HIV-1单纯及合并HCV感染对HIV-1特异性细胞免疫应答的影响

发布时间：2018-01-04 11:03

本文关键词：HIV-1单纯及合并HCV感染对HIV-1特异性细胞免疫应答的影响　出处：《广州医学院》2012年硕士论文　论文类型：学位论文

【摘要】：人类免疫缺陷病毒1型（Human Immunodeficiency Virus Type1，HIV-1）与丙型肝炎病毒（Hepatitis C Virus，HCV）合并感染的临床治疗和相关致病机制研究是目前临床面临的难题和研究热点。由于HIV-1和HCV的传播途径相同，均通过血液及血液制品、性途径、母婴途径传播，HIV-1/HCV合并感染的现象非常普遍。随着高效抗逆转录病毒治疗(Highly Active Anti-RetroviralTherapy,HAART)在艾滋病中的广泛和规范应用，机会性感染的发病率和死亡率都已显著下降，而合并HCV感染所致的肝损害已经成为艾滋病患者死亡的主要原因之一；抗HCV治疗的低有效率和HAART治疗中不良反应的增加；中国国内流行的HCV基因型及准种复杂而又难治，这些都使得对HIV-1/HCV合并感染者的治疗更加棘手。但究其原因，至今并不完全明了，很有必要从病毒学和免疫学角度对HIV-1/HCV合并感染者进行深入研究。既往很多研究已证实细胞因子在HIV-1感染疾病进展过程中发挥了重要的作用，有研究表明：在HIV-1感染过程中IL-4可抑制体内的病毒水平，，IL-7对维持淋巴细胞的存活发挥了重要的作用，IL-17可能参与HIV-1病毒的免疫反应，IL-21可以促使CD8+T细胞增殖及保持活性，IFN-γ是机体免疫系统抵抗病毒感染的重要细胞因子之一。我们推测合并HCV感染会对HIV-1感染者上述细胞因子产生影响，而有关HCV合并感染对HIV-1感染者上述细胞因子的影响阐述得并不多，所以有必要对HCV合并感染对HIV-1感染者上述细胞因子的分泌状况展开研究。既往有关HIV-1感染和HCV感染的特异性细胞免疫应答的研究，结果均提示特异性CTL反应对HIV-1感染和HCV感染的控制均具有重要作用，国内外也有少部分关于HIV-1/HCV合并感染者的特异性细胞免疫应答的研究报道。但由于地域和种族的差异，有必要对华南地区HIV-1/HCV合并感染者的特异性细胞免疫应答进行研究。前期我们用ELISPOT方法检测HIV-1感染者的CD8+T细胞对HIV-1合成表位的免疫主导应答发现：HIV-1Gag区域肽段诱导产生的CD8+T细胞的IFNγ分泌细胞频率最高。故我们选用HIV-1P24区域（Gag133~191）的氨基酸序列人工合成的12个重叠肽段组成的肽段库作为特异性肽段表位，对HIV-1/HCV合并感染者和HIV-1感染者的HIV-1特异性CTL应答进行研究。实验一HIV-1单纯及合并HCV感染的CD4+T细胞、CD8+T细胞及PBMC内IL-7、IL-21、IFN-γmRNA水平测定实验对象：以中国华南地区的已HAART治疗的25例HIV-1单纯感染和22例HIV-1/HCV合并感染者，及20例健康对照者为研究对象。实验方法：分别采用免疫磁珠分选技术、实时荧光定量PCR方法，以上述HIV-1P24区域重叠肽段库作为HIV-1特异性肽段表位，刺激从上述三组研究对象的外周血单个核细胞（PBMC）分选出的CD4+T淋巴细胞，CD8+T淋巴细胞，分别检测CD4+T淋巴细胞、CD8+T淋巴细胞及PBMC在刺激前后IL-7、IL-21、IFN-γ，β-actin的mRNA水平，统计IL-7、IL-21、IFN-γ与β-actin的比值，并用单因素方差分析方法进行统计分析。实验结果：已接受HAART治疗的HIV-1单纯感染组和HIV-1/HCV合并感染组及健康对照组细胞内IFN-γ、IL-7、IL-21的mRNA水平比较 P24抗原表位刺激培养后，HIV-1单纯感染组CD8+T细胞的IFN-γ mRNA水平明显高于CD4+T细胞（P0.05）。无论有无P24特异性抗原表位刺激，健康对照组PBMC中IFN-γ的mRNA水平均明显高于HIV-1单纯感染组和HIV-1/HCV合并感染组（P0.05）； P24抗原表位刺激后，HIV-1/HCV合并感染组PBMC中IL-7mRNA水平明显高于HIV-1单纯感染组（P0.05）；无论有无P24特异性抗原表位刺激，各组PBMC中IL-21mRNA水平相比均无明显差异（P0.05）。实验二ICS检测HIV-1单纯及合并HCV感染者分泌IL-4，IL-17A，IL-21，IL-21R，IFN-γ的CD3+T淋巴细胞比例实验对象：以未HAART治疗的24例HIV-1单纯感染者和21例HIV-1/HCV合并感染者，及20例健康对照组为研究对象。实验方法：采用细胞内细胞因子染色法（intra-cellular CK staining，ICS），以上述HIV-1P24区域重叠肽段库作为特异性肽段表位，刺激上述研究对象的PBMC，检测使用与未使用P24抗原表位刺激后细胞内分泌IL-4、IL-17A、IL-21、IL-21R、IFN-γ的CD3+T淋巴细胞比例，并用单因素方差分析方法进行统计分析。实验结果：未接受HAART治疗的HIV-1单纯感染组和HIV-1/HCV合并感染组及健康对照组细胞内分泌IL-4、IL-17A、IFN-γ、IL-21、IL-21R的T淋巴细胞比例比较比较使用与未使用P24抗原表位刺激后的结果显示，三组T淋巴细胞内的分泌IL-4、IL-17A、IFN-γ、IL-21、IL-21R的比例变化不显著（P0.05）。