【作者】 刘黎星；

【导师】 郑鸣；

【作者基本信息】 福建医科大学 ， 病理学与病理生理学， 2013， 博士

【摘要】 1、 ABCA1基因单核苷酸多态性分布及其与子痫前期脂代谢异常的关联性研究目的：采用cases-controls方法检测ABCA1基因2个候选单核苷酸多态位点（SNPs）:R219K和I883M的基因型、等位基因及单倍型在子痫前期组和正常妊娠组中的频率，探明ABCA1基因SNPs在福建汉族妇女中的分布，探索ABCA1基因与子痫前期发生、发展的相关性，并揭示这2个基因多态位点在子痫前期脂代谢异常中的作用。方法：选取研究对象621例，均为福建地区汉族人群，其中子痫前期患者组305例，正常妊娠组316例。采用聚合酶链-限制性片段长度多态性反应(PCR-RFLP)的技术手段，并结合琼脂糖凝胶电泳技术检测ABCA1基因2个SNP位点在中国福建地区汉族妇女中的等位基因以及基因型的频率分布特点，通过与之相关联的血脂等临床资料，探索ABCA1基因2个SNPs位点与子痫前期及其血脂代谢的遗传相关性。采用SHEsis在线分析软件和SPSS17.0软件包进行相关数据的统计分析。结果：(1)子痫前期组和正常妊娠组ABCA1基因上述2个SNP位点基因型分布符合Hardy-Weinberg平衡定律，P值均大于0.05，样本具有群体代表性。(2)ABCA1基因R219K多态位点K等位基因频率和RK+KK基因型频率在子痫前期病例组明显降低，差异有统计学意义(P<0.05)。I883M多态位点三种基因型频率和等位基因频率在子痫前期病例组和对照组间未发现显著性差异。(3)子痫前期组(包括轻度组及重度组)血清TG、TC、LDL、ApoB、HDL、ApoAI水平分别与对照组比较,差异均有统计学意义(P<0.05)。(4)ABCA1基因上述2个SNPs的基因型与血脂水平的关联分析，发现子痫前期病例组内R219K位点K等位基因携带者（RK+KK基因型）血清甘油三酯（TG）浓度低于RR基因型，而HDL-C浓度刚好相反，差异有显著的统计学意义(P<0.05)。子痫前期病例组I等位基因携带者（MI+II基因型）较MM基因型携带者血液中HDL-c及ApoA1浓度明显增高，差异有显著性(P<0.05)。(5)采用SHEsis在线分析软件对ABCA1基因上述2个SNPs位点间进行两两配对连锁不平衡分析，发现这两个SNPs位点间不存在较强的连锁不平衡关系，故无法构建单倍体。2、子痫前期产妇血清和胎盘组织中ABCA1表达及其与血脂代谢的关联研究目的：实验检测子痫前期组和正常妊娠组的血脂浓度，血清和胎盘组织中ABCA1表达水平，研究ABCA1基因及蛋白表达与血脂异常代谢之间存在的相关性，阐明血脂代谢的异常在子痫前期病理机制中的重要作用，并进一步探索ABCA1在子痫前期患者体内可能是通过参与血脂异常代谢、动脉粥样硬化性改变，从而对子痫前期的发病起着重要作用。方法：选择同期住院分娩的子痫前期组52名（其中轻度子痫前期产妇28名，重度子痫前期产妇24名）、正常妊娠组30例，酶法检测血脂浓度，ELISA法检测血清ABCA1浓度，实时荧光定量RT-PCR法和免疫印迹法检测胎盘组织中ABCA1基因和ABCA1蛋白表达水平。结果：(1)子痫前期组血清TG、TC、LDL浓度较正常妊娠组明显升高，而HDL浓度则明显降低，且与病情的严重程度成正相关，上述差异均有显著的统计学意义(P<0.05)，表明子痫前期患者母体自身动脉硬化指数增高，血管病变风险性增加。(2)子痫前期组血清ABCA1浓度及胎盘组织ABCA1基因和蛋白表达水平较正常妊娠组均显著降低，且与病情严重程度成正相关，差异具有显著性(P<0.05)。(3)子痫前期组血清ABCA1浓度与胎盘组织ABCA1蛋白表达水平呈正相关(r=0.384, P<0.05),与血清低密度脂蛋白(LDL)浓度呈负相关（r=-0.279, P<0.05),而与高密度脂蛋白(HDL)呈正相关(r=0.318, P<0.05)。3、正常妊娠与子痫前期血清ABCA1及OX-LDL表达特点及对内皮细胞损伤的影响目的：研究正常妊娠妇女及子痫前期患者血清中氧化低密度脂蛋白（OX-LDL）的表达及OX-LDL/ABCA1比值的差异，以及不同血清干预对内皮细胞损伤的影响，探讨子痫前期血清中OX-LDL及OX-LDL/ABCA1表达变化与内皮细胞损伤的关系，进一步明确ABCA1在子痫前期发病中的作用及其作用机制。方法：研究对象同第二部分，ELISA法定量检测上述正常妊娠及子痫前期组血清中OX-LDL的表达水平；原代培养脐静脉内皮细胞，分别用上述不同组别血清体外干预人脐静脉内皮细胞生长,利用:荧光标记的牛血清白蛋白检测内皮细胞的单层屏障功能；咪噢兰比色法(MTT)检测内皮细胞的增殖能力；硝酸还原酶法检测内皮细胞的一氧化氮(NO)合成能力，以评估正常妊娠及子痫前期患者血清对内皮细胞功能的影响。结果：(1)子痫前期组患者血清中OX-LDL表达水平较正常妊娠组明显升高，与血清中ABCA1蛋白表达水平呈负相关（r=-0.681, P<0.05)，且与病情的严重程度相关联。(2)与正常妊娠组相比,子痫前期组血清可使脐静脉内皮细胞功能明显受损，包括：通过单层内皮细胞的牛血清白蛋白浓度升高，即内皮细胞的单层屏障功能受损；内皮细胞增殖能力明显被抑止；培养基中NO浓度明显降低。上述改变与病情严重程度成正比，且差异有显著的统计学意义。(3)子痫前期组血清中OX-LDL/ABCA1比值较正常妊娠组明显升高，且与脐静脉内皮细胞功能损伤程度成正相关，差异有显著的统计学意义。且病情越严重，上述改变越明显。综上所述，本实验从整体、细胞和分子等多个层面揭示了ABCA1基因及其蛋白在子痫前期发病中的重要作用，其可能的作用机制包括：影响脂质代谢、促进动脉粥样硬化、产生氧化应激、损伤血管内皮细胞，从而引发一系列子痫前期病理变化和临床症状，为子痫前期的预测、及时采取有效的预防及未来可能的临床个体化治疗和基因治疗提供有价值的研究资料。更多还原

【Abstract】 1The distribution of ABCA1gene single nucleotide polymorphisms and it’srelation to serum lipid levels of preeclampsiaObjective：Through detecting the R219K and M883I of ABCA1gene’s two singlenucleotide polymorphic sites (SNPs) in genotype, allele and haplotype frequency inpreeclampsia(PE) and normal pregnancy group, to explore the association betweenABCA1gene and PE and to reveal the role of two SNPs in PE patients’ abnormallipid metabolism.The aim of our study is to provide valuable research information inprevention, diagnosis, treatment and possible future clinical individualized therapyand gene therapy of PE.Methods：In this study,621subjects of Fujian Han population in total were recruited，consisting