高血压靶器官（脑、心）损害基因表达变化的基础与临床研究
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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AT_1表达、Ca~(2+)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
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
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AT_1表达、Ca~(2+)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
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
引文
1． Kanayama N, Khatun S, Belayet H, et al. Chronic local cold stress to the soles induces hypertension in rats. Am J Hypertens. 1999 Nov; 12(11 Pt 1) : 1124-9
2． Roukoyakina NI, Chefer SI, Rifkind J, et al. Cold acclimation-induced increase of systolic blood pressure in rats is associated with volume expansion. Am J Hypertens 1999; 12: 54-62
3． Hatton DC, McCarron DA. Dietary salt and hypertension. Curr Opin in Nephro and Hypertens 1996; 5: 166-169
4． Weinberger MH. Salt sensitivity: does it play an important role in the pathogenesis and treatment of hypertension. Curr Opin in Nephro and Hypertens 1996; 5: 205-208
5． Kanayama N, Tsujimura R, She L, et al. Cold-induced stress stimulates the sympathetic nervous system, causing hypertension and proteinuria in rats. J Hypertens. 1997 Apr;15(4) :383-9
6． Fujiwara T, Kawamura M, Nakajima J, et al. Seasonal difference in diurnal blood pressure of hypertensive patients living in stable environmental temperature. J Hypertens 1995; 13:1747-1752
7． Minami J, Kawano Y, Ishimitsu T, et al. Seasonal variations in office, home and 24h ambulatory blood pressure in patients with essential hypertension. J Hypertens 1996; 14: 1421-1425
8． Papanek PE, Wood CE, Fregly MJ. Role of the sympathetic nervous system in cold-induced hypertension in rats. J Appl Physiol 1991; 71(1) : 300-306
9． Siani A, Guglielmucci F, Farinaro E, et al. Increasing evidence for the role of salt and salt-sensitivity in hypertension. Nutr Metab Cardiovasc Dis. 2000 Apr;10(2) :93-100．
10． Giner V, Poch E, Bragulat E, et al. Renin-angiotensin system genetic polymorphisms and salt sensitivity in essential hypertension. Hypertension. 2000 Jan;35(l Pt 2) :512-7．
11． Strehlow K, Nickenig G, Roeling J, et al. AT(1) receptor regulation in salt-sensitive hypertension. Am J Physiol. 1999 Nov;277(5 Pt 2) :H1701-7．
12． Hamet P, Pausova Z, Adarichev V, et al. Hypertension: genes and environment. J Hypertens 1998; 16(4) : 397-418
13． Benfante R. Studies of cardiovascular disease and cause-specific mortality trends in Japanese-American men living in Hawaii and risk factor comparisons with other Japanese populations in the Pacific region: a review. Hum Biol 1992 Dec;64(6) :791-805
14． Yamori Y, Nara Y, Mizushima S, et al. Gene-environment interaction in hypertension, stroke and atherosclerosis in experimental models and supportive findings from a world-wide cross-sectional epidemiological survey: a WHO-cardiac study. Clin Exp Pharmacol Physiol Suppl 1992;20:43-52
15． Gale CR, Martyn CN, Winter PD, et al. Vitamin C and risk of death from stroke and coronary heart disease in cohort of elderly people. BMJ 1995 Jun 17;310(6994) : 1563-6
16． Stout RW, Crawford V. Seasonal variations in fibrinogen concentrations among elderly people. Lancet 1991 Jul 6;338(8758) :9-13
17． Jaillard AS, Hommel M, Mazetti P. Prevalence of stroke at high altitude (3380 m) in Cuzco, a town of Peru. A population-based study. Stroke 1995 Apr;26(4) :562-8
2． Roukoyakina NI, Chefer SI, Rifkind J, et al. Cold acclimation-induced increase of systolic blood pressure in rats is associated with volume expansion. Am J Hypertens 1999; 12: 54-62
3． Hatton DC, McCarron DA. Dietary salt and hypertension. Curr Opin in Nephro and Hypertens 1996; 5: 166-169
4． Weinberger MH. Salt sensitivity: does it play an important role in the pathogenesis and treatment of hypertension. Curr Opin in Nephro and Hypertens 1996; 5: 205-208
5． Kanayama N, Tsujimura R, She L, et al. Cold-induced stress stimulates the sympathetic nervous system, causing hypertension and proteinuria in rats. J Hypertens. 1997 Apr;15(4) :383-9
6． Fujiwara T, Kawamura M, Nakajima J, et al. Seasonal difference in diurnal blood pressure of hypertensive patients living in stable environmental temperature. J Hypertens 1995; 13:1747-1752
7． Minami J, Kawano Y, Ishimitsu T, et al. Seasonal variations in office, home and 24h ambulatory blood pressure in patients with essential hypertension. J Hypertens 1996; 14: 1421-1425
8． Papanek PE, Wood CE, Fregly MJ. Role of the sympathetic nervous system in cold-induced hypertension in rats. J Appl Physiol 1991; 71(1) : 300-306
9． Siani A, Guglielmucci F, Farinaro E, et al. Increasing evidence for the role of salt and salt-sensitivity in hypertension. Nutr Metab Cardiovasc Dis. 2000 Apr;10(2) :93-100．
10． Giner V, Poch E, Bragulat E, et al. Renin-angiotensin system genetic polymorphisms and salt sensitivity in essential hypertension. Hypertension. 2000 Jan;35(l Pt 2) :512-7．
11． Strehlow K, Nickenig G, Roeling J, et al. AT(1) receptor regulation in salt-sensitive hypertension. Am J Physiol. 1999 Nov;277(5 Pt 2) :H1701-7．
12． Hamet P, Pausova Z, Adarichev V, et al. Hypertension: genes and environment. J Hypertens 1998; 16(4) : 397-418
13． Benfante R. Studies of cardiovascular disease and cause-specific mortality trends in Japanese-American men living in Hawaii and risk factor comparisons with other Japanese populations in the Pacific region: a review. Hum Biol 1992 Dec;64(6) :791-805
14． Yamori Y, Nara Y, Mizushima S, et al. Gene-environment interaction in hypertension, stroke and atherosclerosis in experimental models and supportive findings from a world-wide cross-sectional epidemiological survey: a WHO-cardiac study. Clin Exp Pharmacol Physiol Suppl 1992;20:43-52
15． Gale CR, Martyn CN, Winter PD, et al. Vitamin C and risk of death from stroke and coronary heart disease in cohort of elderly people. BMJ 1995 Jun 17;310(6994) : 1563-6
16． Stout RW, Crawford V. Seasonal variations in fibrinogen concentrations among elderly people. Lancet 1991 Jul 6;338(8758) :9-13
17． Jaillard AS, Hommel M, Mazetti P. Prevalence of stroke at high altitude (3380 m) in Cuzco, a town of Peru. A population-based study. Stroke 1995 Apr;26(4) :562-8