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

doi:10.16431/j.cnki.1671-7236.2022.05.024

中国畜牧兽医 ›› 2022, Vol. 49 ›› Issue (5): 1840-1851.doi: 10.16431/j.cnki.1671-7236.2022.05.024

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

唐琳¹, 张军芳¹, 王英¹, 孙斌¹, 王恩泽¹, SHIN Jongsuh², 郭盼盼¹, 金鑫¹, 严昌国¹, 李香子¹, 李强^1,3

1. 延边大学, 东北寒区肉牛科技创新教育部工程研究中心, 吉林省肉牛科学与产业技术重大需求协同创新中心, 延吉 133002;
2. 韩国江原大学校动物生命科学系, 春川 24341;
3. 延边大学农学院动物医学系, 延吉 133002

收稿日期:2021-09-22 出版日期:2022-05-05 发布日期:2022-04-29
通讯作者: 李强 E-mail:0000008569@ybu.edu.cn
作者简介:唐琳,E-mail:1422906017@qq.com。
基金资助:
国家自然科学基金资助项目（31660667）；吉林省教育厅科学技术研究项目（JJKH20210590KJ）；延边大学博士启动基金（602020078）

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

TANG Lin¹, ZHANG Junfang¹, WANG Ying¹, SUN Bin¹, WANG Enze¹, SHIN Jongsuh², GUO Panpan¹, JIN Xin¹, YAN Changguo¹, LI Xiangzi¹, LI Qiang^1,3

1. Ministry of Education, Jilin Beef Cattle Science and Industrial Technology Major Demand Collaborative Innovation Center, Engineering-Research Center of North-East Cold Region Beef Cattle Science & Technology Innovation, Yanbian University, Yanji 133002, China;
2. Department of Animal Science, Kangwon National University, Chuncheon 24341, Korea;
3. Department of Veterinary Medicine, Agriculture College of Yanbian University, Yanji 133002, China

Received:2021-09-22 Online:2022-05-05 Published:2022-04-29

摘要/Abstract

摘要： 【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、20 μg/mL脂多糖(LPS)，10、100 mmol/L雨蛙肽(Caerulein)以及10 μg/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，10 μg/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。

关键词: 犬脂肪来源的间充质干细胞(cAd-MSCs); 重症急性胰腺炎(SAP); 胰腺腺泡细胞(PACs); 雨蛙肽; 脂多糖(LPS); 内质网应激

Abstract: 【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】 ①Type Ⅰ collagenase 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 type Ⅰ collagenase.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 20 μg/mL lipopolysaccharide (LPS),10 and 100 mmol/L Caerulein,and 10 μg/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-α gene 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-1α genes 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 10 μg/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-α gene in P group was extremely significantly increased (P<0.01).Compared with P group,the expression of the TNF-α gene 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.

Key words: canine adipose-derived mesenchymal stem cells (cAd-MSCs); severe acute pancreatitis (SAP); pancreatic acinar cells (PACs); Caerulein; lipopolysaccharide (LPS); endoplasmic reticulum stress

中图分类号:

S829.2

唐琳, 张军芳, 王英, 孙斌, 王恩泽, SHIN Jongsuh, 郭盼盼, 金鑫, 严昌国, 李香子, 李强. 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[J]. 中国畜牧兽医, 2022, 49(5): 1840-1851.

TANG Lin, ZHANG Junfang, WANG Ying, SUN Bin, WANG Enze, SHIN Jongsuh, GUO Panpan, JIN Xin, YAN Changguo, LI Xiangzi, LI Qiang. Anti-apoptotic Effect of Canine Adipose-derived Mesenchymal Stem Cells on Endoplasmic Reticulum Stress Model of Severe Acute Pancreatitis in vitro[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1840-1851.

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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

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