犬脂肪来源间充质干细胞对重症急性胰腺炎体外内质网应激模型的抗凋亡作用

Anti-apoptotic Effect of Canine Adipose-derived Mesenchymal Stem Cells on Endoplasmic Reticulum Stress Model of Severe Acute Pancreatitis in vitro

【目的】探究犬脂肪组织来源的间充质干细胞(cAd-MSCs)对重症急性胰腺炎(SAP)体外模型的抗凋亡作用,以期为利用干细胞治疗胰腺炎提供理论指导。【方法】①用Ⅰ型胶原酶消化分离cAd-MSCs,用流式细胞术鉴定其干细胞标志物CD29、CD34、CD44、CD45、CD73和CD90的表达,用成脂、成骨和成软骨分化来鉴定其多向分化潜能;②用Ⅰ型胶原酶从小鼠胰腺组织中分离胰腺腺泡细胞(PACs),用实时荧光定量PCR检测PACs及胰腺组织中胰腺导管特异性基因CK19、β-胰岛细胞特异性细胞基因Insulin-1、α-胰岛细胞特异性基因Glucagon及PAC特异性基因PTF-1α、CPA-1、AMY2B的表达;③以10、20μg/mL脂多糖(LPS),10、100 mmol/L雨蛙肽(Caerulein)以及10μg/mL LPS+100 mmol/L Caerulein处理PACs,不添加药物培养的细胞为对照组,培养24 h后使用CCK-8检测PACs存活率,筛选体外构建内质网应激模型的最佳处理组(即模型组,P);用CCK-8检测对照组(Naive)及模型组(P)细胞0、2、4、8、12和24 h的存活率,Western blotting检测P组细胞内质网应激相关蛋白的相对表达量;④为确定cAd-MSCs对PACs的作用方式,试验分为PAC组(仅PACs,Naive)、P组、间接共培养组(IC)、直接共培养组(构建PAC模型时与cAd-MSCs直接共培养,DC),用实时荧光定量PCR检测肿瘤坏死因子(TNF-α)基因在PACs中的表达水平;⑤在间接共培养系统中,将细胞分为空白对照组(仅PACs,Naive)、对照组(PACs与cAd-MSCs共培养,C)、P组及试验组(药物处理的PACs与cAd-MSCs细胞共培养,T),细胞培养12 h后,通过实时荧光定量PCR、Western blotting检测各组细胞内质网应激相关基因及蛋白表达水平的变化,并用TUNEL法检测各组细胞的凋亡情况。【结果】①分离培养的cAd-MSCs呈现成纤维样细胞形态,高表达干细胞标志物CD29、CD44、CD73及CD90,不表达CD34和CD45,且具备成脂、成骨、成软骨分化能力;②分离的原代PACs呈鹅卵石样,与胰腺组织相比较,AMY2B、CPA1、PTF1α基因的相对表达量均显著增加(P<0.05),CK19、Glucagon、Insulin-1基因的相对表达量均极显著降低(P<0.01)。③与对照组相比,10μg/mL LPS+100 mmol/L Caerulein组细胞存活率极显著降低(P<0.01),因此选为构建内质网应激模型的最佳处理组。与对照组相比,4 h时P组PACs的细胞存活率显著降低(P<0.05),8、12、24 h均极显著降低(P<0.01);Western blotting检测结果显示,Grp78、CHOP、Caspase-12蛋白的表达水平自4 h开始均极显著增加(P<0.01)。④与Naive组相比,P组TNF-α基因的表达水平极显著增加(P<0.01);与P组相比,IC和DC组TNF-α基因表达水平均极显著降低(P<0.01),后续用间接共培养系统进行试验。⑤在间接共培养系统中,与P组相比,T组Grp78、Caspase-12和CHOP mRNA及蛋白的相对表达量均极显著降低(P<0.01)。TUNEL检测结果显示,T组阳性细胞数明显减少。【结论】本试验成功构建SAP体外内质网应激模型,且证明cAd-MSCs对PACs内质网应激具有调控及保护作用。

