高血压靶器官（脑、心）损害基因表达变化的基础与临床研究

【作者】 王先梅；

【导师】 祝善俊；

【作者基本信息】 第三军医大学 ， 内科学， 2001， 博士

【摘要】 背景与目的 高血压病为临床常见病与高发病，其发病机理仍未完全阐明，目前多认为由环境因素与遗传因素相互作用引起。在我国，冬季心脑血管疾病急症也明显增加。另外。在盐敏感性方面也存在个体差异，敏感者过多的钠盐摄入可使血压升高。脑卒中是高血压病最严重的并发症之一。但引起脑血管病的原因很多，遗传因素和环境因素均起重要作用。环境因素的作用使得基因的表达谱发生改变。在众多环境因素中，高盐和寒冷环境对脑血管功能调节起重要作用。因此，我们认为高盐饮食和特殊的环境温度可能对我国脑卒中的发生有重要影响。在以往的研究中，我们选择了高盐与冷应激作用于正常Wistar大鼠，第二到三周大鼠血压即明显升高。血RAAS、平滑肌AT₁表达、Ca²⁺及动脉壁内皮NOS/NO均有明显改变。但在上述两因素诱导的高血压模型上脑卒中的发病率如何，该模型是否可以实验模拟人类高血压诱发的脑卒中的实际情况，仍未进一步深入研究。 最近，我们建立了非外科手术和非遗传因素，单纯由环境因素诱发的大鼠卒中模型，发现给予Wistar大鼠高盐饮食（8％，G/G）加冷刺激（4±2℃，每天4小时）共8周后，仅10～17％的大鼠出现卒中样发作。上述结果提示在相同的种属遗传背景下，是否出现卒中，可能存在环境因素诱导的卒中易感基因。由于方法学的原因，已有的研究仍局限在某个候选基因上，脑卒中致病基因的大规模筛选，尤其与环境因素诱发的高血压相联系，国内外还未见报道。 除了大脑以外，心脏同样是高血压危害的重要靶器官之一。高血压心脏损害易并发心肌缺血、心律失常、心力衰竭和狩死。心肌细胞同样是高度依赖线粒体供能的细胞之一。线粒体基因3243位点突变可引起人类多种疾病。 鉴于上述问题，本研究主要：1．探讨环境因素对大鼠血压的影响。2．筛选环境因素造成脑卒中的易感基因。3．探讨线粒体基因3243位点多态性与高血压靶器宫损害的关系。 方法 3月龄的雄性 Wistar大鼠 100只分为四组：（l）冷刺激组（28只）：每天给予冷刺激（4土ZOC，每天4小时）；（）复合组（28只）：给予冷刺激（4上 2 oC，每天 4小时）及高盐饮食（8％，G／G）；（）高盐组 （8只）：每天给予高盐饮食（8％，G／G）；（4）正常对照组（16只）。实验时间共8周。每周测鼠尾压、心率；．运用复合环境因素造成脑卒中的鼠脑和正常WIStar大鼠鼠脑作为组织来源，建立两个差减文库；将两个文库进行前向和反向杂交，PCR扩增出差异片段，并对这些片段进行测序；利用NCBI网络资源，对差异片段作生物学信息分析；收集临床高血压患者、脑卒中患者及高血压伴有心脏患者的血液标本，提取DNA，应用限制性酶切片段长度多态性方法，观察患者线粒体基因突变。 结果 1．血压与心率：第一周末，寒冷、高盐、复合组的尾压、心率比对照组均明显升高（P＜0刀1），三周末血压达到最高（分别为127刀土6刀，125．9t5，5，131t6刀mmHg）。 2．生物学信息分析：每组随机挑取288个白色菌落，两组共576个。随后对这些克隆进行了序列测定。其中正常组共有230个序列可用，卒中组共有226个序列可用，其余的不可用的序列为测不通的序列或载体自连。全部可用序列均通过互联网在NCBI序列检索中心进行BLAST比较分析，其中正常组 119个克隆在 nr中有明显的同源序列，同源性均在 90％以上，占 51．7％，被认为是己知基因；95个克隆在 dbEST中有明显的同源序列，占41．3％，被认为是已被发现的EST；剩余的16个克隆在nr或dbEST均未发现同源序列，被认为是新基因片段，占 7％。而卒中组 126 Vlll个克隆在nr中有明显的同源序列，同源性均在90％以上，占55．8％，被认为是已知基因；78个克隆在dbEST中有明显的同源序列，占34．5％，被认为是己知的EST；剩余的22个克隆在nr或dbEST均未发现同源序列，被认为是新基因片段，占9．7％。与dbEST同源的序列及克隆的新片段目前功能仍然未知。从功能分类来看，脑卒中时线粒体基因（43个克隆与线粒体基因高度同源，Prto刀1）表达明显上调，而细胞与机体防御基因表达明显下调0 刀1人 3．线粒体基因3243位点多态性：BSp 1201（G”GGCCC）酶切PCR产物，病例及对照组PCR产物酶切后琼脂糖iB凝胶电泳均呈单一条带，未见 2个条带。说明不能被 BSp 120切开。 结论 1．应用复合环境因素成功地复制了大鼠高血压模型；环境因素对血压有明显影响。 2．应用SSH，我们筛选了576个克隆，并对其进行分析，其中正常组 119个克隆被认为是已知基因，占引尸％；95个克隆被认为是已被发现的EST，占41．3％；剩余的16个克隆被认为是新基因片段，占7％。而卒中组126个克隆被认为是已知基因，占55．8％；78个克隆被认为是已知的EST，占34．5％；剩余的22个克隆被认为是新基因片段，占9．7％。从功能分类来看，脑卒更多还原

