【作者】 薛云；

【导师】 李继承；

【作者基本信息】 浙江大学 ， 细胞生物学， 2010， 博士

【摘要】 结核病(Tuberculosis, TB)是一种由结核分枝杆菌(Mycobacterium tuberculosis,MTB)引起的慢性消耗性传染病,曾被称为“白色瘟疫”。尽管全世界约有1／3的人曾感染结核分枝杆菌,但被感染人群中仅5-15%会在一生中患结核病(即发展成活动性结核),提示遗传因素造成的个体差异可能与结核病的发病倾向,即易感性,具有显著关联。遗传学研究表明,结核病的发病是多基因与环境因素共同作用的结果。中国具有远高于世界平均水平的结核病发病率,但中国汉族人群结核病的易感相关性研究却鲜见报道。因此,筛选结核病易感相关的基因,揭示其在中国汉族人群结核病发病中的作用,具有重要意义。单核／巨噬细胞系统在结核分枝杆菌的早期识别和结核病的发生中起着关键作用,我们推测单核／巨噬细胞表面受体基因多态性很有可能与结核病的易感性相关。因此,我们研究了单核／巨噬细胞表面分子Toll样受体及其信号通路位于胞外区的蛋白(最早直接接触结核分枝杆菌抗原的蛋白)——CD14、TLR2和TLR4／MD-2的基因多态性与中国汉族人群结核病的易感相关性,筛选中国汉族人群的结核病易感高风险因子。并进一步在体外试验和体内试验中探讨这些高风险因子引起结核病易感性增加的分子基础。本研究从宿主免疫系统分子遗传学的角度探讨结核病的发病机理,也提供了一种区分结核高风险人群,预防和综合控制结核病传播的新思路。本研究收集了符合中国卫生部结核诊断标准的中国汉族结核病例累计432例,健康对照累计404例。利用生物信息数据库查找“可疑”结核病相关基因多态性位点,共选取TLR2基因外显子区域的4个单核苷酸多态性位点(single nucleotide polymorphisms, SNPs)、内含子区域的1个微卫星多态性位点；TLR4基因外显子区域的2个SNPs位点；CDl4基因启动子区域的全部5个SNPs位点和LY96基因(编码MD-2蛋白)启动子区域的全部6个SNPs位点。采用PCR联合DNA序列测定法,对所选SNPs和微卫星位点进行基因型分型。然后,利用统计学方法分析基因多态性位点和结核病相关性,包括,对照组基因型Hardy-Weinberg遗传平衡检验；等位基因及基因型频率在结核病患与健康对照组中的分布差异；单一SNP与结核病之间在不同遗传模式下的相关性分析。由此筛选出结核病易感高风险基因多态性位点。结果表明：①TLR2基因编码区的4个多态性位点,Arg677Trp在中国人群中只有一种基因型(C／C),Arg753G1n出现频率仅为0.5%,且结核组与对照组没有显著差异(P>0.05)。余下2个位点Asn199Asn和Ser450Ser在病例和对照组中均属于多态位点(MAF>1%)。但这2个多态性位点的相关等位基因频率及基因型在健康组与结核病组间的分布不存在显著性差异(P>0.05),与中国汉族人群TB发病可能不存在显著相关性。TLR2基因微卫星多态性位点Mint2位点S、M和L等位基因频率在健康组与结核病患组间的分布不存在显著性差异(P>0.05)。但是,进一步的基因型分析显示S／M基因型在结核病组中的分布频率远高于健康对照组(P=0.01),S／L基因型在健康对照组中的分布频率远高于结核病组(P=0.007)。提示S／M基因型为中国汉族人群结核病的易感高风险因子,而S／L型则为中国汉族人群结核病的保护性因子。②TLR4外显子区域Asp299G1y和Thr399Ile位点在中国汉族人群中没有多态性分布,与中国汉族人群TB发病可能不存在显著相关性。③CD14基因C-159T和G-1145A等位基因频率已在健康组与结核病患组间的分布显示了显著差异(分别为P=0.01和P＜0.0001)。进一步的基因型分析也表明-159TT和-1145GG基因型在结核病组中的分布频率远高于健康对照组(分别为P=0.027和P＜0.0001)。提示CD14基因-159TT和-1145GG基因型为中国汉族人群结核病的易感高风险因子。④LY96基因启动子全部6个SNPs位点中,T-538G和T-475A在中国人群中只有一种基因型,其余4个位点在病例和对照组中均属于多态位点(MAF>1%)。但这4个多态性位点的等位基因及基因型频率在健康组与结核病组间的分布不存在显著性差异(P>0.05),与中国汉族人群结核病易感性可能不存在显著相关性。在进行了单个SNP与结核病的相关分析后,我们继续进行了基因内和基因间不同SNPs标记之间的连锁不平衡分析；基因内和基因间不同SNPs标记之间构建单体型,单体型频率估算以及不同单体型与结核病的相关性分析。由此筛选出结核病易感高风险基因多态性位点的单体型。结果表明：①TLR2基因Arg753G1n与Asn199Asn和Ser450Ser之间存在显著连锁(D’>0.75)。CD14基因G-1145A和C-159T位点之间存在一定的连锁相关性(D’=0.6685)。LY96基因C-1625G、C-1201G、G-1174T和A-1064G位点之间存在很强的连锁相关性(D’>0.75)。②根据连锁不平衡分析结果,我们构建了SNPs组成的单体型,并分析了单体型与结核病的相关性。结果显示：TLR2基因Arg753G1n与Asn199Asn和Ser450Ser构成的单体型,LY96基因C-1625G、C-1201G、G-1174T和A-1064G构成的单体型均与结核病不存在显著关联。CD14基因G-1145A和C-159T构成的单体型与结核病存在极显著关联(P<0.0001)。③没有发现基因间SNPs存在强连锁关系,构成的单体型与结核病也没有显著关联。针对筛选出的结核病相关高风险基因多态性位点,采用流式细胞技术和酶联免疫吸附实验检测目的基因在结核病患及健康对照血液单核细胞表面和血清中的表达。分析结核病易感高风险SNPs位点各基因型及单体型影响基因体内表达,最终影响结核病易感性的分子机理。结果表明：①对于筛选出的结核病显著相关位点TLR2基因Mint2位点,在结核病患组和健康对照组中,S／M基因型样本TLR2在CD14+PMBCs膜上的表达量均值显著低于S／L基因型(P=0.001,P=0.002)和M／M基因型(P=0.016,P=0.038)。②对于结核病显著相关位点CD14基因CDl4 C-159T和G-1145A位点,在结核病患组和健康对照组中,尽管单一SNP位点各基因型样本CD14在血清中和PMBCs膜上的表达量均值没有表现出显著差异(P＞0.05)。但这2个位点构成的TA和CG单体型样本CD14在血清中和PMBCs膜上的表达量均值显著高于CA单体型(P＜0.05)。结核病患组的CD14表达量均值也显著高于健康对照组(P<0.001)。针对显示了与结核病易感性极其显著相关性的CD14 C-159T和G-1145A位点单体型(如CG；OR=13.7,P<0.0001),我们进一步在体外构建含各单体型的CD14启动子的表达质粒,转染U937细胞,利用荧光素酶报告基因的表达,分析CD14单体型影响CD14启动子的功能,最终影响结核病易感性的分子机理。结果表明：pGL3-TA和pGL3-CG能够有效刺激荧光报告基因的表达,荧光强度显著高于pGL3-CA(分别为P=0.013和P=0.0015)。提示TA、CG单体型CD14基因启动子强度显著高于CA单体型,并充分支持了体内实验结果。结论：(1)本研究首次对TLR2基因外显子区域的4个SNPs位点、内含子区域的1个微卫星多态性位点与中国汉族人群结核病的易感性进行关联研究。结果显示与国外人群不同,中国汉族人群外显子区域4个SNPs与TB发病没有显著关联。但基因统计学试验和临床样本实验表明,内含子区域的1个微卫星多态性位点与中国汉族人群结核病的易感性具有显著关联。(2)首次对TLR4基因外显子区域的2个SNPs位点与中国汉族人群结核病的易感性进行关联研究,结果显示与国外人群不同,中国汉族人群外显子区域2个SNPs与TB发病没有显著关联。(3)首次研究了LY96基因启动子区域全部6个SNPs位点与结核病的相关性,结果发现LY96启动子区域基因多态性与结核病没有显著相关性。(4)首先发现CD14基因G-1145ASNP位点为1个新的结核病易感相关位点,且与另一个结核病易感相关位点CD14C-159T具有联合效应,构成的单体型与结核病易感性具有极显著的相关性(P<0.001)。进一步的体内实验(临床样本)和体外实验(基因转染)结果表明,CD14基因G-1145A SNP位点的单体型通过影响基因启动子强度,影响CD14在结核病患体内表达水平,最终影响了结核病的易感性。更多还原

