研究论文 RESEARCH PAPER

山羊胰腺腺泡细胞体外分离、纯化、培养和鉴定

  • 张天西 ,
  • 燕爱飞 ,
  • 杨超 ,
  • 程艳 ,
  • 贺志雄 ,
  • 谭支良
展开
  • 1. 中国科学院亚热带农业生态研究所, 亚热带农业生态过程重点实验室, 畜禽养殖污染控制与资源化技术国家工程实验室, 湖南省畜禽健康养殖工程技术研究中心, 中国科学院亚热带农业生态研究所公共技术中心, 长沙 410125;
    2. 中国科学院大学, 北京 100049
张天西(1995—),男,贵州六盘水人,硕士研究生,从事反刍动物营养与胰腺外分泌功能研究。E-mail:zhangtianxi20@mails.ucas.ac.cn

收稿日期: 2022-03-22

  网络出版日期: 2022-10-17

基金资助

中国科学院战略性先导科技专项(XDA26040304);创新省份建设专项(2019RS3021)

Isolation, Purification, Culture and Identification of Goat Pancreatic Acinar Cells in Vitro

  • ZHANG Tianxi ,
  • YAN Aifei ,
  • YANG Chao ,
  • CHENG Yan ,
  • HE Zhixiong ,
  • TAN Zhiliang
Expand
  • 1. Public Technology Center of Institute of Subtropical Agriculture, Chinese Academy of Sciences, Hunan Research Center of Livestock & Poultry Sciences, National Engineering Laboratory for Pollution Control and Waste Utilization in Livestock and Poultry Production, Key Laboratory for Agro-Ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha 410125, China;
    2. University of Chinese Academy of Sciences, Beijing 100049, China

Received date: 2022-03-22

  Online published: 2022-10-17

摘要

本试验旨在建立湘东黑山羊胰腺腺泡细胞的原代培养方法,为研究反刍动物胰腺外分泌功能提供细胞模型。试验利用胶原酶Ⅳ分离山羊胰腺腺泡细胞,开展了胶原Ⅳ的适宜浓度和消化时间、与0.25%胰蛋白酶消化效率对比、腺泡细胞纯化以及鉴定研究。结果表明:1)采用1.0 mg/mL胶原酶Ⅳ对湘东黑山羊胰腺组织消化20 min时,腺泡细胞分离效果最佳。应用此条件消化得到2.275×106个/mL总细胞数及91%的细胞活率。2)采用差时消化、差速贴壁及细胞刮取等方法纯化原代细胞,得到活性良好且形态均一成簇生长呈"鹅卵石"状的山羊胰腺腺泡细胞。3)纯化后腺泡细胞能够稳定传5代以上,且形态不发生改变。4)通过免疫荧光技术运用兔抗羧肽酶A1(CPA1)作为一抗对第1代和第5代山羊胰腺腺泡细胞进行鉴定,结果显示阳性。综上所述,本方法快速地分离了山羊胰腺腺泡细胞,并成功地纯化、鉴定和稳定培养了分离的山羊胰腺腺泡细胞。

本文引用格式

张天西 , 燕爱飞 , 杨超 , 程艳 , 贺志雄 , 谭支良 . 山羊胰腺腺泡细胞体外分离、纯化、培养和鉴定[J]. 动物营养学报, 2022 , 34(9) : 6088 -6096 . DOI: 10.3969/j.issn.1006-267x.2022.09.061

Abstract

The purpose of this study was to establish a primary culture method of pancreatic acinar cells in Xiangdong black goats, and to provide a cell model for the study of pancreatic exocrine function in ruminants. In this experiment, collagenase Ⅳ was used to isolate goat pancreatic acinar cells, and the optimum concentration and digestion time of collagenase Ⅳ, comparison with 0.25% trypsin digestion efficiency, purification and identification of acinar cells were studied. The results showed as follows:1) when 1.0 mg/mL collagenase Ⅳ was used to digest 20 min in the pancreatic tissue of Xiangdong black goats, the isolation effect of acinar cells was the best. The total cell number of 2.275×106 cells/mL and the cell viability of 91% were obtained by digestion under this condition. 2) The primary cells were purified by differential digestion, differential adhesion and cell scraping, and the goat pancreatic acinar cells with good activity and "cobblestone" shape were obtained. 3) After purification, the acinar cells can be stably passed on for more than 5 generations, and the morphology does not change. 4) The rabbit anti-carboxypeptidase A1 (CPA1) was used as the first antibody to identify the first and fifth generation goat pancreatic acinar cells by immunofluorescence technique, and the results were positive. To sum up, the goat pancreatic acinar cells are quickly isolated by this method, and the isolated goat pancreatic acinar cells are successfully purified, identified and stably cultured.

