骨髓间充质干细胞对胰岛移植物的保护作用

发布时间：2018-05-11 11:05

本文选题：胰岛移植 + 骨髓间充质干细胞　；参考：《上海交通大学》2015年硕士论文

【摘要】：胰岛移植自2000年来已成为治疗不稳定性胰岛素依赖型糖尿病的重要手段,同时这也给移植领域带来了新的研究方向。不同于其它终末期疾病所进行的器官移植,胰岛移植的目的更注重改善患者的生存质量,所以更理想的方法是诱导移植免疫耐受状态,即在不使用免疫抑制剂的情况下,维持同种异体移植物功能良好,同时保持机体对其它外来抗原正常的宿主反应。因此,如何诱导同种异体胰岛移植物的免疫耐受成为临床胰岛移植研究发展的方向。骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BM-MSCs)有强大的免疫调节作用,可通过分泌多种可溶性分子或通过其与免疫细胞的相互接触等途径抑制抗原特异性或非特异性的淋巴细胞增殖,抑制抗原提呈细胞的分化、成熟,诱导抗原特异性调节性T细胞(T regulatory cell,Treg)的形成,调节机体免疫应答向抗炎症方向偏移,在器官移植、替代治疗等领域具有巨大的临床应用价值。有研究表明:间充质干细胞联合胰岛移植可提高胰岛移植物存活时间,调节胰岛移植物免疫耐受,其作用机制与细胞接触或其分泌的细胞因子有关,但因BM-MSCs功能复杂,目前暂无统一定论。亦有研究表明:滤泡辅助性T细胞(T follicular helper cell,Tfh)作为独立分化来源的新CD4+T细胞亚群,已在多项研究中证实其在自身免疫性疾病、移植免疫与肿瘤免疫等多方面起到重要的调节作用,但是否参与I型糖尿病发生发展尚未可知?因此,我们考虑Tfh细胞是否参与I型糖尿病的发生发展?BM-MSCs是否通过调节Tfh细胞的分化水平从而调节免疫内环境稳态,起到对胰岛移植物的保护作用?本实验就以上问题进行了初步研究。首先在Gotoh胰岛分离技术的基础上进行了改进,使得胰岛分离过程更为简单和有效;同时采用全骨髓培养法分离BM-MSCs细胞并通过流式细胞仪进行鉴定,为BM-MSCs联合胰岛移植提供实验基础。在此基础上,建立非肥胖糖尿病鼠(Non-obese Diabetic mice,NOD)胰岛移植动物模型,研究了不同BM-MSCs联合胰岛移植方案的效果,且初步研究BM-MSCs对Tfh细胞的调控作用,旨在说明MSC是否通过调节Tfh细胞而起到移植胰岛保护作用。第一部分小鼠胰岛的分离与纯化目的:探讨C57BL/6小鼠胰岛分离、纯化的较优方案,并评价分离所得胰岛的生物学特性。方法:小鼠麻醉开腹后,于胰管十二指肠开口处逆行插入24G套管针,先慢后快地注入3m L胰腺消化液,分离胰腺组织,经消化、洗涤、过筛后使用Histopaque-1077溶液与Hanks溶液进行梯度离心,取两溶液中间界面胰岛组织于培养皿中,显微镜下筛选。筛选后的胰岛经双硫腙(DTZ)染色鉴定胰岛细胞,AO/PI染色法鉴定胰岛细胞活性,葡萄糖刺激试验检测胰岛细胞胰岛素分泌功能,ELISA法检测胰岛素水平。结果:经镜下筛选后每只小鼠纯化后获得的胰岛数为195.00±14.95 IEQ/只,纯度90.00%;双硫腙(DTZ)染色后胰岛细胞团呈现猩红色,AO/PI染色后显示,分离的胰岛细胞团内活细胞比例超过95.00%;胰岛素分泌功能检测结果为,高糖刺激下,分离的胰岛释放胰岛素含量(23.00±3.90 ng.ml-110islet-1.45min-1)为低糖浓度(10.10±1.40 ng.ml-110islet-1.45min-1)刺激下的2倍以上,说明分离胰岛具有较高的胰岛素释放能力。结论:胰管十二指肠开口处逆行插管进行消化液灌注与传统的胆总管插管灌注相比可降低因胆管走形较长而出现的刺破胆总管风险,提高手术成功率,Histopaque-1077溶液与Hanks溶液进行密度梯度离心操作简单,提高了实验效率,实验结果证实分离的胰岛功能良好,可用于各项胰岛相关实验。第二部分小鼠骨髓间充质干细胞的分离、培养及鉴定目的:探索小鼠骨髓间充质干细胞(BM-MSCs)原代分离培养方法,鉴定BM-MSCs的生物学特性并为进一步实验提供基础。方法:小鼠消毒处理后分离双侧股骨及胫骨,抽取骨髓腔内骨髓于培养皿中培养,每日于倒置显微镜下观察细胞形态及数量等生长情况。取指数期生长的BM-MSCs细胞,以CD29、CD117、CD44、CD31、Sca-1为细胞特异性标记进行流式细胞仪检测鉴定。结果:每只小鼠平均BM-MSCs获得率为(5.35±0.40)×105个/只,细胞培养状态良好,细胞特异性表达CD29(阳性率96.04%)、CD44(阳性率96.21%)及Sca-1(阳性率95.72%),不表达CD117(阳性率0.13%)及CD31(阳性率0.23%)。结论:全骨髓培养法分离小鼠骨髓间充质干细胞操作较为简单,BM-MSCs获得率高,培养后细胞状态良好,为后续的实验奠定基础。第三部分骨髓间充质干细胞对同种异体胰岛移植物的保护作用及机制目的:研究BM-MSCs联合胰岛移植对胰岛移植物的保护作用,及BM-MSCs对Tfh的调节作用并诱导胰岛移植免疫耐受的机制。方法:实验动物共分为5组。A组(Group A)NOD正常血糖组(非手术组);B组(Group B)糖尿病组(非手术组);C组(Group C)单纯胰岛移植组;D组(Group D)胰岛细胞与BM-MSCs共同移植组;E组(Group E)BM-MSCs尾静脉注射+BM-MSCs/胰岛联合移植组。手术方式为肾包膜下胰岛移植,移植术后检测血糖变化,术后第7天行糖耐量检测。取术后第7天各组小鼠脾脏及外周血行ELISA法检测各组中糖尿病自身抗体GAD65Ab、IAA表达水平,流式细胞仪检测各组外周血及脾脏Tfh细胞水平,Western blot发检测ICOS及Bcl-6表达水平,取移植处肾脏组织行HE染色,免疫组化及免疫荧光染色,观察移植物形态及胰岛素分泌情况。结果:D组(23.67±2.77天)和E组(34.00±4.51天)受鼠平均生存时间较C组(11.67±2.27天)明显延长;C、D、E组受鼠分别从第14.0天、26.0天及42.0天开始表现为移植胰岛功能失活,差异显著;术后第2天C组出现明显低血糖(3.5±1.3 mmol/L),而D组(6.5±1.4 mmol/L)和E组(7.1±1.4 mmol/L)血糖仍在正常范围(5.0~20.0 mmol/L)内;术后第7天糖耐量检测实验结果显示,三个移植组较糖尿病组均有较好的血糖控制,而血糖峰值D组(18.4±0.9 mmol/L)和E组(18.0±0.7 mmol/L)较C组(21.8±1.3 mmol/L)血糖控制更稳定,D组和E组两组之间血糖控制差异不显著;受体血清中糖尿病自身抗体GAD65(A:0.40±0.02;B:1.49±0.03;C:1.20±0.09;D:0.82±0.03;E:0.42±0.03 OD 450nm)及IAA(A:0.31±0.03;B:1.31±0.06;C:1.03±0.07;D:0.70±0.04;E:0.32±0.03 OD450nm)水平差异显著,CD4+CXCR5+Tfh细胞比例分别为A组(4.35±1.54%)、B组(24.55±5.41%)、C组(23.87±6.67%)、D组(9.67±1.34%)及E组(4.27±0.59%),Western blot检测ICOS及Bcl-6表达D组和E组较B组和C组明显降低,差异显著;三手术组移植物病理学检测结果显示:与C组合D组相比,E组胰岛移植物形态更为完整,胰岛融合不明显,淋巴细胞侵润较少,胰岛素分泌水平较高。结论:BM-MSCs通过调节Tfh细胞比例,降低体内糖尿病自身抗体水平,从而起到诱导胰岛移植免疫耐受的效果,对胰岛移植物起到保护作用;且BM-MSCs尾静脉注射+BM-MSCs/胰岛联合移植的方案较单纯胰岛移植及BM-MSCs联合胰岛移植的方案更为有效。
