【作者】 邓燕；

【导师】 秦雪；

【作者基本信息】 广西医科大学 ， 临床检验诊断学， 2016， 博士

【摘要】 第一部分糖蛋白CD44基因多态性与肝癌遗传易感性研究目的:CD44是一种分布极其广泛,具有高度异质性的单链跨膜糖蛋白粘附分子,在肿瘤细胞的增殖、分化、侵袭、浸润以及转移中发挥着重要的作用。本研究通过检测CD44基因rs8193、rs10836347和rs13347三个位点的基因型及等位基因频率分布情况,探讨CD44基因rs8193、rs10836347和rs13347三个位点的基因多态性与肝癌(HCC)患病风险的相关性。方法:本研究共纳入204例肝癌患者和210例对照者,采用聚合酶链反应-限制性片段长度多态性技术及DNA直接测序法,检测CD44基因rs8193、rs10836347和rs13347三个位点的基因多态性。采用拟和优度χ2检验分析各SNP位点在肝癌组和对照组中基因型频率的分布是否符合哈-温平衡(Hardy-Weinberg equilibrium,HWE)。采用Binary logstic回归通过校正性别、年龄、吸烟和饮酒四个影响因素计算比值比(Odds ratios,OR)和95%可信区间(Confidence Intervals,CI),对基因多态性和肝癌的患病风险进行关联分析。结果:1.CD44基因三个SNP基因型频率在肝癌组和对照组中的分布符合哈-温平衡,P值均大于0.05,说明选择的样本具有群体代表性。2.本研究发现rs8193位点共有CC、CT和TT三种基因型。以CC基因型为参考基因型,未发现CT和TT基因型与肝癌的患病风险有相关性(P值均大于0.05)。以C等位基因为参考基因,T基因型与肝癌的患病风险无相关性(P>0.05)。无论是在显性模型还是隐性模型分析时,均未发现有基因型与肝癌的患病风险有相关性(P值均大于0.05)。3.本研究发现rs10836347位点共有CC、CT两种基因型。以CC基因型为参考基因,未发现CT基因型与肝癌的患病风险有相关性(P=0.253)。以C等位基因为参考基因,T基因型与肝癌的患病风险无相关性(P=0.271)。4.本研究发现rs13347位点共有CC、CT和TT三种基因型。以CC基因型为参考基因,携带CT基因型的个体可以增加肝癌的患病风险至1.626倍(95%CI:1.057-2.500,P=0.027)。携带TT基因型的个体可以增加肝癌的患病风险至1.965倍(95%CI:1.043-3.702,P=0.037)。以C等位基因为参考基因,T基因型可以增加肝癌的患病风险至1.461倍(95%CI:1.091-1.956,P=0.011)。在显性模型分析中,CT+TT与CC基因型相比,可以增加肝癌的患病风险至1.697倍(95%CI:1.130-2.549,P=0.011)。在分析隐性模型时,未发现其与肝癌的患病风险有相关性(P=0.549)。5.对性别、年龄、吸烟和饮酒等危险因素进行分层分析时发现,女性人群中,rs13347位点的CT基因型可以增加肝癌的患病风险至2.675倍(95%CI:1.004-7.132,P=0.049),TT基因型可以增加肝癌的患病风险至5.922倍(95%CI:1.083-33.164,P=0.040);在年龄≥50岁组中,rs13347位点的CT基因型可以显著增加肝癌的患病风险至2.157倍(95%CI:1.113-4.181,P=0.023),TT基因型显著增加肝癌的患病风险至3.405倍(95%CI:1.263-9.178,P=0.015);在饮酒人群中,rs10836347位点的CT基因型显著增加肝癌的患病风险至4.552倍(95%CI:1.205-17.192,P=0.025),rs13347位点的CT基因型与肝癌的患病风险无相关性(P=0.179)。TT基因型显著增加肝癌的患病风险至4.549倍(95%CI:1.244-16.635,P=0.022)。6.在进行单倍型分析时发现,TTT单倍型显著增加了肝癌的患病风险(OR=207.16,P=0.014),表明TTT单倍型可能是肝癌的一个危险因素。结论:CD44基因rs8193、rs10836347位点的基因多态性与肝癌的患病风险无相关性。Rs13347位点的T等位基因的基因突变可增加肝癌的患病风险。第二部分肝癌血清糖蛋白聚糖谱 目的:糖基化修饰是蛋白质翻译后最常见的修饰之一。同一蛋白质由于糖链结构的差异可能会导致功能的差异。男性肝癌的发病率明显高于女性,而女性的预后生存率却明显高于男性。本部分研究通过凝集素芯片技术对男性和女性在肝癌发生过程中的血清糖蛋白谱进行检测,旨在研究其糖蛋白糖谱特征性变化,筛选出男女肝癌差异性的糖谱标志,为男女肝癌发病差异研究提供新思路及实验基础。方法:实验主要包含以下步骤:(1)血清低丰度蛋白的收集:等体积混合4组(男性对照组、男性肝癌组、女性对照组和女性肝癌组)患者血清,使用高丰度蛋白去除柱收集血清低丰度蛋白;(2)将收集的低丰度蛋白经除盐、超滤、测定浓度后处理为待标记缓冲液;(3)待测低丰度蛋白CY5标记;(4)凝集素芯片的点制及芯片的杂交;(5)芯片扫描、数据提取;(6)数据统计采用SPSS 13.0,符合正态分布且方差齐性的数据采用两独立样本的均数t检验,方差不齐者采用非参数秩和检验。(7)凝集素印迹。结果:在男性肝癌组和对照组中共筛选出12种差异的凝集素亲和信号,它们是AAL、Con A、DSL、ECL、LCA、MNA-G、MNA-M、PHAE、PSA、SNA,EBL、SNA-I、WFA,WFL。提示在男性肝癌的发生过程中血清糖蛋白聚糖谱中末端核心岩藻糖、双天线结构、α1,6连接的岩藻糖半乳糖胺、半乳糖、甘露糖残基、平分型Glc NAc等糖链结构增加,β-1,4半乳糖-N-乙酰葡糖胺和Gal NAcα/β-1,3/6Gal结构减少。在女性肝癌组和对照组中共筛选出13种差异的凝集素亲和信号,它们是AAL、CALSEPA、Con A、DSL、LCA、LTL、MNA-M、PHA-E、RCA I,RCA120、SNA,EBL、SNA-I、WFA,WFL、WGA,提示在女性肝癌的发生过程中血清糖蛋白聚糖谱中末端核心岩藻糖、双天线甘露糖结构、α1,6连接的岩藻糖半乳糖胺、平分型Glc NAc等糖链结构增加,高甘露糖、双天线结构和Gal NAcα/β-1,3/6Gal结构减少。在男性肝癌组和女性肝癌组中共筛选出7种差异的凝集素亲和信号,提示男性肝癌患者血清中含有较多的双天线、N-乙酰基-D-半乳糖胺、半乳糖、甘露糖残基、唾液酸等结构,而平分型结构和Gal NAcα/β-1,3/6Gal结构较女性肝癌患者减少。结论:不同性别肝癌患者血清蛋白糖谱存在差异,男性肝癌的高发病率及高病死率可能和糖链结构的改变有关。第三部分血清DKK1糖蛋白对肝癌诊断价值交META分析目的:系统评价血清DKK1对肝癌的诊断价值。方法:计算机检索外文数据库:Pub Med、EMBASE、Science Direct、Elsevier、Springer、EBSCO、Cochran e library。