人脐带间充质干细胞向胰岛素分泌细胞分化的初步研究
发布时间：2018-10-20 13:13
【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(P0.05)。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,P0.05。 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。
[Abstract]:Aim: to explore the potential of human umbilical cord mesenchymal stem cells (Human Umbilical cord Wharton's Jelly-derived Mesenchymal Stem Cells, HUMSCs) to differentiate into insulin-secreting cells in the microenvironment of rat islet cell growth and in diabetic rats. Methods: 1 Experimental materials: SD rats were provided by Experimental Animal Center of Guangzhou University of traditional Chinese Medicine. The disposal of animals meets the standards of animal ethics. The umbilical cord was taken from the fetus of the second affiliated Hospital of Shantou University School of Medicine, Obstetrics and Gynecology, healthy full-term pregnancy and caesarean section. The pregnant women and their families gave informed consent to the experiment. Methods: human umbilical cord mesenchymal stem cells were isolated and cultured, and rat islet cells were isolated by collagenase digestion. HUMSCs and rat islet cells were cultured separately by semi-permeable membrane to observe the morphological changes of HUMSCs. The insulin secreted by HUMSCs was detected by radioimmunoassay and the expression of Pdx-1 gene in co-cultured HUMSCs was detected by RT-PCR method on the 7th and 14th day of co-culture. Unco-cultured HUMSCs was used as control. HUMSCs was transplanted into diabetic model rats with fluorescent dye Hoechst33258 labeled HUMSCs, through tail vein. The pancreas of rats was harvested one month later to make rapid frozen sections. The colonization of HUMSCs in pancreatic tissue was observed under fluorescence microscope, and the expression of human insulin gene was detected by semi-quantitative RT-PCR after 60 days. Results: (1) HUMSCs in co-culture condition gradually became round from long spindle shape and gathered into clusters. 2 the results of radioimmunoassay showed that the amount of insulin secreted by HUMSCs in co-culture group increased obviously with the prolongation of co-culture time. The amount of insulin secreted by HUMSCs increased with the increase of glucose-stimulated concentration (P0.05). The RT-PCR results of 3Pdx-1 gene showed that the uncultured HUMSCs did not express Pdx-1. Expression of Pdx-1, in HUMSCs from 3 days after co-culture, HUMSCs from 7 days of co-culture and Pdx-1, from 14 days to 14 days, HUMSCs did not express Pdx-1.. In vivo study: (4) under fluorescence microscope, HUMSCs labeled with Hoechst33258 was seen as a uniform dot in the pancreas of rats, emitting blue fluorescence. The results of RT-PCR of 5Insulin showed that the pancreas of rats transplanted with HUMSCs could detect human Insulin gene at day 60. However, the pancreas of untransplanted HUMSCs rats did not express human Insulin gene. 6 the survival time of diabetic rats transplanted with HUMSCs was significantly longer than that of the control group (P0.05). Conclusion: human umbilical cord mesenchymal stem cells have the potential to differentiate into extroverted insulin-secreting cells in vivo, and human umbilical cord mesenchymal stem cells can be used as a source of islet cells.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R587.1;R329
本文编号：2283288
[Abstract]:Aim: to explore the potential of human umbilical cord mesenchymal stem cells (Human Umbilical cord Wharton's Jelly-derived Mesenchymal Stem Cells, HUMSCs) to differentiate into insulin-secreting cells in the microenvironment of rat islet cell growth and in diabetic rats. Methods: 1 Experimental materials: SD rats were provided by Experimental Animal Center of Guangzhou University of traditional Chinese Medicine. The disposal of animals meets the standards of animal ethics. The umbilical cord was taken from the fetus of the second affiliated Hospital of Shantou University School of Medicine, Obstetrics and Gynecology, healthy full-term pregnancy and caesarean section. The pregnant women and their families gave informed consent to the experiment. Methods: human umbilical cord mesenchymal stem cells were isolated and cultured, and rat islet cells were isolated by collagenase digestion. HUMSCs and rat islet cells were cultured separately by semi-permeable membrane to observe the morphological changes of HUMSCs. The insulin secreted by HUMSCs was detected by radioimmunoassay and the expression of Pdx-1 gene in co-cultured HUMSCs was detected by RT-PCR method on the 7th and 14th day of co-culture. Unco-cultured HUMSCs was used as control. HUMSCs was transplanted into diabetic model rats with fluorescent dye Hoechst33258 labeled HUMSCs, through tail vein. The pancreas of rats was harvested one month later to make rapid frozen sections. The colonization of HUMSCs in pancreatic tissue was observed under fluorescence microscope, and the expression of human insulin gene was detected by semi-quantitative RT-PCR after 60 days. Results: (1) HUMSCs in co-culture condition gradually became round from long spindle shape and gathered into clusters. 2 the results of radioimmunoassay showed that the amount of insulin secreted by HUMSCs in co-culture group increased obviously with the prolongation of co-culture time. The amount of insulin secreted by HUMSCs increased with the increase of glucose-stimulated concentration (P0.05). The RT-PCR results of 3Pdx-1 gene showed that the uncultured HUMSCs did not express Pdx-1. Expression of Pdx-1, in HUMSCs from 3 days after co-culture, HUMSCs from 7 days of co-culture and Pdx-1, from 14 days to 14 days, HUMSCs did not express Pdx-1.. In vivo study: (4) under fluorescence microscope, HUMSCs labeled with Hoechst33258 was seen as a uniform dot in the pancreas of rats, emitting blue fluorescence. The results of RT-PCR of 5Insulin showed that the pancreas of rats transplanted with HUMSCs could detect human Insulin gene at day 60. However, the pancreas of untransplanted HUMSCs rats did not express human Insulin gene. 6 the survival time of diabetic rats transplanted with HUMSCs was significantly longer than that of the control group (P0.05). Conclusion: human umbilical cord mesenchymal stem cells have the potential to differentiate into extroverted insulin-secreting cells in vivo, and human umbilical cord mesenchymal stem cells can be used as a source of islet cells.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R587.1;R329
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