源于体外培养胰岛中表达Ngn3的细胞是胰腺内分泌前体细胞的新证据  

In vitro evidence for pancreatic lineage: Ngn3 positive cells are endocrine progenitors derived from cultured islets

作　　者：宋陆军[1] 秦新裕[1] 牛伟新[1] 沈坤堂[1] 刘凤林[1] K.A.Andreoni D.A.Gerber J.H.Fair L.Rice A.Pleasant J.Wang 

机构地区：[1]复旦大学附属中山医院普外科,上海200032 [2]Transplantation and Stem Cell Lab,Department of Surgery,University of North Carolina at Chape lHill,USA Jian Wang

出　　处：《中华外科杂志》2005年第1期42-45,共4页Chinese Journal of Surgery

基　　金：国家自然科学基金资助项目 (3 0 3 713 88)

摘　　要：目的　评估表达Ngn3的细胞对成体胰岛维护和更新的作用及其意义。方法　用 6～ 8周龄的C5 7BL/ 6小鼠分离胰岛。胆总管内插管成功后 ,切除胰腺组织并用浓度为 2 5mg/ml胶原蛋白酶V灌注消化。手工拣取胰岛后 ,置于 6 0mm培养皿内 ,每个培养皿内含 10～ 12个胰岛 ,用RPMI 16 4 0 (12 5mmol/LHEPES ,5 2mmol/L葡萄糖和 2 %胎牛血清 )培养液培养。用A6抗体 ,胰岛素抗体 ,胰高血糖素抗体 ,Nestin抗体 ,Ngn3抗体和 5′ 溴 2 脱氧尿嘧啶 (5 bromo 2′ deoxy uridine,BrdU)抗体进行胰岛细胞免疫荧光染色。结果　在增殖的胰岛细胞中 ,只有不到 15 %的细胞表达Ngn3,其中约有 30 %以上的细胞同时表达A6。在培养的胰岛细胞中存在表达Ngn3的细胞 ,这一结果与其他在胚胎发育和成体胰岛中的研究结果一致。本研究发现培养的胰岛细胞中存在同时表达A6和Ngn3的细胞。A6被认为是可分化为胆管上皮细胞的肝脏前体细胞的标记物。胰岛内表达A6的细胞可能来源于成体胰管 ,并迁移至胰岛内。细胞表达A6的同时表达Ngn3,表明这些细胞在胰岛内可分化为内分泌细胞。结论　培养的胰岛细胞中发现共同表达Ngn3和A6的细胞 ,表明胰岛内的 4种内分泌细胞可能来源于成体胰岛中的同一内分泌细胞系。Objective Further studies have been conducted to evaluate the roles of Ngn3 in adult islet maintenance and renewal. Methods Islets were isolated from 6 8 week old male C57BL/6 mice. After common bile duct cannulation, the pancreas was resected and digested in collagenase V (2 5 mg/ml). Islets were then handpicked and 10 12 islets were plated in 60 mm culture dish and cultivated with RPMI 1640, which contained 12 5 mmol/L HEPES, 5 2 mmol/L glucose and 2% fetal bovine serum (FBS). Islet cells were analyzed by immunocytochemistry methods for A6, insulin, glucagon, nestin, Ngn3 and 5 bromo 2′ deoxy uridine (BrdU). Results The results of these studies indicated that less than 15 percent of proliferated islet cells were Ngn3 expressing cells, in which about one third of the Ngn3 positive cells co expressed A6. The existence of Ngn3 in cultured islet cells is consistent with the results from other′s findings both in embryogenesis and adult islet studies. A significant finding of our study is that the existence of A6 and Ngn3 co expressing cells in the cultured islet. A6 is a marker for identifying bile duct epithelial cell oriented hepatic progenitor cells. Islet derived A6 cells are possibly born in the adult pancreatic duct and migrate into islets. A6 cells co express Ngn3 when these cells commit to endocrine lineage within the islets. More interestingly, islet derived A6 positive cells have the potential to transdifferentiate into hepatic cells. Conclusion The presence of Ngn3 (+) and A6 (+) cells in the cultured islets suggests that the four established islet cell types arise from a common endocrine lineage residing within the adult islets. A6 and Ngn3 are useful markers for understanding intra islet adult stem cell lineages in our future studies. This approach may allow for significant advances in understanding the IPC proliferation and differentiation, and open the possibility of using intra islet adult stem cells for diabetes treatment.

关 键 词：胰岛细胞 细胞表达 抗体 前体细胞 胰腺 标记物 治疗 成体干细胞 培养皿 内分泌细胞 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]