胎儿胰岛中干细胞的分离与鉴定

Isolation and identification of stem cells from fetal islets

目的:已证实胰腺来源的干细胞或祖细胞在体内外均能分化为胰岛素分泌细胞,但成人胰腺来源的干细胞增殖潜能较差。实验旨在分离并鉴定胎儿胰岛样细胞团中的干细胞,为糖尿病细胞移植治疗寻找可用的种子细胞。方法:实验于2006-10/2007-07在解放军军事医学科学院输血研究所干细胞与再生医学研究室完成。①对象:所用胰腺标本取自流产胎儿,胎龄14～20周,由解放军总医院妇产科提供,胎儿组织的使用得到孕妇本人的知情同意,实验经医院医学伦理委员会批准。②实验方法:无菌条件下取出胎儿胰腺,37℃下用0.5g/L的V型胶原酶进行消化,分离得到胎儿胰岛样细胞团,悬浮培养,加入含体积分数为0.1胎牛血清、1mmol/L丙酮酸钠、1mmol/L谷氨酰胺、71.5μmol/Lβ-巯基乙醇、100U/mL青霉素和100mg/L链霉素的RPMI1640培养基,每日转皿除去贴壁细胞,72h后挑选出悬浮生长的胰岛样细胞团予以贴壁培养,待长成上皮样单层细胞克隆时传代。③实验评估:将悬浮培养的胰岛样细胞团行双硫腙染色。另将少部分胰岛样细胞团及第4代细胞种植于24,96孔板中,采用免疫荧光染色检测细胞表面分子标志物增殖细胞核抗原、胰十二指肠同源盒基因1、Nestin、角蛋白19、波形蛋白、胰岛素、胰高血糖素、CD29的表达情况。MTT法分析第6代细胞的生长曲线。结果:①悬浮及贴壁培养的胰岛形态:刚分离出的胰岛呈簇状,边缘不规整,细胞排列较疏松。贴壁后胰岛样细胞团由簇状逐渐展开形成上皮样单层细胞克隆。传代后细胞形态发生改变,呈梭形或分支样,增殖较快。②悬浮培养的胰岛双硫腙染色检测:分离得到的胎儿胰岛样细胞团双硫腙染色呈阳性表达。③细胞表面标志免疫荧光染色检测:贴壁的第1代细胞大部分呈增殖细胞核抗原、胰十二指肠同源盒基因1、Nestin、波形蛋白阳性,极少数细胞呈胰岛素、胰高血糖素阳性,而角蛋白19为阴性。传代后仍呈增殖细胞核抗原、胰十二指肠同源盒基因1、Nestin、波形蛋白阳性,角蛋白19弱阳性,而胰岛素、胰高血糖素为阴性,所有细胞均不表达CD29。④细胞生长曲线:传代第3天细胞进入对数生长期,第6天进入平台期,细胞生长曲线呈"S"形。结论:由胎儿胰岛成功分离并培养出具有自我更新和良好增殖能力的干细胞,稳定表达多种胰腺干细胞特征性标志,而不表达胰岛细胞标志物,提示该细胞可作为候选的胰岛干细胞用于糖尿病细胞移植治疗。

AIM： Stem cells or progenitor cells from the pancreas can differentiate into insulin-producing cells in vitro or in vivo, but the stem cells from adult pancreas had bad proliferation. We aimed to isolate and identify stem cells from in fetal islet-like cell clusters, and to find available cell resource for diabetes cell-transplantation therapy. METHODS： Experiments were performed at the Laboratory of Stem Cell and Regeneration, Beijing Institute of Transfusion Medicine, Military Medical Academy of Science from October 2006 to July 2007. （1）Human fetal pancreata at 14-20 gestational weeks were provided by Department of Obstetrics and Gynecology, General Hospital of Chinese PLA after the termination of pregnancy, Pregnants signed an informed consent. The experimental procedures were approved by hospital ethical committee. （2） The human fetal pancreata were taken out in sterile station and were digested by 0.5 g/L collagenase type V at 37℃, then the fetal islet-like cell clusters were obtained for suspension culture. Islet-like cell clusters were plated on Petri dish in RPMI 1640 medium containing 0.1 volume fraction of fetal calf serum, 1 mmol/L sodium pyruvate, 1 mmol/L glutamine, 71.5 B mol/L 13 -mercaptoethano, 100 U/mL penicillin and 100 mg/L streptomycin, and the dishes were changed everyday to remove the attached cells. After cultured in suspension for 72 hours, the islet-like cell clusters were handpicked out and plated on tissue culture dishes, when the cells grew out form islet-like cell clusters and became monolayer epithelium-like cell clones the cells were passaged. （3）Islet-like cell clusters were treated with dithizone staining. A small quantity of islet-like cell clusters and the fourth passage cells were plated on 24- and 96-well plates respectively. The expression of several kinds of molecular markers such as proliferating cell nuclear antigen, PDX-1, Nestin, keratin-19, Vimentin, Insulin, Glucagon and CD29 were identified by immunofluorescence staining. The growth curve of the 6th passage cells was measured by MTT. RESULTS： （1）The isolated islet showed cluster-shape, with irregular edge, and islet-like cells were scattered. Monolayer epithelium-like cell colonies were obtained in islet-like cell clusters. After passage, cells were fusiform or branch-shape, and amplified rapidly. （2）The isolated islet-like cell clusters were DTZ-staining positive. （3）Majority of the attached first passage cells expressed proliferating cell nuclear antigen, PDX- 1, Nestin and Vimentin, whereas minority of them expressed Insulin and Glucagon, but none of them expressed keratin-19. After passage, the cells still expressed proliferating cell nuclear antigen, PDX-1, Nestin and Vimentin, slenderly expressed keratin-19, but did not express Insulin and Glucagon. None of the cells express CD29. （4）The third passage cells were in logarithmic growth phase. At day 6, the cells entered into plateau phase. The growth curve shape was ＂S＂. CONCLUSION： The self-renewable cells can be obtained from fetal islet, which express multifold characteristic markers of pancreatic stem cells, but not islet markers. It is indicated that islet-like cell clusters can be used as pancreatic stem cells in the treatment of diabetes mellitus.