【作者】 刘征涛；

【导师】 彭涛；

【作者基本信息】 广西医科大学 ， 外科学， 2014， 博士

【摘要】 第一章第一部分影响广西防城港地区男性人群谷丙转氨酶水平的流行病学因素及其相互作用分析目的：在普通人群中，谷丙转氨酶(alanine aminotransferase,ALT)升高通常提示显著的或者潜在的肝脏疾病，甚至会累计对全身健康造成威胁。因此，基于广西防城港地区男性人群健康调查（The Fangchenggang AreaMale Healthy and Examination Survey, FAMHES）的结果，我们评估了影响该地区成年男性人群血清中ALT水平的流行病学因素以及他们之间的相互作用。方法：在4304例受试者中，我们总共纳入了2119例在2009年9月至2009年12月完成了完整的体格检查，实验室检查，肝脏超声检查以及调查问卷的符合纳入标准的受试者数据。结果：在纳入人群中，非酒精性脂肪肝（non-alcoholic fatty liverdisease, NAFLD）和代谢综合征（metabolic syndrome, MetS）的患病率显著相关于ALT水平的升高（P <0.05）。NAFLD和MetS的患病率密切相关（r=0.991，P=0.009）不正常的MetS各组分比例随着ALT水平的升高成比例的明显增加（P <0.01）。在随后进行的多元线性回归分析中，腹部肥胖指标体重指数（body mass index, BMI）和高脂血症和ALT水平显著相关（P <0.01）。在乙肝表面抗原（hepatitis B virus surface antigen, HBsAg）和MetS的交互作用研究中，我们并未发现其对于ALT水平有明显交互作用。HBsAg和MetS之间对ALT影响的协同作用系数(synergy index,SI)为0.74，95%可信区间（confidence interval, CI）:0.71-0.80。结论：在防城港地区男性人群中，NAFLD和MetS的患病率显著相关于ALT水平。腹部肥胖和高脂血症是导致ALT水平升高的独立危险因素。这一发现提示在这一地区男性人群中，有必要在检测ALT水平的过程中校正这些可能导致ALT水平升高的危险因素。第一章第二部分针对前瞻性研究的循证医学研究发现谷丙转氨酶水平升高显著相关于代谢综合征的发生目的：代谢综合征（metabolic syndrome, MetS）的发病率在世界范围内显著升高且与谷丙转氨酶（alanine aminotransferase,ALT）活动水平紧密联系。但是，ALT活动水平对MetS发生率的影响在已发表文献中的报道并不一致。因此，我们针对前瞻性队列研究通过荟萃分析的方法估算了ALT活动水平升高和MetS发生率之间的关系。方法：收集所有已发表在Pubmed, Embase,和ISI数据库中的研究ALT活动水平和MetS发生率之间关系的前瞻性队列研究（文献发表在2014年1月10日之前）。包括研究的国别，性别组成，纳入人群的年龄，随访时间，MetS定义，校正因素，相对危险度（relative risk,RR）等具体指标直接或间接从最终纳入文献中提取或计算。我们将分别估计二分类变量的合并RR值及其95%可信区间（confidence interval, CI）（RR定义为高ALT水平组人群MetS发生率/低ALT水平组人群MetS发生率）和连续变量的合并RR值及95%CI(RR定义为ALT水平每升高5单位每升[unit perliter,U/l]人群中MetS发生率变化的比值)。如果组间异质性不明显（P值>0.05以及I2<50%）,在计算合并RR值的过程中则采用固定效应模型；如果组间有显著的差异性(P值≤0.05或I2≥50%)，则采用随机效应模型。结果：总共七篇前瞻性队列研究，包含了31545例受试者和2873例在随访期间发生MetS的病例被纳入本次研究。在比较高值ALT水平和低值ALT水平人群MetS发生率的差异中，合并估计的RR值为1.81(95%CI:1.49-2.14)。而在剂量-反应计算估计ALT水平每升高5U/l导致人群MetS发生率的变化中，合并估计的RR值为1.13（95%CI:1.11-1.16）。亚组分析发现性别差异可能是整个研究中异质性的主要来源。在以性别为标准的亚组分析中，亚组之间剂量-反应(每5U/l)估算的合并RR值比较P=0.007。女性有比男性更高的剂量反应RR（1.38,95%CI:1.20-1.55）。进一步的，敏感度分析证实了结果的可靠性。本次荟萃分析并未发现显著的发表偏倚。结论：目前基于前瞻性研究的循证医学证据支持ALT水平升高增加MetS发生风险的观点。且这种关联在女性人群中表现得更加紧密，一致性更好。这一关系有待更多的研究证实。同时ALT水平升高对MetS发生的潜在机制研究也有待进一步探索。第二章全基因组关联研究发现新的影响广西防城港地区男性人群中血清谷丙转氨酶水平的单核苷酸多态性位点及其生物信息学意义分析目的：谷丙转氨酶（alanine aminotransferase, ALT）是评估肝脏疾病和全身健康的重要的生物标志物。ALT水平受到环境因素和遗传因素的共同影响。因此我们实施了一项全基因组关联研究（genome-wideassociation studies，GWAS），结合前期防城港地区男性健康调查（Fangchenggang Area Male Healthy and Examination Survey,FAMHES）结果，找出影响防城港地区男性健康人群的ALT水平的常见遗传学变异及其与环境因素的交互作用。方法：采集研究人群的外周静脉血检测血清提取脱氧核糖核酸（deoxyribonucleic acid, DNA）样本。在前期FAMHES纳入的人群中，总共1999例健康男性的外周血样进行全基因组单核苷酸多态性（SingleNucleotide Polymorphisms,SNPs）检测，平台为Illumina Omni One。在全基因组SNPs分型的基础之上，采用校正了年龄，体重指数（body massindex, BMI）,甘油三酯（triglycerides,TG）变量的多元线性回归模型，寻找可能影响该地区男性人群ALT水平的常见遗传变异标志。进一步地，全基因组关联分析在以乙肝表面抗原（hepatitis B surface antigen，HBsAg）为分类标准的亚组中进行比较。全基因组关联分析统计学检验显著性水平的检验标准为P<1*10‐5,在乙肝表面抗原（hepatitis B surface antigen，HBsAg）阳性人群中的统计学检验显著性水平的检验标准为P<0.05。基因分型的质量控制标准：满足哈‐温平衡（Hardy‐Weinberg Equilibrium，HWE检验P＞0.001）;最小等位基因频率>0.01；基因分型成功率(call rate)＞0.95。采用非参数检验（Mann‐Whitney Test）的方法比较以HBsAg和MetS分类的亚组中不同SNP基因型之间的ALT值。最后，采用交互作用的流行病学模型，结合潜在遗传变异和常见的影响ALT水平的表型因素（包括代谢综合征[metabolic syndrome, MetS]和乙肝病毒[hepatitis B virus, HBV]感染）,评估遗传变异和表型因素之间的交互作用。结果：本次GWAS总共发现位于5个基因区域的7个SNPs经全基因组关联分析统计学检验后，显著性水平P<1*10-5,包括：位于SLC35F3基因1q42.2上的rs10157061(P=7.46*10-7),rs10752781(P=1.29*10-6),rs1878544(P=2.16*10-6)；位于CUX2基因12q24.12上的rs4766453(P=1.11*10-6)；位于SHANK1基因19q13.3上的rs4801847(P=3.26*10-6)；位于WWOX基因上16q23.3上的rs2010020(P=7.73*10-6)；位于CYP1A2基因15q24.1的rs3743484(P=8.20*10-6)。