【作者】 孙志军；

【导师】 赵彦艳；

【作者基本信息】 中国医科大学 ， 遗传学， 2004， 博士

【摘要】 目的 高血压病是当今世界最严重的社会公共健康复杂疾病之一，能引起一系列严重后果，如心衰、肾衰、心源性休克等。其发病受多种遗传因素和环境因素共同作用，其中遗传因素在高血压的发病机制中起重要作用。随着人类基因组测序的完成，研究与疾病表型的基因序列变异已成为医学遗传学的一个主要课题。单个核苷酸多态(single-nucleotide polymorphism，SNP)作为第三代遗传标记，具有数量大、分布广的特点。系统地在群体水平上筛查鉴定SNPS，特别是编码区和基因调控区的SNPs，通过病例-对照组间的比较和统计学分析，就有可能获得与疾病相关联的SNPs位点或单倍型，这对于阐明某些复杂多基因疾病的遗传机制具有重要意义。WNK4(with no K=lysine kinase)基因是新近分离克隆的一种蛋白激酶基因。该基因第7外显子(exon7)和第17外显子(exon17)突变可导致常染色体显性遗传疾病假性低醛固酮血症(Pseudohypoaldosteronism type Ⅱ，PHAⅡ)。PHAⅡ主要表现为高血钾、代谢性酸中毒和高血压，而且WNK4基因已被定位于血压控制热点区域17q12-21上，因此WNK4基因可能与原发性高血压密切相关。为了阐明WNK4基因与原发性高血压的关系及其在原发性高血压发病机制中的作用，本研究在中国东北汉族高血压高发地区对WHK4基因位于编码区内的两个SNP进行了大样本RFLP筛查。同时对WNK4基因的表达调控机制及其与原发性高血压的关系进行初步研究。 方法 1．通过文献报道和小样本测序确定WNK4基因cSNP，之后进行大样本限制片断长度多态(restricted fragment long polymorphism，RFLP)检测，并对我国东北汉族高血压高发地区人群进行群体病例对照分析研究，探讨cSNP与原发性高血压的相关性。同时根据文献报道比较我国东北汉族人群与欧洲高加索人、非洲人及美国黑人等种族cSNP基因型频率和等位基因频率分布，了解不同种族间WNK4基因cSNP的种族分布差异。 2.RT一PCR方法分析WNK4基因表达谱:Trizol一步法提取胎儿不同组织RNA，以其为模板进行RT一PCR反应分析WNK4基因表达谱。 3.不同处理因素对WNK4基因表达的影响:分别以重组人雌激素、生长激素、胰岛素、地塞米松、血管紧张素n及空白对照处理COS一细胞24小时，Trizol一步法提取细胞RNA，分别进行RT一PCR及Northem杂交检测WNK4基因mRNA表达变化。 4.WNK4基因转录起始点确定:以人肾Marathon cDNA为模板，以5‘Marathon RACE反应确定WNK4基因转录起始点，根据基因组数据库所获得的WNK4基因序列设计3’基因特异性引物(3’GSP)，引物序列:5‘TGGGTCTCCATGTCCTCCrl’l，I’r3’。以聚合酶链反应方法在人Marathon cD-NA文库中进行扩增获得对应的cDNA片段，再一次扩增，以前次产物稀释50倍为模板，巢式PCR确定转录起始点。 5.构建WNK4基因启动子pCAT报道表达载体:以基因组DNA为模板，上游引物:5‘CACTGACCTCTCCGTTCGGC3’，下游引物:5’CAGATCAT-TCAGGGCAAAGAC3’，95℃预变性smin，然后按94℃lmin、60℃1而n、72℃lmin3Os进行35个循环，最后72℃延伸10min，扩增WNK4基因启动子序列。PCR产物纯化后，连接到pMD18克隆载体，质粒大量转化并提取质粒，Hindlll和Xbd双酶切，将纯化的酶切产物连接到pCAT载体构建wNK4基因启动子pCAT一WNK4一promoter报道表达载体，测序鉴定证实没有突变发生且符合阅读框架。 6.EMSA方法鉴定WNK4基因启动子区域顺式作用元件及反式作用因子。提取成人肾组织核蛋白并以Bradford比色法测定核蛋白浓度。人工合成含有转录因子API、SPI的双链寡核昔酸序列SA，以T4多核昔酸激酶标记探针后与核蛋白冰浴中连接结合反应，之后8%PAGE凝胶电泳约10小时，放射性自显影。 7.wNK4基因启动子序列G甩的功能鉴定:WNK4基因启动子pCAT-wNK4一promoter报道表达载体转化质粒JM109感受态细胞，无内毒素型超纯质粒DNA提取法提取质粒，酶切鉴定后，脂质体转染法转染COS一细胞，分别以InM，10nM浓度地塞米松处理细胞24小时，同时设置一空白对照，收集细胞，酶联免疫吸附试验(e川守me一hnkedi~unosorbant assay，EllsA)检测CAT蛋白质的表达。结果 1，小样本测序检测到WNK4基因exon7一个新SNP(G1662A)，大样本RFLP筛查证实该SNP和另一个位于exons的cSNP(G1816T)的等位基因频率在高血压及正常人群间存在显著性差异，但基因型频率无显著性差异。两者与我国东北汉族高血压发病存在密切相关。同时通过对不同种族人群的对比分析发现我国东北汉族人群WNK4基因exons GI 8 16T多态的基因型频率和等位基因频率与高加索人、非洲人比较差异明显，具有统计学意义(p<0.001)，但与美国黑人相近，没有显著性差异(p>0.05)。 2.WNK4基因表达谱分析结果显示WNK4基因在胎儿肾脏有高强度表达，在脑、肺、心、脾、肠等组织中有表达，但在肝组织中却无表达。 3.以重组人生长激素(hGH，终浓度为6IunoFL)、重组人雌激素(p一EZ，终浓度为1林m0FL)、血管紧张素n(Angn，终浓度为5林m0FL)、地塞米松(Dexamethson，终浓度为1 nmoFL)及?更多还原

