本文選題：人類免疫缺陷病毒1型 + 細(xì)胞毒性T淋巴細(xì)胞　； 參考：《廣州醫(yī)學(xué)院》2010年碩士論文

【摘要】：目前獲得性免疫缺陷綜合癥(AIDS)已成為嚴(yán)重威脅全人類健康的傳染病之一。高效抗逆轉(zhuǎn)錄病毒療法(HAART)能夠抑制人類免疫缺陷病毒(HIV)復(fù)制,延緩疾病進(jìn)程,是至今為止最有效的控制和治療AIDS的方法。既往研究結(jié)果表明特異性細(xì)胞毒性T淋巴細(xì)胞(CTL)在控制人HIV復(fù)制中起關(guān)鍵作用,但該領(lǐng)域的研究主要是針對(duì)未接受HAART的HIV-1感染者,而有關(guān)HAART對(duì)HIV-1特異性CTL免疫應(yīng)答的影響的國(guó)內(nèi)外研究報(bào)道很少,研究HAART對(duì)特異性CTL免疫應(yīng)答的影響對(duì)于了解HAART后感染者體內(nèi)的細(xì)胞免疫功能狀態(tài)及探討免疫功能重建的內(nèi)在機(jī)理具有十分重要的意義。 以往研究已經(jīng)明確人類白細(xì)胞抗原(HLA)-I等位基因影響HIV-1特異性CTL應(yīng)答及其疾病進(jìn)展,但是這些研究資料都以非中國(guó)人為研究主體的研究結(jié)果,國(guó)內(nèi)尚未見(jiàn)此方面研究報(bào)道。而不同人群其HLA-I等位基因頻率表現(xiàn)出豐富的多態(tài)性,中國(guó)人群HLA-I等位基因的分布頻率明顯不同其它種族。因此對(duì)我國(guó)人群進(jìn)行HLA-I等位基因頻率與特異性CTL免疫應(yīng)答相關(guān)性的研究十分必要,這不僅可以了解中國(guó)HIV-1感染者的特異性CTL應(yīng)答頻率與HLA-I多態(tài)性的相關(guān)性,同時(shí)還可發(fā)現(xiàn)影響HIV-1特異性CTL應(yīng)答頻率和疾病進(jìn)展的特異性HLA-I等位基因。 本研究以中國(guó)廣東地區(qū)的HIV-1感染者為研究對(duì)象,采用酶聯(lián)免疫斑點(diǎn)實(shí)驗(yàn)(ELISPOT)技術(shù),以HIV-1 P24區(qū)域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,刺激16例未接受HAART治療和84例接受HAART治療的HIV-1感染者外周血單個(gè)核細(xì)胞(PBMC),檢測(cè)γ-干擾素(IFN-γ)分泌細(xì)胞頻率,分析特異性CTL免疫應(yīng)答水平,并用PCR-SSP方法檢測(cè)所有感染者HLA-I類等位基因分型,研究HAART和HLA-I類等位基因?qū)IV-1特異性CTL免疫應(yīng)答的影響。 主要研究?jī)?nèi)容和結(jié)果如下: 1.檢測(cè)100例HIV-1感染者特異性CTL免疫應(yīng)答水平后發(fā)現(xiàn),48例感染者的PBMC能夠識(shí)別HIV-1肽段表位并產(chǎn)生免疫應(yīng)答,52例感染者不能識(shí)別HIV-1肽段表位且未產(chǎn)生免疫應(yīng)答,應(yīng)答組CD4+T細(xì)胞計(jì)數(shù)大于無(wú)應(yīng)答組(P=0.01)。 2.將HIV-1感染者按照HAART治療時(shí)間分為未治療組、治療時(shí)間小于12個(gè)月組和治療時(shí)間大于12個(gè)月組。對(duì)這三組的特異性CTL免疫應(yīng)答強(qiáng)度進(jìn)行比較發(fā)現(xiàn),治療時(shí)間大于12個(gè)月組的CTL應(yīng)答強(qiáng)度明顯大于治療時(shí)間小于12個(gè)月組和未治療組(P=0.048;P=0.027);進(jìn)一步對(duì)這三組的CD4+ T細(xì)胞數(shù)進(jìn)行檢測(cè)發(fā)現(xiàn),治療時(shí)間大于12個(gè)月組的CD4+T細(xì)胞數(shù)明顯大于治療時(shí)間小于12個(gè)月組(P=0.0003)。 3.不管HAART治療與否,將HIV-1感染者按照CD4+T細(xì)胞計(jì)數(shù)分為CD4+T細(xì)胞數(shù)200 cell /μL組,CD4+T細(xì)胞數(shù)200～350 cell/μL組,CD4+T細(xì)胞數(shù)350 cell /μL組。其中CD4+T細(xì)胞數(shù)200 cell /μL組特異性CTL陽(yáng)性應(yīng)答率為19.4%(6/31),CD4+T細(xì)胞數(shù)200～350 cell /μL組特異性CTL陽(yáng)性應(yīng)答率為61.8%(21/34),CD4+T細(xì)胞數(shù)350 cell /μL組特異性CTL陽(yáng)性應(yīng)答率為60.0%(21/35)。對(duì)這三組特異性CTL陽(yáng)性應(yīng)答率進(jìn)行比較發(fā)現(xiàn),CD4+T細(xì)胞數(shù)200 cell /μL組特異性CTL陽(yáng)性應(yīng)答率明顯低于CD4+T細(xì)胞數(shù)200～350 cell /μL組、CD4+T細(xì)胞數(shù)350 cell /μL組(P=0.001;P=0.001)。對(duì)三組HIV-1感染者的CTL應(yīng)答強(qiáng)度進(jìn)行比較發(fā)現(xiàn),CD4+T細(xì)胞數(shù)200 cell /μL組的特異性CTL應(yīng)答強(qiáng)度顯著低于CD4+T細(xì)胞數(shù)200～350 cell /μL組、CD4+T細(xì)胞數(shù)350 cell /μL組(P=0.0003;P=0.009)。