参考文献

[1] HARMON D L, YAMKA R M, ELAM N A.Factors affecting intestinal starch digestion in ruminants:a review[J].Canadian Journal of Animal Science, 2004, 84(3):309-318.  
[2] OWENS F N, ZINN R A, KIM Y K.Limits to starch digestion in the ruminant small intestine[J].Journal of Animal Science, 1986, 63(5):1634-1648.  
[3] MORISSET J.Seventy years of pancreatic physiology:take a look back[J].Pancreas, 2014, 43(8):1172-1184.  
[4] SINGH L, BAKSHI D K, VASISHTA R K, et al.Primary culture of pancreatic (human) acinar cells[J].Digestive Diseases and Sciences, 2008, 53(9):2569-2575.  
[5] GOUT J, POMMIER R M, VINCENT D F, et al.Isolation and culture of mouse primary pancreatic acinar cells[J].Journal of Visualized Experiments, 2013, 78:e50514.
[6] ZHAO X, HAN J, TANG C.Primary culture of porcine pancreatic acinar cells[J].Journal of the Pancreas, 2001, 2(2):78-82.
[7] JAYAVENI S, NITHYANANDHAM K, ROSE C.In vitro secretion of zymogens by bovine pancreatic acini and ultra-structural analysis of exocytosis[J].Biochemistry and Biophysics Reports, 2016, 5:237-245.
[8] MORISSET J, LAINÉ J, BOURASSA J, et al.Presence and localization of CCK receptor subtypes in calf pancreas[J].Regulatory Peptides, 2003, 111(1/2/3):103-109.
[9] ANTONUCCI L, FAGMAN J B, KIM J Y, et al.Basal autophagy maintains pancreatic acinar cell homeostasis and protein synthesis and prevents ER stress[J].Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(45):E6166-E6174.
[10] SANS M D, BRUCE J, WILLIAMS J A.Regulation of pancreatic exocrine function by islet hormones[Z].Pancreapedia:Exocrine Pancreas Knowledge Base, 2020.
[11] HIROTA M, OHMURAYA M, BABA H.The role of trypsin, trypsin inhibitor, and trypsin receptor in the onset and aggravation of pancreatitis[J].Journal of Gastroenterology, 2006, 41(9):832-836.  
[12] WILLIAMS J A.Isolation of rodent pancreatic acinar cells and acini by collagenase digestion[Z].Pancreapedia:Exocrine Pancreas Knowledge Base, 2010.
[13] TRIA S, JIMISON L H, HAMA A, et al.Sensing of EGTA mediated barrier tissue disruption with an organic transistor[J].Biosensors, 2013, 3(1):44-57.  
[14] AMSTERDAM A, JAMIESON J D.Structural and functional characterization of isolated pancreatic exocrine cells[J].Proceedings of the National Academy of Sciences of the United States of America, 1972, 69(10):3028-3032.  
[15] LOGSDON C D, JI B A.The role of protein synthesis and digestive enzymes in acinar cell injury[J].Nature Reviews.Gastroenterology & Hepatology, 2013, 10(6):362-370.  
[16] GUO L, TIAN H B, YAO J H, et al.Leucine improves α-amylase secretion through the general secretory signaling pathway in pancreatic acinar cells of dairy calves[J].American Journal of Physiology:Cell Physiology, 2020, 318(6):C1284-C1293.
[17] ASSI M, DAUGUET N, JACQUEMIN P.DIE-RNA:a reproducible strategy for the digestion of normal and injured pancreas, isolation of pancreatic cells from genetically engineered mouse models and extraction of high quality RNA[J].Frontiers in Physiology, 2018, 9:129.
[18] WALDSCHMIDT-LEITZ E, PURR A.Vber proteinase und carboxy-polypeptidase aus pankreas (Ⅹ Ⅴ Ⅱ. Mitteilung zur Spezifität tierischer Proteasen)[J].Berichte der Deutschen Chemischen Gesellschaft(A and B Series), 1929, 62(8):2217-2226.  
[19] UHLIG R, CONTRERAS H, WEIDEMANN S, et al.Carboxypeptidase A1(CPA1) immunohistochemistry is highly sensitive and specific for acinar cell carcinoma (ACC) of the pancreas[J].The American Journal of Surgical Pathology, 2022, 46(1):97-104.  
[20] SAID S, KURTIN P, NASR S, et al.CPA1 and REG1a as new markers for pancreatic acinar cell carcinoma[C]//Laboratory investigation.New York:Nature Publishing Group, 2020.
文章导航

/