本文選題：間充質(zhì)干細(xì)胞 + 實(shí)驗(yàn)性自體免疫性葡萄膜炎��； 參考：《天津醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的 葡萄膜炎是一類最常見的眼部自體免疫性疾病,其中多種類型具有反復(fù)發(fā)作、并發(fā)癥多、治療困難以及預(yù)后較差和致盲率較高等特點(diǎn)。間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)能夠抑制多種免疫細(xì)胞(包括T淋巴細(xì)胞、B淋巴細(xì)胞、NK細(xì)胞以及樹突狀細(xì)胞)的增殖和功能。本研究采用MSCs治療大鼠實(shí)驗(yàn)性自體免疫性葡萄膜炎(experimental autoimmune uveoretinitis, EAU),檢測(cè)MSCs在患有葡萄膜炎的大鼠體內(nèi)能否發(fā)揮免疫調(diào)節(jié)作用,以及其可能的作用機(jī)制,試圖為臨床治療葡萄膜炎提供新的思路以及可供選擇的治療方法。方法1.原代培養(yǎng)Lewis或Wistar大鼠骨髓間充質(zhì)干細(xì)胞,達(dá)90%融合時(shí)傳代培養(yǎng)。用流式細(xì)胞儀檢測(cè)培養(yǎng)細(xì)胞的表型,并做體外誘導(dǎo)分化實(shí)驗(yàn),以備后續(xù)實(shí)驗(yàn)使用。2.使用光感受器間維生素A類結(jié)合蛋白(interphotoreceptor retinoid-binding protein, IRBP)與含有結(jié)核桿菌H37RA的完全弗氏佐劑等體積混合,充分乳化后,在雙后足部皮下免疫Lewis大鼠,24小時(shí)內(nèi)經(jīng)腹腔注射百日咳毒素,制作大鼠EAU動(dòng)物模型。將動(dòng)物隨機(jī)分組,為了觀察在免疫同時(shí)輸注MSCs對(duì)EAU的預(yù)防作用以及MSCs對(duì)已經(jīng)發(fā)生的EAU的治療作用,從不同時(shí)間開始連續(xù)三天(免疫后0,1,2天,EAU發(fā)生前；免疫后第9,10,11天,EAU初發(fā)期；免疫后第12,13,14,EAU高峰期；及免疫后第16,17,18天,EAU慢性期)靜脈輸注自體或異體來源的間充質(zhì)干細(xì)胞,空白對(duì)照組輸注PBS。裂隙燈下定期進(jìn)行臨床觀察,20天后進(jìn)行病理學(xué)觀察,參照Caspi分級(jí)對(duì)臨床體征和病理學(xué)改變進(jìn)行評(píng)分,觀察葡萄膜炎的發(fā)生和轉(zhuǎn)歸情況。3.收集各組大鼠脾臟以及腹股溝淋巴結(jié),分離單個(gè)核細(xì)胞,在IRBP刺激下孵育72小時(shí)后,采用BrdU試劑盒檢測(cè)各組大鼠免疫應(yīng)答強(qiáng)度。4.取IRBP刺激下各組大鼠脾臟和淋巴結(jié)單個(gè)核細(xì)胞孵育72小時(shí)后的上清液,采用ELISA試劑盒檢測(cè)Thl類和Th2類細(xì)胞因子分泌情況。5.免疫20天后取大鼠外周血,流式細(xì)胞儀測(cè)定CD4+CD25+T細(xì)胞占CD4+T細(xì)胞的百分比。 6.收集各組大鼠脾臟以及淋巴結(jié),分離單個(gè)核細(xì)胞,提取總RNA,采用RT-PCR測(cè)定FOXP3 mRNA表達(dá)水平。 結(jié)果 1.成功分離并鑒定MSCs。 2.建立了EAU動(dòng)物模型。 3.采用MSCs治療EAU,與對(duì)照組相比,免疫的同時(shí)、免疫后第9天及免疫后第12天給EAU大鼠輸注MSCs后,臨床癥狀均顯著減輕(P0.05),在免疫同時(shí)注射MSCs的預(yù)防效果尤為明顯,而免疫后第16天,在疾病慢性期注射MSCs則無作用(P0.05)。與臨床表現(xiàn)一致,除外免疫后第16天注射MSCs組,病理學(xué)顯示其他三組實(shí)驗(yàn)組大鼠視網(wǎng)膜結(jié)構(gòu)損害明顯較對(duì)照組輕(P0.05)。 4.體外和體內(nèi)T細(xì)胞增殖實(shí)驗(yàn)表明,MSCs可以顯著抑制致病性T細(xì)胞增殖(P0.05)。 5. ELISA結(jié)果顯示,實(shí)驗(yàn)組Th1細(xì)胞因子分泌顯著降低(P0.05),而Th2細(xì)胞因子分泌升高(P0.05),表明MSCs可以調(diào)節(jié)免疫應(yīng)答平衡,導(dǎo)致Thl向Th2應(yīng)答漂移。 6.與對(duì)照組相比,MSCs治療組大鼠體內(nèi)調(diào)節(jié)性T細(xì)胞比例顯著增高(P0.05),實(shí)時(shí)定量PCR檢測(cè)也發(fā)現(xiàn)FOXP3-mRNA在MSCs治療組表達(dá)量顯著高于對(duì)照組(P0.05),這些結(jié)果表明MSCs可能通過上調(diào)調(diào)節(jié)性T細(xì)胞來發(fā)揮作用。 結(jié)論 MSCs可以通過抑制T細(xì)胞免疫應(yīng)答、改變Th1/Th2應(yīng)答平衡以及上調(diào)調(diào)節(jié)性T細(xì)胞,有效預(yù)防并治療EAU。本研究證實(shí)了通過調(diào)節(jié)機(jī)體免疫系統(tǒng),MSCs對(duì)免疫性疾病具有治療作用。
[Abstract]:objective
Uveitis is one of the most common autoimmune diseases of the eye. Many of them have recurrent attacks, many complications, difficult treatment, poor prognosis and high blindness. Mesenchymal stem cells (MSCs) can inhibit a variety of immune cells (including T lymphocytes, B lymphocytes, NK cells, and trees. This study used MSCs to treat experimental autoimmune uveitis (experimental autoimmune uveoretinitis, EAU) in rats, and to detect the immune regulation of MSCs in rats with uveitis, as well as its possible mechanism to provide new clinical treatment for uveitis. Methods and alternative treatment methods. Methods 1. Lewis or Wistar rat bone marrow mesenchymal stem cells were cultured in the primary generation, and the cells were cultured at the time of 90% fusion. The phenotype of the cultured cells was detected by flow cytometry, and the induction of differentiation in vitro was done in vitro, in order to prepare the use of.2. to use the vitamin A binding protein (interphotorecep). Tor retinoid-binding protein, IRBP) mixed with complete Freund's adjuvant containing H37RA of Mycobacterium tuberculosis, after full emulsification, immunized Lewis rats under the subcutaneous of two hind feet, and intraperitoneally injected with pertussis toxin within 24 hours to make the rat EAU animal model. The animals were randomly