在无P24抗原表位刺激培养后，HIV-1单纯感染组的CD3+IL-4+/T淋巴细胞明显高于健康对照组（P0.05），HIV-1/HCV合并感染组CD3+IL-4+/T淋巴细胞也高于健康对照组，但差异不显著（P0.05）；而在P24抗原表位刺激培养后，三组CD3+IL4+/T淋巴细胞差异不显著（P0.05）。无论有无P24抗原表位刺激培养，HIV-1单纯感染组CD3+IL17-A+/T淋巴细胞HIV-1/HCV合并感染组健康对照组，且HIV-1单纯感染组明显高于其他两组（P0.05）； HIV-1单纯感染组CD3+IFN-γ+/T淋巴细胞 HIV-1/HCV合并感染组健康对照组，三组差异不显著（P0.05）；三组CD3+IL-21+/T淋巴细胞差异不显著（P0.05）；HIV-1单纯感染组CD3+IL-21R+/T淋巴细胞比例健康对照组 HIV-1/HCV合并感染组，HIV-1单纯感染组明显高于其他两组（P0.001）。实验三ELISPOT检测HIV-1单纯及合并HCV感染的HIV-1特异性细胞免疫应答实验对象：以上述实验一和实验二中的单纯感染者和合并感染者为研究对象。实验方法：采用酶联免疫斑点吸附技术（enzyme-linked immunosorbent spot，ELISPOT），以上述HIV-1P24区域重叠肽段库作为特异性肽段表位，分别检测研究对象PBMC中HIV-1特异性CTL应答频数，并用卡方检验方法进行统计分析。分析合并HCV感染对HIV-1感染细胞免疫的影响，HAART对HIV-1感染细胞免疫的影响。实验结果：HIV-1单纯感染组和HIV-1/HCV合并感染组HIV-1特异性CTL应答比较已接受HAART治疗HIV-1单纯感染组HIV-1特异性细胞免疫应答率（13/25）明显高于HIV-1/HCV合并感染组（5/22）（P0.05）；未接受HAART治疗HIV-1单纯感染组HIV-1特异性细胞免疫应答率（8/22）高于HIV-1/HCV合并感染组（6/21），但差异不显著（P0.05）；已治疗单纯感染组的HIV-1特异性细胞免疫应答率（13/25）高于未治疗单纯感染组（8/22），但差异不显著（P0.05）；已治疗合并感染组HIV-1特异性细胞免疫应答率（5/22）与未治疗合并感染组（6/21）的相比无明显差异（P0.05）。结论： 1.合并HCV感染可能使HIV-1感染者HIV-1特异性细胞免疫应答水平下降，这可能是HIV-1/HCV合并感染者HAART治疗疗效差的原因之一。 2.细胞因子IL-7、IL-17、IL-21R在HIV-1特异性细胞免疫中发挥重要作用。HIV-1感染还可以通过Th17和Tfh途径影响感染者的细胞免疫，而HCV合并感染会对此产生干扰；IL-7在HIV-1/HCV合并感染者的细胞免疫中起重要作用。
[Abstract]:Human immunodeficiency virus type 1 (HIV-1 Human Immunodeficiency Virus Type1, and hepatitis C virus (Hepatitis) C Virus, HCV) and clinical research related to the pathogenesis of infection is the problem facing clinical and research hotspot. Because the HIV-1 and HCV channels are the same, through blood and blood products, sexual contact. Mother to child transmission, HIV-1/HCV infection is very common. With highly active antiretroviral therapy (Highly Active, Anti-RetroviralTherapy, HAART) in the wide application and standardization of AIDS, the opportunity to morbidity and mortality of infection has decreased significantly, while the liver injury caused by HCV infection has become one of the main causes of death in patients with AIDS a; increase the adverse reaction in the treatment of low efficiency and HAART anti HCV therapy; HCV genotype China popular and quasi species complex It is difficult to treat, which makes the treatment of HIV-1/HCV complicated with infection more intractable. However, the reason is not clear yet. It is quite necessary to further study the HIV-1/HCV co infection from the perspective of Virology and immunology.