of305PE patients and316normal pregnant women. We detected the twosingle nucleotide polymorphic loci genotypes in Han Chinese women in FujianProvince with the method of polymerase chain reaction-restriction fragment lengthpolymorphism (PCR-RFLP) combined with agarose gel electrophoresis. We exploredthe genetic correlation between the two SNPs and PE associated with lipids and otherclinical data.The statistical analysis of data with the tools SHEsis-online software andSPSS17.0.Results：(1) The frequencies of different genotypes of the two SNPs were inagreement with Hardy-Weinberg equilibrium in our study(P>0.05)．(2) The frequency of allele K and genotype RK+KK were significantly lower inpatients with preeclampsia than in the control group patients, the differences werestatistically significant (P <0.05). The genotype and allele frequencies of M883I SNPof ABCA1gene, without significant difference between the PE group and normalpregnancy group,were statistically analysed by SPSS17.0(P>0.05).(3) Preeclampsia (mild group and severe group) serum TG, TC, LDL, ApoB, HDL, of ApoAI levels were compared with the control group, which showed significantdifferences(P <0.05).(4) We analysed the association of the ABCA1gene2SNPs and serum lipid levels,and found that the concentration of triglyceride was significantly lower in those withgenotype RK+KK than in those with genotype RR in terms of patients withpreeclampsia.The situation was the opposite of that in individuals with aconcentration of high density lipoprotein-cholesterol (HDL-C) and Apolipo proteinA1（ApoA1）; the differences were statistically significant (P<0.05). We also foundthat the concentrations of high density lipoprotein-cholesterol (HDL-C) and Apolipoprotein A1（ApoA1）were significantly higher in those with genotype MI+II than inthose with genotype MM in terms of patients with preeclampsia, the differences werestatistically significant (P<0.05).(5) We analysed the linkage disequilibrium in all the two SNPs with SHEsis software,demonstrated that there is no strong linkage disequilibrium between the two loci,therefore unable to consist the haplotype by paired SNPs.2Expression and clinical significance of ABCA1in serum and placental tissuefrom Chinese patients with preeclampsiaObjective：To investigate lipids levels and ABCA1levels of serum and placenta inpreeclampsia women; to study the relativity between hyperlipemia and ABCA1; todiscuss the effectiveness of hyperlipemia in the pathogenesis of preeclampsia; toilluminate ABCA1possibly has a significant role in the pathogenesis of the casethrough participating in lipid abnormal metabolism.Methods：52cases of preeclampsia (which include28mild preeclampsia women and24severe preeclampsia women) and30normal pregnant women which delived in thesame period were analyzed by experimentation. The lipids levels of serum weredetected by enzymic method. The ABCA1levels of serum were detected by ELISA.The mRNA or protein expression of ABCA1in placentas were detected respectivelyby RT-PCR or Western blot.Results：(1) The preeclampsia serum TG, TC, LDL concentrations were significantlyhigher than normal pregnant group, which lowering HDL levels, the differences were statistically significant (P <0.05).These women whose atherogenic index was highersignificantly were high risk people who have arteriosclerosis diseases.