摘要
目的
缺血性脑卒中是导致人类死亡的三大疾病之一,目前认为缺血性脑卒中的发病是由多种因素决定的,遗传因素对缺血性脑卒中的易感性起到决定性的作用,有关基因多态性和基因突变对缺血性脑卒中发病的潜在影响的研究成为当前缺血性脑卒中研究的热点。通过对易感基因多态性的研究,将有助于从分子水平上认识缺血性脑卒中的发病机制,有望为缺血性脑卒中从分子水平上彻底治疗开辟崭新的途径。动脉粥样硬化是缺血性脑卒中的基础病因,炎症反应是动脉粥样硬化性疾病发生发展的关键环节,循环中的炎症细胞在内皮细胞上的黏附及向血管外迁移是炎症反应的起始环节。血小板内皮细胞黏附分子-1 (platelet endothelial cell adhesion molecule-1, PECAM-1)是黏附在内皮上的白细胞进行跨膜转运,向血管外游走的必要条件。本研究探讨血小板内皮细胞黏附分子-1(PECAM-1)基因Leu125Val与Asn563Ser位点的基因多态性与中国北方人群(辽宁地区)缺血性脑卒中的关系。
方法
1.选择169例缺血性脑卒中患者(病例组)及年龄、性别相匹配的186例正常者(对照组),测定与缺血性脑卒中相关的临床指标,包括血脂,血糖,高血压,BMI等。
2.受试者禁食12小时后,患者于入院第二天清晨、体检者于体检当日,抽取外周静脉血2ml, EDTA抗凝。
3.用DNA提取试剂盒提取基因组DNA,并进行DNA定量和纯度分析。
4.用premier5.0设计引物,并由生物公司制备引物。
5.用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测其PEC AM-1基因型。PCR产物直接测序,验证PCR-RFLP的结果。此步骤由上海生工生物工程技术服务有限公司完成。
6.比较两组基因型及等位基因频率分布及与缺血性脑卒中的关系。并分析两组之间上述临床资料和实验指标。
结果
1.缺血性脑卒中组PECAM-1 L125V位点LL、LV、VV基因型的频率分别为：17.2％、51.5％、31.3％,对照组分别为23.1％、56.5％、20.4％,基因型频率符合Hardy-Weinberg平衡,病例组与对照组PECAM-1 LI25V基因型分布比较具有统计学差异(X2=6.082, P=0.048)。PECAM-1S563A多态性位点AA, AS, SS基因型的频率分别为29.0％,51.5％,19.5％。对照组分别为23.7％,53.2％,23.1％。基因型频率符合Hardy-Weinberg平衡。病例组和对照组PECAM-1 S563A基因型分布比较不具有统计学意义(X2=1.385,p=0.5)。
2.缺血性脑卒中组PECAM-1 L125V的V等位基因频率为57.1%,对照组为48.7％,V等位基因在缺血性脑卒中组和对照组的分布差异具有显著性(X2=5.066,p=0.024)。病例组PECAM-1 S563A的A等位基因频率为54.7%,对照组A等位基因频率为50.3％。病例组和对照组的等位基因频率也不具有统计学意义(X2=1.258,p=0.262)。
3.Logistic回归分析结果表明PECAM-1L125V多态性位点VV基因型是缺血性脑卒中的独立危险因素(OR=2.467,95%CI：1.518-4.009,p=0.000)。
结论
PECAM-1基因Leu125Val位点的基因多态性与缺血性脑卒中相关,VV基因型可能是中国北方人群缺血性脑卒中的遗传易感因子。
Ischemic stoke is one of three diseases leading to death.It is considered a variety of factors leading to ischemic stroke. Genetic factors play a decisive role in the susceptibility of ischemic stroke. The study of potential impacts of gene polymorphism and gene mutation to the incidence of ischemic stroke is a hot spots. Through the study of gene polymorphism,it will help to understand the molecular level of pathogenesis of ischemic stroke, which is expected to open up a completely new way in ischemic stroke therapy at molecular level. Atherosclerosis is the basis etiology of ischemic stroke, is a key point in the occurrence and development of atherosclerosis disease,the beginning link of inflammatory reaction is circulating inflammatory cells adhesion in endothelial cell and migration extravascular. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a necessary condition for the adhesion of leukocytes to transport the endothelium and transmigrate to foreign vessels.The aim of this study is to pinpoint whether single nucleotide polymorphisms(SNPs) in the genes coding Leu125Val and Asn563Ser for PECAM-1 are associated with CIS susceptibility in northern population of China(liaoning area).