其中位于2个基因区域的4个SNPs(位于SLC35F3基因1q42.2上的rs10157061,rs10752781,rs1878544；位于CUX2基因12q24.12上的rs4766453)经全基因组关联分析统计学检验后显著性水平P<3*10-6。在HBsAg阳性人群中，位于SLC35F3基因1q42.2上的rs10157061,rs10752781,rs1878544；位于WWOX基因上16q23.3上的rs2010020；位于CYP1A2基因15q24.1的rs3743484同时达到在HBsAg阳性人群中的统计学效力（P<0.05）。在结合SNP基因型和MetS，HBsAg表型分组的的亚组分析中，各亚组间人群ALT值仍然SNP基因型的变化而有所差异(P<0.05)。我们发现潜在意义较大(P<3*10-6)的候选SNPs（包括位于SLC35F3基因1q42.2上的rs10157061, rs10752781, rs1878544；位于CUX2基因12q24.12上的rs4766453）和阳性HBsAg之间对ALT水平的影响的协同效应系数[synergy index, SI]及其95%可行区间（confidenceinterval，CI）均大于1。结论：因此，一项针对防城港地区成年男性人群的GWAS发现了新的影响人群血清中ALT水平的常见遗传变异标记。其中SLC35F3和CUX2可能是影响该地区成年男性血清ALT水平的易感基因。但是这种效应可能因为人群的肝炎病毒感染背景不同而有所不同。SNPs对人群ALT水平的影响可能会在HBV感染的背景下扩大。候选SNPs及其所在基因区域为个体化医学诊断和治疗提供了潜在的遗传学标记。同时为下一步的验证ALT水平的遗传学机制提供了有意义的靶点。潜在SNPs及其所在基因区域对ALT水平的影响机制有待进一步研究证实。第三章第一部分全基因组关联研究发现的新的影响防城港地区男性人群血清谷丙转氨酶水平的单核苷酸多态性位点在永生化肝细胞株中的验证目的：基于前期针对防城港地区男性健康调查（Fangchenggang AreaMale Healthy and Examination Survey,FAMHES）人群全基因组关联研究（genome-wide association study,GWAS）的结果，我们发现了新的影响防城港地区健康男性血清谷丙转氨酶（alanine aminotransferase, ALT）水平的单核苷酸多态性位点（single nucleotide polymorphisms,SNPs）。但是GWAS发现的SNPs与表型特征之间可能存在假关联。因此，我们选取永生化肝细胞株为研究对象其中候选SNPs与其上清液ALT水平之间的关联关系。同时对SNPs坐落的基因，GPT基因（肝脏ALT编码基因）及其编码蛋白表达水平进行检测，在细胞株层面探讨候选SNPs坐落基因及其编码蛋白的表达水平与GPT基因及编码蛋白表达水平及各细胞株表型（即各细胞株上清液ALT水平）之间存在的关系。方法：选取BEL-7402，BEL-7404，HL-7702，SMMC-7721，Hep-G2，MHCC-97H,MHCC-97L共7株永生化肝细胞株作为研究对象，通过细胞培养繁殖，消化贴壁细胞后提取细胞用做以下实验：1.提取细胞脱氧核糖核酸（deoxyribonucleic acid，DNA），针对目的SNPs设计普通聚合酶链式反应（polymerase chain reaction,PCR）引物,通过PCR反应获得扩增产物。产物以sanger双向测序法获得目的SNPs（左右约50个碱基对[base-pairs,BP]）片段序列,从而获得目的SNPs的基因型结果；2.提取细胞株上清液，采用酶联免疫吸附测定（enzyme-linked immuno sorbentassay，elisa）的方法获取各细胞株上清液ALT值；3.针对候选SNPs坐落基因，设计特异性实时荧光定量PCR（real-time PCR）引物,检测不同基因型的肝细胞株间目的基因和ALT基因的表达情况；4.提取细胞的总蛋白，设计针对目的基因编码蛋白的特异性抗体，采用蛋白印迹（western-blot）的方法观察特异蛋白的表达情况；4.将消化细胞接种到无菌玻璃片上，采用特异性抗体免疫组化（Immunohistochemistry，IHC）的方法观察各特异蛋白在不同细胞株中的表达部位以及表达量变化。细胞实验均采取三次重复，计量指标采用平均值±标准差（mean±standard deviation [SD]）表示。各细胞株间上清液ALT值，目的基因表达量及目的蛋白表达量之间比较采用非参数检验Mann-Whitney U检验的方法进行。表达量相关性比较采用pearson相关系数计算。P<0.05被认为有显著统计学差异。结果：总共7个永生化肝细胞株中，在其中3个基因区域的5个SNPs上表现出多态性，所有候选SNPs在细胞株中均表现为纯合子。7个细胞株按照其携带的不同SNPs的高值等位基因数目分布的不同分为3组：其中A组细胞株MHCC-97H和MHCC-97L在位于SLC35F3基因1q42.2区域上的rs10157061,rs10752781, rs1878544；位于CUX2基因12q24.12区域上的rs4766453；位于SHANK1基因19q13.3上的rs4801847共5个SNPs上均携带高值等位基因，B组细胞株SMMC-7721在位于SHANK1基因上的rs4801847SNP上携带高值等位基因，C组细胞株（包括BEL-7402，BEL-7404，HepG2，HL-7702）在总共位于5个基因区域的7个SNPs上均携带低值等位基因。经比较，A组MHCC-97H和MHCC-97L细胞株在上清液ALT值显著高于其他各组细胞株ALT值，B组SMMC-7721细胞株与C组细胞株间上清液ALT值差异无统计学显著性。实时荧光定量PCR的结果提示A组GPT基因表达水平高于B组，C组，但SLC35F3的表达水平和GPT基因表达水平呈现显著负相关（r2=-0.672，P=0.001），CUX2的表达水平在不同基因型分组中，组间表达差异无统计学显著性（P>0.05）。实时荧光定量PCR结果得到了蛋白印迹和免疫组化结果的印证。在蛋白印迹实验中，A组SLC35F3编码蛋白表达水平显著低于B组，C组，差异有统计学显著性（P<0.05）。A组GPT编码蛋白表达水平显著高于B组，C组，差异有统计学显著性（P<0.05）。CUX2编码蛋白组间表达无统计学差异（P>0.05）。各组间SLC35F3和GPT蛋白表达水平存在明显负相关（r=-0.527， P=0.002）。免疫组化实验印证了蛋白印迹结果。结论：基于FAMHES的GWAS研究中发现的影响男性人群血清ALT水平的SNPs，我们结合体外细胞学验证得出：1.位于SLC35F3基因1q42.2上的显著影响人群血清ALT水平的候选SNPs（包括rs10157061，rs10152781,rs1878544）同时显著影响SLC35F3基因和蛋白表达水平。高值等位基因对应SLC35F3基因和编码蛋白的低表达。2. SLC35F3基因可能是影响肝细胞ALT分泌水平的易感基因。SLC35F3的基因和编码蛋白的表达水平的上调与GPT基因的低表达及细胞ALT的低水平相关。