發(fā)布時(shí)間：2018-01-21 20:04

  本文關(guān)鍵詞： 細(xì)胞凋亡 同種皮膚移植 移植排斥反應(yīng) 樹突狀細(xì)胞 調(diào)節(jié)性T細(xì)胞　出處：《華中科技大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:以異基因小鼠皮膚移植為模型,探討供者來源凋亡胸腺細(xì)胞輸注誘導(dǎo)同種異體移植物免疫耐受的效應(yīng)、細(xì)胞學(xué)基礎(chǔ)與分子機(jī)制,為設(shè)計(jì)防治移植排斥反應(yīng)的新策略提供線索。 方法:用地塞米松誘導(dǎo)供者小鼠(Balb/c,H-2d)胸腺細(xì)胞凋亡:采用DNA Ladde凝膠電泳法以及Annexin V / PI細(xì)胞染色后流式細(xì)胞術(shù)檢測細(xì)胞凋亡;BALB/c小鼠胸腺細(xì)胞以PKH67染色,誘導(dǎo)凋亡后,經(jīng)尾靜脈過繼輸注MHC型別完全不匹配的受者小鼠(C57BL/6, H-2b),12個(gè)小時(shí)后,取其脾細(xì)胞以抗CD11c-PE抗體標(biāo)記樹突狀細(xì)胞,激光共聚焦顯微鏡觀察樹突狀細(xì)胞吞噬凋亡的胸腺細(xì)胞;細(xì)胞過繼7天后,將BALB/c小鼠皮膚移植給C57BL/6小鼠,觀察移植物存活時(shí)間;皮膚移植后7天,取移植皮片,組織學(xué)檢測移植物組織中炎性細(xì)胞浸潤;混合淋巴細(xì)胞反應(yīng)評價(jià)供者脾細(xì)胞刺激受者小鼠T細(xì)胞增殖的作用;RT-PCR檢測受者小鼠脾臟細(xì)胞TGF-β、IL-10、Foxp3的mRNA表達(dá)水平;流式細(xì)胞術(shù)檢測受者小鼠脾臟樹突狀細(xì)胞亞類。 結(jié)果: 一、誘導(dǎo)供者胸腺細(xì)胞凋亡并過繼輸注,及受者DC吞噬凋亡胸腺細(xì)胞的作用 1.DNA Ladder試驗(yàn)和Annexin V / PI染色檢測證實(shí),地塞米松可顯著增強(qiáng)小鼠胸腺細(xì)胞凋亡:經(jīng)地塞米松誘導(dǎo)細(xì)胞凋亡率達(dá)58.7%,未經(jīng)誘導(dǎo)的胸腺細(xì)胞作為對照,在體外培養(yǎng)相同時(shí)間凋亡率為32.4%。 2.BALB/c小鼠胸腺細(xì)胞以PKH67預(yù)先染色,誘導(dǎo)凋亡后經(jīng)尾靜脈輸注至C57BL/6小鼠體內(nèi),12個(gè)小時(shí)后,取其脾細(xì)胞以抗CD11c-PE抗體標(biāo)記DC,激光共聚焦顯微鏡下可見PKH67+CD11c+,提示凋亡胸腺細(xì)胞被DC吞噬。 二、供者凋亡胸腺細(xì)胞過繼輸注延長異基因小鼠皮膚移植物存活時(shí)間及其機(jī)制 以BALB/c小鼠為供者,C57BL/6小鼠為受者,進(jìn)行背部皮膚移植。移植術(shù)前實(shí)驗(yàn)組通過尾靜脈輸注2.5×10~7供者凋亡胸腺細(xì)胞,并以PBS、地塞米松和第三方小鼠(C3H小鼠)作為對照。 1.移植物存活時(shí)間 供者凋亡細(xì)胞過繼輸注,能明顯延長皮膚移植物存活時(shí)間:凋亡細(xì)胞處理組平均存活時(shí)間為17.750±3.956天,PBS組為10.250±1.389天,地塞米松組為15.625±1.685天,C3H組為10.500±1.927天。(n=8,p㩳0.01) 2.移植皮片組織學(xué)檢查 移植術(shù)后7天,取移植皮片制石蠟切片,常規(guī)HE染色,觀察移植物組織中炎性細(xì)胞浸潤。與PBS對照組和C3H組相比,凋亡胸腺細(xì)胞組移植組織中單個(gè)核細(xì)胞浸潤明顯減少,未出現(xiàn)毛細(xì)血管淤血、間質(zhì)出血、動(dòng)脈炎和靜脈炎等現(xiàn)象;地塞米松組移植組織中單個(gè)核細(xì)胞浸潤也比PBS對照組和C3H組略為減少。 3.受者淋巴細(xì)胞增殖功能 移植術(shù)后7天,取供者脾細(xì)胞再次刺激受者脾臟T細(xì)胞,檢測后者對供者同種抗原的再次應(yīng)答能力。結(jié)果顯示:凋亡胸腺細(xì)胞組受者脾臟T細(xì)胞對供者脾細(xì)胞的增殖反應(yīng)顯著低于PBS對照組和C3H組。 4.