本文關(guān)鍵詞：HIV-1單純及合并HCV感染對(duì)HIV-1特異性細(xì)胞免疫應(yīng)答的影響　出處：《廣州醫(yī)學(xué)院》2012年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 人免疫缺陷病毒1型 丙型肝炎病毒 合并感染 特異性細(xì)胞免疫 細(xì)胞因子

【摘要】：人類(lèi)免疫缺陷病毒1型（Human Immunodeficiency Virus Type1，HIV-1）與丙型肝炎病毒（Hepatitis C Virus，HCV）合并感染的臨床治療和相關(guān)致病機(jī)制研究是目前臨床面臨的難題和研究熱點(diǎn)。由于HIV-1和HCV的傳播途徑相同，均通過(guò)血液及血液制品、性途徑、母嬰途徑傳播，HIV-1/HCV合并感染的現(xiàn)象非常普遍。隨著高效抗逆轉(zhuǎn)錄病毒治療(Highly Active Anti-RetroviralTherapy,HAART)在艾滋病中的廣泛和規(guī)范應(yīng)用，機(jī)會(huì)性感染的發(fā)病率和死亡率都已顯著下降，而合并HCV感染所致的肝損害已經(jīng)成為艾滋病患者死亡的主要原因之一；抗HCV治療的低有效率和HAART治療中不良反應(yīng)的增加；中國(guó)國(guó)內(nèi)流行的HCV基因型及準(zhǔn)種復(fù)雜而又難治，這些都使得對(duì)HIV-1/HCV合并感染者的治療更加棘手。但究其原因，至今并不完全明了，很有必要從病毒學(xué)和免疫學(xué)角度對(duì)HIV-1/HCV合并感染者進(jìn)行深入研究。 既往很多研究已證實(shí)細(xì)胞因子在HIV-1感染疾病進(jìn)展過(guò)程中發(fā)揮了重要的作用，有研究表明：在HIV-1感染過(guò)程中IL-4可抑制體內(nèi)的病毒水平，，IL-7對(duì)維持淋巴細(xì)胞的存活發(fā)揮了重要的作用，IL-17可能參與HIV-1病毒的免疫反應(yīng)，IL-21可以促使CD8+T細(xì)胞增殖及保持活性，IFN-γ是機(jī)體免疫系統(tǒng)抵抗病毒感染的重要細(xì)胞因子之一。我們推測(cè)合并HCV感染會(huì)對(duì)HIV-1感染者上述細(xì)胞因子產(chǎn)生影響，而有關(guān)HCV合并感染對(duì)HIV-1感染者上述細(xì)胞因子的影響闡述得并不多，所以有必要對(duì)HCV合并感染對(duì)HIV-1感染者上述細(xì)胞因子的分泌狀況展開(kāi)研究。 既往有關(guān)HIV-1感染和HCV感染的特異性細(xì)胞免疫應(yīng)答的研究，結(jié)果均提示特異性CTL反應(yīng)對(duì)HIV-1感染和HCV感染的控制均具有重要作用，國(guó)內(nèi)外也有少部分關(guān)于HIV-1/HCV合并感染者的特異性細(xì)胞免疫應(yīng)答的研究報(bào)道。但由于地域和種族的差異，有必要對(duì)華南地區(qū)HIV-1/HCV合并感染者的特異性細(xì)胞免疫應(yīng)答進(jìn)行研究。前期我們用ELISPOT方法檢測(cè)HIV-1感染者的CD8+T細(xì)胞對(duì)HIV-1合成表位的免疫主導(dǎo)應(yīng)答發(fā)現(xiàn)：HIV-1Gag區(qū)域肽段誘導(dǎo)產(chǎn)生的CD8+T細(xì)胞的IFNγ分泌細(xì)胞頻率最高。故我們選用HIV-1P24區(qū)域（Gag133~191）的氨基酸序列人工合成的12個(gè)重疊肽段組成的肽段庫(kù)作為特異性肽段表位，對(duì)HIV-1/HCV合并感染者和HIV-1感染者的HIV-1特異性CTL應(yīng)答進(jìn)行研究。 