[Abstract]:Islet transplantation has become an important means for the treatment of unstable insulin dependent diabetes since 2000, and it has also brought new research directions in the field of transplantation. Different from other end-stage diseases, the purpose of islet transplantation is to improve the patient's quality of life, so the more ideal method is to induce migration. The state of immune tolerance is to maintain the allograft function well without the use of immunosuppressive agents, and to maintain the host response to other external antigens. Therefore, how to induce the immune tolerance of allograft islet graft is the direction of the research and development of the clinical islet transplantation. B One Marrow Mesenchymal Stem Cells, BM-MSCs) has powerful immune regulation effect. It can inhibit antigen specific or non specific lymphocyte proliferation by secreting a variety of soluble molecules or through their contact with immune cells, inhibit antigen presenting cell differentiation, maturation, and induce antigen specific regulatory T cells (T). The formation of regulatory cell, Treg, which regulates the immune response of the body to the direction of anti inflammatory, is of great clinical value in the fields of organ transplantation and alternative therapy. Contact or its secreted cytokine are related, but because of the complex function of BM-MSCs, there is no general theory at present. There are also studies showing that follicular auxiliary T cells (T follicular helper cell, Tfh), as a new CD4+T cell subgroup of independent differentiation origin, have been confirmed in a number of studies in autoimmune diseases, transplantation immunity and tumor immunity. Is it an important regulatory role, but is it not known to be involved in the development of type I diabetes? Therefore, we consider whether Tfh cells are involved in the development of type I diabetes? Does BM-MSCs regulate the homeostasis of the immune environment by modulating the level of differentiation of Tfh cells to protect the islet grafts? The above experiment A preliminary study was carried out. First, an improvement was made on the basis of the Gotoh islet isolation technology to make the islet separation process more simple and effective. At the same time, the isolation of BM-MSCs cells by full bone marrow culture and identification by flow cytometry were used to provide experimental basis for BM-MSCs combined with islet transplantation. On this basis, non obesity was established. Non-obese Diabetic mice (NOD) pancreas islet transplantation animal model, studied the effect of different BM-MSCs combined islet transplantation, and preliminarily studied the regulation effect of BM-MSCs on Tfh cells. It was aimed to explain whether MSC could protect the pancreas by regulating Tfh cells. The purpose of isolation and purification of islet in the first part of mice was to isolate and purify the islets of the pancreas. The optimal scheme of isolation and purification of the islets of C57BL/6 mice was discussed and the biological characteristics of the isolated islets were evaluated. Methods: after the mice were anesthetized, the 24G cannula was inserted into the pancreatic duct and duodenum open at the opening of the pancreatic duct. The pancreatic juice was injected slowly and quickly