摘要目的 探讨骨髓间充质干细胞(bone marrow mesenchymal stem cells,mMSCs)在实验性ANP并发多脏器功能障碍综合征(MODS)中的作用.方法 50只体重180～220 g SD雌性大鼠按完全随机法分为5组,每组10只.对照组不做任何处理;ANP组由腹腔注射L-精氨酸诱导;自体mMSCs回输组在ANP诱导后1 d将核染料Hoechst 33258标记的自体mMSCs回输到自体骨髓腔;异体mMSCs移植组在造模前3 d通过尾静脉移植雄性mMSCs;粒细胞集落因子(G-CSF)组在造模前3 d将标记的自体mMSCs回输到自体骨髓腔,并连续3 d注射G-CSF 40μg/kg体重.造模后3 d处死大鼠,观察胰腺、肝脏、肠道大体及组织学改变,部分组织冷冻切片,选择有黄绿色荧光的切片.对胰腺、肝脏切片行CK19免疫荧光染色;对肠道切片行Pan Cytokemtin免疫荧光染色.记录造模后3 d内的死亡率.结果 对照组组织结构正常,无死亡.ANP组和自体mMSCs回输组造模后3 d见大量血性腹水,胰周脂肪皂化,胰腺结构破坏、坏死、炎细胞浸润;肝脏及肠道等多脏器受累、坏死;死亡率均为40%.异体mMSCs移植组和G-CSF组在造模后3 d见腹水量明显减少,胰腺轻度水肿,腺泡小叶完整、间质无渗出、出血坏死减轻、炎细胞浸润较轻;肝脏及肠道等多脏器损伤减轻;死亡率均为10%.对照组和ANP组的胰腺、肝脏、肠道切片未见黄绿色荧光;自体mMSCs回输组偶见黄绿色荧光,但免疫荧光染色阴性;异体mMSCs移植组和G-CSF组黄绿色荧光多见,且胰腺、肝脏组织中可见CK19阳性细胞,肠道组织中可见PanCytokeratin阳性细胞.结论 mMSCs参与ANP并发MODS时的病理修复,异体mMSCs移植和自体mMSCs动员对其具有保护作用.

abstractsObjective To investigate the role of bone marrow mesenchymal stem cells (MSC) in early acute necrotizing pancreatitis (ANP) complicated with multiple organ dysfunction (MODS). Methods Fifty Sprague-Dawley (SD) rats weighing 180 ～ 220 g were randomly assigned into 5 groups (n = 10). Group A was the normal negative control without any treatment, ANP was induced in Group B rats by intraperitoueal injections with L-arginine 2.5 g/kg body weight twice, Group C received Hoechst33258 labeled autologous bone marrow mMSCs one day after ANP model induction, Group D was the group of mMSCs transplantation, in which the mice were given the isolated mMSCs via the tail vein 3 days prior to the ANP induction, Group E was the stem cell mobilized group treated by the injection of granulocyte-colony stimulating factor (G-CSF) into rats 33258 and transplanted into the arigiual cavity or via the tail vein. Three days after the injury was induced, the rats were sacrificed, the tissues of pancreas, liver and intestine were harvested and the morphological changes were examined. A part of samples were snap-frozen and the presence of labeled MSC in the cryostat prepared was examined directly by fluorescence microscopy. The positive sections were chosen for further immunofluorescence assay. Anti-CK19 immunofluorescence staining was performed in pancreatic and liver sections;and Pan Cytokeratin immunofluorescence staining were performed in intestinal sections. The mortality rates within 30 days were recorded. Results The control group had normal tissue structures, with no death. 3 hour after ANP induction, there were mass hemorrhagic ascites, pefi-pancreas saponification, pancreatic disorganization, necrosis, phlogocyte infiltration;liver and intestine involvement and necrosis in rats in Group B and C with a mortality rate 40%. 3 hour after ANP induction, there were less ascites, mild pancreatic edema, intact acinns lobula, no interstitial tissue exudation, less pancreatic hemorrhage and necrosis, less phlogocyte infiltration;less liver and intestine injuries in rats in Group D and E with a mortality rate 10%. The pancreatic, liver, and intestinal sections in the control group and ANP group had no flavo green fluorescence;while the sections in Group C had some flavo green fluorescence but they were negative for immunofluorescence staining;in addition, the sections in Group D and E had plenty of flavo green fluorescence and CK19 (+) cells were present in pancreatic and liver tissues and Pan Cytokeratin (+) cells were present in intestinal tissues. Conclusions MSC played an important role in the process of pathological repair in ANP complicated with MODS, autologous or transplanted MSC had protective effects.