中文数据库:中国期刊网全文数据库(CNKI)、万方数据库(WF)、中国生物医学文献数据库(CMCC)和中文科技期刊全文数据库(VIP),检索时间截止至2016年1月。按照改来的文献进行严格的筛选,提取所需要的纳入文献的基本资料信息。使用Meta-Di Sc软件进行统计分析,根据异质性特点选择相应的效应模型进行计算合并的敏感度、特异度、阳性似然比、阴性似然比和诊断比值比(DOR),绘制汇总受试者工作曲线(SROC),并计算曲线下面积(AUC)。结果:共检索到452篇相关文献,最终纳入11篇文献,提供了13个DKK1诊断肝癌的研究结果。Meta分析数据显示DKK1诊断肝癌的合并敏感度为72%,合并特异度为88%,DOR为25.95,AUC为0.872。其中的6篇文献,提供8个DKK1联合AFP诊断肝癌的研究结果,汇总DKK1联合AFP诊断肝癌的合并敏感度为81%,合并特异度为85%,DKK1联合AFP检测的DOR为31.28,AUC为0.913。结论:血清DKK1在肝癌的诊断中具有较高的敏感度。采用DKK1联合AFP检测,可显著提高两者对肝癌的诊断效能。更多还原

【Abstract】 CHAPTER ⅠAssociation between Glycoprotein CD44 Gene Polymorphism and Risk of Susceptibility to Hepatocellular CarcinomaObjective: CD44 is a widely distributed and with a high degree of heterogeneity single-chain transmembrane glycoprotein adhesion molecules, playing an important role in tumor cell proliferation, differentiation, invasion and metastasis. The study detected genotype and allele frequencies distribution of CD44 gene rs8193, rs10836347 and rs13347 to explore the association between CD44 gene rs8193, rs10836347 and rs13347 polymorphism and the risk of HCC.Method: A total of 204 HCC and 210 controls were included in this study, restriction fragment length polymorphism and DNA sequencing were used to detect the polymorphism of CD44 gene rs8193, rs10836347 and rs13347. The χ2 test was used to analyze whether the distribution of the SNP genotype frequencies in HCC group and the healthy controls group consistent with Hardy-Weinberg equilibrium(HWE). Binary logstic regression adjusted for sex, age, smoking and drinking four factors were used to calculate the odds ratio(OR) and 95% confidence intervals(CI) between the genetic polymorphisms and the risk of HCC.Results:1. The genotype frequencies in the HCC group and controls group of the three SNPs of CD44 gene were found to be in HWE(all P>0.05), indicating that the sample selected had group representation.2. The study found that the rs8193 had CC, CT and TT three genotypes. Using the CC genotype as a reference genotype, the CT and TT genotype were not associated with HCC risk(all P>0.05). Using the C allele as a reference genotype, T allele was not associated with HCC risk(P>0.05). Both in a dominant model and recessive model analysis, not genotype was found to be associated with the risk of HCC(all P>0.05).3. The study found that rs10836347 had CC and CT genotypes. Using the CC genotype as a reference genotype, TT genotype were not associated with HCC risk(P=0.253). Using the C allele as a reference genotype, T allele was not associated with HCC risk(P=0.271).4. The study found that rs13347 had CC, CT and TT three genotypes. Using the CC genotype as a reference genotype, patients with CT genotype showed 1.626-fold(95% CI: 1.057-2.500, P=0.027) higher risks of HCC. Patients carrying TT genotype showed 1.965-fold(95% CI: 1.043-3.702, P=0.037) higher risks of HCC. Using C allele as reference genotype, T allele showed 1.461-fold(95% CI: 1.091-1.956, P=0.011) higher risks of HCC. In the