位于1q42.2上的强连锁不平衡区域的rs10157061，rs10152781,rs1878544可能通过影响SLC35F3的基因和蛋白表达水平间接地影响和调控了肝细胞ALT水平。相关机制有待进一步研究证实。第三章第二部分低浓度乙醇暴露下永生化肝细胞株MHCC-97H和BEL7402中SLC35F3基因表达研究背景和目的：基于前期细胞株验证的初步结果，我们发现SLC35F3基因可能对体外永生化肝细胞株中谷丙转氨酶（glutamic-pyruvictransamine GPT）基因表达水平，上清液谷丙转氨酶（alanineaminotransferase,ALT）水平存在影响。SLC35F3基因及其编码蛋白的低表达关联于GPT基因的高表达和ALT值的升高。但永生化肝细胞株在外界环境压力下，SLC35F3和GPT的表达情况仍不得而知。因此，我们设计了低浓度乙醇干预实验，观察体外永生化肝细胞株SLC35F3，GPT基因及其编码蛋白表达情况，及其相互关系。方法：选取永生化肝细胞株BEL-7402和MHCC-97H作为研究对象。分别以50mmol/l,100mmol/l,和200mmol/l的乙醇浓度干预永生化肝细胞株，并观察永生化肝细胞株在3小时，6小时，12小时，24小时不同时间节点的ALT分泌值，SLC35F3，GPT基因及其编码蛋白表达值选取BEL-7402，MHCC-97H两株永生化肝细胞株作为研究对象，通过细胞培养繁殖，消化吹散后均匀接种于24孔板中，贴壁后观察细胞，分别以50mmol/l,100mmol/l,和200mmol/l的乙醇浓度干预永生化肝细胞株，并观察永生化肝细胞株在3小时，6小时，12小时，24小时不同时间节点对永生化肝细胞株进行以下处理：1.提取细胞株上清液，采用酶联免疫吸附测定（enzyme-linked immuno sorbent assay，elisa）的方法获取各细胞株上清液ALT值；2.针对SLC35F3和GPT基因，设计特异性实时荧光定量PCR（real-time PCR）引物,检测不同细胞株不同时间-浓度乙醇干预水平下SLC35F3和GPT基因表达水平；3.提取细胞株在不同时间-浓度乙醇干预水平下细胞的总蛋白，设计针对SLC35F3和GPT基因编码蛋白的特异性抗体，采用蛋白印迹（western-blot，WB）的方法观察特异蛋白的表达情况；4.将不同时间-浓度乙醇干预水平下的细胞消化后，接种到无菌玻璃片上，采用特异性抗体免疫组化（Immunohistochemistry，IHC）的方法观察各SLC35F3和GPT特异蛋白在不同细胞株中的表达部位以及表达量变化。细胞实验均采取三次重复，计量指标采用平均值±标准差（mean±standard deviation [SD]）表示。组间比较统计学方法使用遵循以下原则：如数据符合正态分布，则比较选用方差分析（one-way ANOVA）;如数据不符合正态分布，则比较选用非参数检验（Mann-Whitney U test）。除非特殊说明，P<0.05被认为是有显著统计学差异。结果：我们检测了不同时间-浓度乙醇干预浓度下BEL-7402和MHCC-97H两株细胞的上清液分泌值，SLC35F3，GPT两基因表达，编码蛋白表达情况。具体结果如下：1. BEL-7402ALT达峰时间稳定在6小时，而MHCC-97H ALT达峰时间由100mmol/l乙醇干预下的12小时，缩短至200mmol/l乙醇干预下的3小时。不同浓度酒精干预下，BEL-7402的ALT峰值水平差别无统计学显著性（P>0.05），MHCC-97H的ALT峰值水平随着乙醇干预浓度的加大而升高，差别有统计学显著性（P<0.05）；2.不同时间-浓度乙醇干预下BEL-7402和MHCC-97H的SLC35F3及GPT基因表达水平存在差异。BEL-7402的SLC35F3基因和GPT基因分别在12小时和6小时达到峰值，相对稳定，且随乙醇暴露浓度的增加峰值水平变化不大,差别无统计学显著性（P>0.05）。而MHCC-97H的SLC35F3基因和GPT基因表达水平的达峰时间和峰值表达水平随着不同干预浓度的变化较大，其达峰时间从50mmol/l乙醇暴露下的24小时，提前到200mmol/l乙醇暴露下的3小时。峰值水平随着乙醇暴露浓度的升高而升高，差异有统计学显著性（P<0.05）。3.蛋白印迹半定量结果显示：BEL-7402SLC35F3和GPT基因编码蛋白表达达峰时间稳定在12小时和6小时，而MHCC-97H SLC35F3和GPT基因编码蛋白表达达峰时间随着乙醇暴露浓度的增加而提前，由200mmol/l乙醇暴露下SLC35F3和GPT编码蛋白表达水平高于相应的50mmol/l和100mmol/l乙醇干预浓度下的表达水平（P<0.05）。免疫组化结果与蛋白印迹观察结果一致。结论：在应激环境下，基础高表达SLC35F3基因的BEL-7402表现出在体外环境下表现出比低表达SLC35F3基因的MHCC-97H细胞株更强的自身稳定性和对乙醇的低度易感性，及其更小的损伤。SLC35F3基因的基础表达水平可能与乙醇干预下肝细胞自身调节及ALT代谢机制有关。在基础状态下SLC35F3基因的高表达有利于在应对乙醇刺激时，降低乙醇损伤的易感性，减轻乙醇所致的肝脏损伤。更多还原

【Abstract】 CHAPTER IPART ICombinative analysis of factors influence serum alanineaminotransferase activity in adult male population from southernChinaObjective: Abnormal alanine aminotransferase (ALT) activity isindicative of liver disease even a burden of overall health. Therefore, based onthe investigation of Fangchenggang Area Male Healthy and ExaminationSurvey (FAMHES), We assessed the factors associated with ALT activity andtheir internal relationships in a male population from southern China.Methods: Data of physical examinations, laboratory tests, hepaticultrasounds and standardized questionnaire were collected from2119malesparticipating in a population-based survey from September2009to December2009.Results: Nonalcoholic fatty liver disease (NAFLD) and metabolicsyndrome (MetS) were associated with the elevation of ALT levels (P<0.05).Prevalence of NAFLD was correlated to MetS (r=0.991, P=0.009). The levels of abnormal metabolic syndrome components increased in proportion with theALT elevation (P<0.01). Obesity and hyperlipidemia were associated with theALT levels