【作者】 闫晓华；

【导师】 高立芬；

【作者基本信息】 山东大学 ， 免疫学， 2008， 硕士

【摘要】 背景:脂肪性肝病(fatty liver disease,FLD)简称脂肪肝,是一种由多种病因引起的、病变主要在肝小叶,以肝细胞内甘油三酯异常储积为主要表现的临床病理综合征,近年来,随着人们生活饮食结构的变化,FLD的发病率有进一步增高的趋势,有统计,在校正了年龄、性别和居住地区等因素后,脂肪肝、酒精性脂肪肝(alcoholic fatty liver disease,AFLD)和非酒精性脂肪肝(nonalcoholic fatty liverdisease,NAFLD)总的标准发病率为11.3%,2.2%和9.1%(成人为14.5%,2.9%和11.7%),日趋超越病毒性肝炎,是导致肝脏相关性致残和死亡的重要原因,已成为全球普遍关注的医学问题和社会问题。脂肪肝的发病与多种因素有关,是多种因子相互作用的结果。目前,凋亡因子在肝脂肪变过程中的作用已引起许多学者的关注。肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor related apoptosisinducing ligand,TRAIL),是1995年由Wiley发现并命名的,人TRAIL基因定位于染色体3q26,由5个外显子组成,编码1.77 kb mRNA,并可编码281个氨基酸残基组成的分子量为32.5 KDa的Ⅱ型跨膜蛋白。TRAIL属于TNF超家族成员,其结构与该家族的其他成员相仿。TRAIL可通过与其受体结合,诱导细胞凋亡,或发挥其他生物学作用。近几年通过临床资料及动物实验研究,发现TRAIL的表达水平与人外周血脂质水平呈现相关性,并且TRAIL在病毒感染和摄入酒精后可诱导肝脂肪变,但TRAIL在肝脂肪变中的具体作用尚不明了。FLD的发病与多种凋亡相关因子的多态性有关,但TRAIL的多态性是否与FLD的发生有关尚未见报道。目的:通过检测人外周血TRAIL、转氨酶、血糖、血脂等生化指标和检测非酒精性脂肪肝、酒精性脂肪肝、健康对照和嗜酒无脂肪肝四组人群TRAIL基因第五外显子3’-非编码区1525G/A、1595C/T位点基因多态性,推测脂肪肝与TRAIL及其等位基因和基因型的关联性。方法:采用病例对照的方法,对体检人群用B超筛选出163例脂肪肝患者,其中NAFLD组84例,AFLD组79例,同时选择同期体检的102例对照者,其中80例健康对照者,22例嗜酒无脂肪肝者。全部选择对象均空腹检测转氨酶、血糖、血脂等各项生化指标,从中随机选择44例NAFLD患者、35例健康对照者,用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)法检测血清可溶性TRAIL的水平。通过聚合酶链式反应-限制性内切酶片段长度多态性(polymerase chain reaction-Restriction fragment length polymorphism,PCR-RFLP)法检测四组人群TRAIL基因第五外显子3’-非编码区(3’-untranslatedregion,3’-UTR)1525/1595两位点基因多态性。同时从中选择部分对象通过基因测序验证PCR-RFLP法的正确性。结果:(1)非酒精性脂肪肝组血清可溶性TRAIL的表达量明显高于健康对照组(p=0.017,p＜0.05)。(2)血清可溶性TRAIL浓度与甘油三酯(triglyceride,TG)含量呈正相关(r=0.368,P=0.014,P＜0.05)。(3)中国汉族人群存在TRAIL基因1525G/A、1595C/T突变。(4)对中国汉族265名人员TRAIL基因第五外显子3’-UTR区1525、1595位点进行多态性分析,发现同一个体两位点G/A、C/T基因突变情况完全一致。(5)NAFLD、AFLD组1525/1595位点基因型分布与健康对照组比较均具有显著性差异(p=0.016,p＜0.05;p=0.013,p＜0.05);NAFLD组1525/1595位点G/C等位基因频率明显高于健康对照组(p=0.017,p＜0.05);AFLD组1525/1595位点G/C等位基因的频率明显高于健康对照组(p=0.022,p＜0.05);(6)NAFLD组1525/1595位点GG/CC基因型TG量显著高于AA/TT基因型个体的TG量(p=0.035,p＜0.05);(7)NAFLD组血清丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartateaminotransferase,AST)、γ-谷氨酰转肽酶(glutamyl transpeptidase,GGT)、血糖(Blood Glucose,GLU)、胆固醇(cholesterol,CH)、TG血清浓度高于健康对照组(无饮酒史)有统计学意义(p＜0.05);(8)AFLD组ALT、GLU、CH、TG血清浓度高于嗜酒无脂肪肝病组(p＜0.05);(9)AFLD组ALT、AST、GGT、GLU、CH、TG水平高于健康对照组(无饮酒史)(p＜0.05);(10)非条件多因素Logistic回归分析身体质量指数(body mass index,BMI)、腰臀比(waist hipratio,WHR)、高血脂、GGT均是NAFLD的危险因素。结论:PCR-RFLP方法检测TRAIL基因型特异性高,成熟,稳定;中国汉族人群存在TRAIL基因第五外显子3’-非编码区1525G/A、1595C/T突变。(1)研究发现NAFLD患者血清TRAIL水平与肝脂肪病变有关,血清sTRAIL浓度与血清TG水平呈正相关。(2)发现中国汉族人群TRAIL基因第五外显子3’-UTR1525G、1595C等位基因是与脂肪肝发病相关联的基因。(3)研究发现TRAIL基因第五外显子3’-UTR各位点基因突变一致,即一些相互邻近的多态位点趋向于在一起共同遗传,属于一种单体型。(4)TRAIL 1525/1595位点为GG/CC基因型的个体易患高血脂。(5)肥胖、高血脂、高GGT水平是非酒精性脂肪肝的危险因素。更多还原

