本文選題：大鼠 + 骨髓��； 參考：《復(fù)旦大學(xué)》2007年博士論文

【摘要】： 器官移植的治療目的是向人體引入異體的細(xì)胞、組織或器官，從而代替喪失相應(yīng)功能的細(xì)胞、組織或器官，并使受體接受，不被機(jī)體免疫系統(tǒng)排斥，而且移植物也不排斥宿主。目前器官移植術(shù)后，受體多須終身使用免疫抑制劑，降低受體的全身免疫功能，減少或抑制受體對(duì)移植物的免疫排斥反應(yīng)以達(dá)到維持移植物長(zhǎng)期存活的目的。但是長(zhǎng)期使用用免疫抑制劑導(dǎo)致感染和腫瘤高發(fā)，而且各種免疫抑制劑藥物的毒副作用影響受體的生存質(zhì)量，嚴(yán)重者甚至導(dǎo)致慢性移植物功能衰竭的發(fā)生。此外，即使受體長(zhǎng)期服用并能耐受免疫抑制劑，移植物長(zhǎng)期存活率也并不理想。誘導(dǎo)受體對(duì)供者器官特異性免疫耐受是解決排斥反應(yīng)最理想的措施。也就是說(shuō)在長(zhǎng)期不使用免疫抑制劑的情況下，移植物既不被受體排斥，發(fā)生宿主抗移植物反應(yīng)；同時(shí)也不致發(fā)生移植物抗宿主反應(yīng)，但是仍保留對(duì)除移植物以外的正常免疫應(yīng)答。 間充質(zhì)干細(xì)胞是中胚層來(lái)源的具有多向分化能力的干細(xì)胞，主要存在于全身結(jié)締組織和器官間質(zhì)中，以骨髓組織中含量最豐富。間充質(zhì)干細(xì)胞具有多向分化能力，可以分化為骨、軟骨、脂肪等；具有較低的免疫原性，在體內(nèi)可以逃避免疫系統(tǒng)的攻擊；同時(shí)發(fā)現(xiàn)具有免疫調(diào)節(jié)作用，可以抑制同種異體T細(xì)胞增殖，研究表明間充質(zhì)干細(xì)胞可以延長(zhǎng)皮膚移植物的存活時(shí)間，減少移植物抗宿主病的發(fā)生。近來(lái)對(duì)間充質(zhì)干細(xì)胞免疫調(diào)控功能的研究，提示其在誘導(dǎo)器官移植免疫耐受中的應(yīng)用前景。本研究從大鼠骨髓中分離出間充質(zhì)干細(xì)胞，建立大鼠同種異位心臟移植模型，旨在研究骨髓間充質(zhì)干細(xì)胞在心臟移植中誘導(dǎo)免疫耐受，減少免疫排斥的作用，并探討其可能機(jī)制。本研究分為四部分。 第一部分：大鼠骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)和生物學(xué)鑒定 第二部分大鼠骨髓間充質(zhì)干細(xì)胞對(duì)同種T細(xì)胞的作用及其機(jī)制 第三部分大鼠同種異體心臟移植模型的建立 第四部分大鼠骨髓間充質(zhì)干細(xì)胞對(duì)同種異體心臟移植的作用及其機(jī)制 第一部分大鼠骨髓間充質(zhì)干細(xì)胞的分離培養(yǎng)和生物學(xué)鑒定 【目的】探討體外分離、純化大鼠骨髓間充質(zhì)干細(xì)胞的方法，并分析其表型特點(diǎn)；評(píng)價(jià)大鼠骨髓間充質(zhì)干細(xì)胞進(jìn)行同種異體移植的安全性。 【方法】用密度梯度離心法分離純化大鼠骨髓MSCs，傳代擴(kuò)增，，形態(tài)學(xué)觀察，流式細(xì)胞儀分析測(cè)定細(xì)胞表面抗原，DAPI標(biāo)記。20只Wistar大鼠，體重200—250g。隨機(jī)分為2組：A空白對(duì)照組，B MSCs移植組。觀察移植術(shù)后大鼠一般情況，術(shù)后7天處死取骨髓涂片，免疫熒光鏡下觀察。 【結(jié)果】MSCs是骨髓細(xì)胞中的單個(gè)核細(xì)胞，密度梯度離心法能有效分離純化大鼠骨髓MSCs，MSCs在含10％胎牛血清的L-DMEM中生長(zhǎng)性狀相對(duì)穩(wěn)定，細(xì)胞呈均一的成纖維細(xì)胞樣，均一表達(dá)CD90，不表達(dá)CD34、CD45。MSCs移植組可以長(zhǎng)期存活，術(shù)后7天骨髓涂片可見存活的MSCs。 【結(jié)論】本實(shí)驗(yàn)建立了一種體外分離純化、培養(yǎng)擴(kuò)增大鼠骨髓MSCs的方法，MSCs穩(wěn)定表達(dá)CD90，不表達(dá)CD45、CD34；同種異體來(lái)源的MSCs可在宿主體內(nèi)存活、定居，并未發(fā)生免疫排斥。 