in following multivariate regression analysis (P<0.01). There was nosynergic effect of hepatitis B virus surface antigen (HBsAg) and MetS on theALT levels (synergy index [SI]=0.74,95%confidence interval [CI]:0.71–0.80).Conclusions: NAFLD and MetS were associated with ALT levels in amale population from Fangchenggang area, southern China. Obesity andhyperlipidemia were independent MetS components contributing to elevatedALT (e-ALT). This finding might suggest necessity on justification of theseconfounding factors when detecting ALT levels among this population. CHAPTER IPART IIElevated Alanine Aminotransferase Is Strongly Associated withIncident Metabolic Syndrome: A MetaAnalysis of ProspectiveStudiesObjective: The incidence of metabolic syndrome (MetS) is rapidlyincreasing worldwide and associated with alanine aminotransferase (ALT)activity. However, the impact of ALT activity on MetS incidence is inconsistentin published literature. We therefore estimated the association between elevatedALT activity and incident MetS through a meta-analysis of prospective cohortstudies.Methods: All published prospective cohort studies on the associationbetween elevated ALT activity and incident MetS were retrieved from Pubmed,Embase, and the Institute for Scientific Information (ISI)(literatures publishedbefore January10th,2014). Nationality, gender, age of the enrolled population,follow-up duration, definition of MetS, adjusted covariates, relative risk (RR)are retrieved or calculated directly or indirectly from enrolled reference. We willevaluated the pooled results of dichotomous RR and their95%confidenceinterval(CI)(presented as the incidence of MetS in population with highestALTvalue/the incidence of MetS in population with lowest ALTvalue).Meanwhile, the continuous RR and95%CI was also evaluated (presented as theRR of MetS per5U/l of ALT increment)。If there was no significantheterogeneity (P-value>0.05and I2<50%), the fixed-effect model will be chosento estimate the overall RR and95%CI. Otherwise, the random-effect model willbe used. Results: In all, seven prospective cohort studies, with31545participantsand2873cases of incident MetS were recruited. The calculated RR was1.81(95%CI:1.49–2.14) when the incidence of MetS was compared between thehighest versus the lowest classification of ALT activities. The pooled RR was1.13(95%CI:1.11–1.16) in dose-response analysis with5U/l of ALTincrement. Subgroup analysis suggested that gender disparity might be the mainorigin of heterogeneity in overall analysis (P=0.007between RRs ofgender-specific subgroups evaluated with5U/l increments of ALT). Women hada higher dose-response risk of MetS incidence (1.38,95%CI:1.20–1.55per5U/l of ALT elevation) than men. Furthermore, sensitivity analysis confirmed thestability of results. No publication bias was found in our meta-analysisConclusion: Current evidence from prospective studies supports theassociation between ALT elevation and increasing MetS incidence. Thisassociation is closer and more consistent in female population. Further studiesare needed to confirm this association and to investigate the potentialmechanism of ALT activity on MetS occurrence. CHAPTER IIA genome-wide association study identified two novel loci onalanine aminotransferase level