【Abstract】 ObjectiveHypertension is one of the most serious complex diseases nowadays that threaten public health and may lead to a series severe results such as cardiac and renal failure and shock. Hypertension is a complex disease resulting from the interaction of the cumulative effect of multiple genetic and environmental factors with genetic ones being important. Along with the completion of human genome project, it has been a key aim to study the sequence variations that correlate with certain diseases. As the third generation of genetic markers, single-nu-cleotide polymorphism (SNP) is characterized as large numbering and wide distributing. SNPs or haplotypes that are related to diseases may be obtained through systematically identifying SNPs especially those in coding and regulating region and the case-control study. This will play an essential role in probing the genetic mechanisms of some complex polygenic diseases. WNK4 gene is a newly cloned protein kinase gene with the mutation in exon 7 and exon 17, which may cause Pseudohypoaldosteronism type II (PHA II) , an autosomal dominant inheritance disease. The main symptoms of PHA II are hyperkalemia, metabolic aci-dosis and hypertension. WNK4 locates on 17ql2-21, a hot locus of blood pressure regulation. Therefore WNK4 is very likely to correlate with essential hypertension. In order to investigate the relation between WNK4 gene and hypertension and its effect in hypertension pathogenesis, two SNPs in coding region of WNK4 gene were assessed in this study and also the expression regulation mechanisms and its correlations with hypertension were studied.Methods1. cSNPs were identified by relative reports and small-sample sequencing and were examined by RFLP. Then the case-control study in a large group wascarried out to determine the relation between cSNPs and hypertension.2. WNK4 gene expression levels detected by RT-PCR; RNA was extracted from fetal tissues by TRIzol reagent and was used as templates to perform RT-PCR and analyze WNK4 expression level.3. Effects of different factors on WNK4 expression; COS-7 cells were cultured 24 hours with different treatments such as recombinant human estrogen, growth hormone, Insulin, dexamethasone, angiotensinll. Then mRNA of these cells was prepared and expression of WNK4 gene in transcriptional level was detected by RT-PCR and Northern blot.4. Identification of WNK4 transcription initiation site; WNK4 transcription initiation site was identified by 5’ Marathon RACE with human renal Marathon cDNA as template. 3’specific primer was designed according to WNK4 sequence in GenBank: 5 TGGGTCTCCATGTCCTCCTTTD ’. Corresponding cDNA fragments were obtained by amplifying human Marathon cDNA library by PCR method. Using this PCR product diluted 50 times as template, a nested PCR was performed to identify the transcription initiation site.5. Construction of WNK4 gene pCAT-promoter reporter expression vectors: genomic DNA as template, forward primer; 5 ’ CACTGACCTCTCCGTTCGGC 3’ ,reward primer; 5 ’ CAGATCATTCAGGGCAAAGAC 3 ’. WNK4 promoters were amplified by PCR under the condition of 95 C 5min for pre-denaturation, 35cycles of 94C lmin,60C lmin,72 C IminSOs, and then 721 10 min for extension. After purification, PCR products were ligated to pMD18 clone vectors. After sequencing, the plasmid was digested by Hind III and Xbal. Purified products were ligated to pCAT vector to construct pCAT-WNK4-promoter reporter vectors.6. Cis-acting elements and trans-acting factors of WNK4 gene promoters were identified by EMSA.7. Identification of function of WNK4 gene promoter GRE; COS-7 cells were transfected through lipofectin transfection method. Cells were harvested after treatments with InM, 10nM dexamethasone and blank control respectively. CAT protein expression level was detected by enzyme-linked immunosorbant assay (ELISA).Results1. A new SNP ( G1662A) was found in exon 7 of WNK4 gene through sequencing in small samples. The genotype frequencies of G1662A and another cSNP in exon 8更多还原