【作者】 王晓辉；

【导师】 聂绍发；

【作者基本信息】 华中科技大学 ， 流行病与卫生统计学， 2011， 博士

【摘要】 研究目的1.以中国汉族HIV-1暴露而血清阴性者(HIV-1-exposed seronegative subjects, ESNs人群)为研究对象,以健康对照人群和人类免疫缺陷病毒I型(Human Immunodeficiency Virus Type 1, HIV-1)携带者为对照,探讨三组人群DC-SIGN和DC-SIGNR基因多态性与HIV-1不易感性的关系。2.分析三组人群HIV-1感染相关受体的表达差异,从蛋白质表达水平上寻找与HIV-1不易感性相关的受体。3.从基因多态性的角度解释与HIV-1不易感性密切相关的受体差异表达原因。研究方法应用扩增片段长度多态性(PCR-ALFP)的方法进行DC-SIGN和DC-SIGNR基因多态性分析；应用流式细胞术分析HIV-1感染相关受体的表达差异；应用序列特异引物PCR (sequence-specific primers, PCR-SSP)分析HLA基因多态性；应用SPSS13.0统计软件分析三组人群之间受体表达和基因型分布的差异。主要研究结果1. DC-SIGN在三组人群中以7/7型为主,三组人群共检出9个非7/7基因型,但DC-SIGN各基因型分布差异无统计学意义(P=0.648); DC-SIGNR具有高度多态性,7/5、5/5和6/7基因型的频率从ESN人群、健康对照人群和HIV-1携带者逐渐降低,同时5和6等位基因的频率也呈现均匀下降,但各基因型分布差异无统计学意义(P=0.782)；2. ESNs人群T淋巴细胞HLA-DR+CD4和HLA-DR+CD8均显著性低于健康对照人群(P=0.024,0.009),这两项指标同时呈现显著性正相关(P<0.001,r=0.960)；利用HLA-DR+CD8的双峰分布特征可以将健康对照人群分为高低表达两群；3.确切概率法卡方检验结果显示,三个基因座的多态性位点中只有A*03和B*55在三组人群中的分布差异有显著性。健康对照人群HLA基因型的分析发现,A*02和A*24基因型在HLA-DR+CD8表达高低两组的分布有显著性差异(P=0.020,0.038)；等位基因分析也验证了这个多态性变异分布的差异(P=0.007,0.009),DRB1*09等位基因分布也有显著性差异(P=0.028)；A*02多态性变异与HLA-DR在CD8阳性细胞表面的表达有显著性负相关(P=0.008)；A*24多态性变异与HLA-DR在CD8阳性细胞表而的表达有显著性正相关(P=0.045)。研究结论1.我国汉族人群DC-SIGN基因多态性变异很低,没有继续研究的意义；DC-SIGNR基因多态性对于HIV-1不易感性的意义需要大样本的验证或细胞生物学实验数据的支持；2.对于中国汉族人群,T淋巴细胞低活化状态与中国汉族人群HIV-1不易感性密切相关,从统计学分析和生物学意义上可以将中国汉族人群按照HLA-DR+CD8活化高低分为HIV-1易感人群和不易感人群；3. HLAA*02多态性变异可能是中国汉族人群HIV-1感染的保护性因素,而HLAA*24多态性变异可能是感染的危险因素。创新性1.首次在国内ESNs人群的研究中设定了定量的的入选标准；2.在国内首次报道HLA-DR的高低表达与中国汉族人群HIV-1不易感性密切相关；3.首次报道HLA-A*02多态性变异可能是中国汉族人群HIV-1感染的保护性因素,而HLA-A*24多态性变异可能是感染的危险因素。更多还原

【Abstract】 Objective1. Setting HIV-1-exposed seronegative subjects (ESNs) of Han Chinese as the research objective, healthy people and Human Immunodeficiency Virus Type 1(HIV-1) carriers as the control population, to explore the relationship between the gene polymorphism of DC-SIGN, DC-SIGNR and HIV-1 low susceptibility.2. To analyzing the expression difference of HIV-1 infection related co-receptor, from the level of protein expression to search the co-receptor related with HIV-1 low susceptibility. 3. To clarify the reason of expression difference of co-receptor from the view of gene polymorphism.MethodsPCR-ALFP method was applied to analyze DC-SIGN, DC-SIGNR gene polymorphism. Flow cytometer was used to analyze the expression difference of HIV-1 infection related co-receptor, PCR-SSP was used to analyze HLA gene polymorphism, SPSS 13.0 was applied to analyze the difference of co-receptor expression and genotype distribution.Results1. Genotype 7/7 was the primary genotype of DC-SIGN. Only 9 non 7/7 genotype was tested in three groups. There was no significant difference between 7/7 and non-7/7 genotypes(P=0.648).DC-SIGNR genetic polymorphism was high, and the frequency of 5/5 and 6/7 genotypes among ESN, healthy control and HIV-1 carriers gradually decrease. The allele frequency of 5 and 6 also had the same trend. But there were no significant difference in genotypes (P=0.782) and gene alleles between three groups.2. We found the ESNs had significantly lower percentage of HLA-DR+ CD4 T cells and HLA-DR+ CD8 T cells than healthy controls and HIV-1 carriers (P=0.024,0.009). The two index also had significant positive correlation (P<0.001, r=0.960). Healthy controls could be clearly divided into low expression group and high expression group according to HLA-DR+ CD8 T cells expression.3. According to Fisher’s exact Chi-square test, only A*03 and B*55 genotype distribution had statistic significance. In healthy controls, A*02 and A*24 genotype distribution shown significant difference between HLA-DR+CD8 low and high expression groups (P=0.020, 0.038). The analysis of allele distribution also confirm above results of the difference between the two gene polymorphism (P=0.007,0.009). DRB1*09 also show significant difference in allele distribution. The distribution of A*02 allele showed significant negative correlation with HLA-DR+CD8 expression (P=0.008), whereas the distribution of A*24 displayed a significant positive correlation with HLA-DR+CD8 expression (P=0.045).Conclusions1. Han Chinese has very low mutation frequency in DC-SIGN. It is not necessary to analyze DC-SIGN polymorphism in future study. We need large sample or data of cell biology to conform the relation between HIV-1 susceptibility and DC-SIGNR polymorphism.2. To Han Chinese, the low activation status of T lymphocyte has close correlation with HIV-1 low susceptibility. Han Chinese can be divided into low and high susceptibility groups according to activation status of HLA-DR+CD8 from the aspects of statistic analysis and biological significance.3. HLA-A*02 polymorphism mutation may be the protect factor of HIV-1 infection and HLA-A*24 polymorphism mutation may be the risk factor of HIV-1 infection to Han Chinese. Innovative Point1. Firstly set quantitive enrollment standard in ESNs research in China.2. Firstly report the close relationship between HLA-DR high/low expression and HIV-1 susceptibility of Han Chinese.3. Firstly report the result that HLA-A*02 polymorphism mutation may be the protect factor of HIV-1 infection and HLA-A*24 polymorphism mutation may be the risk factor of HIV-1 infection to Han Chinese.更多还原