胰岛联合骨髓间充质干细胞治疗糖尿病大鼠的研究
发布时间：2018-12-26 15:55
【摘要】：以标准的糖尿病大鼠为模型,探索同种异体的SD大鼠胰岛联合骨髓间充质干细胞(Bone Marrow Mesenchymal Cells,BMSCs)能否提高治疗糖尿病的远期疗效,为临床开展胰岛联合BMSCs移植治疗糖尿病的提供实验依据,并观察同种异体大鼠联合BMSCs移植后细胞在体内的分布、归巢情况,为临床细胞移植后细胞的定植、归巢提供理论依据。链脲佐菌素(Streptozotocin,STZ)构建大鼠糖尿病模型;贴壁法分离、培养BMSCs,免疫荧光法和流式细胞鉴定BMSCs的表面抗原(CD44、CD34、CD90、CD45、CD29);胶原酶法分离大鼠胰岛,双硫腙法(Dithizone,DTZ)鉴定胰岛细胞;将细胞分为对照组;BMSCs培养组;单独胰岛组;胰岛与BMSCs共培养组,于培养后的第1、3、7天取其上清液进行ELISA法检测各组的胰岛素与C-肽的分泌;18F-FDG体外标记胰岛与BMSCs,随机将糖尿病大鼠分成:对照(n=15)、单独胰岛移植组(n=15)、单独BMSCs移植组(n=15)、胰岛与BMSCs联合移植(n=15),经门静脉移植分别移植(对照组0.2ml生理盐水;胰岛组0.2ml胰岛500IEQ/100g;干细胞组0.2ml的3*10^5/100g个BMSCs;联合组0.1ml胰岛500IEQ/100g+0.1ml 3*10^5/100g个BMSCs)。移植后的第1、3、7、14、30天每组每次均随机处死3只大鼠,取肝脏、血液,ELISA测量移植后各组大鼠血清的胰岛素与C-肽的含量及检测大鼠的血清的肝功能,免疫荧光法了解肝组织内胰岛素的表达情况,HE检测肝脏的病理情况,PET-CT示踪移植入体内的细胞分布情况。经STZ法诱导糖尿病大鼠期间有95%的大鼠血糖浓度在一周内有3天连续高于16.8mmol/L的,伴随体重下降,总胆红素、谷丙转氨酶、谷草转氨酶均升高;经全贴壁法培养的BMSCs大多是呈长梭形生长,免疫荧光法的检测结果显示BMSCs为CD44阳性,CD34阴性,流式细胞仪测量结果表明BMSCs是高表达CD90、CD29,低表达CD45的;胶原酶法能够很有效的分离出胰岛细胞,且被DTZ染成猩红色的就是胰岛细胞,未被染成猩红色的就是外分泌腺;体外共培养中的胰岛形态与单独胰岛的形态相比保存的更好,存活的时间也更长,胰岛素与C-肽的分泌也较单独胰岛的高;移植后联合组的血糖在正常血糖内波动一个月,单独胰岛组的血糖水平只能在正常血糖范围波动3天,单独BMSCs组、对照组的大鼠在移植后的血糖水平未明显降低。联合组在移植后的第1、7、14、21、30天的血糖明显低于单独胰岛组,差异有统计学意义(P0.05);联合组比胰岛组能有效的修复肝脏的功能;联合组的胰岛素与C-肽的分泌也比单独胰岛组高;联合组和单独胰岛组的炎细胞侵润都不明显。PET-CT结果显示,BMSCs主要均匀分布在肝脏的右肝,胰岛主要聚集在肝门静脉末端分支,且BMSCs围绕在胰岛的周围,对照组的肝脏无明显的显影。同种异体的胰岛联合BMSCs治疗大鼠糖尿病的效果优于单独胰岛移植。PET-CT能直观的揭示胰岛与BMSCs经门静脉移植入后在肝内的分布。
[Abstract]:To explore whether islet of islets combined with bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal Cells,BMSCs) of allogeneic SD rats can improve the long-term curative effect of diabetic rats. To provide the experimental basis for the clinical treatment of diabetes mellitus with islet and BMSCs transplantation, and to observe the distribution and homing of cells after allograft combined with BMSCs transplantation, and to provide the theoretical basis for the implantation and homing of the cells after clinical cell transplantation. Streptozotocin (Streptozotocin,STZ) was used to establish diabetic model in rats, and the surface antigen (CD44,CD34,CD90,CD45,CD29) of BMSCs was identified by BMSCs, immunofluorescence assay and flow cytometry. Rat islets were isolated by collagenase and identified by dithizone (Dithizone,DTZ). The cells were divided into control group, BMSCs culture group, isolated islet group; The supernatants of islet and BMSCs were taken from the supernatant on the 7th day after culture and the secretion of insulin and C-peptide was detected by ELISA method. 18F-FDG labeled islets and BMSCs, were randomly divided into three groups: control group (n = 15), islet transplantation group (n ~ 15), BMSCs transplantation group (n ~ 15), islet / BMSCs transplantation group (n ~ (15). Portal vein transplantation (control group, 0.2ml saline); Islet group 0.2ml islet 500 IEQ / 100g; stem cell group 0.2ml 3n10 ^ 5 / 100g BMSCs; + 0.1ml islet 500IEQ/100g 0.1ml 10 ^ 5100g BMSCs). Three rats were randomly killed in each group on the 1st day after transplantation. The liver and blood were taken. ELISA was used to measure the contents of insulin and C-peptide in serum and the liver function of the rats. The expression of insulin in liver tissue was detected by immunofluorescence, the pathological changes of liver were detected by HE and the distribution of cells transplanted into body by PET-CT tracer. During the period of diabetes induced by STZ, 95% of the diabetic rats had higher blood glucose concentration than that of 16.8mmol/L for 3 days in one week. With the decrease of body weight, total bilirubin, alanine aminotransferase and alanine aminotransferase were all increased. The results of immunofluorescence assay showed that BMSCs was CD44 positive and CD34 negative. Flow cytometry showed that BMSCs expressed high expression of CD90,CD29, and low expression of CD45. Collagenase method can effectively isolate islet cells, and the scarlet cells stained by DTZ are islet cells and exocrine glands that are not stained scarlet. The morphology of islets in vitro was better than that of isolated islets, the survival time was longer, and the secretion of insulin and C-peptide was higher than that of isolated islets. After transplantation, the blood glucose of the combined group fluctuated within the normal blood glucose for one month, the blood glucose level of the islet group only fluctuated within the normal blood glucose range for 3 days, the blood glucose level of the BMSCs group and the control group did not decrease significantly after transplantation. The blood glucose in the combined group was significantly lower than that in the islet group after transplantation on the 1st day after transplantation (P0.05), and the liver function in the combined group was more effective than that in the islet group. The secretion of insulin and C-peptide in the combined group was also higher than that in the islet group alone. The results of PET-CT showed that BMSCs mainly distributed in the right liver of liver, pancreatic islets mainly concentrated in the branches of portal vein of liver, and BMSCs was around the islets. There was no obvious development of liver in the control group. The effect of islet allograft combined with BMSCs in the treatment of diabetic rats was better than that of islet transplantation alone. PET-CT could intuitively reveal the distribution of islets and BMSCs in the liver after portal vein transplantation.