dominant model analysis, CT+TT compared to CC genotype showed 1.697-fold(95% CI: 1.13-2.549, P=0.011) higher risks of HCC. However, in the recessive model analysis, it has found no correlation between the polymorphisms and the risk of HCC(P=0.549).5. Stratified analysis according to the risk factors of age, sex, smoking and alcohol consumption, in women subgroup, patients with CD44 rs13347 CT genotype exhibited 2.675-fold(95% CI: 1.004-7.132, P=0.049) and TT genotype exhibited 5.922-fold(95% CI: 1.083-33.164, P=0.040) higher risks of HCC than those of patients with the CC genotype. In age≥50 years subgroup, patients with CD44 rs13347 CT genotype exhibited 2.157 fold(95% CI: 1.113-4.181, P=0.023) and TT exhibited 3.405-fold(95% CI: 1.263-9.178, P=0.015) higher risks of HCC than those of patients with the CC genotype. In drinking subgroup, for the rs10836347 site, CT genotype significantly increased the risk of HCC to 4.552-fold(95% CI: 1.205-17.192, P=0.025). For the rs13347 site, using the CC genotype as reference, CT genotype was no associated with the risk of HCC(P=0.179), patients with CD44 rs13347 TT genotype significantly increased the risk of HCC to 4.549-fold(95% CI: 1.244-16.635, P=0.022);6. The haplotype distributions of CD44 rs8193, rs10836347 and rs13347 were evaluated. The TTT haplotype significantly increased the risk of HCC(OR=207.16, P=0.014), indicating that TTT haplotype maybe a risk for HCC.Conclusion: The polymorphism of CD44 gene rs8193, rs10836347 was not associated with the risk of HCC. T mutant allele of rs13347 may increase the risk of HCC.CHAPTER Ⅱ Comparative serum glycoprotein of Hepatocellular Carcinoma with different sexObjective: Glycosylation is one of the most common protein posttranslational modification. The same protein with different sugar chain structure may present different function. The incidence of HCC in male is higher than that in female, while survival in female is better than that in male. In this part, we use lectin microarray technology to detect the serum glycoprotein spectrum between male and female in the process of HCC. The aim of this study is to find its characteristic changes of glycoprotein spectrum and the different sugar spectrum signs between male and female in the process of HCC, providing new ideas and experimental basis for the study of difference in the incidence of HCC between male and female.Method: Experiment process was as followed(1) low-abundance serum proteins were collected: Mixing the equal volume serum of four groups(control group of male, HCC group of male, control group of female and HCC group of female), low-abundance protein was collected using the high-abundance protein removal kits.(2) After desalting and ultrafiltration, detect the concentration of the low abundance proteins.(3) The target proteins were labeled with Cy5 using Lightning-Link kits.(4) Prepare the lectin microarray and hybridization.(5)Lectin microarray was scaned and data was collected.