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

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C57BL／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60mm培养皿内,每个培养皿内含10—12个胰岛,用RPMI-1640(12．5mmol／L HEPES,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。A6和Ngn3是对胰岛内成体干细胞研究很有帮助的标记物。这可以让我们进一步了解IPC的增殖和分化,并且有可能通过胰岛内成体干细胞来治疗糖尿病。

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

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.