【作者】 王虎林；

【导师】 司良毅；

【作者基本信息】 第三军医大学 ， 老年医学， 2010， 博士

【摘要】 研究背景Klotho基因具有潜在抗衰老作用。小鼠Klotho表达缺陷可出现几乎全部与人类衰老类似的表型。人类Klotho基因与小鼠具有同源性,二者之间蛋白序列有86%的相似性,提示Klotho基因变异可能参与人类衰老进程。已有研究表明人类Klotho基因单核苷酸多态性位点与多种增龄性疾病密切相关。Klotho缺陷小鼠两个显著的变化是动脉硬化和内皮功能障碍,这与人类高血压的病理特点非常相似。但目前为止汉族人群中Klotho基因SNP与高血压的相关性研究未见报道。研究目的本研究拟通过病例对照研究,观察Klotho基因启动子区G-395A单核苷酸多态性(SNP)与原发性高血压的相关性。通过双荧光素酶报告基因实验进行启动子缺失突变,对G-395A位点在启动子区的功能进行定位,结合定点突变,了解该位点G/A替换对Klotho基因转录调控的影响。方法1、本研究选择2006年2月至2008年5月期间西南医院老年科门诊、住院及体检的高血压患者215例及非高血压对照220例为研究对象,各研究对象之间无亲缘关系。运用Taqman等位基因特异性杂交分析对各研究对象进行SNP分型,对高血压组和对照组间年龄、性别、吸烟史、糖尿病、高血脂、体重指数等高血压的传统影响因素及基因型分布进行比较,并进一步针对年龄、性别和吸烟史进行亚组分析。运用logistic回归模型分析了各个危险因素对高血压的相对危险度。为排除G-395A多态性在人群中可能造成的生存效应,对G-395A基因型在各年龄段之间的分布也进行了比较。2、全部研究对象中,有232例检测了臂踝脉搏波传导速度(baPWV)和血浆一氧化氮(NO)的影响,以初步了解G-395A多态性对血管功能的影响。3、在生物信息学预测的基础上,采用双荧光素酶报告基因实验,对Klotho基因启动子序列进行缺失突变分析。将包含Klotho启动子区-975bp～-8bp、-506bp～-8bp、-320bp～-8bp、-145bp～-8bp四个片断克隆入pGL3-Basic报告基因质粒,和pRL-TK内参质粒共转染HEK293细胞,检测双荧光素酶活性,分析各片断的启动活性,对G-395A位点进行启动子区的功能定位。4、将包含G-395A位点且具有启动子活性的-506bp～-8bp片断克隆入pGL3-Enhancer荧光素酶报告基因质粒,进一步证实其启动子活性。5、运用重叠延伸PCR技术对包含-506bp～-8bp片断的pGL3-Basic及pGL3-Enhancer重组质粒进行-395G/A定点突变,将各重组质粒分别和内参质粒pRL-TK共转染HEK293细胞,比较G/A替换对报告基因活性的影响。结果1、在435例研究对象中存在GG、GA、AA三种基因型,分别为GG型288例(66.2%),GA型130例(29.9%),AA型17例(3.9%),A等位基因频率为0.191,G等位基因为0.809,符合Hardy-Weinberg平衡(χ2=0.107,P=0.948)。2、高血压组和对照组间年龄、吸烟史、体重指数、糖尿病患病率有差异,而性别、高血脂症在两组间无差异。G-395A多态性基因型的分布在两组之间存在差异,有统计学意义(χ2=6.883,P=0.032)。亚组分析显示在大于60岁亚组、女性亚组、非吸烟亚组中G-395A基因型分布有差异(P = 0.039, P = 0.001及P = 0.010),而年龄小于60岁亚组、男性亚组及吸烟亚组基因型分布无统计学差异。Logistic回归分析显示调整年龄、性别、吸烟史、体重指数、糖尿病、高血脂症等传统危险因素后,G-395A多态性仍然与高血压有相关性,-395A等位高血压呈负相关(OR=0.593,95% CI=0.376～0.935,P=0.024)。3、G-395A多态性在研究人群中各年龄段之间的分布无统计学差异(P=0.071),提示G-395A多态性在本研究的人群中未造成明显生存效应。4、-395A等位携带者平均baPWV较非携带者显著降低(1463.8±297.0 vs. 1572.6±347.1,P=0.014)。-395A等位携带者平均血浆NO与非携带者之间无统计学差异(11.8±6.6 vs. 11.1±6.7,P=0.448)。5、成功将Klotho基因5′侧翼-975bp～-8bp、-506bp～-8bp、-320bp～-8bp、-145bp～-8bp四个片断克隆入pGL3-Basic报告基因质粒。通过RT-PCR证实HEK293细胞可表达Klotho基因。双报告基因实验结果显示-975bp～-8bp、-506bp～-8bp片断均具有启动子活性(和pGL3-Basic空质粒相比,P=0.014及P<0.001)。将-506bp～-8bp片断克隆入pGL3-Enhancer质粒,其报告基因活性较相对应pGL3-Basic重组质粒明显增强(P<0.001)。提示G-395A位点可能参与Klotho基因核心启动子构成或接近核心启动子区。6、成功对包含Klotho基因-506bp～-8bp片断的pGL3-Basic和pGL3-Enhancer重组质粒进行了-395G/A突变。双报告基因活性分析显示-395G/A替换增加了pGL3-Basic重组质粒中相对荧光素酶活性,但其影响无统计学意义(0.043±0.09 vs. 0.028±0.012; P=0.094),而在pGL3-Enhancer重组质粒中-395G/A替换后显示出更高的相对荧光素酶活性(0.251±0.084 vs. 0.103±0.013; P=0.013)。结论本研究在重庆汉族人群中发现Klotho基因G-395A SNP与高血压易感性相关,-395A等位有可能是高血压遗传学保护因素。在人群中-395A等位携带者动脉僵硬度较非携带者低,提示-395A等位有潜在改善动脉功能的作用。Klotho基因G-395A位点可通过G/A替换提高Klotho基因转录活性起到抗高血压作用,是潜在的调节性SNP位点。更多还原