【Abstract】 Tuberculosis (TB) is a serious infectious disease caused by Mycobacterium tuberculosis (MTB). This chronic wasting disease was once called "white plague", leading the death rates among all infectious diseases around the world. Although about a third of the world’s population is infected with Mycobacterium tuberculosis, only 5-15% of those develop clinical active TB during their lifetime. Some evidence suggests that certain genetic factors may involve in innate immunity and play important roles in susceptibility to TB. China has a much higher incidence of tuberculosis than the world average level. Whether the genetic factors increase the TB disease susceptibility in Chinese or not is still less known. The aim of the present study was to investigate whether genes are associated with the susceptibility to TB in a Chinese Han population.Monocyte/macrophage system plays a key role in the early identification of Mycobacterium tuberculosis and the incidence of tuberculosis. Therefore, we studied the monocyte/macrophage cell surface molecules-Toll-like receptors and their signaling pathway proteins in the extracellular which directly contact with the protein antigen of Mycobacterium tuberculosis in the frist stage. CD14, TLR2 and TLR4/MD-2 gene polymorphisms which concern with the susceptibility to TB were selected and identified as risk factors for TB in the Chinese Han population. Then further trials were done on the molecular basis of these high risk factors causing the increase of susceptibility to TB. Characteristic of this study was viewing molecular genetics in the host immune system to understand the pathogenesis of TB. It also provides a distinction between high-risk population tuberculosis, and a new idea in prevention and control of TB.Blood samples were obtained from 432 unrelated cases, diagnosed with TB. All patients were histological confirmed with criteria of the Chinese Ministry of Health on Tuberculosis. Control DNAs were obtained from 404 unaffected individuals of Han Chinese. According to references, suspicious TB-related gene polymorphisms were selected by using biological information database。In this study, four SNPs in the exon region of TLR2 gene, a microsatellite polymorphism in the intron regions of TLR2 gene; two SNPs in the exon region of TLR4 gene; all the 5 SNPs in the promoter region of CD 14 gene; and all the 6 SNPs in the promoter region of LY96 gene (encoding MD-2 protein) were selected. The polymorphisms were detected by polymerase chain reaction (PCR) method and followed by direct sequencing. Statistical analysis was as follows: The deviation from Hardy-Weinberg equilibrium (HWE) was examined in controls by the x2 test. Based on the logistic regression method, the case-control association of genotypes in five inheritance models (codominant, dominant, recessive, overdominant, log-additive) was tested for all the single SNPs. Our data showed that:①Different from foreign reports, the TLR2 Arg677Trp polymorphism, was not observed in either group. The TLR2 Arg753Gln polymorphism