in a male population fromFangchenggang area and their bioinformatic analysisObsjective: Alanine aminotransferase (ALT) plays an important role inliver disease detection and health evaluation. ALT level is influenced by geneticand environmental factors. Therefore, a genome-wide association study (GWAS)was performed in2018healthy Chinese men, combining with the previousepidemiology data from the Fangchenggang Area Male Healthy andExamination Survey (FAMHES), we are trying to find out the common geneticvariation that influence the ALT level in male population from Fangchenggangarea and their interaction with environmental covariates.Methods: Venous blood of enrolled subjects were sampled to extract theDNA. A genome-wide association study (GWAS) was performed in1999healthy male subjects enrolled in FAMHES to check the genotype ofgenome-wide Single Nucleotide Polymorphisms (SNPs), platform illuminaomini one。GWAS was adjusted the body mass index (BMI), triglycerides ascovariates in multi-variate regression model. Further, GWAS was performed inthe subgroup with positive hepatitis B surface antigen (HBsAg). The statisticalsignificance in GWAS of the whole participants was P<1*10-5. The statisticalsignificance in GWAS of the positive HBsAg participants was P<0.05. We thenapplied the following QC criteria to filter SNPs: P <0.001for theHardy-Weinberg equilibrium (HWE) test, minor allele frequency(MAF)<0.01and genotype call rate <95%. The ALT level was compared between positive HBsAg or MetS subgroups classified by variousSNPs with non-parametric analysis (Mann-Whitney Test). Finally, we adoprtedthe synergic effect model to assess the interaction between genetic variation andenvironmental covariates.Results: As results,7SNPs in5genetic loci showed statisticalsignificance after GWAS on the level of P<1*10-5, including the rs10157061(P=7.46*10-7),rs10752781(P=1.29*10-6), rs1878544(P=2.16*10-6) at1q42.2ofSLC35F3gene； rs4766453(P=1.11*10-6) at12q24.12of CUX2gene；rs4801847(P=3.26*10-6)at19q13.3of SHANK1gene；rs2010020(P=7.73*10-6)at16q23.3of WWOX gene；rs3743484(P=8.20*10-6) at15q24.1of CYP1A2gene. Among them,4SNPs in2gene loci (rs10157061(P=7.46*10-7),rs10752781(P=1.29*10-6), rs1878544(P=2.16*10-6) at1q42.2ofSLC35F3gene；rs4766453(P=1.11*10-6) at12q24.12of CUX2gene)showedmore significance with ALT level (P<3*10-6).In population with positiveHbsAg, the rs10157061,rs10752781, rs1878544at1q42.2of SLC35F3gene,rs4801847at19q13.3of SHANK1gene；rs2010020at16q23.3of WWOXgene；rs3743484at15q24.1of CYP1A2gene still reached the statisticalsignificance (P<0.05). Combining with the potential SNPs and MetS or HbsAgstatus, we found the ALT level is significantly different followed with thechange of SNP genotype in different subgroups(P<0.05). We found significantsynergic effects on ALT variation between candidate SNPs(rs10157061,rs10752781, rs1878544at1q42.2of SLC35F3gene,andrs4766453at12q24.12of CUX2gene) and positive HbsAg, the synergic indexsand their95%confidence interval (CI)>1.Conclusions: In conclusion a GWAS found new genetic variation that influence the variation of ALT level in adult males from Fangchenggang area.Among them, SLC35F3and CUX2might be the susceptible gene that influencethe ALTlevel in adult male of this area. However, these effects