發(fā)布時間：2018-04-27 23:41

  本文選題：人獲得性免疫缺陷病毒 + 獲得性免疫缺陷綜合征　； 參考：《中國人民解放軍醫(yī)學(xué)院》2016年博士論文

【摘要】：目的：評價不同人免疫缺陷病毒(Human immunoddficiency virus, HIV)感染階段淋巴結(jié)(Lymph nodes, LNs)纖維化情況以及導(dǎo)致纖維化的可能致病機制,同時,探討HIV感染后影響LNs內(nèi)微環(huán)境的趨化因子的改變,以及因HIV感染導(dǎo)致的微生物易位可能對濾泡輔助T細胞(T follicular helper cells, Tfh)功能的影響。方法：第一個研究選擇HIV感染者43例,分為HIV感染無癥狀組和獲得性免疫缺陷綜合征(Acquired immunodeficiency syndrome, AIDS)組,留取外周淺表LNs活體組織檢查(活檢)組織;另外選擇非HIV感染者12例作為健康對照組,同樣取其外周淺表LNs活檢組織。利用免疫組織化學(xué)方法檢測研究對象LNs中CD4+T淋巴細胞、Ⅰ型膠原蛋白和白介素(Interleukin,IL)-7定量和分布情況。第二個研究收集HIV感染者活檢外周淺表LNs32例,其中HIV感染無癥狀者組10例,AIDS組22例。將LNs分為兩部分,一部分4%多聚甲醛固定,石蠟包埋,另一部分研磨淋巴細胞,分別進行免疫組化,流式細胞檢測的實驗,檢測不同T細胞亞群中轉(zhuǎn)化生長因子(Transformi ng growth factor, TGF)-β1的表達。同時以不同TGF-β 1濃度刺激淋巴系統(tǒng)成纖維細胞,以高表達TGF-β1的CD8+T淋巴細胞與后者共培養(yǎng),利用Western Blot法和免疫熒光法檢測成纖維細胞分泌膠原蛋白情況。第三個研究選取不同HIV感染階段的患者43例,分為HIV感染無癥狀組和AIDS組,利用免疫組織化學(xué)方法檢測其LNs中CCL19、CCL21、CXCL12、 CXCL13的分泌及分布情況,另選非HIV感染者淺表LNs活檢標本12例為對照。第四個研究選取非HIV感染者外周淺表LNs共20例,分別以脂多糖(lipopolysaccharide, LPS)和R848刺激,或者在HIV-1感染同時以LPS和R848刺激,利用流式細胞方法檢測Tfh細胞表達Ki67.CXCR5和BcL6情況。結(jié)果：第一個研究中：1.隨著病程進展,HIV感染者外周淺表LNs中膠原沉積逐漸增加,AIDS組高于無癥狀組,無癥狀組高于健康對照組,差異均有統(tǒng)計學(xué)意義;2.HIV感染無癥狀者外周淺表LNs中CD4+ T淋巴細胞計數(shù)與健康對照組相比差異無統(tǒng)計學(xué)意義,而AIDS患者則顯著減少;3.HIV感染者外周淺表LNs中CD4+ T淋巴細胞計數(shù)與膠原沉積量呈負相關(guān),與外周血中CD4+T淋巴細胞計數(shù)呈正相關(guān)：4.3組IL-7的表達水平差異無統(tǒng)計學(xué)意義,而AIDS組部分患者LNs中IL-7呈局部聚集性分泌。第二個研究中：1.在非HIV感染者、HIV感染無癥狀組和AIDS患者LNs內(nèi)大量細胞表達TGF-β1,但在感染者LNs中TGF-β1陽性細胞分布紊亂；2.HIV感染無癥狀組和對照組LNs中調(diào)節(jié)性T細胞(Regulatory T cells, Tregs)頻數(shù)無明顯差異,但前者LNs中TGF-β1+Tregs頻數(shù)更高。AIDS患者組Tregs頻數(shù)與其他兩組相比明顯下降,TGF-β 1+Tregs頻數(shù)雖也均較其他兩組偏低,但差異僅與HIV感染無癥狀組相比有統(tǒng)計學(xué)意義;3.三組中均有一群高表達TGF-β1的T細胞,其中以HIV感染無癥狀組頻數(shù)最高,AIDS組次之,對照組最低,在感染者中,這群細胞的主要組成部分是CD8+T淋巴細胞,而非感染者則以CD4+T淋巴細胞為主;4.TGF-β1的致纖維化作用有計量依賴性,高表達TGF-β1的CD8+T淋巴細胞可能是HIV感染后LNs纖維化的重要致病機制之一;5.與非HIV感染者比,HIV感染無癥狀者與AIDS患者LNs中表達CD38或PD-1的CD8+T淋巴細胞頻數(shù)顯著升高,表達CD127的CD8+T淋巴細胞顯著減少。兩組相比差異無統(tǒng)計學(xué)意義。第三個研究中：1.HIV感染無癥狀者組與對照組相比,外周淺表LNs內(nèi)僅CCL21表達偏低,差異有統(tǒng)計學(xué)意義,而CCL19、CXCL12及CXCL13差異均無統(tǒng)計學(xué)意義;2.AIDS組與對照組相比,外周淺表LNs中CCL19、CXCL13表達升高,差異有統(tǒng)計學(xué)意義,而CCL21、CXCL12差異均無統(tǒng)計學(xué)意義,與無癥狀組相比,相關(guān)趨化因子表達均升高,差異有統(tǒng)計學(xué)意義;3.HIV感染至AIDS期后,外周淺表LNs中各個趨化因子的分布沒有明顯的區(qū)域界線。第四個研究中：1.LPS和R848的刺激可以促使Tfh細胞擴張并高表達BcL6,雖然R848相對于LPS起效晚,但作用效果更強;2.HIV-1感染可顯著下調(diào)Tfh細胞表達BcL6, LPS可協(xié)助HIV進一步抑制Tfh細胞BcL6的表達。因此,HIV本身可能是導(dǎo)致Tfh細胞功能異常的根本原因,而LPS可以起到協(xié)同作用;3.LPS和R848刺激可導(dǎo)致Tfh細胞下調(diào)CXCR5表達,使Tfh細胞接受CXCL13趨化定位于淋巴濾泡中的能力降低。結(jié)論：HIV感染后外周淺表LNs中膠原沉積逐漸增加導(dǎo)致結(jié)構(gòu)破壞,可能是CD4+T淋巴細胞進行性減少的一個重要原因,雖然IL-7有隨病程進展而分泌增加的趨勢,但仍不足以彌補LNs結(jié)構(gòu)破壞對CD4+T淋巴細胞的影響。慢性炎癥所導(dǎo)致的高表達TGF-β1的CD8+T淋巴細胞在慢性HIV感染LNs增多,是LNs纖維化的重要致病機制之一。HIV感染者外周淺表LNs中相關(guān)趨化因子的改變可能是機體代償和LNs結(jié)構(gòu)破壞的結(jié)果,而這種變化又進一步促進了免疫功能的破壞和CD4+T淋巴細胞的減少。HIV感染后消化道微生物產(chǎn)物的漏入可能刺激了Tfh細胞的擴張,但同時導(dǎo)致了其功能的受損,而HIV本身可能是導(dǎo)致Tfh細胞功能異常的根本原因。因此,抑制感染后大量微生物產(chǎn)物的易位,可能改善感染者體液免疫功能,進而提高針對HIV的清除能力,降低機會性感染。
[Abstract]:Objective: To evaluate the fibrosis of Lymph nodes (LNs) and the possible pathogenesis of fibrosis in Human immunoddficiency virus (HIV) infection stage, and to explore the changes in chemokines that affect the microenvironment of LNs after HIV infection, and the possible bacterial translocation caused by HIV infection. The effect of T follicular helper cells (Tfh) on the function of T cells. Methods: the first study selected 43 cases of HIV infection, divided into HIV infection asymptomatic group and acquired immunodeficiency syndrome (Acquired immunodeficiency syndrome, AIDS) group, and left peripheral superficial superficial tissue examination (biopsy) tissue; and the other non infected persons. 