rats. The 200 μL cell suspension of BMSCs were transfused into the portal vein with number of 5×10^5, 1 ×10^6, 2×10^6, and 3×10^6 separately in rats of later 4 groups, and rats of blank control group were injected with phosphate buffered saline of equal volume. At 24 hours after cell transplantation, blood samples were collected to test aspartate aminotransferase （AST） and alanine aminotransferase （ALT）, liver tissues were obtained to test malonaldehyde （MDA）, superoxide dismutase （SOD）, and nuclear factor-κB （NF-κB） p65 protein. Liver tissues were also used to perform HE staining to observe the pathological changes. Results Compared with blank control group, 5 ×10^5 group, and 3 ×10^6 group, the levels ofAST, ALT, and MDA were lower （P〈0.05） while activity levels of SOD were higher （P〈 0. 05） in 1×10^6 group and 2×10^6 group, and expression levels of NF-κB p65 protein were lower with the pathological injury of liver tissue improved, but there were no significant differences on levels of AST, ALT, MDA, and SOD （P 〉 0.05）, and both of the 2 groups had the similar pathological change. ConclusionThe optimal dosage of the BMSCs transplantations after hepatic ischemia-reperfusion injury is 1×10^6.