第二部分大鼠骨髓間充質(zhì)干細(xì)胞對(duì)同種T細(xì)胞的作用及其機(jī)制 【目的】探討骨髓間充質(zhì)干細(xì)胞(MSCs)在混和淋巴細(xì)胞反應(yīng)中對(duì)同種異體T淋巴細(xì)胞免疫應(yīng)答反應(yīng)的影響，并初步探討其作用機(jī)制。 【方法】建立MSCs和同種異體淋巴細(xì)胞共培養(yǎng)體系，反應(yīng)體系總量250μl。以SD大鼠的脾T淋巴細(xì)胞為刺激細(xì)胞，以Wistar大鼠的脾T淋巴細(xì)胞為反應(yīng)細(xì)胞，分為6組。組Ⅰ：對(duì)照組，1×10~5／50μl刺激細(xì)胞和1×10~5／50μl反應(yīng)細(xì)胞共同培養(yǎng)；組Ⅱ：1×10~5／50μl反應(yīng)細(xì)胞與1×10~4／50μl SD大鼠的MSCs共同培養(yǎng)；組Ⅲ：1×10~5／50μl刺激細(xì)胞和1×10~5／50μl反應(yīng)細(xì)胞并加入1×10~4／100μl SD大鼠的MSCs共同培養(yǎng)；組Ⅳ：細(xì)胞種類及數(shù)量同第三組，，另加1-甲基色氨酸(1—MT)(終濃度1mmol／ml)；組Ⅴ：細(xì)胞種類及數(shù)量同第三組，另加植物刺激素(終濃度2μg／ml)；組Ⅵ：每孔加入反應(yīng)細(xì)胞和刺激細(xì)胞各50μl(各含細(xì)胞數(shù)1×10~5個(gè))及MSC0.01ml(含細(xì)胞數(shù)1×10~3個(gè))。混合培養(yǎng)120小時(shí)，結(jié)束培養(yǎng)前13小時(shí)，每孔加入~3H-TdR 20ul，以液閃測(cè)定儀測(cè)定各組的CPM值。反相高效液相色譜法檢測(cè)MSC和MLR共培養(yǎng)體系中色氨酸含量。 【結(jié)果】MSCs可以抑制MLC體系中T淋巴細(xì)胞增殖，并呈現(xiàn)出劑量依賴關(guān)系；同時(shí)MSCs和MLR共培養(yǎng)體系中色氨酸含量明顯降低。1-MT可以阻斷這一作用。 【結(jié)論】MSCs在體外可抑制同種異體T淋巴細(xì)胞的免疫應(yīng)答，IDO參與了這種免疫抑制作用。 第三部分同種異體大鼠異位心臟移植模型的建立 【目的】以改良Ono法進(jìn)行大鼠腹部心臟移植，建立動(dòng)物模型。 【方法】以體重200～250gSD大鼠為供體，體重200～250g的Wistar大鼠為受體，進(jìn)行大鼠腹部異位心臟移植手術(shù)。SD大鼠經(jīng)麻醉、肝素化后，打開胸腔，阻斷主動(dòng)脈，經(jīng)主動(dòng)脈根部灌注心肌保護(hù)液，將供體的心臟取下。Wistar大鼠麻醉后，打開腹腔，將供體的主動(dòng)脈與受體的腹主動(dòng)脈行端側(cè)吻合，將供體的肺動(dòng)脈和受體的下腔靜脈行端側(cè)吻合。 【結(jié)果】大鼠異位心臟移植模型成功率達(dá)100％�？偸中g(shù)時(shí)間約60～75min，平均(70.1±4.9min)；供心摘取時(shí)間約8～12min，平均(10.5±2.3min)；受體準(zhǔn)備時(shí)間約11～15min，平均(13.7±2.0min) 【結(jié)論】改良Ono法進(jìn)行大鼠腹部心臟移植模型可靠易行，可重復(fù)率高 第四部分大鼠骨髓間充質(zhì)干細(xì)胞對(duì)同種異體心臟移植的作用及其機(jī)制 【目的】研究靜脈輸注骨髓間充質(zhì)干細(xì)胞(MSCs)對(duì)心臟移植術(shù)后急性排斥反應(yīng)的影響及其機(jī)制。 【方法】64只Wistar大鼠隨機(jī)分成4組，每組16只，作為受體。組Ⅰ：空白對(duì)照組經(jīng)腰靜脈注射PBS；組Ⅱ術(shù)中經(jīng)腰靜脈注射供體大鼠骨髓間充質(zhì)干細(xì)胞1×10~6／ml；組Ⅲ術(shù)后7天每天使用環(huán)胞霉素5mg／Kg；組Ⅳ術(shù)中供心經(jīng)腰靜脈注射供體大鼠骨髓間充質(zhì)干細(xì)胞1×10~6／ml，術(shù)后7天每天使用環(huán)胞霉素5mg／Kg。