成年大鼠胰腺导管干细胞的体外分离、培养及鉴定  

Isolation,cultivation and identification of adult rats pancreatic duct stem cells in vitro

作　　者：陈海燕[1] 张强[1] 吴英[1] 柴怡[1] 李娟[1] 杨涛[1] 

机构地区：[1]南京医科大学第一附属医院内分泌科,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2008年第3期273-277,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金青年基金资助项目(30400219)

摘　　要：目的:建立成年大鼠胰腺导管干细胞的体外分离、培养及鉴定的方法。方法:V型胶原酶溶液灌注消化成年大鼠胰腺组织,并经过Ficoll密度梯度离心去除胰岛组织,培养于含10%胎牛血清的RPMI1640培养液,而后加入表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)继续培养,7天可形成单层细胞,用0.25%胰酶-EDTA消化并传代培养。取第2代细胞利用免疫荧光染色和RT-PCR方法检测CK19、Pdx-1、Nestin、insulin及glucagon的表达。结果:经胶原酶灌注消化胰腺导管上皮,再经过Ficoll密度梯度离心去除胰岛组织,可使胰腺导管细胞得到较好的纯化。免疫荧光结果示:胰腺导管细胞CK19、Pdx-1和Nestin染色呈阳性,阳性细胞率分别为(88.6±6.2)%、(84.6±8.6)%和(79.3±10.5)%,而insulin及glucagon染色为阴性。RT-PCR结果显示该细胞表达CK19、Pdx-1和Nestin基因。结论:该方法可较好的分离出胰腺导管细胞,经鉴定该法培养所获细胞具有胰腺干细胞的特性。Objective：To explore the method of isolation,cultivation and identification of adult rats pancreatic duct stem cells in vitro. Methods：The pancreas of adult rats were digested by injecting collagenase V solution and purified by removing the islets with Ficoll density gradient centrifugation. The islet-depleted tissue was cultured in RPMI1640 with 10% fetal calf serum,and the medium was supplemented with EGF and bFGF for 7 days, then the cells could form a monolayer. 0.25% Trypsin-EDTA were used to detach the cells, which were serial-subcultivation. Cells from the second passage （P2） were used to detect the expression of CK19,Pdx-1, Nestin, insulin and glucagon with methods of immunofluorescence staining and RT-PCR. Results： Through collagenase solution injecting and digesting the pancreatic duct,and depleting the islet tissue after Ficoll density gradient centrifugation,the pancreatic duct cells could get a better purification. The results of immunofluorescence staining revealed that the expression of CK19,Pdx-1 and Nestin of P2 cells were positive, and the rate of positive cells were（88.6±6.2）%, （84.6 ± 8.6）% and （79.3± lO.5）%,respectively, while insulin and glucagon staining were negative. The results of RT-PCR demomstrated that the cells also expressed CK19,Pdx-1 and Nestin. Conclusion：This method can isolate the pancreatic duct cells well, and the cells obtained have the character of stem cells through identification.

关 键 词：胰腺 导管细胞 干细胞 细胞培养 

分 类 号：Q813.1[生物学—生物工程]