成年大鼠胰腺导管干细胞的体外分离?培养及鉴定
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Isolation,cultivation and identification of adult rats pancreatic duct stem cells in vitro
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    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-

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    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 ± 10.5)%,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.

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陈海燕,张 强,吴 英,柴 怡,李 娟,杨 涛.成年大鼠胰腺导管干细胞的体外分离?培养及鉴定[J].南京医科大学学报(自然科学版),2008,28(3):273-277

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  • 收稿日期:2007-10-08
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