动力性三维细胞培养系统建立大鼠胰腺导管来源干细胞系的研究  

Culture of pancreatic duct-derived stem cells of rats in vitro in the dynamic three-dimension cell culturesystem

作　　者：陈旭春[1] 程颖[1] 李桂臣[1] 石蕊[1] 李晓航[1] 李达[1] 刘永锋[1] 

机构地区：[1]中国医科大学附属第一医院普通外科教研室器官移植科 ,沈阳110001

出　　处：《中华器官移植杂志》2012年第6期371-375,共5页Chinese Journal of Organ Transplantation

基　　金：卫生部行业专项基金（201002004）;教育部科学技术研究重点项目（210048）;教育部高等学校博士学科点专项科研（20092104110006）;辽宁省教育厅重点实验室项目（2008s245）

摘　　要：目的利用动力性三维细胞培养技术建立大鼠胰腺导管来源干细胞（PDSC）系。方法以大鼠胰腺组织为材料，采用V型胶原酶原位消化分离大鼠胰腺组织，通过不连续密度梯度离心使胰腺导管细胞与胰岛细胞初步分离，采用动力性三维培养技术进行培养，获得原代PDSC，并进行扩增培养、连续传代和纯化，建立PDSC系。对原代分离的PDSC进行鉴定，包括形态学的鉴定及表达特性的鉴定。结果通过胶原酶消化、不连续密度梯度离心、动力性二三维细胞培养，可分离培养出大鼠PDSC，并经过动力性三维培养，可以建立大鼠PDSC系。形态学方面，PDSC呈单个核；生长行为方面，PDSC在三维载体上沿纤维贴壁生长；表达特性方面，通过流式细胞仪分选结果显示，PDSC高表达CD29、CD73、CD90、CD105，不表达CD14、CD19、CD34、CD45，证实PDSC为问充质干细胞。结论通过采用原位胶原酶消化法、不连续密度梯度离心、动力性三维细胞培养系统，可分离培养出大鼠PDSC，成功建立PDSC细胞系。Objective To isolate, purify and identify pancreatic duct-derived stem cells （PDSCs） from the pancreatic duct of rats, and culture in the three-dimension cell culture system. Methods Adult male Wistar rats underwent perfusion with collagenase V via the pancreatic duct, then the pancreas was surgically procured, digested, followed by discontinued density gradient centrifuge to isolate ductal tissue from islets. The aeinar and ductal tissue was cultivated in serum containing medium in the three-dimension cell culture to obtain adherent cells, as PDSCs, which were expanded by consecutive passages. The morphology and characterization of PDSCs on phenotype were examined. Results PDSCs could be obtained through in situ collagenase V digestion, discontinued density gradient centrifuge, and culture in the three-dimension cell culture system. Morphologically, PDSCs had remarkable size, most with one nucleus. PDSCs grew in many layers in three-dimension cell culture system. PDSCs was revealed to express CD29, CD73, CD90, CD105, but not CD14, CD19, CD34, CD45 by FACS, in agreement with MSCs. Conclusion PDSCs of rats could be obtained through in situ collagenase V digestion, discontinued density gradient centrifuge, and culture in the three-dimension cell culture system. PDSCs lines were successfully established.

关 键 词：大鼠 胰腺管 干细胞 细胞培养技术 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]