【摘要】 目的:观察人的骨髓间充质干细胞多分化潜能及在糖尿病治疗领域的价值。方法:实验于2005-07/2006-07在青岛大学医学院附属医院内分泌科完成。骨髓来源于非造血系统疾病的16岁儿童胸骨骨髓血(查体以排除造血系统疾病,结果显示为健康体质),经得家属同意。Percoll淋巴细胞分层液分离骨髓间充质干细胞,取第3代细胞,等密度接种于培养瓶中,经CD44抗体、CD45抗体、CD34抗体鉴定。取其第4代细胞,诱导其向脂肪细胞及神经细胞分化,利用碱性成纤维细胞生长因子预处理先获得巢蛋白阳性细胞,分别用两种方法诱导其向胰岛祖细胞的转化:化学物质诱导和共培养法诱导,免疫荧光检测胰岛祖细胞标志-胰十二指肠同源异型盒基因(蛋白的表达,电化学发光法检测其是否表达胰岛素)。胶原酶消化法获取胰腺间充质干细胞,鉴定,用添加碱性成纤维细胞生长因子的无血清低糖DMEM使其增殖。将胰腺间充质干细胞接种于底层已接种骨髓间充质干细胞的6孔板共培养,共培养法诱导骨髓间充质干细胞向胰岛祖细胞的初步转化。结果:成脂诱导及成神经诱导可获得油红O染色阳性细胞以及巢蛋白阳性细胞。化学法向胰岛祖细胞诱导后可检测到PDX-1免疫反应阳性细胞。共培养法诱导也可获得PDX-1免疫反应阳性细胞。新生儿胰腺具有巢蛋白、CK-19阳性的胰腺间充质干细胞,体外高糖诱导可形成胰岛样细胞团。结论:骨髓间充质干细胞在体外具有诱导分化为脂肪细胞、神经元样细胞及胰岛祖细胞的潜能。新生儿胰腺间充质干细胞向胰岛细胞分化过程中所分泌的一些物质对骨髓间充质干细胞向胰岛祖细胞的转化具有促进作用。更多还原

【Abstract】 AIM: To observe the multitude differentiating potency of human marrow mesenchymal stem cells (MSCs) and the value in the field of diabetes mellitus therapy.METHODS: This experiment was performed from July 2005 to July 2006 in Department of Endocrinology, Affiliated Hospital of Medical College of Qindao University. The bone marrow was gained from bone marrow of sternum blood in child aged 16 years without disease of hematopoietic system under the permission of the family (investigation aiming to preclude disease of hematopoietic system, and the results showed healthy state). The human MSCs were isolated by Percoll lymphocyte laminating liquor. The third passage cells were inoculated in culture flask after being identified by CD44 antibody, CD45 antibody and CD34 antibody. The fourth passage MSCs nerve cells were induced to differentiate into adipocyte. Nestin positive cells were gained by bFGF pretreatment and they were induced towards pancreatic islet progenitor cells by chemical materials method and co-culture method, respectively. Islet progenitor cells label-pancreafic duodenal homeobox-1 (PDX-1) (expression of protein, insulin was analyzed by electrochemiluminescence) was detected by immunofluorescence. Pancreatic mesenchymal stem cells were obtained by collagenase enzyme digestion, which was proliferated by serum-free low glucose-DMEM medium containing bFGF. Pancreatic mesenchymal stem cells were inoculated into insert, and then the insert was put into the transclear plate which underlayer was covered with MSCs. Co-culture method was used to induce MSCs to differentiate into pancreatic islet progenitor cell initially. RESULTS: Adipocytes which could be stained by oil red and neuron-like cells which could be identified by nestin staining were gained. Both chemical method induction and co-culture induction could induce MSCs to differentiate into PDX-1 immunological reaction positive cells. Pancreatic cells containing nestin and CK-19 positive cells could be induced to form island-like organ in vitro.CONCLUSION: MSCs can differentiate into adipocyte, neuron-like cells and pancreatic islet progenitor cell in vitro. Some substances secreted during differentiation of pancreatic mesenchymal stem cell towards pancreatic cells have active effect on the differentiation of MSCs towards pancreatic islet progenitor cell initially.更多还原