人免疫缺陷病毒感染对淋巴结结构和功能的影响

发布时间：2018-04-27 23:41

本文选题：人获得性免疫缺陷病毒 + 获得性免疫缺陷综合征　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：目的：评价不同人免疫缺陷病毒(Human immunoddficiency virus, HIV)感染阶段淋巴结(Lymph nodes, LNs)纤维化情况以及导致纤维化的可能致病机制,同时,探讨HIV感染后影响LNs内微环境的趋化因子的改变,以及因HIV感染导致的微生物易位可能对滤泡辅助T细胞(T follicular helper cells, Tfh)功能的影响。方法：第一个研究选择HIV感染者43例,分为HIV感染无症状组和获得性免疫缺陷综合征(Acquired immunodeficiency syndrome, AIDS)组,留取外周浅表LNs活体组织检查(活检)组织;另外选择非HIV感染者12例作为健康对照组,同样取其外周浅表LNs活检组织。利用免疫组织化学方法检测研究对象LNs中CD4+T淋巴细胞、Ⅰ型胶原蛋白和白介素(Interleukin,IL)-7定量和分布情况。第二个研究收集HIV感染者活检外周浅表LNs32例,其中HIV感染无症状者组10例,AIDS组22例。将LNs分为两部分,一部分4%多聚甲醛固定,石蜡包埋,另一部分研磨淋巴细胞,分别进行免疫组化,流式细胞检测的实验,检测不同T细胞亚群中转化生长因子(Transformi ng growth factor, TGF)-β1的表达。同时以不同TGF-β 1浓度刺激淋巴系统成纤维细胞,以高表达TGF-β1的CD8+T淋巴细胞与后者共培养,利用Western Blot法和免疫荧光法检测成纤维细胞分泌胶原蛋白情况。第三个研究选取不同HIV感染阶段的患者43例,分为HIV感染无症状组和AIDS组,利用免疫组织化学方法检测其LNs中CCL19、CCL21、CXCL12、 CXCL13的分泌及分布情况,另选非HIV感染者浅表LNs活检标本12例为对照。第四个研究选取非HIV感染者外周浅表LNs共20例,分别以脂多糖(lipopolysaccharide, LPS)和R848刺激,或者在HIV-1感染同时以LPS和R848刺激,利用流式细胞方法检测Tfh细胞表达Ki67.CXCR5和BcL6情况。结果：第一个研究中：1.随着病程进展,HIV感染者外周浅表LNs中胶原沉积逐渐增加,AIDS组高于无症状组,无症状组高于健康对照组,差异均有统计学意义;2.HIV感染无症状者外周浅表LNs中CD4+ T淋巴细胞计数与健康对照组相比差异无统计学意义,而AIDS患者则显著减少;3.HIV感染者外周浅表LNs中CD4+ T淋巴细胞计数与胶原沉积量呈负相关,与外周血中CD4+T淋巴细胞计数呈正相关：4.3组IL-7的表达水平差异无统计学意义,而AIDS组部分患者LNs中IL-7呈局部聚集性分泌。第二个研究中：1.在非HIV感染者、HIV感染无症状组和AIDS患者LNs内大量细胞表达TGF-β1,但在感染者LNs中TGF-β1阳性细胞分布紊乱；2.HIV感染无症状组和对照组LNs中调节性T细胞(Regulatory T cells, Tregs)频数无明显差异,但前者LNs中TGF-β1+Tregs频数更高。AIDS患者组Tregs频数与其他两组相比明显下降,TGF-β 1+Tregs频数虽也均较其他两组偏低,但差异仅与HIV感染无症状组相比有统计学意义;3.三组中均有一群高表达TGF-β1的T细胞,其中以HIV感染无症状组频数最高,AIDS组次之,对照组最低,在感染者中,这群细胞的主要组成部分是CD8+T淋巴细胞,而非感染者则以CD4+T淋巴细胞为主;4.TGF-β1的致纤维化作用有计量依赖性,高表达TGF-β1的CD8+T淋巴细胞可能是HIV感染后LNs纤维化的重要致病机制之一;5.与非HIV感染者比,HIV感染无症状者与AIDS患者LNs中表达CD38或PD-1的CD8+T淋巴细胞频数显著升高,表达CD127的CD8+T淋巴细胞显著减少。两组相比差异无统计学意义。第三个研究中：1.HIV感染无症状者组与对照组相比,外周浅表LNs内仅CCL21表达偏低,差异有统计学意义,而CCL19、CXCL12及CXCL13差异均无统计学意义;2.AIDS组与对照组相比,外周浅表LNs中CCL19、CXCL13表达升高,差异有统计学意义,而CCL21、CXCL12差异均无统计学意义,与无症状组相比,相关趋化因子表达均升高,差异有统计学意义;3.HIV感染至AIDS期后,外周浅表LNs中各个趋化因子的分布没有明显的区域界线。第四个研究中：1.LPS和R848的刺激可以促使Tfh细胞扩张并高表达BcL6,虽然R848相对于LPS起效晚,但作用效果更强;2.HIV-1感染可显著下调Tfh细胞表达BcL6, LPS可协助HIV进一步抑制Tfh细胞BcL6的表达。因此,HIV本身可能是导致Tfh细胞功能异常的根本原因,而LPS可以起到协同作用;3.LPS和R848刺激可导致Tfh细胞下调CXCR5表达,使Tfh细胞接受CXCL13趋化定位于淋巴滤泡中的能力降低。结论：HIV感染后外周浅表LNs中胶原沉积逐渐增加导致结构破坏,可能是CD4+T淋巴细胞进行性减少的一个重要原因,虽然IL-7有随病程进展而分泌增加的趋势,但仍不足以弥补LNs结构破坏对CD4+T淋巴细胞的影响。慢性炎症所导致的高表达TGF-β1的CD8+T淋巴细胞在慢性HIV感染LNs增多,是LNs纤维化的重要致病机制之一。HIV感染者外周浅表LNs中相关趋化因子的改变可能是机体代偿和LNs结构破坏的结果,而这种变化又进一步促进了免疫功能的破坏和CD4+T淋巴细胞的减少。HIV感染后消化道微生物产物的漏入可能刺激了Tfh细胞的扩张,但同时导致了其功能的受损,而HIV本身可能是导致Tfh细胞功能异常的根本原因。因此,抑制感染后大量微生物产物的易位,可能改善感染者体液免疫功能,进而提高针对HIV的清除能力,降低机会性感染。
[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.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R512.91

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