實(shí)驗(yàn)一HIV-1單純及合并HCV感染的CD4+T細(xì)胞、CD8+T細(xì)胞及PBMC內(nèi)IL-7、IL-21、IFN-γmRNA水平測(cè)定 實(shí)驗(yàn)對(duì)象：以中國(guó)華南地區(qū)的已HAART治療的25例HIV-1單純感染和22例HIV-1/HCV合并感染者，及20例健康對(duì)照者為研究對(duì)象。 實(shí)驗(yàn)方法：分別采用免疫磁珠分選技術(shù)、實(shí)時(shí)熒光定量PCR方法，以上述HIV-1P24區(qū)域重疊肽段庫(kù)作為HIV-1特異性肽段表位，刺激從上述三組研究對(duì)象的外周血單個(gè)核細(xì)胞（PBMC）分選出的CD4+T淋巴細(xì)胞，CD8+T淋巴細(xì)胞，分別檢測(cè)CD4+T淋巴細(xì)胞、CD8+T淋巴細(xì)胞及PBMC在刺激前后IL-7、IL-21、IFN-γ，β-actin的mRNA水平，統(tǒng)計(jì)IL-7、IL-21、IFN-γ與β-actin的比值，并用單因素方差分析方法進(jìn)行統(tǒng)計(jì)分析。 實(shí)驗(yàn)結(jié)果：已接受HAART治療的HIV-1單純感染組和HIV-1/HCV合并感染組及健康對(duì)照組細(xì)胞內(nèi)IFN-γ、IL-7、IL-21的mRNA水平比較 P24抗原表位刺激培養(yǎng)后，HIV-1單純感染組CD8+T細(xì)胞的IFN-γ mRNA水平明顯高于CD4+T細(xì)胞（P0.05）。無(wú)論有無(wú)P24特異性抗原表位刺激，健康對(duì)照組PBMC中IFN-γ的mRNA水平均明顯高于HIV-1單純感染組和HIV-1/HCV合并感染組（P0.05）； P24抗原表位刺激后，HIV-1/HCV合并感染組PBMC中IL-7mRNA水平明顯高于HIV-1單純感染組（P0.05）；無(wú)論有無(wú)P24特異性抗原表位刺激，各組PBMC中IL-21mRNA水平相比均無(wú)明顯差異（P0.05）。 實(shí)驗(yàn)二ICS檢測(cè)HIV-1單純及合并HCV感染者分泌IL-4，IL-17A，IL-21，IL-21R，IFN-γ的CD3+T淋巴細(xì)胞比例 實(shí)驗(yàn)對(duì)象：以未HAART治療的24例HIV-1單純感染者和21例HIV-1/HCV合并感染者，及20例健康對(duì)照組為研究對(duì)象。 實(shí)驗(yàn)方法：采用細(xì)胞內(nèi)細(xì)胞因子染色法（intra-cellular CK staining，ICS），以上述HIV-1P24區(qū)域重疊肽段庫(kù)作為特異性肽段表位，刺激上述研究對(duì)象的PBMC，檢測(cè)使用與未使用P24抗原表位刺激后細(xì)胞內(nèi)分泌IL-4、IL-17A、IL-21、IL-21R、IFN-γ的CD3+T淋巴細(xì)胞比例，并用單因素方差分析方法進(jìn)行統(tǒng)計(jì)分析。 實(shí)驗(yàn)結(jié)果：未接受HAART治療的HIV-1單純感染組和HIV-1/HCV合并感染組及健康對(duì)照組細(xì)胞內(nèi)分泌IL-4、IL-17A、IFN-γ、IL-21、IL-21R的T淋巴細(xì)胞比例比較 比較使用與未使用P24抗原表位刺激后的結(jié)果顯示，三組T淋巴細(xì)胞內(nèi)的分泌IL-4、IL-17A、IFN-γ、IL-21、IL-21R的比例變化不顯著（P0.05）。