12 cases were taken as the healthy control group, and the peripheral superficial LNs biopsy tissue was also taken. The quantitative and distribution of CD4+T lymphocytes in LNs, Interleukin, IL (IL) -7 in the study subjects were detected by immunohistochemistry. The second studies collected the superficial LNs32 cases of the live detection of HIV infection, in which HIV infected the asymptomatic group. 10 cases and 22 cases in group AIDS were divided into two parts, one part of 4% polyformaldehyde fixed, paraffin embedded, and another part of lapping lymphocytes, immunohistochemistry and flow cytometry were carried out to detect the expression of transforming growth factor (Transformi ng growth factor, TGF) - beta 1 in different T cell subgroups. At the same time, the concentration of different TGF- beta 1 was stimulated. The lymphoid cells were cultured with high expression of TGF- beta 1 CD8+T lymphocyte and the latter. The collagen secretion in fibroblasts was detected by Western Blot and immunofluorescence. 43 patients with different HIV infection stages were selected and divided into HIV infection asymptomatic group and AIDS group, which were detected by immunohistochemical method. The secretion and distribution of CCL19, CCL21, CXCL12 and CXCL13 in LNs were measured, and 12 cases of superficial LNs biopsy specimens from non HIV infected persons were selected as control. Fourth studies selected 20 cases of peripheral superficial LNs in non HIV infected persons, with lipopolysaccharide (lipopolysaccharide, LPS) and R848 stimulus, or the flow fines were used to stimulate the infection at the same time. Cell method detected the expression of Ki67.CXCR5 and BcL6 in Tfh cells. Results: in the first study: 1. with the progress of the course of the disease, the collagen deposition in the peripheral superficial LNs of the HIV infected persons increased gradually, the AIDS group was higher than the asymptomatic group, the asymptomatic group was higher than the healthy control group, and the difference was statistically significant; CD4+ T in the peripheral superficial LNs of the asymptomatic 2.HIV infected persons was in the 2.HIV infection. There was no statistical difference between the control group and the control group, but the AIDS patients decreased significantly, and the CD4+ T lymphocyte count in the peripheral LNs of the 3.HIV infected people was negatively correlated with the amount of collagen deposition, and was positively correlated with the CD4+T lymphocyte count in the peripheral blood: the 4.3 groups of IL-7 were not statistically significant, but the AIDS part was not significant. In patients with LNs, IL-7 was locally aggregated. In second studies, 1. in non HIV infected persons, a large number of cells expressed TGF- beta 1 in HIV infection asymptomatic group and AIDS patients, but TGF- beta 1 positive cells in LNs of infected persons were disordered; 2.HIV infection asymptomatic group and control group LNs regulated T cells in LNs. The difference was obvious, but the frequency of TGF- beta 1+Tregs in the former LNs was higher than that of the other two groups, and the frequency of TGF- beta 1+Tregs was lower than the other two groups, but the difference was only statistically significant compared with that of the asymptomatic group with HIV infection. 