大鼠骨髓间充质干细胞联合大承气汤对重症急性胰腺炎模型大鼠的保护作用及机制

Therapeutic effects of rat bone marrow-derived mesenchymal stem cells combined with Dachengqi decoction in rats with severe acute pancreatitis

目的:探讨间充质干细胞(mesenchymal stem cells,MSCs)联合大承气汤(dachengqi decoction,DcqD)对重症急性胰腺炎(severe acute pancreatitis,SAP)大鼠的治疗效果及机制.方法:将60只SD♂大鼠随机分为假手术组(sham-operated,SO),模型组(model control,MC),MSCs治疗组(MSC),DcqD治疗组(DD)和MSCs联合DcqD治疗组(MSCDD)(n=12),用1.5%脱氧胆酸钠胆胰管内逆行注射诱导SAP模型.通过Percoll密度梯度离心法与全骨髓贴壁分离法分离、培养MSCs.MSCs经大鼠尾静脉注入前用DAPI标记.24h后,荧光显微镜观察MSCs体内分布,流式细胞仪检测中性粒细胞凋亡率,测量各组血清淀粉酶、脂肪酶、肿瘤坏死因子α(tumour necrosis factor-α,TNF-α)、白介素6(interleukin-6,IL-6)、IL-10及C D4+C D25+调节性T细胞(CD4^+CD25^+regulatory T cells,CD4^+CD25^+Tregs)含量.记录各组大鼠死亡率,观察并记录各组大鼠胰腺组织病理变化及组织学评分.结果:各治疗组大鼠死亡率低于MC.荧光显微镜下,MSC胰腺管腔可见大量蓝色荧光,MC未发现.中性粒细胞凋亡指数,DD较MC显著升高(P<0.05).血清TNF-α、IL-6、淀粉酶与脂肪酶水平,MSCDD较SAP、MSC及DD显著降低(P<0.05).IL-10与CD4^+CD25^+Tregs含量,MSCDD较SAP、MSC及DD组显著升高(P<0.05),MSCDD与SO之间无统计学差异(P>0.05).各治疗组均能改善组织病理变化及降低组织学评分,尤其是MSCDD.结论:MSCs联合DcqD应用于SAP早期时优于单独治疗,并可能引起协同效果.其潜在机制可能是通过参与胰腺损伤修复,促进中性粒细胞及腺泡细胞由坏死转向凋亡,以及抑制炎症反应.

AIM：To investigate the therapeutic effects of mesenchymal stem cells（MSCs） combined with Dachengqi decoction（DcqD） in rats with severe acute pancreatitis（SAP） and to explore the underlying mechanism.METHODS：Sixty male Sprague-Dawley rats were randomly divided into sham-operated（SO）,model-control（MC）,MSCs-treatment（1.0 ×10^6 MSCs;MSC group）,DcqD-treatment（1 mL/ 100 g;DD group）,and MSCs-plusDcqD-treatment（MSCDD group） groups（n= 12）.SAP was induced in rats by retrograde infusion of 1.5%sodium deoxycholate into the biliopancreatic duct.Isolation and culture of MSCs were performed by Percoll density gradient centrifugation and plastic adherence separating.Ahead of infusion,MSCs were labelled with DAPI via the tail vein.After 24 h of administration,distribution of MSCS in vivo was observed by fluorescence microscopy.Neutrophil apoptosis was identified by flow cytometry.Serum levels of amylase,lipase,tumour necrosis factor-α（TNF-α）,interleukin（IL）-6,IL-10 and blood CD4^＋CD25^＋regulatory T cells（CD4^＋CD25^＋Tregs） percentages were determined.Mortality,pathological changes in the pancreas,and histological scores were assessed.RESULTS：The mortality rate of SAP rats was significantly lower in the treatment groups.Under a fluorescence microscope,the lumen tissue in the pancreatic sections of the MSC group exhibited bright blue fluorescence,whereas that in the MC group did not.Compared with the MC group,the MSC,DD and MSCDD groups had a significant decrease in TNF-α,IL-6,amylase,and lipase（P〈0.05）.IL-10 and CD4^＋CD25^＋Tregs percentage were significantly higher in the MSCDD group than in the SAP,MSC and DD groups.However,there was no significant difference between the MSCDD and SO groups in IL-10 or CD4^＋CD25^＋Tregs percentage（P〉0.05）.The pancreatic pathological changes and histopathologic scores were attenuated in the treatment groups,especially in the MSCDD group.CONCLUSION：The combined therapy proved to be more effective than either MSC or DD alone and may cause synergistic effects in the early stage of SAP.The potential mechanisms that might account for the favourable effects include participating in injured pancreas repair,switching from neutrophils or acinar cell necrosis to apoptosis and inhibiting overinflammatory reaction.