胰腺干细胞负向筛选分子的鉴定  

Identification of marker molecules for negative screening of pancreatic stem cells

作　　者：冯锐成[1] 郎延贺[1] 梁洋[1] 吴莹[1] 滕春波[1] 

机构地区：[1]东北林业大学生命科学学院,黑龙江省哈尔滨市150040

出　　处：《中国组织工程研究》2013年第10期1785-1792,共8页Chinese Journal of Tissue Engineering Research

基　　金：黑龙江省自然科学基金面上项目(C201215);哈尔滨市科技创新人才研究专项优秀学科带头人基金资助项目(2012RFXXS048)~~

摘　　要：背景:利用胰腺干/祖细胞体外增殖分化形成β细胞是细胞替代疗法治疗糖尿病的潜在细胞来源之一。由于缺少有效的筛选分子,胰腺干细胞的分离及特性研究进展缓慢。目的:观察植物凝集素花生凝集素、菜豆凝集素和热稳定抗原对胰腺细胞的标记情况,以期找到胰腺干细胞的负向筛选分子。方法:采用荧光组织化学方法对植物凝集素(花生凝集素、菜豆凝集素)和细胞表面分子热稳定抗原抗体在成体小鼠胰腺、胚胎胰腺及切除后再生胰腺组织中进行化学及荧光组织化学检测;流式细胞仪分析负向筛选分子对胰腺细胞分群情况的影响;应用细胞培养方法对不同群细胞进行体外培养和观察。结果与结论:花生凝集素对胚胎胰腺中分化的腺泡细胞能较好标记,但不标记未分化的胰腺干/祖细胞及前体细胞;菜豆凝集素对未分化和分化的胚胎期胰腺细胞均有标记,热稳定抗原能标记正在分化的胚胎胰腺祖细胞、前体细胞和已分化胰腺细胞。在成体胰腺中,花生凝集素和菜豆凝集素均能标记胰腺腺泡和部分导管细胞,但不能标记胰岛细胞;热稳定抗原能特异标记腺泡细胞、导管细胞、血管内皮细胞、血细胞及再生胰腺新生区的导管样结构。PNA-/lowHSA-/low细胞占总细胞量的1.6%,对其培养观察发现,其中富含体积较小、三角形或多角形的上皮细胞,这些细胞可连续传代。提示协同使用花生凝集素与热稳定抗原可以筛除大部分分化的胰腺细胞及部分非胰腺细胞,从而富集胰腺干细胞。BACKGROUND： Pancreatic stem/progenitor cells are a multipotent self-renewing population and are the potential surrogates in the cell replacement therapy of diabetes mellitus. However, lack of effective cell markers hampers the identification and characterization of pancreatic stem cells. OBJECTIVE： To explore whether plant lectins peanut agglutinin （PNA） and phaseolus vulgaris agglutinin （PHA） as well as the heat stable antigen （HAS） act as negative screening markers of pancreatic stem cells. METHODS： The binding of plant lectins （PNA, PHA） and HSA antibody on the cell surface of adult pancreas, developing pancreas and regenerating pancreas was studied through histochemistry and immunofluorescent staining method. The clustering of pancreatic cells according to negative screening molecules was analyzed through flow cytometry. The cell types got from different gates by fluorescence-activated cell sorting were studied through cell culture and observation. RESULTS AND CONCLUSION： PNA marked pancreatic acinar cells in embryonic and neonatal pancreata, but they did not mark undifferentiated progenitors and precursors, PHA bound most of the undifferentiated and differentiated embryonic pancreatic cells, and HSA marked differentiating and differentiated cells in embryonic pancreata. In adults, both PNA and PHA marked pancreatic acinar cells and partial ductal cells, but not insular cells. HSA could recognize acinar, ductal, vascular endothelial cells, blood cells, and ductal-like cells in the regenerating pancreata. PNA - /lowHSA - /low cells accounted for 1.6% of the total pancreatic cells. Through culturing the population of double low cells, they are rich in smaller, triangular or polygonal epithelial cells, and they could be continuously sub-cultured. Our results indicate that PNA and HSA as negative markers can get rid of most differentiated pancreatic cells and part of non-pancreatic cell types, thus enrich pancreatic stem cells.

关 键 词：干细胞 干细胞培养与分化 植物凝集素 花生凝集素 菜豆凝集素 热稳定抗原 胰腺干细胞 负向筛选 标志分子 荧光组织化学 省级基金 干细胞图片文章 

分 类 号：R394.2[医药卫生—医学遗传学]