【作者】 王爱红；

【导师】 马廉；

【作者基本信息】 汕头大学 ， 儿内科， 2008， 硕士

【摘要】 目的:探索人脐带间充质干细胞(Human Umbilical cord Wharton’s Jelly-derived Mesenchymal Stem Cells ,HUMSCs)在大鼠胰岛细胞生长的微环境中及在糖尿病大鼠体内向胰岛素分泌细胞分化的潜能。方法:①实验材料: SD大鼠由广州中医药大学实验动物中心提供。对动物处置符合动物伦理学标准。脐带取自汕头大学医学院第二附属医院妇产科健康足月妊娠剖宫产的胎儿,产妇及家属对实验知情同意,并经医院伦理委员会批准。②实验方法:分离培养人脐带间充质干细胞,并采用胶原酶消化法分离大鼠胰岛细胞。将HUMSCs与大鼠胰岛细胞以半透膜相隔共同培养,观察HUMSCs的形态变化,在共培养的第3,7,14天采用放射免疫法检测HUMSCs分泌的胰岛素水平,RT-PCR方法检测共培养的HUMSCs中的Pdx-1基因的表达,以未共培养的HUMSCs做对照。用荧光染料Hoechst33258标记HUMSCs,经尾静脉将HUMSCs移植入糖尿病模型大鼠体内,一个月后取大鼠胰腺制作快速冰冻切片,荧光显微镜下观察HUMSCs在胰腺组织中的定植情况。60天后取移植鼠的胰腺组织,采用半定量RT-PCR方法检测人insulin基因的表达。结果:体外研究:①共培养条件下的HUMSCs由长梭形逐渐变圆,并聚集成团。②放射免疫法结果表明,共培养组HUMSCs随着共培养时间的延长分泌的胰岛素量明显增多,当HUMSCs有聚集成团倾向时可以随葡萄糖刺激浓度的增高而分泌的胰岛素量也增高,与对照组相比差异显著(P<0.05)。③Pdx-1基因的RT-PCR结果表明未经共培养的HUMSCs不表达Pdx-1 ,共培养3d的HUMSCs开始表达Pdx-1,共培养7d的HUMSCs仍表达Pdx-1,共培养14d的HUMSCs未表达Pdx-1。体内研究:④荧光显微镜下看到移植标记Hoechst33258的HUMSCs在大鼠的胰腺中呈均匀的点状分部,发出蓝色荧光,表明移植入体内的HUMSCs可以在大鼠胰腺内定植。⑤Insulin的RT-PCR结果表明移植HUMSCs的大鼠胰腺在第60天时能够检测出人Insulin基因,而未经移植HUMSCs的大鼠胰腺不表达人Insulin基因。⑥移植HUMSCs的糖尿病大鼠与对照组相比能明显延长生存期,P<0.05。结论:人脐带间充质干细胞具有在体内外向胰岛素分泌细胞分化的潜能,人脐带间充质干细胞可以作为胰岛细胞的来源。更多还原

【Abstract】 Objective: We co-cutured HUMSCs with pancreatic cells of neonatal rat and transplanted HUMSCs into the body of diabetic rat. To explore the possibility of inducing HUMSCs to differentiate into insulin-producing cells in microenviroment of pancreatic cells in neonatal rat and in body of diabetic rat.Methods:①Subjects: SD rats were bought from Traditional Chinese Medicine University Of Guangzhou. According to the animal ethical standard, animals were disposed in the experiment. The human umbilical cords were obtained from healthy caesarean delivered uterogestation foetus from The Second Affiliated Hospital of Shantou University. The fact of the experiment was known by the lying-in mother and her family member, and was be approved by hospital Ethics Committee.②Experiment Methods: The pancreatic cells of fetal rat were isolated by enzyme dispelling therapy and human umbilical cord mesenchymal stem cells were also isolated and cultured, and then HUMSCs were co-cultured with pancreatic cells. On the 3rd、7th and 14th day during co-culture, Insulin release after glucose challenge was tested with radio-immunity, Human pancreatic and duodenal homeobox 1(PDX-1) was detected with reverse transcription-polymerase chain reaction (RT-PCR) method, meanwhile Control Group lonely cultured was set up. Last Human umbilical cord mesenchymal stem cells were marked by Hoechst33258, which is a fluorescent dye. Then human umbilical cord mesenchymal stem cells were transplanted into the body of diabetic rats through vena caudalis. Under the fluorescence microscope, to observe whether stem cells field in the pancreas, and then human insulin gene were detected by RT-PCR.Results:①The morphology of human umbilical cord mesenchymal stem cells under co-culturing gradually changed from fibroblast to round and had the tend of forming clusters.②The results of radio-immunity manifested that the insulin quantity secreted by co-cultured HUMSCs could increase following the prolongation of co-culture. And the insulin quantity secreted by co-cultured HUMSCs could increase following different density of glucose when the cells have the tend of gathering to lumping. There was significant difference compared with control group(P<0.05).③RT-PCR analysis indicated that Un-cocultured HUMSCs didn’t express Pdx-1; Co-cultured HUMSCs could express Pdx-1 on third day;Pdx-1 can also be detected on seventh day. But on fourteenth day, PDX-1 wasn’t be expressed.④The cells marked with Hoechst33258 distribute in uniformity in the pancreas under the fluorescent microscope. The un-transplanted diabetic rat pancreas didn’t express human insulin gene, but the human insulin gene could be detected in the rat pancreas after transplanted HUMSCs on sixty day.⑤The transplantation results showed that rats treated with HUMSCs significantly increased their lifespan, as compared with untreated rats.Conclusion: In this study, we successfully differentiated HUMSCs into mature islet-like cell clusters in microenviroment, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. HUMSCs in Wharton’s Jelly of the umbilical cord seem to be the preferential source of stem cells to convert into insulin-producing cells, because of the large potential donor pool, its rapid availability, no risk of discomfort for the donor, and low risk of rejection.更多还原