胎猪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 porcine fetus pancreatic duct stem cells in vitro

作　　者：李云龙[1] 郭欣[2] 杨晓波[1] 黄跃南[1] 

机构地区：[1]哈尔滨医科大学附属第二医院普外科,黑龙江哈尔滨150081 [2]哈尔滨医科大学附属第一医院普外科,黑龙江哈尔滨150001

出　　处：《哈尔滨医科大学学报》2012年第3期222-226,共5页Journal of Harbin Medical University

基　　金：黑龙江省自然科学基金资助项目(D2007-05)

摘　　要：目的建立胎猪胰腺导管干细胞的体外分离、纯化、培养及鉴定的方法。方法采用胰酶和胶原酶Ⅳ、P消化法分离获得细胞,在含10%胎牛血清的M199培养基中行导管上皮细胞原代培养,而后将血清浓度提高到20%以上继续培养,3～4天后至每个小圆细胞形成一个集落;采用逆转录-多聚酶链反应(RT-PCR)的方法对获得的细胞检测CK19、Pdx-1、NeuroD/Beta2、insulin、Glut-2等基因的表达。结果成功分离获得胎猪胰腺导管细胞,并证实该细胞为胎猪导管源胰腺干细胞。经检测,细胞表达CK19,Pdx-1,Glut-2,NeuroD/Beta2等胰腺干细胞的标志,而insulin、Neurogenin3阴性表达。结论该方法可较好地分离纯化出胎猪胰腺导管细胞,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 porcine fetus pancreatic duct stem cells in vitro. Methods The pancreatic tissues were digested by injecting pancreatin or collagenase IV, P solution and purified by removing the islets with Ficoll density gradient centrifugation. The islet-depleted tissue was cultured in M199 with 10% fetal calf serum, then these cells proliferated quickly when medium was changed to M199 with 20% serum and formed cell clones after 3～4 days. The obtained cells were detected expression of CK19, Pdx-1, NeuroD/Beta2, insulin, Glut-2 and Neurogenin3 by reverse polymerase chain reaction （RT-PCR）. Results The porcine pancreatic duct ceils were successfully separated, and demonstrated to be porcine fetus pancreatic duet stem cells. The results of RT-PCR showed that the expression of CK19 ,Pdx-1, Glut-2 and NeuroD/Beta2 of ceils were positive,while insulin and Neurogenin3 were negative. Conclusion This method can isolate the pancreatic duct cells well, and the obtained cells have the characteristics of stem cells.

关 键 词：胎猪 导管细胞 干细胞 胰岛素 

分 类 号：R-332[医药卫生] R587.1