【Abstract】 Background:Fatty liver disease(FLD) also called fatty liver,is a clinical pathological syndrome caused by a variety of causations.The lesion of FLD mainly lies in liver foliole,and it is characterized as triglycerides accumulation superfluously in hepatocytes.In recent years,with the improvement of people’s lives and the change of diet structure,the incidence rate of FLD has further increased.According to statistics, in the correction of age,sex and residential area,and other factors,the standard incidence rate of FLD,alcoholic fatty liver disease(alcoholic fatty liver disease,AFLD) and non-alcoholic fatty liver(nonalcoholic fatty liver disease,NAFLD) were 11.3%,2.2%and 9.1%separately(in adult,they were 14.5%,2.9%and 11.7% separately),so the incidence rate of FLD has the growing trend beyond viral hepatitis.In addition,FLD may lead to liver-related disability and deaths,which has become a global medical issues of common concern and social problems.Fatty liver disease is the result of a variety of factors interaction.At present,the relationships between apoptotic factors and hepatocyte steatosis aroused the concern by many scholars.Tumour necrosis factor(TNF)-related apoptosis inducing ligand(TRAIL) was found and named by Wiley in 1995,TRAIL is mapped to the long arm of chromosome 3q26 in humans and is composed of five exons.It encodes approximately 1.77 kb mRNA,32.5 KDa transmembrane(typeⅡ) glycoprotein composed of 281 amino acid.TRAIL belongs to TNF-super family,and its structure is similar to other TNF family members.By combining with its receptor,TRAIL can induce apoptosis or play other biological effects.It has been reported that levels of TRAIL are related to lipid levels of human peripheral blood.In recent years,it is found that TRAIL can induce hepatocyte steatosis after virus infection and after alcohol intaking.However,the mechanism of TRAIL involved in FLD is unclear.It is believed that the occurrence of FLD are related to polymorphisms of many apoptotic factors,but reports on the relation between FLD and TRAIL polymorphisms have not been appeared in the literature.Objective:To observe the serum concentration of soluble TRAIL(sTRAIL), aminotransferase,Glucose,lipid among NAFLD,AFLD,healthy controls and excessive drinking controls without liver disease.To observe the polymorphism and gene frequency of TRAIL gene 1525G/A and 1595C/T of the 3’-UTR in Chinese Han nationality population.To further explore the association of TRAIL polymorphism with susceptibility to fatty liver.Methods:This study followed a case-control design.A total of 163 FLD subjects(84 NAFLD and 79 AFLD) were analyzed according to generally accepted diagnosis criteria for example B ultrasonic.102 healthy blood donors(80 healthy controls and 22 excessive drinking controls without liver disease) were used as controls.All targets were chosen fasting.The levels of sTRAIL in serum were determined by ELISA(44 NAFLD patients and 35 healthy controls).The polymorphisms of TRAIL gene were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods in 80 patients of alcohol fatty liver disease,79 patients of nonalcoholic fatty liver disease,80 healthy controls and 22 healthy controls with alcohol.At the same time,we selected 10 samples to sequence randomly in order to validate the foregoing of PCR-RFLP results.Results:(1) The NAFLD group showed significantly higher levels of sTRAIL in serum than those of healthy control group(p=0.017,p＜0.05);(2) Serum sTRAIL concentrations positively correlated with triglyceride(TG) significantly(r=0.368,P =0.014,P＜0.05).(3)There were TRAIL gene 1525G/A,1595C/T polymorphisms in Chinese Hans.(4)The same mutation happened at position 1525,1595 in exon 5 of TRAIL gene in 265 Chinese Han.(5) The genotype distributions among NAFLD, AFLD and healthy control(without alcohol) were significantly different(p=0.016,p ＜0.05;p=0.013,p＜0.05);1525G allele and 1595C allele were more frequent in FLD patients than in controls(without alcohol)(p=0.017,p＜0.05).(6) In NAFLD patients,the TG concentration of individuals with GG/CC genotype at 1525/1595 was higher than that of AA/TT genotype(p=0.035,p＜0.05).(7) The serum concentration of alanine aminotransferase(ALT),aspartate aminotransferase(AST), glutamyl transpeptidase(GGT),blood Glucose(GLU),cholesterol(CH),TG in NAFLD group were higher than those in healthy control(without alcohol),the differences were statistically significant(p＜0.05).(8) The serum concentration of ALT,GLU,CH,TG in AFLD group were higher than those in excessive drinking controls without liver disease(p＜0.05).(9) The serum concentrations of ALT,AST, GGT,GLU,CH,TG in AFLD group were higher than those in healthy control(without alcohol)(p＜0.05).(10) Obesity,hyperlipidemia,High level of GGT are risk factors of NAFLD by Non-conditions more Logistic regression analysis.Conclusion:The PCR-RFLP can detect the TRAIL genotype accurately.There are TRAIL gene 1525G/A,1595C/T polymorphisms in Chinese Hans.(1) The study indicated that sTRAIL concentrations in serum were related to hepatic steatosis and sTRAIL concentrations were positively correlated with TG concentrations.(2)The results showed Chinese Hans people with alleles 1525G,1595C in the 3’-UTR of exon 5 of TRAIL are susceptibility to FLD.(3) There is the same genetic variation of TRAIL at position 1525G/A and 1595C/T in Chinese populations.Some nearby polymorphic loci tend to with the common genetic,and it is one type of haplotype.(4) People with GG/CC genotype at 1525/1595 sites of TRAIL are susceptible to high blood lipid.(5) Obesity,hyperlipidemia,High level of GGT are risk factors of NAFLD.更多还原