骨髓干细胞移植和动员对重症急性胰腺炎肾损伤的保护作用

目的 探讨骨髓间充质干细胞(MSC)移植和动员对重症急性胰腺炎(SAP)大鼠急性肾损伤的保护作用.方法 240只SD大鼠按照随机数字表法分为假手术组、模型组、干细胞移植组、干细胞动员组和联合组,每组48只.腹腔注射L-精氨酸制作SAP大鼠模型.假手术组在制作SAP大鼠模型后腹腔注射生理盐水,干细胞移植组制作SAP大鼠模型后6h经股静脉注入自体MSC 1.2 ml,干细胞动员组制作SAP大鼠模型前连续3d皮下注入重组人粒细胞集落刺激因子(G-CSF) 40 μg/kg,联合组则联合应用MSC和G-CSF.各组大鼠再按术后不同时相点分为12、24、48、72 h亚组,每组12只.在术后相应时相点观察各组大鼠的存活情况,肾脏组织的病理变化,肾小管上皮细胞Bax和Bcl-2蛋白的表达情况和细胞凋亡指数,检测血清中TNF-α、IL-6、血清尿素氮(BUN)、肌酐(Cr)、LDH、C反应蛋白(CRP)的含量.采用单因素方差分析各组间的指标,两两比较用SNK-q检验,大鼠存活情况用Fisher确切概率法.结果 假手术组大鼠全部存活.模型组大鼠术后48、72 h分别存活11只和8只.干细胞移植组、干细胞动员组和联合组术后48 h前未见大鼠死亡,术后72 h分别存活11、10和11只,与模型组比较,差异无统计学意义(P＞0.05).各治疗组术后肾脏组织病理变化均较模型组减轻,联合组损伤减轻最为明显.术后12 ～72 h肾小管上皮细胞Bax蛋白、Bcl-2蛋白、肾小管细胞凋亡指数变化情况:模型组分别为12.80±1.78 ～20.30±2.40、4.34±1.20 ～3.03±1.06、12.65％±2.31％ ～35.10％±5.54％;干细胞移植组分别为9.68±2.11～17.01±2.54、5.57±1.35～4.13±1.05、6.20％±1.53％ ～ 17.50％±2.80％;干细胞动员组分别为10.05±2.17～16.81±2.55、5.49±1.48～4.19±1.05、6.41％±1.64％ ～ 17.14％±2.27％;联合组分别为8.33±2.06～14.03±2.27、6.60±2.11 ～5.63±1.52、5.80％±1.52％～12.30％±2.43％.联合组术后24、72 h Bax蛋白,48、72 h Bc1-2蛋白,24、48、72 h凋亡指数与干细胞移植组和干细胞动员组比较,差异有统计学意义(P＜0.05);干细胞移植组与干细胞动员组比较,差异无统计学意义(P＞0.05);各治疗组术后12 ～72 h炎症因子及肾功能指标较模型组不同程度降低,以联合组最明显.联合组术后72 hTNF-α含量,48、72 h IL-6含量,48、72 h BUN含量,48、72 h Cr含量,24、48、72 h LDH含量,72 h CRP含量与干细胞移植组和干细胞动员组比较,差异有统计学意义(P＜0.05);干细胞移植组与于细胞动员组比较,差异无统计学意义(P＞0.05).结论 自体MSC移植与动员能有效减轻SPA大鼠的肾损伤,可能与MSC参与组织的病理再生修复、抗炎症及抑制细胞凋亡等机制有关.