【Objective】The aim of this study was to investigate the anti-apoptotic effect of canine adipose-derived mesenchymal stem cells(cAd-MSCs)on in vitro model of severe acute pancreatitis(SAP),in order to provide a theoretical guide for the treatment of pancreatitis with stem cells.【Method】①TypeⅠcollagenase digestion method was used to separate cAd-MSCs,the expression of stem cell markers CD29,CD34,CD44,CD45,CD73 and CD90 was identified by flow cytometry,and its multidirectional differentiation potential was identified by adipogenic,osteogenic and chondrogenic differentiation.②Pancreatic acinar cells(PACs)were isolated from mouse pancreatic tissue with typeⅠcollagenase.The expression of pancreatic duct-specific gene CK19,β-islet cell-specific gene Insulin-1,α-islet cell-specific gene Glucagon and PAC-specific genes PTF-1α,CPA-1 and AMY2B in PACs and pancreatic tissues were detected by Real-time quantitative PCR.③PACs were treated with 10 and 20μg/mL lipopolysaccharide(LPS),10 and 100 mmol/L Caerulein,and 10μg/mL LPS+100 mmol/L Caerulein,the cells cultured without drugs were used as the control group.The survival rate of PACs was detected by CCK-8 at 24 h to screen the optimal treatment group for constructing endoplasmic reticulum(ER)model in vitro(model group,P).The survival rate of control group(Naive)and P group were detected by CCK-8 at 0,2,4,8,12 and 24 h,the expression of ER stress-related proteins in P group were detected by Western blotting.④To determine the mode of action of cAd-MSCs on PACs,the experiment was divided into PAC group(only PACs,Naive),P group,indirect co-culture group(IC)and direct co-culture group(DC).The expression of TNF-αgene was detected by Real-time quantitative PCR.⑤In the IC system,cells were divided into blank control group(PACs only,Naive),control group(PACs co-cultured with cAd-MSCs),P group and experimental group(drug-treated PACs co-cultured with cAd-MSCs,T).The expression of ER stress-related genes and proteins were detected by Real-time quantitative PCR and Western blotting at 12 h.The apoptosis of cells in each group was detected by TUNEL assay.【Result】①The isolated and cultured cAd-MSCs showed fibroblast-like cell morphology,highly expressed stem cell markers CD29,CD44,CD73 and CD90,did not express CD34 and CD45,and had the ability of adipogenic,osteogenic and chondrogenic differentiation.②The isolated PACs showed cobblestone-like morphology,and compared with pancreatic tissue,the expression of AMY2B,CPA1,PTF-1αgenes were significantly increased(P<0.05),CK19,Glucagon,and Insulin-1 were extremely significant decreased(P<0.01).③Compared with control group,the cell viability in 10μg/mL LPS+100 mmol/L Caerulein group was extremely significant decreased(P<0.01),which was selected as the best treatment group for building an ER stress model.Compared with control group,the cell viability of PACs in P group was significantly decreased at 4 h(P<0.05),and extremely significant decreased at 8,12 and 24 h(P<0.01).Western blotting results showed that the expression of Grp78,CHOP and Caspase-12 protein were increased significantly from 4 h(P<0.01).④Compared with Naive group,the mRNA expression of TNF-αgene in P group was extremely significantly increased(P<0.01).Compared with P group,the expression of the TNF-αgene in IC and DC groups was extremely significantly decreased(P<0.01).The subsequent experiments were carried out with IC system.⑤In the IC system,compared with P group,the relative mRNA and protein expressions of Grp78,Caspase-12 and CHOP in T group were extremely significantly decreased(P<0.01).The TUNEL results showed that the number of positive cells in T group was obviously reduced.【Conclusion】In this experiment,ER stress model of SAP in vitro were sucessfully constructed,and it was confirmed that cAd-MSCs could protect the ER stress of PACs.