受者脾臟細(xì)胞因子mRNA表達(dá) 移植術(shù)后7天,取供者脾細(xì)胞和淋巴結(jié),提取總RNA,借助RT-PCR檢測TGF-β、IL-10、Foxp3的mRNA表達(dá)水平。結(jié)果顯示:凋亡胸腺細(xì)胞組脾臟TGF-β、IL-10、Foxp3的mRNA表達(dá)水平均較對照組升高,差異有顯著性;地塞米松組脾臟IL-10、Foxp3的mRNA表達(dá)水平增高。(p㩳0.05) 5.受者脾臟中DC亞類檢測 移植術(shù)后7天,取供者脾細(xì)胞流式細(xì)胞術(shù)檢測DC亞類。結(jié)果顯示:凋亡胸腺細(xì)胞組CD11c+B220+DC比例顯著增高;凋亡胸腺細(xì)胞組和地塞米松組CD11c+CD45RB+DC比例均增高。 結(jié)論: 供者來源凋亡胸腺細(xì)胞過繼輸注,可促進(jìn)受者體內(nèi)產(chǎn)生具有免疫抑制功能的DC和調(diào)節(jié)性T細(xì)胞,促進(jìn)IL-10、TGF-β表達(dá);導(dǎo)致受者同種反應(yīng)性T細(xì)胞增殖活性下降,移植物組織中單個(gè)核細(xì)胞浸潤減少,并顯著延長異基因小鼠皮膚移植物存活時(shí)間。該作用具有供者抗原特異性。本研究成果提示,供者凋亡胸腺細(xì)胞過繼輸注可抑制同種移植物排斥反應(yīng),具有潛在的臨床應(yīng)用價(jià)值。
[Abstract]:Objective: To investigate the effects of donor derived thymocyte infusion on allograft immune tolerance, based on allogeneic skin transplantation in mice, and to provide clues for designing new strategies to prevent graft rejection.
Methods: donor mice induced by dexamethasone (Balb/c, H-2d) on thymocyte apoptosis: using DNA Ladde gel electrophoresis and Annexin V / PI cells after staining by flow cytometry to detect cell apoptosis; BALB/c mouse thymus cells were stained with PKH67 staining, apoptosis induction, intravenous infusion of MHC type adoptive do not match the mice (C57BL/6, H-2b), after 12 hours, with anti CD11c-PE antibody labeled dendritic cells from the spleen cells, confocal microscopy observation of dendritic cells phagocytosis of apoptotic thymocytes; cell adoptive 7 days later, the BALB /c mouse skin transplanted to C57BL/6 mice, observe the survival time of the grafts; 7 days after skin transplantation take, skin graft, infiltration of inflammatory cells in histological examination of tissue graft; mixed lymphocyte reaction evaluation of donor spleen cells stimulated the proliferation of T cells in mice; RT-PCR recipient mice spleen fine detection The mRNA expression level of TGF- beta, IL-10, Foxp3, and splenic dendritic cells in the recipient mice were detected by flow cytometry.