into the pancreatic juice of 3M L, and the pancreatic tissue was separated, and the Histopaque-1077 solution was used after digestion and after sieving. A gradient centrifugation was carried out with the Hanks solution. The islet tissue in the middle interface of the two solution was selected in a culture dish and screened under microscope. The islet cells were identified by Dithizone (DTZ) staining after screening. The activity of islet cells was identified by AO/PI staining. The glucose stimulation test was used to detect the secretory function of islet cells, and the level of insulin was detected by ELISA. Fruit: after the screening, the number of isolated islets was 195 + 14.95 IEQ/, the purity was 90%. The islet cell masses were scarlet after dithizone (DTZ) staining, and AO/PI staining showed that the proportion of living cells in the isolated islet cell group was more than 95%; the insulin secretion function test result was the isolated islets under the high sugar stimulation. The release of insulin (23 + 3.90 ng.ml-110islet-1.45min-1) was more than 2 times more than that of low glucose (10.10 + 1.40 ng.ml-110islet-1.45min-1), indicating that the isolated islets had higher insulin release ability. Conclusion: the retrograde intubation of the pancreatic duct and duodenum at the opening of the duodenum is compared with the traditional intubation of the common bile duct. Reducing the risk of choledochal puncture caused by long bile duct shape and increasing the success rate of the operation, the density gradient centrifugation operation of Histopaque-1077 solution and Hanks solution is simple, and the experimental efficiency is improved. The experimental results confirm that the isolated islet function is good and can be used in various islet related experiments. Second part of mouse bone marrow mesenchymal stem cells are used. The purpose of separation, cultivation and identification: To explore the primary separation and culture method of mouse bone marrow mesenchymal stem cells (BM-MSCs), to identify the biological characteristics of BM-MSCs and to provide the basis for further experiments. Methods: the mice were isolated from bilateral femur and tibia after disinfection, and the bone marrow of the bone marrow was extracted from the bone marrow in the culture dish, and daily under the inverted microscope. Observe the growth of cell morphology and quantity. BM-MSCs cells grown in the exponential period were detected by flow cytometry with CD29, CD117, CD44, CD31, Sca-1 as cell specific markers. Results: the average BM-MSCs acquisition rate of each mouse was (5.35 + 0.40) x 105 / only, the cell culture state was good and the cell specificity expressed CD29 (positive rate 96.04%). CD44 (positive rate 96.21%) and Sca-1 (positive rate 95.72%) did not express CD117 (positive rate 0.13%) and CD31 (positive rate 0.23%). Conclusion: total bone marrow culture method for isolation of mouse bone marrow mesenchymal stem cells is relatively simple, BM-MSCs obtains high rate, and the cell status is good after culture. Third parts of bone marrow mesenchymal stem cells are established. Protective effect and mechanism of allograft islet graft: study the protective effect of BM-MSCs combined islet transplantation on islet graft, and the mechanism of BM-MSCs regulating Tfh and inducing immune