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

Abstract

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

CLC Number:

S829.2

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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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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