(2) The ABCA1levels of serum and placenta were decreased significantly in thepreeclampsia women compared to the normal pregnant women, which is related to theseverity of the disease,the differences were statistically significant (P <0.05).(3) Correlation analysis revealed that ABCA1levels of serum were positively relatedto that of placenta (r=0.384, P<0.05）, and negatively related to that of serum LDL (r=-0.279, P<0.05）,and positively related to that of serum HDL (r=0.318, P<0.05）inthe preeclampsia women.3The expression of ABCA1and OX-LDL in serum of normal Pregnancy andPreeclampsia and the effect of serum on endothelial cell dysfunction.Objective： To investigate the expression of OX-LDL and the ratio ofOX-LDL/ABCA1in serum obtained from normal pregnancy and preeclampsiawoman,and the correlation to endothelial cell dysfunction.Methods：The level of OX-LDL protein in serum samples of the study objects whichare belong to the said second part were determined by performing enzyme-linkedimmunosorbent assay(ELISA). Primary cultured human umbilical vein endothelialcells(HUVEC) were interfered with serum samples as deseribed previously.The effectof serum on endothelial cell dysfunction was determined on the basis of followingaspects:(1) monolayer barrier function was evaluated by transferringfluorescently-labeled BSA across HUVEC monolayer;(2) cell proliferation functionwas evaluated by performing methl thiazolyl tetrazolium(MTT);(3) level of secretednitric oxide(NO) was estimated by nitrate reductase assay.Results：(1) The level of OX-LDL protein in serum of preeclampsia woman wassignificantly higher than that of normal pregnancy woman,whereas the level ofABCA1protein was significantly lower.There was obvious negative correlationbetween the level of OX-LDL and ABCA1in preeclampsia（r=-0.681, P<0.05). Aboveassociated with the severity of the disease.(2) Compared to that of normal pregnaney woman,the serum of preeclampsia woman could lead to endothelial cell dysfunction,including:①high transfer of fluorescentlylabeled BSA across HUVEC monolayer indicated the damage of monolayer barrierfunction;②the proliferation ability of HUVEC was markedly decreased;③the levelof secreted NO was low as compared to that of normal pregnancy group. Aboveassociated with the severity of the disease.The differences were statisticallysignificant (P <0.05).(3) There was positive correlation between endothelial cell dysfunction and the levelof OX-LDL/ABCA1in serum of preeclampsia,the differences were statisticallysignificant (P <0.05). Above associated with the severity of the disease.In summary, based on various levels of bodies, cells and molecules, the resultsreveal that ABCA1gene and its protein plays an important role in the pathogenesis ofpreeclampsia. The possible mechanisms for this was fourfold: lipid metabolism,promote atherosclerotic sclerosis, oxidative stress, vascular endothelial cells, causinga series of preeclampsia pathological changes and clinical symptoms, for theprevention of preeclampsia, prediction, treatment and possible future individualizedtreatment and gene therapy provide valuable research data.更多还原