山羊胰腺腺泡细胞体外分离、纯化、培养和鉴定

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

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。

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,collagenaseⅣwas used to isolate goat pancreatic acinar cells,and the optimum concentration and digestion time of collagenaseⅣ,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 collagenaseⅣwas 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.