發(fā)布時(shí)間：2018-05-11 11:05

  本文選題：胰島移植 + 骨髓間充質(zhì)干細(xì)胞�。� 參考：《上海交通大學(xué)》2015年碩士論文

【摘要】：胰島移植自2000年來(lái)已成為治療不穩(wěn)定性胰島素依賴(lài)型糖尿病的重要手段,同時(shí)這也給移植領(lǐng)域帶來(lái)了新的研究方向。不同于其它終末期疾病所進(jìn)行的器官移植,胰島移植的目的更注重改善患者的生存質(zhì)量,所以更理想的方法是誘導(dǎo)移植免疫耐受狀態(tài),即在不使用免疫抑制劑的情況下,維持同種異體移植物功能良好,同時(shí)保持機(jī)體對(duì)其它外來(lái)抗原正常的宿主反應(yīng)。因此,如何誘導(dǎo)同種異體胰島移植物的免疫耐受成為臨床胰島移植研究發(fā)展的方向。骨髓間充質(zhì)干細(xì)胞(Bone Marrow Mesenchymal Stem Cells,BM-MSCs)有強(qiáng)大的免疫調(diào)節(jié)作用,可通過(guò)分泌多種可溶性分子或通過(guò)其與免疫細(xì)胞的相互接觸等途徑抑制抗原特異性或非特異性的淋巴細(xì)胞增殖,抑制抗原提呈細(xì)胞的分化、成熟,誘導(dǎo)抗原特異性調(diào)節(jié)性T細(xì)胞(T regulatory cell,Treg)的形成,調(diào)節(jié)機(jī)體免疫應(yīng)答向抗炎癥方向偏移,在器官移植、替代治療等領(lǐng)域具有巨大的臨床應(yīng)用價(jià)值。有研究表明:間充質(zhì)干細(xì)胞聯(lián)合胰島移植可提高胰島移植物存活時(shí)間,調(diào)節(jié)胰島移植物免疫耐受,其作用機(jī)制與細(xì)胞接觸或其分泌的細(xì)胞因子有關(guān),但因BM-MSCs功能復(fù)雜,目前暫無(wú)統(tǒng)一定論。亦有研究表明:濾泡輔助性T細(xì)胞(T follicular helper cell,Tfh)作為獨(dú)立分化來(lái)源的新CD4+T細(xì)胞亞群,已在多項(xiàng)研究中證實(shí)其在自身免疫性疾病、移植免疫與腫瘤免疫等多方面起到重要的調(diào)節(jié)作用,但是否參與I型糖尿病發(fā)生發(fā)展尚未可知?因此,我們考慮Tfh細(xì)胞是否參與I型糖尿病的發(fā)生發(fā)展?BM-MSCs是否通過(guò)調(diào)節(jié)Tfh細(xì)胞的分化水平從而調(diào)節(jié)免疫內(nèi)環(huán)境穩(wěn)態(tài),起到對(duì)胰島移植物的保護(hù)作用?本實(shí)驗(yàn)就以上問(wèn)題進(jìn)行了初步研究。首先在Gotoh胰島分離技術(shù)的基礎(chǔ)上進(jìn)行了改進(jìn),使得胰島分離過(guò)程更為簡(jiǎn)單和有效;同時(shí)采用全骨髓培養(yǎng)法分離BM-MSCs細(xì)胞并通過(guò)流式細(xì)胞儀進(jìn)行鑒定,為BM-MSCs聯(lián)合胰島移植提供實(shí)驗(yàn)基礎(chǔ)。在此基礎(chǔ)上,建立非肥胖糖尿病鼠(Non-obese Diabetic mice,NOD)胰島移植動(dòng)物模型,研究了不同BM-MSCs聯(lián)合胰島移植方案的效果,且初步研究BM-MSCs對(duì)Tfh細(xì)胞的調(diào)控作用,旨在說(shuō)明MSC是否通過(guò)調(diào)節(jié)Tfh細(xì)胞而起到移植胰島保護(hù)作用。第一部分小鼠胰島的分離與純化目的:探討C57BL/6小鼠胰島分離、純化的較優(yōu)方案,并評(píng)價(jià)分離所得胰島的生物學(xué)特性。