might be distichtfor the background of HBVinfection. The influence might be amplified on thebackground of HBVinfection. The candidate SNPs and their located loci providepotential genetic biomarker for the diagnosis and treatment of individualizedmedicine. Meanwhile, it provide a significant a meaningful target for thevalication of genetic foundation of ALTlevel on the next step. And themechanism of these influence needs investigation in future. CHAPTER IIIPART IThe replication in the hepatic cell lines on the new findingsthrough genome-wide association study that significantlyinfluenced the alanine amniotransferase level in adult malepopulation from Fangchenggang areaObsjective: Based on the results of genome-wide association study(GWAS) in the previous Fangchenggang Area Male Healthy and ExaminationSurvey (FAMHES), we found new genetic variation that influence the serumalanine aminotransferase (ALT) level in adult male population fromFangchenggang area. However, false connection exsists in the SingleNucleotide Polymorphisms (SNPs) and phenotypic traits found by GWAS.Therefore, we selected the hepatic cell lines as observation to evaluate theassociation between candidate SNPs and ALT level. Meanwhile, we checked thegene that the SNPs located, the protein expression that the candidate geneencoded. We are trying to investigate the association between candidate SNPs,the expression of their located gene, encoded protein by the candidated gene andthe phenotype of various hepatic cell lines.Methods:7hepatic cell lines including BEL-7402, BEL-7404,HL-7702,SMMC-7721, Hep-G2, MHCC-97H, MHCC-97L were selected for observation.The hepatic cell lines were cultured for the following experiments:1.deoxyribonucleic acid (DNA) extraction, design the SNP-specific primer usingthe Polymerase Chain Reaction (PCR) method ti amplify the products.And thePCR products were tested by Sanger’s methods to get the genotype of potentialSNPs;2. Supernate of hepatic cell lines was extracted, the ALT level of various hepatic cell lines was tested by Enzyme Linked Immunosorbent Assay (elisa);3.design the gene-specific primer for real-time PCR(RT-PCR) to test theexpression of candidate gene in various hepatic cell lines;4. the total proteinextraction, design the protein-specific antibody to test the specific proteinexpression by using the western-blot;5. the digested cell was planted in thegerm-free disinfected glass slides, test the specific protein expression withprotein-specific antibody using the Immunological Histological Chemistrymethod.All the cell experiments was performed three times, measurement datawas expressed by mean±standard deviation (SD). The ALT level in varioushepatic cell lines, expression of potential gene was compared with thenon-parametric test (Mann-Whitney U test). The correlation between expressioncovariates was compared by using pearson index. P<0.05was considered assignificance unless special illustration.Results: In the7hepatic cell lines, polymorphisms were presented in5SNPs located in3loci, all the candidate SNPs in the hepatic cell lines ispresented as homozygote.7hepatic cell lines were divded into3groupsaccording to the genotype distinction among different genotypes: A groupincluded the cell line MHCC-97H and MHCC-97L take