觀察各組供心存活時(shí)間；術(shù)后七天取供心，觀察病理改變，并取脾臟細(xì)胞懸液流式細(xì)胞儀檢測(cè)CD4~+、CD8~+的百分比，計(jì)算并比較各組CD4~+／CD8~+的比值，ELISA法檢測(cè)外周血細(xì)胞因子干擾素-γ(Interferon-γ，IFN-γ)、白細(xì)胞介素—10(interleukin-10，IL-10)水平。 【結(jié)果】移植心臟的存活時(shí)間為組Ⅰ：7.5±0.9天；組Ⅱ：15.5±1.6天；組Ⅲ：17.3±1.3天；組Ⅳ：33.4±3.2天。統(tǒng)計(jì)學(xué)分析顯示，移植心臟存活時(shí)間組Ⅱ比組Ⅰ明顯延長(zhǎng)(p＜0.05)，組Ⅳ比其它三組明顯延長(zhǎng)(P＜0.01)。組Ⅳ移植心臟病理改變較其它三組為輕。受體外周血CD4／CD8比例，組Ⅱ、Ⅲ和Ⅳ均高于組Ⅰ(P＜0.05)；組Ⅳ與其它三組相比亦明顯增高(P＜0.05)。受體血清IFN-γ水平，組Ⅱ、Ⅲ明顯低于組Ⅰ，組Ⅳ低于其他三組(P＜0.05)。血清IL-10水平，組Ⅱ、Ⅲ和Ⅳ均高于組Ⅰ(p＜0.05)，組Ⅳ高于組Ⅰ和組Ⅱ(P＜0.05) 【結(jié)論】骨髓間充質(zhì)干細(xì)胞可減輕大鼠同種異體移植心臟的免疫排斥反應(yīng)并延長(zhǎng)移植心臟的存活時(shí)間。其機(jī)制可能與調(diào)節(jié)同種異體T細(xì)胞功能和細(xì)胞因子分泌有關(guān)。環(huán)胞霉素A與骨髓間充質(zhì)干細(xì)胞聯(lián)合應(yīng)用抑制免疫排斥反應(yīng)有協(xié)同作用。
[Abstract]:The purpose of organ transplantation is to introduce allogenic cells, tissues or organs to the human body instead of the cells, tissues or organs that lose the corresponding function, and to make receptors accept, not be rejected by the immune system, and the grafts do not repel the host. The systemic immune function reduces or inhibits the rejection of the receptor to the graft for the purpose of maintaining the long-term survival of the graft. However, the long-term use of immunosuppressive agents leads to high incidence of infection and tumor, and the toxic side effects of various immunosuppressive drugs affect the quality of the receptor, and the serious may even lead to chronic graft work. In addition, the long-term survival rate of the graft is not ideal even if the receptor is long taken and can tolerate immunosuppressive agents. The most ideal measure to induce the recipient's organ specific immune tolerance is to solve the rejection. That is to say, the graft is not rejected by the receptor in the long term without the use of immunosuppressive agents. The host is resistant to graft reaction, and does not cause graft versus host reaction, but it still maintains normal immune response except for allografts.