方法:小鼠麻醉開(kāi)腹后,于胰管十二指腸開(kāi)口處逆行插入24G套管針,先慢后快地注入3m L胰腺消化液,分離胰腺組織,經(jīng)消化、洗滌、過(guò)篩后使用Histopaque-1077溶液與Hanks溶液進(jìn)行梯度離心,取兩溶液中間界面胰島組織于培養(yǎng)皿中,顯微鏡下篩選。篩選后的胰島經(jīng)雙硫腙(DTZ)染色鑒定胰島細(xì)胞,AO/PI染色法鑒定胰島細(xì)胞活性,葡萄糖刺激試驗(yàn)檢測(cè)胰島細(xì)胞胰島素分泌功能,ELISA法檢測(cè)胰島素水平。結(jié)果:經(jīng)鏡下篩選后每只小鼠純化后獲得的胰島數(shù)為195.00±14.95 IEQ/只,純度90.00%;雙硫腙(DTZ)染色后胰島細(xì)胞團(tuán)呈現(xiàn)猩紅色,AO/PI染色后顯示,分離的胰島細(xì)胞團(tuán)內(nèi)活細(xì)胞比例超過(guò)95.00%;胰島素分泌功能檢測(cè)結(jié)果為,高糖刺激下,分離的胰島釋放胰島素含量(23.00±3.90 ng.ml-110islet-1.45min-1)為低糖濃度(10.10±1.40 ng.ml-110islet-1.45min-1)刺激下的2倍以上,說(shuō)明分離胰島具有較高的胰島素釋放能力。結(jié)論:胰管十二指腸開(kāi)口處逆行插管進(jìn)行消化液灌注與傳統(tǒng)的膽總管插管灌注相比可降低因膽管走形較長(zhǎng)而出現(xiàn)的刺破膽總管風(fēng)險(xiǎn),提高手術(shù)成功率,Histopaque-1077溶液與Hanks溶液進(jìn)行密度梯度離心操作簡(jiǎn)單,提高了實(shí)驗(yàn)效率,實(shí)驗(yàn)結(jié)果證實(shí)分離的胰島功能良好,可用于各項(xiàng)胰島相關(guān)實(shí)驗(yàn)。第二部分小鼠骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)及鑒定目的:探索小鼠骨髓間充質(zhì)干細(xì)胞(BM-MSCs)原代分離培養(yǎng)方法,鑒定BM-MSCs的生物學(xué)特性并為進(jìn)一步實(shí)驗(yàn)提供基礎(chǔ)。方法:小鼠消毒處理后分離雙側(cè)股骨及脛骨,抽取骨髓腔內(nèi)骨髓于培養(yǎng)皿中培養(yǎng),每日于倒置顯微鏡下觀察細(xì)胞形態(tài)及數(shù)量等生長(zhǎng)情況。取指數(shù)期生長(zhǎng)的BM-MSCs細(xì)胞,以CD29、CD117、CD44、CD31、Sca-1為細(xì)胞特異性標(biāo)記進(jìn)行流式細(xì)胞儀檢測(cè)鑒定。結(jié)果:每只小鼠平均BM-MSCs獲得率為(5.35±0.40)×105個(gè)/只,細(xì)胞培養(yǎng)狀態(tài)良好,細(xì)胞特異性表達(dá)CD29(陽(yáng)性率96.04%)、CD44(陽(yáng)性率96.21%)及Sca-1(陽(yáng)性率95.72%),不表達(dá)CD117(陽(yáng)性率0.13%)及CD31(陽(yáng)性率0.23%)。結(jié)論:全骨髓培養(yǎng)法分離小鼠骨髓間充質(zhì)干細(xì)胞操作較為簡(jiǎn)單,BM-MSCs獲得率高,培養(yǎng)后細(xì)胞狀態(tài)良好,為后續(xù)的實(shí)驗(yàn)奠定基礎(chǔ)。