對(duì)三組HIV-1感染者血漿病毒載量的檢測(cè)發(fā)現(xiàn),三組病毒載量之間具有統(tǒng)計(jì)學(xué)差異(P=0.005),其中CD4+T細(xì)胞200 cell /μL的感染組病毒量大于其它兩組(P=0.004;P=0.009)。 4.對(duì)入選的所有HIV-1感染者進(jìn)行HLA-I類等位基因檢測(cè)發(fā)現(xiàn),具有不同HLA-I類等位基因的HIV-1感染者對(duì)于HIV-1肽段表位的免疫應(yīng)答反應(yīng)不同。HLA-A等位基因中陽(yáng)性應(yīng)答最高的為不常見(jiàn)基因(57.1%),其次為HLA-A*33(55.0%)和HLA-A*11(50.8%);HLA-B等位基因中陽(yáng)性應(yīng)答最高的為HLA-B*5(861.9%),其次為HLA-B*40(54.2%)和不常見(jiàn)等位基因(53.8%);HLA-C等位基因中陽(yáng)性應(yīng)答依次為HLA-Cw07(52.8%),HLA-Cw*03(50%),HLA-Cw*01(45%)。 5.HIV-1感染者中,攜帶HLA-B*18、-B*40和-B*58基因型的感染者特異性CTL免疫應(yīng)答強(qiáng),以HLA-B*18/ B*40雜合狀態(tài)的感染者更為明顯;HLA-B*58特異性CTL應(yīng)答強(qiáng)度和陽(yáng)性應(yīng)答率明顯高于HLA-B*51(P=0.041;P=0.031)。表達(dá)不常見(jiàn)HLA-A等位基因的感染者和表達(dá)HLA-A*11、-A*33的感染者之間盡管特異性CTL陽(yáng)性應(yīng)答率和應(yīng)答強(qiáng)度無(wú)差異,但是CD4+T細(xì)胞數(shù)有統(tǒng)計(jì)學(xué)意義(P=0.027;P=0.019)。 結(jié)論 1、如果不考慮HAART治療因素的影響,血中CD4+T細(xì)胞數(shù)越高,HIV-1抗原特異性CTL免疫應(yīng)答越強(qiáng),二者呈正相關(guān)。 2、HAART治療能提升HIV-1感染者的特異性CTL免疫應(yīng)答,延長(zhǎng)治療時(shí)間至一年以上更有助于改善特異性CTL免疫應(yīng)答。 3、HIV-1病毒載量較低的HIV-1感染者擁有較強(qiáng)的特異性CTL免疫應(yīng)答。 4、在我們研究的HIV-1感染者中,HLA-I等位基因分布頻率較高的為HLA-A*11(0.59),HLA-A*02(0.53),HLA-Cw*03(0.50),HLA-Cw*01(0.40),HLA-B*46(0.35)。HLA-B等位基因型別比HLA-A、HLA-Cw等位基因更復(fù)雜多樣。 5、HIV-1感染者的特異性CTL免疫應(yīng)答似乎與HLA-A、B、Cw等位基因分布頻率無(wú)關(guān);擁有不同HLA-B基因型的HIV-1感染者的特異性CTL免疫應(yīng)答存在顯著性差異,而對(duì)于HLA-A基因型和HLA-Cw基因型未發(fā)現(xiàn)此現(xiàn)象。表明HLA-B等位基因?qū)IV-1特異性CTL免疫應(yīng)答影響最大。 6、HLA-B*58、HLA-B*40、HLA-B*18基因型,可能是HIV-1肽段表位的限制性HLA-I位點(diǎn),我們首次發(fā)現(xiàn)HLA-B*40/B*18雜合狀態(tài)的感染者特異性CTL對(duì)HIV-1表位的免疫應(yīng)答可能更具有優(yōu)勢(shì)。
[Abstract]:The acquired immunodeficiency syndrome (AIDS) is one of the infectious disease has become a serious threat to humanity health. Highly active antiretroviral therapy (HAART) can inhibit human immunodeficiency virus (HIV) replication, slow the progression of the disease, control and treatment of AIDS is the most effective method so far. The previous research results show that specific cytotoxic T lymphocytes (CTL) play a key role in controlling HIV replication, but the research in this field is mainly aimed at not receiving HAART HIV-1 infection, but the reports about the effect of HAART on HIV-1 specific CTL immune response of the little research on the influence of HAART on the immune response of specific CTL for the understanding of HAART infected cells the body's immune function and has very important significance to explore the internal mechanism of immune reconstitution.