3. three groups all had a group of T cells with high expression of TGF- beta 1, among which the HIV infection was asymptomatic. The frequency of the group was the highest, the AIDS group was the next, the control group was the lowest. In the infected people, the main component of the group was CD8+T lymphocyte, but the non infected person was mainly CD4+T lymphocyte. The fibrotic effect of 4.TGF- beta 1 was dependent on measurement. The CD8+T lymphoblastic cells with high expression of TGF- beta 1 may be the important pathogenicity of LNs fibrosis after HIV infection. One of the mechanisms: 5. compared with non HIV infected persons, the frequency of CD8+T lymphocytes expressed in LNs with CD38 or PD-1 in the asymptomatic HIV infected and AIDS patients increased significantly, and the CD8+T lymphocyte expressed in CD127 was significantly reduced. There was no statistical difference between the two groups. The third studies: the 1.HIV infected asymptomatic group compared with the control group, the peripheral superficial LNs within Only CCL21 expression was low, the difference was statistically significant, but the differences in CCL19, CXCL12 and CXCL13 were not statistically significant. Compared with the control group, the expression of CCL19 and CXCL13 increased in the peripheral superficial LNs, and the difference was statistically significant, but the differences in CCL21 and CXCL12 were not statistically significant. Compared with the asymptomatic group, the expression of chemokines increased, and the differences were all higher than those in the asymptomatic group. There was statistical significance; there was no obvious regional boundary in the distribution of the chemokines in the peripheral LNs after 3.HIV infection to AIDS. In the fourth studies, 1.LPS and R848 stimulated the expansion of Tfh cells and high expression of BcL6, although R848 had a relatively late effect on the LPS, but the 2.HIV-1 infection could significantly reduce the Tfh cell table. BcL6, LPS can help HIV to further inhibit the expression of BcL6 in Tfh cells. Therefore, HIV itself may be the root cause of the abnormal function of Tfh cells, and LPS can play a synergistic effect; 3.LPS and R848 stimulation can lead to Tfh cells to downregulate CXCR5 expression and reduce the ability to accept chemotaxis in lymphoid follicles. The gradual increase of collagen deposition in the superficial LNs after infection leads to structural damage, which may be an important cause of the progressive reduction of CD4+T lymphocytes. Although IL-7 has a tendency to increase with the progression of the disease, it is still insufficient to make up for the impact of LNs structure destruction on CD4+T lymphocytes. The CD8+ of the high expression of TGF- beta 1 caused by chronic inflammation The increasing LNs of T lymphocytes in chronic HIV infection is one of the important pathogenesis of LNs fibrosis. The changes in chemokine related chemokines in the peripheral superficial LNs may be the result of the body compensation and the destruction of the LNs structure, which further promotes the destruction of the immune function and the decrease of the digestive tract after.HIV infection by CD4+T lymphocytes. The leakage of microbial products may stimulate the expansion of Tfh cells, but also cause damage to their functions, and HIV itself may be the root cause of the abnormal function of Tfh cells. Therefore, the inhibition of the translocation of a large number of microbiological products after infection may improve the humoral immune function of the infected people, and then improve the scavenging ability against HIV and reduce the ability of the machine. Sexual infection.

【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R512.91

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