Methods
1.169 subjects with CIS and 186 controls are enrolled in the study. Determination the clinical indication associated with CIS including blood fat,blood glucose,high blood pressure and BMI.
2.Subjects fast 12 hours later, the patients are admitted the next morning, volunteers are on the examination day, collecting peripheral venous blood 2ml, with EDTA anticoagulant.
3. Using DNA extraction kit to extract genomic DNA, and conducting DNA quantification and purity analysis.
4. Primers are designed by premier5.0, Preparate primers by biotech companies.
5.PEC AM-1 genotype was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Direct sequencing of PCR product to verify PCR-RFLP results. This step is completed by Shanghai Public Health Service Co., Ltd. bio-engineering techniques.
6. Comparison the relationship between the two groups of genotype and allele frequency distribution with ischemic stroke.
Results
1.Cerebral infarction group PEC AM-1 L125 V locus LL, LV, VV genotype frequencies are as follows:17.2%,51.5%,31.3%,in the control group are 23.1%, 56.5%,20.4%, genotype frequencies consistent with Hardy-weinberg balance. Cerebral infarction group and the control group genotype distribution of PECAM-1 LI25V compared with a statistically significant difference (X2= 6.082, P= 0.048). Cerebral infarction group PECAM-1 S125A locusAA, AS,SS genotype frequencies were as follows:29.0%,51.5%,19.5%,in the control group were 23.7%,53.2%, 23.1%,genotype frequencies consistent with Hardy-weinberg balance.There is less statistically significant between case group and control group (X2=1.258, p=0.262).
2. Cerebral infarction group PECAM-1 L125V V-allele frequency is 57.1%, control group 48.7%, V allele in the cerebral infarction group and the control group were significant differences in the distribution of (X2= 5.066, P= 0.024). Cerebral infarction group PECAM-1 S563A A-allele frequency is 54.7%, control group 48.7%, There is also less statistically significant between case group and control group (X2=1.258, p=0.262)
3. Logistic regression analysis shows that PECAM-1 L125V polymorphic VV-based type is a result of atherosclerosis independent risk factor for cerebral infarction (OR=2.467,95%CI:1.518-4.009, P=0.000).
Conclusion
PECAM-1 gene Leu125Val locus gene polymorphism and ischemic stroke-related, VV genotype may be genetic susceptibility factor of ischemic stroke in Chinese population
引文
1 Thomopson RD, Wakelin MW, Larbi KY, et al. Divergent effeets platelet endothe-lial cell adhesion molecule-1 and beta3integrin blockade on Leukocyte transmigration invivo.J Immunol.2000; 165(1):426-434.
2 Sun J, Paddock C, Shubert J, et al.Contributions of the extracellular and cytoplasmic domains of platelet-endothelial cell adhesion moleeule-1(PECAM-1/CD31) inregulating cell localization.J Cell Sci.2000; 113(8):1459-1694.
3 Dimitrijevic I, Axelsson L, Andersson T. The adhesion receptor CD-31 can be primed to rapidly adjust the neutrophil cytoskeleton. Biochem Biophys Res Commun.2002; 292(4):1092-1097.
4 Robson P. Stein P, Zhou B, et al.Inner cellmass-specific expression of a cell adhesion molecule(PECAM-1/CD31) in the mouse blasocyst.DevBiol.2001; 234(2):317-329.