Result:
1. Inducing apoptosis and adoptive infusion of donor thymus cells, and the effect of DC phagocytosis of apoptotic thymocytes
1.DNA Ladder test and Annexin V / PI staining test confirmed that dexamethasone could significantly enhance thymocyte apoptosis in mice: the apoptosis rate induced by dexamethasone was 58.7%, and the apoptosis rate of 32.4%. cells was 32.4%. at the same time when cultured in vitro.
2.BALB/c mice thymocytes were pre stained by PKH67 and induced apoptosis. After tail vein infusion into C57BL/6 mice, 12 hours later, spleen cells were labeled with anti CD11c-PE antibody for DC, and PKH67+CD11c+ was observed under confocal laser scanning microscope, indicating that apoptotic thymocytes were phagocytosis by DC.
Two, adoptive infusion of donor apoptotic thymocytes to prolong the survival time and mechanism of skin graft in allogeneic mice
BALB/c mice served as donors and C57BL/6 mice as recipients. Back skin transplantation was performed. Before transplantation, the experimental group was injected with 2.5 * 10~7 apoptotic thymocytes via tail vein, and PBS, dexamethasone and third party mice (C3H mice) were used as controls.
1. graft survival time
Donor cell apoptotic cells infusion can significantly prolong the survival time of skin graft: the average survival time of the apoptotic cell treatment group is 17.750 + 3.956 days, the PBS group is 10.250 + 1.389 days, the dexamethasone group is 15.625 + 1.685 days, and the C3H group is 10.500 + 1.927 days. (n=8, P 0.01).
Histological examination of 2. skin graft
7 days after transplantation, the graft for paraffin section, stained by HE, the infiltration of inflammatory cells to evaluate the graft tissue. Compared with PBS control group and C3H group, infiltration of mononuclear cells significantly reduced thymocyte apoptosis in the transplanted tissue group, without capillary congestion, interstitial hemorrhage, arteritis and phlebitis the phenomenon; infiltration of mononuclear cells than the PBS control group and C3H group decreased slightly in the dexamethasone group transplanted tissue.
Lymphocyte proliferation of 3. recipients
On the 7 day after transplantation, donor spleen cells stimulated the recipient spleen T cells again, and the ability of the latter to respond to donor allo antigen was detected. The results showed that the proliferation of splenic T cells in donor cells was significantly lower than that in PBS control group and C3H group.
Expression of spleen cytokine mRNA in 4. recipients
7 days after transplantation, donor spleen cells and lymph nodes, the extraction of total RNA by RT-PCR detection of TGF- beta, IL-10, the expression level of Foxp3 mRNA. The results showed: the thymocyte apoptosis group of spleen TGF- beta, IL-10, the expression level of Foxp3 mRNA were higher than those in control group, there was significant difference between dexamethasone group; the spleen of IL-10, increased the expression level of Foxp3 mRNA. (P? 0.05)
Detection of DC subclass in the spleen of 5. recipients
On the 7 day after transplantation, donor subgroup splenocytes and flow cytometry were used to detect DC subclasses. Results showed that the proportion of CD11c+B220+DC in apoptotic thymocytes group increased significantly, and the proportion of CD11c+CD45RB+DC in apoptotic thymocyte group and dexamethasone group increased.
Conclusion:
The source of donor apoptotic thymocytes infusion of adoptive recipients, can promote the production and regulatory T cells, promote the immunosuppressive function of DC IL-10, TGF- beta expression; T cell proliferation caused by decreased activity of allogeneic mononuclear cell infiltration, reduce graft tissues and prolong allogeneic skin of mice the survival time of the grafts. The effect with donor antigen specificity. The results of this study suggest that adoptive donor apoptotic thymocytes infusion can inhibit allograft rejection and potential clinical value.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 簡優(yōu)強(qiáng);記憶性T細(xì)胞在抗-CD45RB抗體和DST誘導(dǎo)的免疫耐受中的作用[D];暨南大學(xué);2013年

，

本文編號：1452439