divided in order to observe the prevention of MSCs against EAU at the same time. The effect and the therapeutic effect of MSCs on the already occurring EAU were three days from time to time (0,1,2 days after immunization, before EAU, 9,10,11 days after immunization, EAU initial onset, 12,13,14, EAU peak after immunization, and 16,17,18 days after immunization, and EAU chronic phase) intravenous infusion of mesenchymal stem cells derived from autologous or allogeneic sources, blank pairs The clinical observation under the PBS. slit lamp was carried out regularly. After 20 days, the pathological observation was carried out. The clinical signs and pathological changes were graded according to the Caspi classification. The occurrence and prognosis of uveitis were observed and.3. collected the spleen and inguinal lymph nodes in each group, isolated mononuclear cells, after incubating for 72 hours under the stimulation of IRBP. The immune response intensity of rats in each group was detected by BrdU kit. The supernatant was incubated in each group of spleen and lymph node mononuclear cells for 72 hours under the stimulation of IRBP, and the secretion of Thl and Th2 cytokines was detected by ELISA kit and.5. immunized for 20 days, and the peripheral blood was taken for 20 days. The flow cytometry was used to determine the number of CD4+T cells in CD4+CD25+T cells. The percentage.
6. the spleen and lymph nodes of rats in each group were collected, the mononuclear cells were isolated, the total RNA was extracted, and the expression level of FOXP3 mRNA was measured by RT-PCR.
Result
1. successful separation and identification of MSCs.
2. the animal model of EAU was established.
3. MSCs was used to treat EAU. Compared with the control group, the clinical symptoms were significantly reduced after immunization, Ninth days after immunization and twelfth days after immunization to EAU rats (P0.05). The preventive effect of MSCs injection at the same time was particularly obvious, while the injection of MSCs in the chronic phase of the disease was no effect (P0.05). The MSCs group was injected sixteenth days after immunization. Pathology showed that the retinal structure damage of the other three groups was significantly lighter than that of the control group (P0.05).
4. in vitro and in vivo T cell proliferation assays showed that MSCs could significantly inhibit the proliferation of pathogenic T cells (P0.05).
5. ELISA results showed that the secretion of Th1 cytokines in the experimental group decreased significantly (P0.05), while the secretion of Th2 cytokines increased (P0.05), indicating that MSCs could regulate the balance of the immune response, leading to the drift of Thl to Th2.
6. compared with the control group, the proportion of regulatory T cells in the MSCs treatment group increased significantly (P0.05). Real-time quantitative PCR detection also found that the expression of FOXP3-mRNA in the MSCs treatment group was significantly higher than that of the control group (P0.05). These results suggest that MSCs may play a role by up regulating the regulatory T cells.
conclusion
MSCs can inhibit the immune response of T cells, change the balance of Th1/Th2 response and up-regulate the regulatory T cells, and effectively prevent and treat EAU.. This study confirmed that MSCs has a therapeutic effect on immune diseases by regulating the body's immune system.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R773

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Transplantation of Human Bone Marrow Mesenchymal Stem Cell Ameliorates the Autoimmune Pathogenesis in MRL/lpr Mice[J];Cellular & Molecular Immunology;2008年06期

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本文編號(hào)：1804818