(6) Data was analysis by the SPSS 13.0, two independent samples t test or non-parametric test was used.(7) Lectin immuno-bloting.Results: Between the control group and HCC group of male, 12 different lectin affinity signals were found, including AAL, Con A, DSL, ECL, LCA, MNA-G, MNA-M, PHA-E, PSA, SNA,EBL, SNA-I, WFA,WFL. In the process of HCC, terminal α Fuc, α-man biantennary, Fucα1-6Glc NAc, Gal, oligomannosyl residues, bisecting Glc NAc and biantennary N-glycans increased, while galactosyl(β-1,4) N-acetylglucosamine and Gal NAcα/β-1,3/6Gal structure decreased. Between the control group and HCC group of female, 13 different lectin affinity signals were found, including AAL, CALSEPA, Con A, DSL, LCA, LTL, MNA-M, PHA-E, RCA I, RCA120, SNA,EBL, SNA-I, WFA, WFL, WGA. In the process of HCC, terminal α Fuc, Fucα1-6Glc NAc, bisecting Glc NAc increased, while high mannose, biantennary and Gal NAcα/β-1,3/6Gal decreased. Between the HCC group of male and HCC group of female, 7 differenct lectin affinity signals were found, indicated that in the HCC group of male, biantennary, N-Acetyl-D-Galactosamine, Gal, oligomannosyl residues increased, while bisecting and Gal NAcα/β-1,3/6Gal decreased compare with female.Conclution: Different sex of HCC patients had different serum protein, sugar chain structure changes associated with the higher incidence and higher mortality in male with HCCCHAPTER Ⅲ Diagnostic Value of DKK1 in Hepatocellular Carcinoma: A Meta-analysisObjective: To evaluate the diagnostic value of serum DKK1 in patients with hepatocellular carcinoma.Method: Pub Med, EMBASE, Science Direct, Elsevier, Springer, EBSCO, Cochrane library, WF, VIP, CMCC and CNKI were searched for relevant reports publish before January 2016. Eligibility studies were identified and determined according to the criteria of inclusion and exclusion. The characteristics of the included articles were appraised and extracted. Meta-Di Sc 1.4 was used to statistic the data. Three methods were used to exam the heterogeneity between the included articles. According to the heterogeneity results, the proper effect model was selected to calculate pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio and diagnostic odds ratio(DOR). Then the summary receiver operating characteristic(SROC) curve was performed and the area under the curve(AUC) was calculated.Results: A total of 452 articles were searched and 11 articles were included in this meta-analysis, providing 13 results about diagnosis value of DKK1 in HCC. The meta-analysis showed that when using DKK1 diagnosis HCC the pooled sensitivity was 72%, the pool specificity was 88%, the DOR was 25.95 and the AUC was 0.872. Using DKK1 combine AFP diagnosis HCC,the pooled sensitivity was 81%, the pool specificity was 85%, the DOR was 31.28 and the AUC was 0.913.Conclusion: The sensitivity of serum DKK1 in diagnosing HCC is high. Using DKK1 combine AFP to diagnosis HCC can significantly improve the diagnostic performance.更多还原