Previous studies have identified human leukocyte antigen (HLA) alleles of -I affect the progress of HIV-1 specific CTL responses and disease, but the results of these research data are non China as the research subject, it has not been reported. The research in this area and different groups of the HLA-I allele frequency showed abundant polymorphism the frequency distribution of HLA-I, China population allele was significantly different from other races. Therefore the study of correlation between HLA-I allele and CTL specific immune response to the population of our country is very necessary, it can not only understand the relationship between specific CTL response frequency and HLA-I polymorphism of Chinese HIV-1 infection, but also found that the influence of progress of HIV-1 specific CTL response frequency and disease specific HLA-I alleles.
In this study, the Guangdong area Chinese HIV-1 infection as the research object, using enzyme-linked immunospot (ELISPOT) technique, 12 overlapping peptides with the amino acid sequence of HIV-1 P24 region of the synthetic composition of the peptide library as specific peptide epitopes, stimulation of 16 patients did not receive HAART treatment and 84 cases accepted HAART treatment of HIV-1 infection in peripheral blood mononuclear cells (PBMC), detection of interferon gamma (IFN- gamma) frequency analysis of secretory cells, specific CTL immune response, and PCR-SSP method to detect all infected HLA-I genotypes, the effect of HAART and HLA-I alleles of HIV-1. The specific CTL immune response.
The main contents and results are as follows:
1. detected 100 cases of HIV-1 infected antigen-specific CTL immune response levels found, 48 cases infected by PBMC can recognize HIV-1 peptide epitopes in immune response, 52 cases of infected people do not recognize HIV-1 peptide epitope without immune responses, response group CD4+T cell count was higher than the non response group (P=0.01).
2. HIV-1 infected persons in accordance with the HAART treatment time divided into untreated group, treatment time is less than 12 months group and treatment group of more than 12 months. The intensity of immune response of specific CTL of the three groups were compared, the treatment time is greater than the CTL response intensity for 12 months group was significantly higher than the treatment time of less than 12 month group and non treatment group (P=0.048; P=0.027); further to the number CD4+ T cells of the three groups were detected, the treatment time is greater than the number of CD4+T cells in 12 month group was significantly higher than the treatment time of less than 12 months group (P=0.0003).
