新生大鼠胰腺干细胞的分离和培养及初步鉴定

发布时间：2018-05-21 13:29

本文选题：胰腺干细胞 + 移植　；参考：《医学综述》2015年02期

【摘要】：目的探讨分离、培养及初步鉴定新生大鼠胰腺干细胞的方法。方法取30只无特定病原体(SPF)级新生大鼠的胰腺组织,用完整胰腺胶原酶消化法分离胰岛。以低糖改良Eagel培养液为基础培养液,于不同时期分别添加血清、碱性成纤维细胞生长因子、表皮细胞生长因子、胰岛素-转铁蛋白-硒添加剂、白血病抑制因子、神经干细胞原代培养的无血清培养液中的营养因子B27、神经元培养的无血清培养液中的营养因子N2等进行培养,倒置相差显微镜下观察其形态学特征及生长特性。应用免疫细胞化学法对培养不同时期的细胞进行巢蛋白(Nestin)及细胞角质蛋白19(CK-19)检测,并进行初步鉴定。结果 1新生大鼠胰腺内纤维组织少,体积小,完整胰腺胶原酶消化法简化了胰岛分离步骤,减少了污染机会,有利于进一步培养。2培养24 h可见细胞贴壁,但贴壁细胞数量较少,改用无血清培养后,贴壁细胞快速生长,10~15 d形成单层细胞汇集,细胞形态较一致。3培养所得细胞一直较多表达胰腺干细胞的标志物Nestin,随培养时间延长,表达CK-19的细胞数量逐渐增多。结论新生大鼠胰腺消化后,获得的细胞于不同时期表达胰腺干细胞的标志物Nestin、CK-19。
[Abstract]:Objective to investigate the method of isolation, culture and identification of pancreatic stem cells in neonatal rats. Methods Pancreatic tissues of 30 neonatal rats without specific pathogen were isolated by collagenase digestion. Serum, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-transferrin selenium additive, leukemia inhibitory factor were added in low glucose modified Eagel medium. The nutrition factor B27 in the serum-free culture medium of neural stem cells and N2 in the serum-free culture medium of neuron culture were cultured. The morphological characteristics and growth characteristics were observed under inverted phase contrast microscope. Nestin (Nestin) and CK-19 (cytokeratin 19) were detected by immunocytochemistry and identified preliminarily. Results 1Intrapancreatic fibrous tissue of neonatal rats was small and small. Collagenase digestion of intact pancreas simplified the separation of pancreatic islets, reduced the chance of contamination, and was beneficial to the adherence of visible cells to the cells cultured for 24 h in the further culture of .2 hours. However, the number of adherent cells was relatively small. After serum-free culture, monolayer cells converged after 10 15 days of rapid growth of adherent cells, and Nestin, a marker of pancreatic stem cells, was expressed more frequently in the cells cultured in a consistent form of 3. 3. The expression of Nestin in pancreatic stem cells was increased with the increase of culture time. The number of cells expressing CK-19 increased gradually. Conclusion after pancreatic digestion, Nestinine CK-19, a marker of pancreatic stem cells, was obtained in different stages of pancreatic digestion in neonatal rats.
【作者单位】：内蒙古自治区人民医院肿瘤外科;内蒙古医科大学中心实验室;
【分类号】：R329.2

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