【Abstract】 BackgroundMice with defects in the Klotho gene exhibit multiple aging phenotypes including a short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysema. Human Klotho, showing 86% amino acid identity with the mouse protein, may be involved in the aging process. In previous studies, more than ten single nucleotide polymorphisms (SNPs) in the human Klotho gene were found to be associated with age-related diseases. Interestingly, two notable changes of Klotho-deficient mice are arteriosclerosis and endothelial dysfunction, which are the fundamental etiological factors of human essential hypertension (EH). However, the association of the Klotho gene SNP with EH in the Chinese population is scarce.ObjectivesIn the present studies, we hypothesized that the G-395A polymorphism in the promoter region of the human Klotho gene may contribute to the prevalence of EH and the G-395A site may be a potential regulatory SNP (rSNP) of the Klotho gene.MethodsThe case-control study was performed in the Chongqing Han population consisting of 215 patients with EH and 220 non-hypertensive subjects. The genotypes of the Klotho gene G-395A SNP were determined by the TaqMan allelic discrimination assay. The distributions of genotype frequencies were analyzed in the two groups and in the subgroups according to age, gender and smoking status. The odds ratios of G-395A SNP and traditional risk factors of EH were computed by the use of a multiple logistic regression model. In addition, the brachial-ankle pulse wave velocity (baPWV) and serum nitric oxide (NO), as the vascular function markers, were compared between the -395A carriers and non-carriers in 232 subjects of the population. By using serial deletion recombinant plasmids of the promoter, the -975bp~-8bp fragment upstream of the Klotho gene was investigated for responsible promoter region in HEK293 cells. Furthermore, whether a G/A substitution at the G-395A site of Klotho gene affected the transcription level in vitro was also tested through the dual-luciferase reporter assay.Results1. In the study subjects, 288 subjects had the GG genotype, 130 subjects had the GA genotype, and 17 subjects had AA genotype. The allele frequencies were 0.809 for the G allele and 0.191 for the A allele. These were in compliance with the Hardy–Weinberg equilibrium (χ2 = 0.107; P = 0.948).2. Differences in the genotype distributions of the G-395A polymorphism between the EH and non-hypertension groups were statistically significant (P = 0.032). There were differential effects of age, gender and smoking status on the association of the G-395A polymorphism with EH; the G-395A polymorphism was significantly associated with EH in subjects over 60 years old, in females and in nonsmokers (P = 0.039, P = 0.001, and P = 0.010, respectively). A multiple logistic regression analysis indicated that the odds ratio for EH in the–395A allele carriers as compared with the control group was 0.593 after adjusting for current traditional risk factors including advanced age, smoking status, being overweight, diabetes mellitus and dyslipidemia (95% CI = 0.376–0.935; P = 0.024).3. There were no statistical differences of G-395A distributions among the different age groups in the study population.4. The ?395A allele carrier of the G-395A polymorphism of the human Klotho gene was found to have a lower baPWV than the non-carrier (1463.8±297.0 vs. 1572.6±347.1, P=0.014), but the serum NO between the two groups were not statistically significant (11.8±6.6 vs. 11.1±6.7; P=0.448).5. The expression of Klotho in HEK293 cells was confirmed by RT-PCR. The dual-luciferase reporter assay of serial deletion of the Klotho promoter revealed that there was a promoter element in the -506bp～-8bp DNA fragment (containing the G-395A site) upstream of the Klotho gene ATG start codon.6. No significant differences of the luciferase activities were seen after the G/A substitution in the pGL3-Basic recombinant plasmid of the Klotho -506bp～-8bp DNA fragment (0.043±0.09 vs. 0.028±0.012 respectively; P = 0.094). However, the–395A carrier in the pGL3-Enhancer recombinant plasmid had a higher relative luciferase activity as compared with the–395G carrier (0.251±0.084 vs. 0.103±0.013 respectively; P = 0.013).ConclusionsThe G-395A polymorphism of the human Klotho gene is associated with EH in Han population and may be a potential regulatory site by altering the transcriptional level of the Klotho gene.更多还原