5 Andreotti F, Porto I, Crea F, et al. Inflammatory gene polymorphisms and ischaemic heart disease:review of population association studie Heart.2002; 87(2):107-112.
6 亢爱春,敖彩卉,郭宏怡,等.血小板内皮细胞粘附分子-1基因多态性及血浆水平与严重冠脉粥样硬化病变的相关性.中华老年多器官疾病杂志.2008；4(7)：84-89.
7 Sasaoka T, Kimura A, Hohta SA, et al.Polymorphisms in the platelet endothelial cell adhision molecule-1 (PECAM-1) gene,Asn563Ser and Gly670Arg,associated with myocardial infarction in the Japanese.Ann NY Acad Sci.2001; 947:259-269.
8 Wei H, Fang L, Sanual H, et al.Platelet endothelial cell adhesion molecule-1 gene polymorphism and its soluble level are associated with severe coronary artery stenosis in the Chinese Singaporean.Clin Biochem.2004; 37:1091-1097.
9 ListiF, Candore G, Lio D, et al.Association between platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31 polymorphisms and acute myocardial infarction:a study in patients from Sicily.Eur J Immunogenet.2004; 31:175-178.
10 Novinska M.S., Pietz B.C., Ellis T.M., et al. The alleles of PECAM-1. Gene.2006; 376(1): 95-101.
11 Robbins FM, Hartzman RJ. CD31/PECAM-1 genotyping and haplotype analyses show population diversity. Tissue Antigens.2007; 69(1):28-37.
12 Elrayess MA, Webb KE, Bellingan GJ.R643G polymorphism in PEC AM-1 influences transendothelial migration of monocytes and is associated with progression of CHD and CHD events. Atherosclerosis.2004; 177(1):127-35.
13 Wei H, Fang L, Chowdhury SH, et al. Platelet-endothelial cell adhesion molecule-1 gene polymorphism and its soluble level are associated with severe coronary artery stenosis in Chinese Singaporean. Clin Biochem.2004; 37(12):1091-1097.
14 Fang L, Wei H, Chowdhury SH, et al.Association of Leu125Val polymorphism of platelet endothelial cell adhesion molecule-1 (PECAM-1) gene& soluble level of PEC AM-1 with coronary artery disease in Asian Indians. Indian J Med Res.2005; 121(2):92-99.
15 Wei YS, Lan Y, Liu YG, et al. Platelet-Endothelial Cell Adhesion Molecule-1 Gene Polymorphism and Its Soluble Level Are Associated with Ischemic Stroke.DNA Cell Biol. 2009 Feb 1.
16 Davis NE.Atherosclerosis--an inflammatory process.J Insur Med.2005; 37(1):72-75.
17 Elkind MS. Inflammation, atherosclerosis, and stroke. Neurologist.2006; 12(3):140-148.
18 Grimaldi MP, Vasto S, Balistreri CR, et al. Genetics of inflammation in age-related atherosclerosis:its relevance to pharmacogenomics. Ann N Y Acad Sci.2007; 1100:123-131.
19 Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med.1999; 340(2):115-126.
20 Andreotti F, Porto I, Crea F, et al. Inflammatory gene polymorphisms and ischaemic heart disease:review of population association studies. Heart.2002; 87(2):107-112.
21 Blankenberg S, Barbaux S, Tiret L. Adhesion molecules and atherosclerosis. Atherosclerosis. 2003; 170:191-203.
22 Muller WA, Weigl SA, Deng X, et al. PECAM-1 is required for transendothelial migration of leukocytes. J Exp Med.1993; 178:449-460.
23 Varon D, Jackson DE, Shenkman B, et al. Platelet-endothelial cell molecule-1 serves as a costimulatory agonist receptor that modulates integrin dependent adhesion and aggregation of human platelets. Blood.1998; 91:500-507.