allele associated withhigher ALT value in rs10157061,rs10752781,rs1878544in SLC35F3,rs4766453in CUX2, rs4801847in SHANK-1; B group included theSMMC-7721take the allele associated with higher ALT level at rs4801847inSHANK-1; C group included the BEL-7402, BEL-7404, Hep-G2, and HL-7702take allele associated with lower ALT level in all the7SNPs located in5geneloci. After comparison, the ALT value in supernate of A group was higher than the ALT value in groups B and C, and no significance was found between groupB and C. RT-PCR indicated that the GPT expression was higher than group Band C, but the SLC35F3expression is negative correlated to the GPT expression(r=-0.672, P=0.001). The CUX2expression had no significant difference indifferent groups (P>0.05). The results from western-blot and IHC confirmedthese results. In western-blot experiments, the SLC35F3encoded proteinexpression in group A was much lower than the group B and C statistically(P<0.05). The GPT protein expression was higher than group B and Cstatistically (P<0.05). No significant difference between CUX2proteinexpression among various hepatic cell lines (P>0.05). The protein expression ofSLC35F3was negative correlated to GPT expression (r=-0.527, P=0.002). TheIHC results confirmed the western-blot results.Conclusions: Based on the SNPs that influence the serum ALT level inadult male population from previous GWAS, the hepatic cell line experiment invitro confirmed:1. The candidate SNPs in1q42.2of SLC35F3(includingrs10157061, rs10752781, rs1878544) significantly influenced the expression ofSLC35F3. Alleles associated with higher ALT level was associated with lowerSLC35F3expression.2. the SLC35F3gene might be a susceptible gene thatinfluence the ALT secrection. The upgrade of SLC35F3expression might becorrelated to the low GPT expression. The quantitative trait loci (QTL) withstrong Linkage disequilibrium (LD) composing by rs10157061, rs10752781,rs1878544influence and regulate the ALT secrection of ALT through regulatingthe expression of SLC35F3. The inner mechanism investigation is waiting to be confiemed. CHAPTER IIIPART IIExpression of SLC35F3gene in hepatic cell lines MHCC-97H andBEL-7402in low exposure of ethanolBackground and Obsjective: Based on the validation in previous study,we found SLC35F3gene influced the alanine aminotransferase (ALT)secrection, GPT gene expression in hepatic cell lines in vitro. The lowexpression of SLC35F3gene and its encoded protein was correlated to thehigher GPT expression and higher ALT secrection. However, the SLC35F3geneexpression in different kinds of hepatic cell lines on the press outside isunknown. Therefore, we designed interaction experiment on low exposure ofethanol, observed the SLC35F3and GPT gene expression of hepatic cell lines,and assess their interaction.Methods: The hepatic cell line BEL-7402and MHCC-97H was selectedfor observation. The expression of SLC35F3, GPT gene and encoded proteinwas tested in different ethanol concentration (50mmol/l,100mmol/l, and200mmol/l) at3hours,6hours,12hours,24hours. The procedure of experimentwas described as follows:1. extracting the supernate of hepatic cell lines andtested the ALT value with Enzyme Linked Immunosorbent Assay (elisa);2.aiming at the SLC35F3and GPT gene, design the