3. HAART regardless of whether or not the therapy, HIV-1 infected CD4+T cell number 200 cell / L group according to CD4+T cell count and CD4+T cell number 200~350 cell/ L group, CD4+T cell number 350 cell / L group. The number of CD4+T cells of 200 cell / L group specific CTL positive response rate was 19.4% (6/31), CD4+T cell number 200~350 cell / L group specific CTL positive response rate was 61.8% (21/34), CD4+T cell number 350 cell / L group specific CTL positive response rate was 60% (21/35). The three group specific CTL positive response rates were compared, CD4+T cell number 200 the cell / L group specific CTL positive response rate was significantly lower than that of CD4+T cells in 200~350 cell / L group, the number of CD4+T cells in 350 cell / L group (P=0.001; P=0.001). The CTL response strength of three groups of HIV-1 infection were compared, specific CTL response intensity of 200 cell / CD4+T cells the L group was significantly lower than that of CD4+T The cell number of 200~350 cell / L group, the number of CD4+T cells in 350 cell / L group (P=0.0003; P=0.009). It is found that detection of plasma viral load in three groups of HIV-1 infection, with statistical difference between the three groups of viral load (P=0.005), of which 200 CD4+T cell cell / L virus infection group than the other two groups (P=0.004; P=0.009).
4. of all HIV-1 infected subjects were selected class HLA-I alleles detected with different HLA-I alleles of HIV-1 infection for the immune response of HIV-1 peptide epitopes of different.HLA-A alleles in the highest positive response to the common gene (57.1%), followed by HLA-A*33 (55%) and HLA-A*11 (50.8%); HLA-B alleles in the highest positive response for HLA-B*5 (861.9%), followed by HLA-B*40 (54.2%) and common alleles (53.8%); HLA-C allele positive response followed by HLA-Cw07 (52.8%), HLA-Cw*03 (50%), HLA-Cw*01 (45%).
5.HIV-1 infection, with HLA-B*18, -B*40 and -B*58 genotypes infected antigen-specific CTL immune response, with HLA-B*18/ B*40 heterozygous infection is more obvious; the HLA-B*58 specific CTL response intensity and the positive response rate was significantly higher than that of HLA-B*51 (P=0.041; P=0.031). Infection unusual expression of HLA-A alleles and the expression of HLA-A*11 between -A*33 infection while there was no difference in specific CTL response magnitude and the positive response rate, but the number of CD4+T cells was statistically significant (P=0.027; P=0.019).
conclusion
1, the higher the number of CD4+T cells in the blood, the stronger the HIV-1 antigen specific CTL immune response without taking into account the effect of HAART treatment factors, and the more positive correlation was found in the two.
2, HAART therapy can enhance the specific CTL immune response of HIV-1 infected people, and prolonging the time of treatment more than one year is more helpful to improve the specific CTL immune response.
3, the HIV-1 infected with low HIV-1 viral load has a strong specific CTL immune response.
4, in the HIV-1 infected people we studied, the distribution frequencies of HLA-I alleles were higher than those of HLA-A*11 (0.59), HLA-A*02 (0.53), HLA-Cw*03 (0.50), HLA-Cw*01 (0.40), HLA-B*46 (0.35).HLA-B allele type than those of HLA-A and HLA-Cw alleles.
5, specific CTL immune response in HIV-1 infected with HLA-A B Cw, it seems, allele frequency independent; there is a significant difference between the specific CTL immune response with different HLA-B genotype HIV-1 infection, and for HLA-A and HLA-Cw genotypes were not found. This phenomenon indicates that the HLA-B allele the greatest impact on the HIV-1 specific CTL immune responses.
6, HLA-B*58, HLA-B*40 and HLA-B*18 genotype may be the restrictive HLA-I sites of HIV-1 peptide epitope. For the first time, we found for the first time that the specific CTL of HLA-B*40/B*18 heterozygous state may have an advantage over HIV-1 epitopes.

【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】：2010
【分類號(hào)】：R392

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