Mesenchymal stem cells are the multidirectional stem cells derived from mesoderm, mainly in the connective tissue and interstitial tissue of the whole body, and are the most abundant in the bone marrow tissue. Mesenchymal stem cells have the ability to differentiate into bone, cartilage and fat, which can be differentiated into bone, cartilage and fat. It has a low immunogenicity and can escape the immune system in the body. At the same time, it is found that the immune regulation can inhibit the proliferation of allogenic T cells. The study shows that mesenchymal stem cells can prolong the survival time of the skin graft and reduce the occurrence of graft versus host disease. Recent studies on the immune regulation function of mesenchymal stem cells suggest that it can induce immune tolerance in organ transplantation. In this study, mesenchymal stem cells were isolated from rat bone marrow, and a rat model of heterotopic heart transplantation was established. The aim of this study was to study the immune tolerance induced by bone marrow mesenchymal stem cells in heart transplantation, reduce immune rejection, and explore its possible mechanism. This study was divided into four parts.
Part one: isolation, culture and biological identification of rat bone marrow mesenchymal stem cells
The second part is the effect and mechanism of rat bone marrow mesenchymal stem cells on homologous T cells.
The third part is the establishment of rat cardiac allograft model.
The effect and mechanism of fourth parts of rat bone marrow mesenchymal stem cells on allograft heart transplantation
Part one: isolation, culture and biological identification of rat bone marrow mesenchymal stem cells
[Objective] to investigate the method of isolation and purification of rat bone marrow mesenchymal stem cells in vitro, and to analyze its phenotypic characteristics, and to evaluate the safety of allograft transplantation of bone marrow mesenchymal stem cells in rats.
[Methods] the density gradient centrifugation was used to separate and purify the rat bone marrow MSCs and be amplified, and the morphological observation and flow cytometry were used to determine the cell surface antigen. The DAPI labeled.20 rats were only Wistar rats, and the weight 200 to 250g. were randomly divided into 2 groups: A blank control group and B MSCs transplantation group. The general condition of the rats after the transplantation was observed and the bone was killed 7 days after the operation. Medullary smears were observed under immunofluorescence.
[results] MSCs is a mononuclear cell in bone marrow cells. Density gradient centrifugation can effectively separate and purify rat bone marrow MSCs. The growth character of MSCs in L-DMEM containing 10% fetal bovine serum is relatively stable, the cells are homogeneous fibroblast like, CD90 is expressed as CD90, and CD34 is not expressed. The CD45.MSCs transplantation group can survive for a long time and the bone marrow is 7 days after the operation. The smear can be seen in the surviving MSCs.
[Conclusion] in this experiment, a method of isolation and purification in vitro was established to culture and amplify the MSCs of rat bone marrow. MSCs expressed CD90 steadily and did not express CD45, CD34. The MSCs of allogeneic origin could survive in the host and settled without immune rejection.
The second part is the effect and mechanism of rat bone marrow mesenchymal stem cells on homologous T cells.
[Objective] to investigate the effect of bone marrow mesenchymal stem cells (MSCs) on the immune response of allogeneic T lymphocyte in mixed lymphocyte reaction and to explore its mechanism.
[Methods] to establish a co culture system of MSCs and allogeneic lymphocyte, the total amount of the reaction system was 250 Mu L., the spleen T lymphocyte of SD rats was used as the stimulating cell, and the spleen T lymphocyte of Wistar rats was divided into 6 groups. Group I: the control group, 1 x 10~5 / 50 micron stimulus cells and 1 x 10~5 / 50 micron l reactive cells co culture; group II: 1 10~5 / 50 mu l reactive cells were co cultured with MSCs of 1 x 10~4 / 50 mu L SD rats; group III: 1 x 10~5 / 50 micron l irritation cells and 1 x 10~5 / 50 micron l reactive cells and 1 x 10~4 / 100 micron l rats. V: the type and number of cells in the same third groups, plus plant stimulating hormone (terminal concentration 2 g / ml), group VI: 50 mu L (each cell number 1 x 10~5) and MSC0.01ml (the number of cells with 1 x 10~3) per pore, mixed culture for 120 hours, 13 hours before culture, ~3H-TdR 20ul per pore, and measured by liquid flash meter The CPM value of each group was determined by RP HPLC. The tryptophan content in MSC and MLR co culture system was detected.
[results] MSCs can inhibit the proliferation of T lymphocytes in the MLC system, and present a dose dependent relationship. At the same time, the content of tryptophan in the co culture system of MSCs and MLR can be significantly reduced by.1-MT to block this effect.
[Conclusion] MSCs can inhibit the immune response of allogeneic T lymphocytes in vitro, and IDO is involved in this immunosuppressive effect.
The third part is the establishment of heterotopic heart transplantation model in rats.
[Objective] to establish an animal model of abdominal heart transplantation in rats by modified Ono method.