第三部分骨髓間充質(zhì)干細(xì)胞對(duì)同種異體胰島移植物的保護(hù)作用及機(jī)制目的:研究BM-MSCs聯(lián)合胰島移植對(duì)胰島移植物的保護(hù)作用,及BM-MSCs對(duì)Tfh的調(diào)節(jié)作用并誘導(dǎo)胰島移植免疫耐受的機(jī)制。方法:實(shí)驗(yàn)動(dòng)物共分為5組。A組(Group A)NOD正常血糖組(非手術(shù)組);B組(Group B)糖尿病組(非手術(shù)組);C組(Group C)單純胰島移植組;D組(Group D)胰島細(xì)胞與BM-MSCs共同移植組;E組(Group E)BM-MSCs尾靜脈注射+BM-MSCs/胰島聯(lián)合移植組。手術(shù)方式為腎包膜下胰島移植,移植術(shù)后檢測(cè)血糖變化,術(shù)后第7天行糖耐量檢測(cè)。取術(shù)后第7天各組小鼠脾臟及外周血行ELISA法檢測(cè)各組中糖尿病自身抗體GAD65Ab、IAA表達(dá)水平,流式細(xì)胞儀檢測(cè)各組外周血及脾臟Tfh細(xì)胞水平,Western blot發(fā)檢測(cè)ICOS及Bcl-6表達(dá)水平,取移植處腎臟組織行HE染色,免疫組化及免疫熒光染色,觀察移植物形態(tài)及胰島素分泌情況。結(jié)果:D組(23.67±2.77天)和E組(34.00±4.51天)受鼠平均生存時(shí)間較C組(11.67±2.27天)明顯延長(zhǎng);C、D、E組受鼠分別從第14.0天、26.0天及42.0天開(kāi)始表現(xiàn)為移植胰島功能失活,差異顯著;術(shù)后第2天C組出現(xiàn)明顯低血糖(3.5±1.3 mmol/L),而D組(6.5±1.4 mmol/L)和E組(7.1±1.4 mmol/L)血糖仍在正常范圍(5.0~20.0 mmol/L)內(nèi);術(shù)后第7天糖耐量檢測(cè)實(shí)驗(yàn)結(jié)果顯示,三個(gè)移植組較糖尿病組均有較好的血糖控制,而血糖峰值D組(18.4±0.9 mmol/L)和E組(18.0±0.7 mmol/L)較C組(21.8±1.3 mmol/L)血糖控制更穩(wěn)定,D組和E組兩組之間血糖控制差異不顯著;受體血清中糖尿病自身抗體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)水平差異顯著,CD4+CXCR5+Tfh細(xì)胞比例分別為A組(4.35±1.54%)、B組(24.55±5.41%)、C組(23.87±6.67%)、D組(9.67±1.34%)及E組(4.27±0.59%),Western blot檢測(cè)ICOS及Bcl-6表達(dá)D組和E組較B組和C組明顯降低,差異顯著;三手術(shù)組移植物病理學(xué)檢測(cè)結(jié)果顯示:與C組合D組相比,E組胰島移植物形態(tài)更為完整,胰島融合不明顯,淋巴細(xì)胞侵潤(rùn)較少,胰島素分泌水平較高。結(jié)論:BM-MSCs通過(guò)調(diào)節(jié)Tfh細(xì)胞比例,降低體內(nèi)糖尿病自身抗體水平,從而起到誘導(dǎo)胰島移植免疫耐受的效果,對(duì)胰島移植物起到保護(hù)作用;且BM-MSCs尾靜脈注射+BM-MSCs/胰島聯(lián)合移植的方案較單純胰島移植及BM-MSCs聯(lián)合胰島移植的方案更為有效。
[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.

【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R657.5

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