specific primer to test theexpression level of SLC35F3and GPT level on different time-concentration ofethanol exposure in various hepatic cell lines with real-time PCR;3. extractingthe total protein of hepatic cell lines in different time-concentration of ethanolexposure,designing the SLC35F3and GPT specific antibody, to check theprotein expression by western-blot method;4. digest the cell in different time-concentration of ethanol exposure, to plant on the germ-free glass slides,the protein expression was checked by protein specific anti-body withImmunological Histological Chemistry (IHC) method.The cell experiments were duplicated for three times. Measurement datawas presented as mean±standard deviation (SD). The statistic comparison wasfollowed the criteria as below: one-way ANOVA was used in comparisonbetween data with normal distribution; non-parametric test (Mann-Whitney Utest) was used in comparison data with un normal distribution. P<0.05wasconsidered as statistical significance unless specific illustration.Results: We have checked the ALT secrection, SLC35F3, GPT gene andencoded protein expression level in different time-concentration ethanolexposure of BEL-7402and MHCC-97H. The specific results is as follows:1.the time-to-peak ALT level in BEL-7402was6hours, while the time-to-peakALT level was shorten from12hours on the exposure of100mmol/l ethanolconcentration to3hours on the exposure of200mmol/l ethanol concentration.In different intervention of various ethanol concentration, no significantdifference for the peak ALT value was observed between different ethanolconcentration exposure (P>0.05), while in MHCC-97H, the peak ALT value wasincreased significantly with the increased ethanol interactive concentrationstatistically (P<0.05);2. significant difference of time-concentration ethanolinteraction between BEL-7402and MHCC-97H on SLC35F3and GPTexpression. The time-to-peak of SLC35F3and GPT in BEL-7402is12hoursand6hours. No significant variation was observed between peak expressionlevel on different ethanol interaction concentration (P>0.05). While inMHCC-97H, the time-to-peak and peak level of SLC35F3and GPT expression varied with different ethanol concentration (P<0.05). The time-to-peak isshorten from24hours on the exposure of50mmol/l to3hours on the exposureof200mmol/l. Significant increasing GPT and SLC35F3expression level wereobserved followed with increasing exposure of ethanol concentration (P<0.05).3. the western-blot showed: the SLC35F3and GPT protein expression werestabilized at12hours and6hours respectively, while the time-to-peak ofSLC35F3and GPT in MHCC-97H was shorten followed with the increasingconcentration of ethanol exposure, the SLC35F3and GPT protein expression inMHCC-97H is higher on200mmol/l of ethanol exposure(P<0.05). Thewestern-blot results were confirmed by IHC results.Conclusions: In the stress environment, the BEL-7402with highexpression of SLC35F3is more susceptible to the ethanol exposure than theMHCC-97H with low expression of SLC35F3. The SLC35F3expression mightbe associated with the self-regulation of hepatic cell and the ALT secrectionmechanism on the ethanol exposure. The low expression of SLC35F3isfacilitated to decrease the susceptibility and injury on the exposure of ethanol.更多还原