pi3k抑制剂ly294002诱导hesc定向分化为胰岛素分泌细胞的分析word论文.doc

优秀毕业论文 精品参考文献资料 摘要 免疫荧光染色分析胰岛细胞特异蛋白的表达；通过体外胰岛素诱导释放实验初 步评价细胞的分化及其功能。 结果： 通过 ITS 条件培养液筛选出巢蛋白阳性细胞，细胞免疫荧光可呈现细胞核的 胰岛素阳性染色和核膜的巢蛋白阳性染色且两者呈共染状态。诱导分化第 4 阶 段添加 bFGF 以后巢蛋白阳性细胞进入快速增长期，第 7 天表现为胰岛素的核膜 染色，细胞核中心逐渐淡染，巢蛋白逐渐呈现出核膜与细胞核的全染状态。诱 导第五阶段 7 天后，LY 组胰岛素免疫荧光单染阳性率为（82.7 ±17.5）%高于 B27 组的（52.6 ±14.6）%（P 0.05），但 LY 组生长抑素和胰高血糖素单染 阳性率，胰岛素/生长抑素共染率和 c-肽/胰高血糖素共染率分别低于 B27 组（P 0.05）。诱导第五阶段 14 天后，LY 组胰岛素免疫荧光单染阳性率与 B27 组胰岛 素单染阳性率差异无统计学意义，但生长抑素和胰高血糖素单染阳性率，胰岛 素/生长抑素共染阳性率仍低于 B27 组（P 0.05）。B27 组和 LY 组各诱导 1 周 以后，在高糖（25mM）DMEM 培养液中培养 1 小时后，LY 诱导组细胞胰岛素 释放量高于 B27 诱 导 组 ，分别为 428.3 ± 43.9μIU/ml/mg 蛋白及 259.2 ± 18.5μIU/ml/mg 蛋白，两组之间差异具有统计学意义 (p 0.05) ；2.5 小时后， LY 诱导组细胞胰岛素释放量 仍 高于 B27 诱导组细胞， 分 别 为 515.3 ± 24.0μIU/ml/mg 蛋白及 258.7±17.8μIU/ml/mg 蛋白，差异具有统计学意义 (p 0.05) ，体外可以检测到胰岛样细胞具有胰岛素释放的功能。 结论： 在无血清培养体系下，磷脂酰肌醇 3 激酶抑制剂 LY294002 能够诱导人胚胎 干细胞在体外分化为更加成熟的胰岛素分泌细胞。 关键词： 人胚胎干细胞， 细 胞 分 化 ， 胰 岛 素 ； 磷脂酰肌醇 3 激 酶 抑 制 剂 II Abstract The study of phosphoinositide-3-kinase inhibitor LY294002 in the differentiation of human embryonic stem cells into more mature insulin-producing cells Postgradute: Ya nan Wang Supervisor: Prof Genhong Mao Department of Obstetrics and Gynecology The Second Clinical College of Zhengzhou University Zhengzhou, Henan 450014, China Abstract Objective: To investigate the effects of phosphoinositide-3-kinase inhibitors LY294002 in the differentiation of human embryonic stem cells (HESC) into more mature insulin-producing cells. Methods: Human embryonic stem cells were introduced to differentiate into insulin- producing cells through five stages. Human embryonic stem cells were cultured in vitro. Embryoid bodies were shaped. Nestining positive cells were screenned by ITSF solution. Nesting positive cells were amplificated by the addition of B27, N2 and bFGF. Nicotinamide and B27 (group B27) were given as the control group or nicotinamide and LY294002 (group LY) were given as study group, which was utilized to induce the nesting positive cells into mature insulin-producing cells. The morphologic