人胰腺星状细胞分离方法改良  

Improvement of a in isolation method for pancreatic stellate cells from human pancreas

作　　者：田蕾[1,2] 陆子鹏[1,2] 吴鹏飞[1,2] 蔡宝宝 赵良涛[1,2] 钱栋[1,2] 徐庆成[1,2] 朱毅[1,2] 张静静[1,2] 杜青[1,2] 蒋奎荣[1,2] 吴峻立[1,2] 苗毅[1,2] 

机构地区：[1]南京医科大学胰腺研究所 [2]南京医科大学第一附属医院胰腺中心普外科实验室,江苏南京210029

出　　处：《南京医科大学学报（自然科学版）》2015年第5期595-599,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金面上项目(81272239);国家自然科学基金青年基金(81300351)

摘　　要：目的 ：探讨人胰腺星状细胞的两种新分离方法。方法 ：通过酶消化-密度梯度离心法进行静止态的人正常胰腺星状细胞的分离,分离获得的细胞在含10%FBS的DMEM/F12继续培养。通过组织块外植法获得激活态的人肿瘤相关胰腺星状细胞（Ca PSCs）,分离获得的细胞在含20%FBS的DMEM/F12继续培养。结果：通过方法改良,1 g人正常胰腺组织能够获得（0.5-5.0）×106个星状细胞,细胞活率约为90%。使用新的组织块外植法后,组织块不易掉落,细胞爬出时间显著缩短,约5-10 d即可获得Ca PSCs。所有细胞贴壁良好,细胞倍增时间约为24 h。正常胰腺星状细胞（normal pancreatic stellate cells,NPSCs）培养早期形态符合典型静止态星状细胞特征,细胞为多边形或圆形,内含丰富脂滴,在荧光显微镜下可观察到320nm激发波长下的蓝绿色自发荧光。NPSCs培养晚期和Ca PSCs呈多触角状,胞内无脂滴,细胞增殖速度明显快于静止态细胞。激活态的细胞均表达α-SMA、Desmin、vimentin、GFAP等胰腺星状细胞特征性标记。结论：新的原代培养方法能够满足静止态和激活态两种状态胰腺星状细胞的分离培养,同时能够增加细胞得率、缩短分离培养时间,所获得细胞的活性和纯度符合进一步实验的要求,为胰腺星状细胞的相关研究提供了便利。Objective：To describe our novel modification in isolating pancreatic stellate cells（PSCs） from normal human pancreas and human pancreatic ductal adenocarcinoma （PDAC） tissue. Methods： Normal PSCs were isolated with enzyme digestion and ladder centrifuge. Isolated PSCs were cultured in DMEM/F12 containing 10% fetal bovine serum. Cancer-associated PSCs were obtained by an outgrowth method. Isolated PSCs were cultured in DMEM/F12 containing 20% fetal bovine serum. Results：With our modifications, normal pancreas tissue from human yields an adequate number of PSCs （approximately 0.5-5 million/g pancreas） for in vitro studies, and the cell viability was about 90%. After new outgrowth method applied ,tissue blocks were attached more tightly and cells grew out earlier compared to the previous method. Primary isolated PSCs were verified with appearance, auto-fluorescence, positive expression of a-SMA,Vimentin, Desmin, GFAP. Conclusion： Our modification for PSCs isolation significantly increase the isolating efficiency with shorter culture period,which can provide great convenience for future researches on PSCs.

关 键 词：胰腺星状细胞 分离方法 改良 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]