【学位授予单位】：电子科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
本文编号：2392347
[Abstract]:To explore whether islet of islets combined with bone marrow mesenchymal stem cells (Bone Marrow Mesenchymal Cells,BMSCs) of allogeneic SD rats can improve the long-term curative effect of diabetic rats. To provide the experimental basis for the clinical treatment of diabetes mellitus with islet and BMSCs transplantation, and to observe the distribution and homing of cells after allograft combined with BMSCs transplantation, and to provide the theoretical basis for the implantation and homing of the cells after clinical cell transplantation. Streptozotocin (Streptozotocin,STZ) was used to establish diabetic model in rats, and the surface antigen (CD44,CD34,CD90,CD45,CD29) of BMSCs was identified by BMSCs, immunofluorescence assay and flow cytometry. Rat islets were isolated by collagenase and identified by dithizone (Dithizone,DTZ). The cells were divided into control group, BMSCs culture group, isolated islet group; The supernatants of islet and BMSCs were taken from the supernatant on the 7th day after culture and the secretion of insulin and C-peptide was detected by ELISA method. 18F-FDG labeled islets and BMSCs, were randomly divided into three groups: control group (n = 15), islet transplantation group (n ~ 15), BMSCs transplantation group (n ~ 15), islet / BMSCs transplantation group (n ~ (15). Portal vein transplantation (control group, 0.2ml saline); Islet group 0.2ml islet 500 IEQ / 100g; stem cell group 0.2ml 3n10 ^ 5 / 100g BMSCs; + 0.1ml islet 500IEQ/100g 0.1ml 10 ^ 5100g BMSCs). Three rats were randomly killed in each group on the 1st day after transplantation. The liver and blood were taken. ELISA was used to measure the contents of insulin and C-peptide in serum and the liver function of the rats. The expression of insulin in liver tissue was detected by immunofluorescence, the pathological changes of liver were detected by HE and the distribution of cells transplanted into body by PET-CT tracer. During the period of diabetes induced by STZ, 95% of the diabetic rats had higher blood glucose concentration than that of 16.8mmol/L for 3 days in one week. With the decrease of body weight, total bilirubin, alanine aminotransferase and alanine aminotransferase were all increased. The results of immunofluorescence assay showed that BMSCs was CD44 positive and CD34 negative. Flow cytometry showed that BMSCs expressed high expression of CD90,CD29, and low expression of CD45. Collagenase method can effectively isolate islet cells, and the scarlet cells stained by DTZ are islet cells and exocrine glands that are not stained scarlet. The morphology of islets in vitro was better than that of isolated islets, the survival time was longer, and the secretion of insulin and C-peptide was higher than that of isolated islets. After transplantation, the blood glucose of the combined group fluctuated within the normal blood glucose for one month, the blood glucose level of the islet group only fluctuated within the normal blood glucose range for 3 days, the blood glucose level of the BMSCs group and the control group did not decrease significantly after transplantation. The blood glucose in the combined group was significantly lower than that in the islet group after transplantation on the 1st day after transplantation (P0.05), and the liver function in the combined group was more effective than that in the islet group. The secretion of insulin and C-peptide in the combined group was also higher than that in the islet group alone. The results of PET-CT showed that BMSCs mainly distributed in the right liver of liver, pancreatic islets mainly concentrated in the branches of portal vein of liver, and BMSCs was around the islets. There was no obvious development of liver in the control group. The effect of islet allograft combined with BMSCs in the treatment of diabetic rats was better than that of islet transplantation alone. PET-CT could intuitively reveal the distribution of islets and BMSCs in the liver after portal vein transplantation.
【学位授予单位】：电子科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1
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