Previous studies have demonstrated that many cytokines play an important role in the progression of the disease course of HIV-1 infection, studies have shown that in the process of HIV-1 infection IL-4 virus can inhibit the in vivo, IL-7 plays an important role in maintaining the survival of lymphocytes, IL-17 immune response involved in HIV-1 virus, IL-21 can induce CD8+T cells keep the proliferation and activity of IFN- gamma is one of the most important cytokines in the immune system against virus infection. We speculate that HCV infection with HIV-1 infection will affect the cell factor, and the HCV infection effect on HIV-1 infection of these cytokines is not much, so it is necessary for the secretion of HCV infection the HIV-1 infected the cytokines studied.
Study on the specific cellular immune response to HIV-1 infection and HCV infection in the past, the results indicated that specific CTL response plays an important role in the control of HIV-1 infection and HCV infection, there are few reports on the specific cellular immune response of HIV-1/HCV infection of both at home and abroad. But because of the difference of regional and ethnic the specific cellular immune response, it is necessary to HIV-1/HCV co infection in Southern China is studied. We use ELISPOT method to detect early HIV-1 infection of CD8+T cells on immune HIV-1 synthetic epitope dominant response found: IFN gamma HIV-1Gag region peptide induced CD8+T cell secretory cells of the highest frequency. So we selected HIV-1P24 region (Gag133~191) 12 overlapping synthetic peptides composed of amino acid sequence of the peptide library as specific peptide epitopes of HIV-1/HCV infection and H The HIV-1 specific CTL response of IV-1 infected people was studied.
Experiment 1 HIV-1 simple and HCV infected CD4+T cells, CD8+T cells and IL-7, IL-21, IFN- gamma mRNA levels in CD8+T cells and PBMC
Participants: 25 cases of HIV-1 infection and 22 cases of HIV-1/HCV infection and 20 healthy controls were treated with HAART in Southern China, China.
Methods: using immunomagnetic separation technology, real-time fluorescence quantitative PCR method, the overlapping HIV-1P24 peptides as HIV-1 specific peptide epitopes, stimulation from the three groups of subjects of peripheral blood mononuclear cells (PBMC) isolated CD4+T cells, CD8+T cells, CD4+T were detected respectively. Lymphocyte, CD8+T lymphocyte and PBMC in IL-21, IL-7 before and after stimulation, IFN- gamma, beta -actin mRNA, IL-7 IL-21, IFN- statistics, the ratio of gamma and beta -actin, and statistical analysis method with single factor analysis of variance.
Experimental results: the comparison of the mRNA levels of IFN- gamma, IL-7, IL-21 in the cells of the simple infection group of HIV-1 and the HIV-1/HCV combined infection group and the healthy control group with HAART treatment.
The epitopes of P24 stimulated HIV-1, simple infection group CD8+T cells IFN- gamma mRNA levels were significantly higher than that of CD4+T cells (P0.05). No P24 specific epitope stimulation group PBMC IFN- gamma mRNA levels were significantly higher than those in healthy control group and HIV-1/HCV HIV-1 infection and infection group (P0.05) P24; antigenic stimulation, HIV-1/HCV infection in patients with IL-7mRNA levels in the PBMC group was significantly higher than that of pure HIV-1 infection group (P0.05); no P24 specific epitope stimulation, the serum level of IL-21mRNA in PBMC showed no significant difference (P0.05).