在無(wú)P24抗原表位刺激培養(yǎng)后，HIV-1單純感染組的CD3+IL-4+/T淋巴細(xì)胞明顯高于健康對(duì)照組（P0.05），HIV-1/HCV合并感染組CD3+IL-4+/T淋巴細(xì)胞也高于健康對(duì)照組，但差異不顯著（P0.05）；而在P24抗原表位刺激培養(yǎng)后，三組CD3+IL4+/T淋巴細(xì)胞差異不顯著（P0.05）。無(wú)論有無(wú)P24抗原表位刺激培養(yǎng)，HIV-1單純感染組CD3+IL17-A+/T淋巴細(xì)胞HIV-1/HCV合并感染組健康對(duì)照組，且HIV-1單純感染組明顯高于其他兩組（P0.05）； HIV-1單純感染組CD3+IFN-γ+/T淋巴細(xì)胞 HIV-1/HCV合并感染組健康對(duì)照組，三組差異不顯著（P0.05）；三組CD3+IL-21+/T淋巴細(xì)胞差異不顯著（P0.05）；HIV-1單純感染組CD3+IL-21R+/T淋巴細(xì)胞比例健康對(duì)照組 HIV-1/HCV合并感染組，HIV-1單純感染組明顯高于其他兩組（P0.001）。 實(shí)驗(yàn)三ELISPOT檢測(cè)HIV-1單純及合并HCV感染的HIV-1特異性細(xì)胞免疫應(yīng)答 實(shí)驗(yàn)對(duì)象：以上述實(shí)驗(yàn)一和實(shí)驗(yàn)二中的單純感染者和合并感染者為研究對(duì)象。 實(shí)驗(yàn)方法：采用酶聯(lián)免疫斑點(diǎn)吸附技術(shù)（enzyme-linked immunosorbent spot，ELISPOT），以上述HIV-1P24區(qū)域重疊肽段庫(kù)作為特異性肽段表位，分別檢測(cè)研究對(duì)象PBMC中HIV-1特異性CTL應(yīng)答頻數(shù)，并用卡方檢驗(yàn)方法進(jìn)行統(tǒng)計(jì)分析。分析合并HCV感染對(duì)HIV-1感染細(xì)胞免疫的影響，HAART對(duì)HIV-1感染細(xì)胞免疫的影響。 實(shí)驗(yàn)結(jié)果：HIV-1單純感染組和HIV-1/HCV合并感染組HIV-1特異性CTL應(yīng)答比較 已接受HAART治療HIV-1單純感染組HIV-1特異性細(xì)胞免疫應(yīng)答率（13/25）明顯高于HIV-1/HCV合并感染組（5/22）（P0.05）；未接受HAART治療HIV-1單純感染組HIV-1特異性細(xì)胞免疫應(yīng)答率（8/22）高于HIV-1/HCV合并感染組（6/21），但差異不顯著（P0.05）；已治療單純感染組的HIV-1特異性細(xì)胞免疫應(yīng)答率（13/25）高于未治療單純感染組（8/22），但差異不顯著（P0.05）；已治療合并感染組HIV-1特異性細(xì)胞免疫應(yīng)答率（5/22）與未治療合并感染組（6/21）的相比無(wú)明顯差異（P0.05）。 結(jié)論： 1.合并HCV感染可能使HIV-1感染者HIV-1特異性細(xì)胞免疫應(yīng)答水平下降，這可能是HIV-1/HCV合并感染者HAART治療療效差的原因之一。 2.細(xì)胞因子IL-7、IL-17、IL-21R在HIV-1特異性細(xì)胞免疫中發(fā)揮重要作用。HIV-1感染還可以通過(guò)Th17和Tfh途徑影響感染者的細(xì)胞免疫，而HCV合并感染會(huì)對(duì)此產(chǎn)生干擾；IL-7在HIV-1/HCV合并感染者的細(xì)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.

【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R392

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