【摘要】：目的:在新疆地區(qū)獻血員中開展大規(guī)模的流行病學橫斷面調查,掌握HHV-8在該人群中的感染率和危險因素,為評價本地血源質量、制定有針對性的防控措施提供參考數(shù)據(jù)。進行HHV-8分子病毒學研究,明確基因型和病毒載量特征并建立與KS臨床表型的相關性,為指導臨床治療提供科學依據(jù)。初步建立本地KS獨有的基因差異表達譜并進行功能分析。方法:研究對象分為三類:1)2006年8月至2007年5月收集進入自治區(qū)血液中心獻血并符合納入標準的標本4461例;2)1980～2007年本院收集的51例KS組織標本;3)KS病灶和正常皮膚“配對標本”共2對。應用本課題國內首建的HHV-8復合抗原ELISA法篩檢獻血員人群,統(tǒng)計感染率并應用多元Logistic回歸模型預測感染相關危險因素。23例KS組織標本的HHV-8 K1基因經(jīng)PCR擴增和雙向測序后,應用MEGA 3.0和phylip 23.63軟件對比GENBANK中同源性高的毒株序列,繪制系統(tǒng)發(fā)生樹以確定其型別、來源等信息。構建HHV-8 ORF26和內參基因β-actin的重組T載體,應用優(yōu)化的實時熒光定量PCR法檢測HHV-8病毒載量。建立上述指標與KS臨床表型的相關性。應用表達芯片檢測2對癌與癌旁標本的差異表達基因,為探索新型的診斷或治療靶點提供資料。結果:HHV-8復合抗原ELISA法其敏感性和特異性分別為81.8%和97.9%,與整病毒免疫熒光法檢出率符合度為90.9%。本地獻血者中HHV-8平均感染率可達20.4%。民族是最終進入方程的唯一危險因素(p,0.000;OR,1.8;95% CI,1.1-4.2)。HHV-8感染和高效價抗體在民族間的分布差異獨立存在。本地HHV-8 K1基因型以A、C為主,且A5、C6和C7為國內首次分離。本地毒株呈現(xiàn)成簇聚集的特征,參考序列主要來自非洲、俄羅斯和中東。C型主要在病程較短的KS中占優(yōu)勢(p,0.046)。HHV-8 ORF26和β-actin起始模板濃度與Ct值之間呈良好的線性關系,相關系數(shù)r2=0.996,且重復性較好。不同個體的病毒拷貝數(shù)可相差107個數(shù)量級。AIDS-KS患者其HHV-8病毒載量顯著低于其它兩型KS(p,0.025),與抗體效價的分布一致。2對標本共同的表達差異基因為117條,主要與HIV感染、增生、皮膚腫瘤、黑色素瘤、炎癥等影響HHV-8感染或KS致瘤過程的已知因素有關。結論:HHV-8復合抗原ELISA法是開展大規(guī)模流行病學研究的有力工具。新疆獻血員中HHV-8感染率遠高于其他省市同類人群,被再次印證屬HHV-8流行區(qū),應開展獻血前抗HHV-8抗體的篩查。民族背景是導致HHV-8感染和高效價抗體產(chǎn)生的最關鍵因素。本地HHV-8基因型的類別和分布與少數(shù)民族的起源和進化密切相關,且不同型別的致病機制可能不同,具備后期開展遺傳易感性研究的基礎。成功建立高靈敏性且特異性好的HHV-8實時熒光定量PCR法,可用于確診臨床感染或可疑病例。本地AIDS-KS中病毒載量的高低不能用于預測腫瘤進展,應區(qū)別分析該指標在不同類型KS中的作用。表達譜分析顯示內質網(wǎng)壓力信號通路可能在KS致瘤中起重要作用。
[Abstract]:Objective: to carry out a large-scale epidemiological cross-section survey among blood donors in Xinjiang to master the infection rate and risk factors of HHV-8 in this population, so as to provide reference data for evaluating the quality of local blood sources and formulating targeted prevention and control measures. The molecular virological study of HHV-8 was carried out to clarify the genotypic and viral load characteristics and to establish the correlation with the clinical phenotype of KS, so as to provide scientific basis for guiding clinical treatment. The differential expression profile of genes unique to local KS was established and functional analysis was carried out. Methods: the subjects were divided into three categories: 1) from August 2006 to May 2007, 4461 blood samples were collected from the blood center of the autonomous region and met the inclusion criteria, 2) 51 KS tissue specimens were collected from 1980 to 2007, and 51 samples were collected from our hospital from 1980 to 2007. 3) there were 2 pairs of KS lesions and matched specimens of normal skin. The HHV-8 complex antigen ELISA method, which was first established in China, was used to screen the blood donors. The infection rate was counted and the risk factors related to infection were predicted by multivariate Logistic regression model. The HHV-8 K1 gene in 23 KS tissue specimens was amplified by PCR and sequenced bidirectionally. MEGA 3.0 and phylip 23.63 software were used to compare the sequences of strains with high homology in GENBANK, and the phylogenetic tree was drawn to determine the type and source of the strains. The recombinant T vector of HHV-8 ORF26 and internal reference gene 尾-actin was constructed and the load of HHV-8 virus was detected by optimized real-time fluorescence quantitative PCR. To establish