【Abstract】 Background Hypertension is one of the most common cardiovascular diseases, its pathogenic mechanism is still unclear. Hypertension is considered a complex disease with significant genetic and environmental components that interact to play a role in blood pressure variation, and hypertension is now as a polygenic disease with complexities such as "gene-gene" and "invironment- gene" interactions. In China, the incidence of cerebrovascular accidents is significantly increased in winter. Moreover, there is such heterogeneity concerning the different responses of blood pressure to dietary salt intake, there are sensitive individuals who respond to a high sodium intake by an increase of blood pressure and others who do not. Clinical and epidemiological studies have provided strong evidence for the relationship between hypertension and stroke. Stroke is a common consequence of hypertension and a complex disorder caused by a combination of genetic and environmental factors. In some previous studies, environmental risk factors (cold stress plus salt loading) induced hypertension rats were used as the model. Investigation in our laboratory has revealed that high salt diet and cold stress can cause blood pressure elevation in the 2nd and 3rd week post exposure, and invoke changes of plasma RAAS levels, AT1 expression and [Ca2+] and NO/NOS system in vascular smooth muscle cells. Recently, we established a hypertensive model without surgical or pharmacological intervention which produced a complication of stroke, and found that only 10-17% Wistar rats had stroke-like episodes. These results III suggested that there may be exist genetic susceptibility. Past effort has mainly engaged in several candidate genes due to the methodological limitation, there is still no reports that at the large-scale gene screening to explore possible genetic sensitivity to stroke particularly related to environmental induced- hypertension. The heart also is one of the hypertensive target organs. Hypertension can produce severely consequences such as myocardium ischemia, arrhythmia, heart failure and sudden cardiac death. The physiological function of myocytes strongly depends on mitochondria because of its high energy demand. The mutation at 3243 locus of mitochondrial DNA can lead to many human diseases. Purposes The present study was designed: I .to explore the role of environmental factors in the development and maintenance of hypertension. 2. to establish a hypertensive model without surgical or pharmacological intervention. 3. to identify the differential gene expression pattern between the two populations, namely control and stroke group. 4. to study the mutation at 3243 locus of mtDNA in the hypertensives and controls using restriction fragment length polymorphism (RFLP). Methods One hundreds male, 2-3-month-old Wistar rats were randomly divided into 4 groups : l.Cold-treated group(C, n28): animals were exposed to cold (4?0C) for 4 hours per day for 8 weeks; 2. High salt-treated group (5, n=28): animals were given 8% NaCI diets for 8 weeks. 3. Cold-treated + high salt-treated group (CS, n=28): animals were given 8% NaCl diets for 8 weeks besides cold exposure. 4. Control group (N, n=16). Systolic blood pressure(SBP, Tail-cuff technique) and heart rate were measured weekly in 4 groups during the experiment. A new technique, suppression subtractive hybridization更多还原