Protective effects of bone marrow mesenchymal stem cells transplantation and mobilization for severe acute pancreatitis with acute renal injury

Objective To investigate the protective effects of bone marrow mesenchymal stem cells transplantation (MSCT) and mobilization on severe acute pancreatitis (SAP) with acute renal injury.Methods A total of 240 SD rats were randomly divided into sham operation group ( n =48 ),model control group ( n =48 ),MSCT group ( n =48),bone marrow mesenchymal stem cells mobilization (MSCM) group ( n =48) and MSCT +MSCM group ( n =48 ) according to the random number table.Rat models of SAP were made by peritoneal injection of L-arginine.Rats in the MSCT group were injected with 1.2 ml of bone marrow mesenchymai stem cells via femoral vein at 6 hours after SAP model establishment; rats in the MSCM group were subcutaneously injected with 40 μg/kg of granulocyte-colony stimulating factor (G-CSF) at 3 days before SAP model establishment; rats in the MSCT + MSCM group were injected with 1.2 ml of MSC and 40 μg/kg of G-CSF simultaneously; rats in the sham operation group were injected with equal volume of normal saline.According to different time points after operation,rats in each group were subdivided into 12 h,24 h,48 h and 72 h groups (n =12).At each time points after operation,the mortality rate,pathological changes of renal tissue,expression of Bax protein,Bcl-2 protein and apoptosis indexes of renal tubular epithelium cells were observed.The contents of tumor necrotic factor-α (TNF-α),interleukin-6 (IL-6),blood urea nitrogen (BUN),creatinine (Cr),lactate dehydrogenase (LDH) and C-reactive protein (CRP) were determined.All data were analyzed by using SNK-q test,Fisher exact probability and analysis of variance.Results All rats in the sham operation group were survived.The numbers of rats in the model control group survived at postoperative 48 hours and 72 hours were 11 and 8,respectively.No rat died at postoperative 48 hours in the MSCT group,MSCM group and MSCT + MSCM group.The numbers of rats survived at postoperative 72 hours in the MSCT group,MSCM group and MSCT + MSCM group were 11,10 and 11,which were not significantly different from the number of survived rats in the model control group (P ＞0.05).The pathological injuries of renal tissues were relieved in the MSCT group,MSCM group and MSCT + MSCM group when compared with model control group.The expression of Bax protein,Bc1-2 protein,renal tubular epithelium cell apoptosis indexes at 12-72 hours were 12.80 + 1.78-20.30 + 2.40,4.34 + 1.20-3.03 ± 1.06,12.65％ ±2.31％-35.10％ ± 5.54％ in the model control group,9.68 ± 2.11-17.01 ± 2.54,5.57 ± 1.35-4.13 + 1.05,6.20％ ± 1.53％- 17.50％ ± 2.80％ in the MSCT group,10.05 ± 2.17-16.81 ± 2.55,5.49 ± 1.48-4.19 ±1.05,6.41％± 1.64％-17.14％±2.27％ in the MSCM group,8.33 ±2.06-14.03 ±2.27,6.60 ±2.11-5.63 ±1.52,5.80％ ± 1.52％-12.30％ ±2.43％ in the MSCT + MSCT group.There were significant differences in the expressions of Bax protein at 24 and 72 hours,Bcl-2 protein at 48 and 72 hours,renal tubular epithelium cell apoptosis index at 24,48 and 72 hours between the MSCT group,MSCM group and MSCT + MSCM group ( P ＜0.05 ),but no significant difference was found between the MSCT group and the MSCM group ( P ＞ 0.05 ).The contents of TNF-α,IL-6,BUN,Cr,LDH,CRP in the MSCT group,MSCM group and MSCT + MSCM group were decreased when compared with those in the model control group,and a significant decrease of the 6 factors was observed in the MSCT + MSCM group.There were significant difference in the content of TNF-α at 72 hours,IL-6,BUN and Cr at 48 and 72 hours,LDH at 24,48 and 72 hours and CRP at 72 hours between the MSCT group,MSCM group and MSCT + MSCM group (P ＜0.05),while no significant difference was observed between the MSCT group and the MSCM group (P ＞ 0.05).Conclusion MSCT and MSCM can significantly protect acute renal injury in the progress of SAP,the probable mechanisms are pathological regeneration,anti-inflammatory effect and apoptosis inhibition of mesenchymal stem cells.