tolerance of islet transplantation. Methods: the experimental animals were divided into 5 groups of.A group (Group A) NOD normal blood glucose group (non operation group); B group (Group B) diabetes mellitus Group (non operation group); group C (Group C) simple islet transplantation group; group D (Group D) islet cells and BM-MSCs co transplantation group; E group (Group E) BM-MSCs caudal vein injection of +BM-MSCs/ islet islet group. The operation method was subcapsular islet transplantation. After transplantation, blood sugar changes were detected and seventh days after operation, the glucose tolerance test was performed. Each group was small after seventh days. The ELISA method of spleen and peripheral blood was used to detect the GAD65Ab, IAA expression level of the autoantibodies in each group. The level of peripheral blood and spleen Tfh cells were detected by flow cytometry. The expression level of ICOS and Bcl-6 was detected by Western blot, HE staining, immunohistochemistry and immunofluorescence staining were taken to observe the morphology of graft and the islets of the pancreas in the transplanted kidney tissue. Results: the average survival time of the rats in group D (23.67 + 2.77 days) and group E (34 + 4.51 days) was significantly longer than that in group C (11.67 + 2.27 days). The group of C, D and E showed the function inactivation of the transplanted islet from 14 days, 26 days and 42 days respectively, and there were significant hypoglycemia (3.5 + 1.3 mmol/L) in the C group on second days after operation (3.5 + 1.3 mmol/L), and D group (6.5 +). 1.4 mmol/L) and group E (7.1 + 1.4 mmol/L) were still in normal range of blood glucose (5.0~20.0 mmol/L). The results of glucose tolerance test on seventh days after operation showed that three transplanted groups had better blood glucose control than those in the diabetic group, while the peak blood glucose peak D group (18.4 + 0.9 mmol/L) and E group (18 + 0.7 mmol/L) were more stable than the C group (21.8 + 1.3 mmol/L), and the D group was more stable. There was no significant difference in blood glucose control between the two groups in the group E, and the levels of diabetes autoantibody GAD65 (A:0.40 + 0.02; B:1.49 + 0.03; C:1.20 + 0.09; D:0.82 + 0.03; E:0.42 + 0.03 OD 450nm) and IAA (A:0.31 + 0.03; B:1.31 + 0.06; 0.04; 0.04; 0.03) Group (4.35 + 1.54%), group B (24.55 + 5.41%), group C (23.87 + 6.67%), group D (9.67 + 1.34%) and E (4.27 + 0.59%), Western blot detection of ICOS and Bcl-6 expression in D group and E group were significantly lower than B group and C group, and the pathological examination results showed that the form of islet graft was more complete and islet melting compared with that of combination group. It is not obvious that the lymphocyte invasion is less and the level of insulin secretion is high. Conclusion: BM-MSCs can induce the immune tolerance of islet transplantation by regulating the proportion of Tfh cells, reducing the level of autoantibody in the body of diabetes, and protecting the islet graft; and the scheme of +BM-MSCs/ islet transplantation by BM-MSCs caudal vein injection. It is more effective than islet transplantation and BM-MSCs combined with islet transplantation.

【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R657.5

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