occur at very low frequency in the patients with TB (0.49%, compared with 0.49% in the control subjects, P= 0.094). No significant genetic association between 2 coding region SNPs (Asn199Asn and Ser450Ser) and TB were observed in the experiment (P> 0.05). No association in allelic polymorphism between control subjects and TB patients was found. However, the S/M genotype of the microsatellite polymorphism was more frequent in TB patients than in healthy controls (P= 0.01), while the S/L genotype was more popular in controls than in TB patients (P=0.007). The data suggests that the S/M genotype of the microsatellite (GT)n polymorphisms in intron 2 of the TLR2 gene may increase the susceptibility to tuberculosis in Chinese people, and the genotype S/L may act as a negative risk factor.②No Asp299Gly or Thr399Ile SNP in TLR4 was found from either the tuberculosis group or the healthy control group, indicating there was no significant association between these SNPs in the TLR4 gene and the susceptibility to TB.③The C allele of CD14 C-159T and G allele of CD14 C-159T and G allele of CD14 G-1145A were significantly associated with TB (P= 0.001 and P< 0.0001, respectively). Moreover, there was also significant association between the CC genotype of CD14 C-159T or GG genotype of CD14 G-1145A and TB (P= 0.027 and P x 0.001, respectively). Our results suggests that SNPs CD14 C-159T and G(-1145)A might be risk factors for the development of TB.④No genetic variation was observed at positions-538 and-475 of LY96 gene. For the orther SNPs-1625,-1201,-1174 and-1064, no significant differences between patients and controls were observed in allele, haplotype or genotype distribution (P> 0.05). Our results suggest that polymorphisms in the LY96 promoter region are not associated with TB, and may not play a major role in susceptibility to TB in a Chinese population.Pairwise Linkage Disequilibrium (LD) was calculated for the cases and controls in Han Chinese population. Constructed the haplotype blocks based on the results of LD analyses.The association analysis of the haplotypes with TB was similar to that of genotypes of single SNP by logistic regression. The significance of all these tests was 0.05.①We found strong LD (D’> 0.75) between some pairs of the markers, such as Arg753Gln, Asn199Asn and Ser450Ser in the TLR2 gene, C-1625G、C-1201G、G-1174T和A-1064G in the LY96 gene. Positions G-1145A and C-159T in the CD14 gene were also found to be in tight linkage disequilibrium (D’= 0.6685).②Therefore, we constructed haplotype blocks consisting of some nearby SNPs which were in strong LD. And the association between these haplotypes and the susceptibility of TB was also analysised. Our data showed that the haplotypes constructed by Arg753Gln/Asn199Asn/Ser450Ser and C-1625G/C-1201G/G-1174T/A-1064G had no significant association with TB. However, haplotype-specific association analysis revealed that the G-1145A/C-159T haplotype block showed significant association with TB (P< 0.0001).③In addition, no significant linkage was found in SNPs between different genes and no significant association was found in haplotype constructed by SNPs between different genes.In order to evaluate the influence of TB-related gene polymorphisms on gene expression in vivo, Flow cytometry and enzyme-linked immunosorbent assay were used to detect target gene expression in blood mononuclear cell surface and serum.