the correlation between the above indexes and the clinical phenotype of KS. Two pairs of differentially expressed genes between cancer and paracancerous specimens were detected by expression chip, which provided data for exploring new diagnostic or therapeutic targets. Results: the sensitivity and specificity of HHV-8 complex antigen ELISA method were 81.8% and 97.9%, respectively, and the coincidence rate with the detection rate of whole virus immunofluorescence was 90.9%. The average infection rate of HHV-8 in local blood donors was 20.4%. Nationality is the only risk factor for entering the equation (p, 0.000 OR, 1.8% CI,1.1-4.2). There are independent differences in the distribution of HHV-8 infection and high valence antibodies among ethnic groups. The local HHV-8 K1 genotype was mainly A, C, and A5, C 6 and C 7 were isolated for the first time in China. The local strains showed the characteristics of cluster aggregation, and the reference sequences were mainly from Africa, Russia and the Middle East. C was mainly dominant in KS with a short course of disease (p, 0.046). There was a good linear relationship between the initial template concentration of HHV-8 ORF26 and 尾-actin and KT value, and the correlation coefficient R2 was 0.996, and the reproducibility was good. The viral copy number of different individuals varied by 107 orders of magnitude. The HHV-8 viral load of AIDS-KS patients was significantly lower than that of the other two types of KS, which was consistent with the distribution of antibody titer. 2 the common expression differentially expressed genes were 117, It is mainly related to HIV infection, proliferation, skin tumor, melanoma, inflammation and other known factors affecting the tumorigenic process of HHV-8 infection or KS. Conclusion: HHV-8 complex antigen Elisa is a powerful tool for large-scale epidemiological research. The infection rate of HHV-8 in Xinjiang blood donors is much higher than that in other provinces and cities. It has been confirmed that it belongs to HHV-8 epidemic area again. The screening of anti-HHV-8 antibody before blood donation should be carried out. Ethnic background is the most key factor leading to HHV-8 infection and high valence antibody production. The types and distribution of local HHV-8 genotypes are closely related to the origin and evolution of ethnic minorities, and the pathogenic mechanisms of different types may be different, which has the basis of genetic susceptibility research in the later stage. A highly sensitive and specific HHV-8 real-time fluorescence quantitative PCR method was successfully established, which can be used to diagnose clinical infection or suspicious cases. The level of viral load in local AIDS-KS could not be used to predict the progression of tumor. The role of the index in different types of KS should be analyzed differentially. Expression profile analysis showed that endoplasmic reticulum pressure signaling pathway may play an important role in the tumorigenicity of KS.
【學位授予單位】：新疆醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2009
【分類號】：R181.3

【引證文獻】

相關期刊論文 前1條

1 冷弘;王媛媛;燕備戰(zhàn);方春霞;馬晶;臧文巧;李敏;趙國強;;洛陽地區(qū)無償獻血人群中皰疹病毒8型的檢測[J];廣東醫(yī)學;2012年20期

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本文編號：2474692