Test two ICS to detect the proportion of CD3+T lymphocyte secreting IL-4, IL-17A, IL-21, IL-21R, IFN- gamma in patients with HIV-1 and HCV infection.
Subjects: 24 cases of simple infection of HIV-1 and 21 cases of HIV-1/HCV with HIV-1/HCV infection and 20 healthy controls were studied.
Methods: using intracellular cytokine staining (intra-cellular CK, staining, ICS, HIV-1P24) in the area of overlapping peptides as specific peptide epitopes, stimulate the research object of PBMC detection with and without antigen epitope of P24 cells after stimulating the secretion of IL-4, IL-17A, IL-21, IL-21R. The percentage of CD3+T cells of IFN- gamma, and statistical analysis method with single factor analysis of variance.
Results: the proportion of T cells in the endocrine IL-4, IL-17A, IFN-, IL-21, IL-21R of the HIV-1 infection group and the HIV-1/HCV co infection group and the healthy control group without HAART treatment were compared.
Compared with and without P24 epitope after stimulation showed that T lymphocytes in the three groups of the secretion of IL-4, IL-17A, IL-21, IFN- gamma, IL-21R ratio did not change significantly (P0.05). In the absence of antigen epitope of P24 stimulated HIV-1, simple infection group CD3+IL-4+/T cells was significantly higher than that of healthy controls group (P0.05), HIV-1/HCV infection group of CD3+IL-4+/T lymphocytes is higher than that of the healthy control group, but the difference was not significant (P0.05); and the antigen epitope of P24 stimulated CD3+IL4+/T lymphocytes, three groups had no significant difference (P0.05). No P24 epitope HIV-1 stimulated CD3+IL17-A+/T lymphocyte HIV-1/HCV with simple infection group the infection group and healthy control group, HIV-1 infection group was significantly higher than the other two groups (P0.05); HIV-1 herpes infection group health control group CD3+IFN- gamma +/T lymphocyte HIV-1/HCV infection, no difference between the three groups Significantly (P0.05); there was no significant difference in CD3+IL-21+/T lymphocyte between the three groups (P0.05); the proportion of CD3+IL-21R+/T lymphocytes in HIV-1 infection group was higher than that in the healthy control group, HIV-1/HCV infection group, HIV-1 infection group was significantly higher than the other two groups (P0.001).
Test three ELISPOT to detect the specific cellular immune response of HIV-1 alone and with HCV infection in HIV-1
Subjects: the subjects of the above experiment one and the second of the experiment were the simple infection and the combined infection.
Methods: the enzyme-linked immunospot (enzyme-linked immunosorbent spot, adsorption technology, ELISPOT) to the HIV-1P24 region overlapping peptides as specific peptide epitopes were detected in HIV-1 PBMC studied in specific CTL response frequency, and the data was analyzed by chi square test method. Analysis of influence on cellular immunity in HIV-1 infection with HCV infection, the effect of HAART on cell immunity of HIV-1 infection.
Experimental results: comparison of HIV-1 specific CTL responses in HIV-1 simple infection group and HIV-1/HCV combined infection group
Have been treated with HAART HIV-1 infection group HIV-1 specific cellular immune response rate (13/25) was significantly higher than that of HIV-1/HCV infection group (5/22) (P0.05); HAART HIV-1 did not accept the treatment of simple infection group HIV-1 specific cellular immune response rate (8/22) than HIV-1/HCV infection group (6/21), but the difference was not significant (P0.05); for HIV-1 specific cellular immune responses in simple infection group (13/25) was higher than the rate of treatment of simple infection group (8/22), but the difference was not significant (P0.05); treated infection group HIV-1 specific cellular immune response rate (5/22) and untreated infection group (6/21) compared with no significant differences (P0.05).
Conclusion:
1., combined with HCV infection, the level of HIV-1 specific cellular immune response in HIV-1 infected patients may be decreased. This may be one of the reasons for the poor efficacy of HAART treatment in HIV-1/HCV infected patients.
2. cell factor IL-7, IL-17, IL-21R in HIV-1 specific cellular immunity play an important role in.HIV-1 infection can also influence cell immune infection through Th17 and Tfh pathway of the HCV infection will have interference; play an important role in IL-7 cell immunity in HIV-1/HCV coinfected.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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