[Methods] the Wistar rats of 200 ~ 250gSD body weight and 200 to 250g were used as receptors, and the rats were anesthetized with.SD rats with abdominal ectopic heart transplantation. After heparinized, the thoracic cavity was opened, the aorta was blocked and the myocardial protective solution was perfused through the aorta root. The donor heart was removed from the.Wistar rats and the abdominal cavity was opened. The abdominal cavity was opened and will be opened. The aorta of the aorta and the recipient's abdominal aorta end to side anastomosis, and the donor pulmonary artery and the recipient's inferior vena cava end to side anastomosis.
[results] the total operation time of the model of heterotopic heart transplantation in rats was about 60 ~ 75min, average (70.1 + 4.9min), and the time of donor heart extraction was about 8 ~ 12min, average (10.5 + 2.3min), and the preparation time of the receptor was 11 ~ 15min, average (13.7 + 2.0min).
[Conclusion] the modified Ono method is reliable and highly repeatable for abdominal heart transplantation in rats.
The effect and mechanism of fourth parts of rat bone marrow mesenchymal stem cells on allograft heart transplantation
[Objective] to study the effect and mechanism of intravenous infusion of bone marrow mesenchymal stem cells (MSCs) on acute rejection after heart transplantation.
[Methods] 64 Wistar rats were randomly divided into 4 groups, with 16 rats in each group. Group I: the blank control group was injected with PBS in the lumbar vein, and the bone marrow mesenchymal stem cells of the donor rats were injected into the lumbar vein during the second operation 1 x 10~6 / ml, and the group used cyclosporin 5mg / Kg every day after the third operation; the group IV was injected into the donor rat via the lumbar vein. Bone marrow mesenchymal stem cells were 1 x 10~6 / ml, 7 days after operation, the donor heart survival time was observed by cyclosporin 5mg / Kg.. The donor heart was observed on the seven day after operation, and the pathological changes were observed and the percentage of CD4~+ and CD8~+ was detected by the splenic cell suspension flow cytometry. The ratio of CD4~+ / CD8~+ in each group was calculated and compared. ELISA method was used to detect the peripheral blood cell cause. The levels of interferon - gamma (Interferon- gamma, IFN- gamma) and interleukin - 10 (interleukin-10, IL-10).
[results] the survival time of the transplanted heart was group I: 7.5 + 0.9 days, group II: 15.5 + 1.6 days, group III: 17.3 + 1.3 days, group IV: 33.4 + 3.2 days. Statistical analysis showed that the survival time group of the transplanted heart group was significantly longer than group I (P < 0.05), group IV was significantly longer than the other three groups (P < 0.01). Group IV transplantation heart pathological changes were compared with those in group IV The three groups were light. Group II, III and IV were higher than group I (P < 0.05). Group IV was also significantly higher than the other three groups (P < 0.05). The level of IFN- gamma in the receptor serum, group II, III was significantly lower than group I, group IV was lower than the other three groups (P < 0.05). Serum IL-10 level, group II, III and IV were higher than group I (P < 0.05), group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV, group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV, group IV group IV, group IV group IV (P < 0.05), group IV Group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV, group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV group IV (P < 0.05), group IV Higher than group I and group II (P < 0.05)
[Conclusion] bone marrow mesenchymal stem cells can relieve the immune rejection of allograft heart and prolong the survival time of transplanted heart. The mechanism may be related to the regulation of the function of allogenic T cells and the secretion of cytokine. The combination of cytosamycin A and bone marrow mesenchymal stem cells combined with the inhibition of immune rejection has a synergistic effect. Use.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類號(hào)】：R654.2;R392.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 康新勤,臧偉進(jìn),宋土生,于曉江,曾菊絨;大鼠骨髓間充質(zhì)干細(xì)胞分離培養(yǎng)及其形態(tài)觀察[J];西安交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2003年05期

2 呼瑩,張麗艷,馬冠杰,姜學(xué)英,趙春華;胎兒骨髓和肝臟間充質(zhì)干細(xì)胞的表型和生物學(xué)性狀研究[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2001年04期

3 鄧為民,韓欽,尤勝國(guó),張偉,葛薇,李長(zhǎng)虹,鄧?guó)櫂I(yè),趙春華;異基因骨髓源間充質(zhì)干細(xì)胞移植形成穩(wěn)定的嵌合體并誘導(dǎo)免疫耐受的研究[J];中華檢驗(yàn)醫(yī)學(xué)雜志;2003年08期

本文編號(hào)：1817376