成人胰腺导管干细胞的体外分离、纯化、培养及鉴定  

Isolation,Purification,Cultivation and Identification of Adult Human Pancreatic Duct Stem Cells in Vitro

作　　者：李娟[1] 陈海燕[1] 吴英[1] 张强[1] 于小竹[1] 王云[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：《现代生物医学进展》2009年第11期2024-2026,2086,F0002,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金资助项目(NO30400219;30671010)

摘　　要：目的:建立人胰腺导管干细胞的体外分离、纯化、培养及鉴定的方法。方法:胶原酶分次消化剪切的人胰腺组织,经过Ficoll密度梯度离心后去除胰岛组织,培养于含10%胎牛血清的CMRL1066培养液中,7-10天可行成单层细胞,用0.25%胰酶-EDTA消化并传代培养。取2-3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:经过胶原酶消化、Ficoll密度梯度离心及后续的培养,去除了胰岛组织及外分泌腺,可获得较纯化的鹅卵石样的胰腺导管细胞。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(87.5±6.2)%、(77.5±8.6)%和(50.9±9.5)%,而Insulin染色为阴性。RT-PCR结果显示该细胞表达CK19、Pdx-1和Nestin基因,而未观察到Insulin及Glucagon基因的表达。结论:该方法可较好的分离纯化出人胰腺导管细胞,经鉴定获得细胞具有胰腺干细胞的特性。Objective： To explore the method of isolation, purification, cultivation and identification of adult human pancreatic duct stem cells in vitro. Methods： The pancreastic tissue were digested by injecting collagenase P solution and purified by removing the islets with Ficoll density gradient centrifugation. The islet-depleted tissue was cultured in CMRL1066 with 10% fetal calf serum, then the cells could form a monolayer after 7-10 days. 0.25% Trypsin-EDTA was used to detach the cells, which were serial-subcultivation. Cells from the second passage （P2-3） 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 digesting the pancreatic tissue, and depleting the islet tissue after Ficoll density gradient centrifugation, the pancreatic duct cells could get a better purification. The results of immunofiuorescence staining revealed that the expression of CK19,Pdx-1 and Nestin of P2-3 cells were positive, and the rate of positive cells were （87.5± 6.2）%,（77.5± 8.6）% and （50.9± 9.5）%, respectively, while insulin and glucagon staining were negative. The results ofRT-PCR demomstrated that the cells also expressed CK19, Pdx-1 and Nestin. Conclusions： This method can isolate the pancreatic duct cells well, and the cells obtained have the character of stem cells through identification.

关 键 词：干细胞 胰腺 导管细胞 细胞培养 

分 类 号：R587.1[医药卫生—内分泌] Q95-3[医药卫生—内科学]