①The expression of TLR2 was lower in healthy volunteers possessing the S/M genotype compared to those with the S/L genotype (P= 0.002) or with the M/M genotype (P= 0.038). The expression of TLR2 was lower in TB patients with the S/M genotype compared to patients with the S/L genotype (P= 0.001) or with the M/M genotype (P= 0.016).②The levels of sCD14 in the sera of TB patients were also higher than those in the control subjects (mean level of concentration, P< 0.001). In neither the patients with pulmonary TB nor the control subjects was there any association between the G-1145A and C-159T genotype and the levels of mCD14 and sCD14 (P> 0.05). However, the TA and CG haplotype of the CD14 gene had much higher expression level than CA haplotype in both PBMCs and serum (P< 0.05). In additon, before the patients with TB started anti-TB treatment, the levels of mCD14 on monocytes were higher than those in the control subjects (P< 0.001, respectively).For the haplotypes which showed highly significant correlation between CD 14 C-159T/G-1145A loci haplotypes and susceptibility to tuberculosis (such as CG; OR= 13.7, P<0.0001), we further constructed the CD14 promoter containing such haplotypes in vitro. The expression plasmids were transfected to U937 cells. By using luciferase reporter gene expression, haplotypes affect the function of CD14 promoter were analysised. The results showed that:pGL3-TA and pGL3-CG can be effective in stimulating the expression of fluorescent reporter gene, whose fluorescence intensity was significantly higher than pGL3-CA (respectively, P= 0.013 and P= 0.0015). Tips CD 14 gene promoter which contain TA, CG haplotypes was significantly higher than that contain CA haplotype. The data fully supported the in vivo test results.Conclusion:(1) This is the first time to study the association between 4 SNPs in the exon region of TLR2 gene, a microsatellite polymorphism in the intron 2 regions of TLR2 gene in Han Chinese population. Different with foreign groups, there were no significant genetic association between 4 SNPs and susceptibility to TB in Han Chinese population. However, significant genetic association between the microsatellite polymorphism in the intron 2 regions of TLR2 gene and TB was observed. (2) This is the first time to study the association between 2 SNPs in the exon region of TLR4 gene in Han Chinese population. Different with foreign groups, there were no significant genetic association between 2 SNPs and susceptibility to TB in Han Chinese population. (3) This is the first time to study the association between 6 SNPs in the promoter region of LY96 gene worldwide. Our results suggest that polymorphisms in the LY96 promoter region are not associated with TB, and may not play a major role in susceptibility to TB. (4) Our results suggest that the CD14 G(-1145)A polymorphism might be a new risk factor for TB. This SNP has strong linkage with CD14 C-159T, and the haplotype constructed by these 2 SNPs has extremly significant association with susceptibility to TB (P< 0.0001). Further in vivo (clinical samples) and the in vitro test (gene transfer) results showed that, CD14 Gene SNP loci haplotypes influenced promoter strength, the expression of CD 14 levels of TB patients, and ultimately the TB susceptibility.更多还原