成年大鼠非胰岛内Nestin阳性细胞的体外分离、培养及分化

目的:建立成年大鼠胰腺非胰岛内Nestin阳性细胞的体外分离培养及分化的方法。方法:采用胰管内注入胶原酶消化法分离3只成年大鼠胰腺组织，并经含10%胎牛血清RPMI 1640液（pH 7.6）体外培养，36 h后弃去悬浮细胞及胰岛，改用无10%胎牛血清RPMI 1640液（pH 7.4）加bFGF、EGF及N₂添加剂培养，18～22 d后收集新生类胰岛样细胞团并传代培养。挑选新分离的胰岛和新生类胰岛样细胞团每次各40个，共3次，采用¹³¹Ⅰ放射免疫法测定分离的胰岛及新生类胰岛样细胞团的胰岛素分泌量，并计算葡萄糖刺激指数（SI）；免疫荧光细胞化学法检查贴壁细胞及传代后的新生类胰岛样细胞团Nestin阳性细胞的表达。 结果:胰管内注入胶原酶消化法可获得功能、形态良好的胰岛；分离后胰腺组织在pH 7.6的10%胎牛血清RPMI 1640液培养条件下，36 h内胰岛细胞不贴壁，贴壁细胞中大多数表达Nestin阳性细胞，无胰岛素和胰高血糖素表达，但Nestin表达不一，培养18～22 d可见新生类胰岛样细胞团出现，新生类胰岛样细胞团传代后仍可见Nestin阳性细胞。新分离的40个胰岛在含糖3.3、16.7 mmol?L^-1培养液中胰岛素分泌量为(63.6±4.0)、(202.2±14.8)mIU?L^-1；而新生的40个类胰岛样细胞团胰岛素分泌量为(3.5±0.2)、(3.3±0.2)mIU?L^-1，差异有显著性（P<0.001）。 结论:胰管内注入胶原酶消化法可获得功能、形态良好的胰岛；在pH 7.6条件下可分离培养出成年大鼠胰腺非胰岛内Nestin阳性细胞，经无血清培养形成的类胰岛样细胞团，可传代培养，并表达胰岛素。胰腺非胰岛内Nestin阳性细胞具有胰腺干细胞特点。

Isolation, culture, and differentiation of Nestin positive cells of non-islet cells in adult rats in vitro

Objective To establish the method of isolation,culture, and differentiation of Nestin positive cells of non-islet cells in adult rats in vitro. Methods The whole pancreas of three adult rats were digested in vitro with the intraductal collagenase distention method, then both islets and non-islets were collected and cultured with 10% fetal bovine serum (pH 7.6) and serum-free RPMI 1640 (pH 7.4).The formation process of new islet-like cell masses were examined by light microscopy,and the glucose-stimulated insulin release (SI) from 40 just isolated islets and 40 new produced islet-like cell masses were tested.The expression of Nestin was tested by immunocytochemistry. Results Functioning islets in good morphology could be obtained from pancreas of adult rats by the intraductal collagenase distention method;there were adherent cells after 36 h culture,but no islets; the Nestin positive cells proliferated fast and formed new islet-like cell masses after 18-22 d. Nestin expression could be confirmed among adherent cells,but no insulin and glucagon, and Nestin positive cells can be seen in passage cells of new produced islet-like cell masses. Insulin secretion levels in 40 new isolated islets were (63.6±4.0), (202.2±14.8)mIU·L~ -1 in 3.3 and 16.7 mmol·L~ -1 glucose, respectively;and (3.5±0.2), (3.3± 0.2)mIU·L~ -1, respectively, in 40 new produced islet-like cell masses (P<0.001).Conclusion Functioning islets can be obtained from the pancreas of adult rats. Nestin positive cells of non-islet cells can be obtained from pancreas of adult rats in pH 7.6 condition through culture in vitro,and can form islet-like cell masses;Nestin positive cells of non-islet cells in pancreas of adult rats possess the characters of islet stem cells.