HIV-1抗原特异性CTL免疫应答影响因素的研究

发布时间：2018-04-16 23:12

本文选题：人类免疫缺陷病毒1型 + 细胞毒性T淋巴细胞　；参考：《广州医学院》2010年硕士论文

【摘要】：目前获得性免疫缺陷综合症(AIDS)已成为严重威胁全人类健康的传染病之一。高效抗逆转录病毒疗法(HAART)能够抑制人类免疫缺陷病毒(HIV)复制,延缓疾病进程,是至今为止最有效的控制和治疗AIDS的方法。既往研究结果表明特异性细胞毒性T淋巴细胞(CTL)在控制人HIV复制中起关键作用,但该领域的研究主要是针对未接受HAART的HIV-1感染者,而有关HAART对HIV-1特异性CTL免疫应答的影响的国内外研究报道很少,研究HAART对特异性CTL免疫应答的影响对于了解HAART后感染者体内的细胞免疫功能状态及探讨免疫功能重建的内在机理具有十分重要的意义。以往研究已经明确人类白细胞抗原(HLA)-I等位基因影响HIV-1特异性CTL应答及其疾病进展,但是这些研究资料都以非中国人为研究主体的研究结果,国内尚未见此方面研究报道。而不同人群其HLA-I等位基因频率表现出丰富的多态性,中国人群HLA-I等位基因的分布频率明显不同其它种族。因此对我国人群进行HLA-I等位基因频率与特异性CTL免疫应答相关性的研究十分必要,这不仅可以了解中国HIV-1感染者的特异性CTL应答频率与HLA-I多态性的相关性,同时还可发现影响HIV-1特异性CTL应答频率和疾病进展的特异性HLA-I等位基因。本研究以中国广东地区的HIV-1感染者为研究对象,采用酶联免疫斑点实验(ELISPOT)技术,以HIV-1 P24区域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,刺激16例未接受HAART治疗和84例接受HAART治疗的HIV-1感染者外周血单个核细胞(PBMC),检测γ-干扰素(IFN-γ)分泌细胞频率,分析特异性CTL免疫应答水平,并用PCR-SSP方法检测所有感染者HLA-I类等位基因分型,研究HAART和HLA-I类等位基因对HIV-1特异性CTL免疫应答的影响。主要研究内容和结果如下: 1.检测100例HIV-1感染者特异性CTL免疫应答水平后发现,48例感染者的PBMC能够识别HIV-1肽段表位并产生免疫应答,52例感染者不能识别HIV-1肽段表位且未产生免疫应答,应答组CD4+T细胞计数大于无应答组(P=0.01)。 2.将HIV-1感染者按照HAART治疗时间分为未治疗组、治疗时间小于12个月组和治疗时间大于12个月组。对这三组的特异性CTL免疫应答强度进行比较发现,治疗时间大于12个月组的CTL应答强度明显大于治疗时间小于12个月组和未治疗组(P=0.048;P=0.027);进一步对这三组的CD4+ T细胞数进行检测发现,治疗时间大于12个月组的CD4+T细胞数明显大于治疗时间小于12个月组(P=0.0003)。 3.不管HAART治疗与否,将HIV-1感染者按照CD4+T细胞计数分为CD4+T细胞数200 cell /μL组,CD4+T细胞数200～350 cell/μL组,CD4+T细胞数350 cell /μL组。其中CD4+T细胞数200 cell /μL组特异性CTL阳性应答率为19.4%(6/31),CD4+T细胞数200～350 cell /μL组特异性CTL阳性应答率为61.8%(21/34),CD4+T细胞数350 cell /μL组特异性CTL阳性应答率为60.0%(21/35)。对这三组特异性CTL阳性应答率进行比较发现,CD4+T细胞数200 cell /μL组特异性CTL阳性应答率明显低于CD4+T细胞数200～350 cell /μL组、CD4+T细胞数350 cell /μL组(P=0.001;P=0.001)。对三组HIV-1感染者的CTL应答强度进行比较发现,CD4+T细胞数200 cell /μL组的特异性CTL应答强度显著低于CD4+T细胞数200～350 cell /μL组、CD4+T细胞数350 cell /μL组(P=0.0003;P=0.009)。对三组HIV-1感染者血浆病毒载量的检测发现,三组病毒载量之间具有统计学差异(P=0.005),其中CD4+T细胞200 cell /μL的感染组病毒量大于其它两组(P=0.004;P=0.009)。 4.对入选的所有HIV-1感染者进行HLA-I类等位基因检测发现,具有不同HLA-I类等位基因的HIV-1感染者对于HIV-1肽段表位的免疫应答反应不同。HLA-A等位基因中阳性应答最高的为不常见基因(57.1%),其次为HLA-A*33(55.0%)和HLA-A*11(50.8%);HLA-B等位基因中阳性应答最高的为HLA-B*5(861.9%),其次为HLA-B*40(54.2%)和不常见等位基因(53.8%);HLA-C等位基因中阳性应答依次为HLA-Cw07(52.8%),HLA-Cw*03(50%),HLA-Cw*01(45%)。 5.HIV-1感染者中,携带HLA-B*18、-B*40和-B*58基因型的感染者特异性CTL免疫应答强,以HLA-B*18/ B*40杂合状态的感染者更为明显;HLA-B*58特异性CTL应答强度和阳性应答率明显高于HLA-B*51(P=0.041;P=0.031)。表达不常见HLA-A等位基因的感染者和表达HLA-A*11、-A*33的感染者之间尽管特异性CTL阳性应答率和应答强度无差异,但是CD4+T细胞数有统计学意义(P=0.027;P=0.019)。结论 1、如果不考虑HAART治疗因素的影响,血中CD4+T细胞数越高,HIV-1抗原特异性CTL免疫应答越强,二者呈正相关。 2、HAART治疗能提升HIV-1感染者的特异性CTL免疫应答,延长治疗时间至一年以上更有助于改善特异性CTL免疫应答。 3、HIV-1病毒载量较低的HIV-1感染者拥有较强的特异性CTL免疫应答。 4、在我们研究的HIV-1感染者中,HLA-I等位基因分布频率较高的为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等位基因型别比HLA-A、HLA-Cw等位基因更复杂多样。 5、HIV-1感染者的特异性CTL免疫应答似乎与HLA-A、B、Cw等位基因分布频率无关;拥有不同HLA-B基因型的HIV-1感染者的特异性CTL免疫应答存在显著性差异,而对于HLA-A基因型和HLA-Cw基因型未发现此现象。表明HLA-B等位基因对HIV-1特异性CTL免疫应答影响最大。 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位点,我们首次发现HLA-B*40/B*18杂合状态的感染者特异性CTL对HIV-1表位的免疫应答可能更具有优势。
[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.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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