24 Litwin M, Clark K, Noack L, et al. Novel cytokine-independent induction of endothelial adhesion molecules regulated by platelet-endothelial cell adhesion molecule (CD31). J Cell Biol.1997; 139:219-228.
25 Paul S, Newman DK, Newman P, et al.Platelet endothelial cell adhesion molecule-1 severe as aninhibitory receptor that modulates platelet responses to collagen.Blood.2001; 97:1727-1732.
26 Auer J, Weber T, Berent R, et al. Genetic polymorphisms in cytokine and adhesion molecule genes in coronary artery disease. Am J Pharmacogenomics.2003; 3(5):317-328.
27 Karen L, Jones, Saseha C, et al.Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet collagen interactions.Blood.2001; 98:1456-1463.
28 Jeron A, Hengstenberg C, Engel S, et al.The D-allete of the ACE polymorphism is related to increased QT dispersion in 609 patienis after mycardial infarction.Eur Heart J.2001; 22: 663-668.
29曾军,洪震,黄茂盛,等.脑卒中患者主要危险因素检测.中华老年心脑血管病杂志.2002；4(3)：173-175.
30 Vilas AP, Veiga MZ, Santos ME, et al.Hemorrhagic stoke:experience of an internal medicine department.Rev Port Cardiol.2001; 20157-20165.
1 Libby P.Inflammation and cardiovascular disease mechanisms.Nutrition.2006; 83(2):456-460.
2 Chamorro A, Hallenbeck J.The Harms and Benefits of Inflammatory and Immune Responsesin Vascular Disease.Stroke.2006; 37:291-293.
3 Togashi H, Sakisaka T, Takai Y.Cell adhesion molecules in the central nervous system.Cell Adh Migr.2009; 3(1). [Epub ahead of print]
4 Qing W, XianNTT, Midori A.The Inflmmatory Responses in Stroke.Neuroimmunol.2007; 184(122):53-68.
5 Dimitrijevic I, Axelsson L, Andersson T. The adhesion receptor CD-31 can be primed to rapidly adjust the neutrophil cytoskeleton. Biochem Biophys Res Commun.2002; 292 (4): 1092-1097.
6 亢爱春,敖彩卉,郭宏怡,等.血小板内皮细胞粘附分子-1基因多态性及血浆水平与严重冠脉粥样硬化病变的相关性.中华老年多器官疾病杂志.2008；4(7)：84-89.
7 Sasaoka T, Kimura A, Hohta SA, et al.Polymorphisms in the platelet-endothelial-cell-adhision-molecule-(PECAM-1)-gene,Asn563Ser and Gly670Arg,associated with myocardial infarction in the Japanese.Ann NY Acad Sci.2001; 947:259-269.
8 Wei H, Fang L, Sanual H, et al.Platelet-endothelial cell adhesion molecule-1 gene polymorphism and its soluble level are associated with severe coronary artery stenosis in the Chinese Singaporean.Clin Biochem.2004; 37:1091-1097.
9 ListiF, Candore G, Lio D, et al.Association between platelet endothelial cellular adhesion molecule-1 (PECAM-1/CD31 polymorphisms and acute myocardial infarction:a study in patients from Sicily.Eur J Immunogenet.2004; 31:175-178.
10 Jackon DE.The unfolding tale of PECAM-1.Febs Letts.2003; 540(1-3):7-14.
11 Wang Y, Sheibani N.Expression pattern of alternatively spliced PECAM-1 isoforms in hematopoietic cells and platelets.J Cell Biochem.2002; 87(4):424-438.
12 Wang Y, Su X, Wu Z, et al.Thrombospondin-1 degicientmice exhibit and altered expression pattern of alternatively spliced PECAM-1 isoforms in retinal vasculature and endothelial cells.J Cell Physiol.2005. [Epub ahead of print]
13 Wang Y, Repyak K, Sheibani N, et al.Expression pattern of alternatively spliced PECAM-1 isoforms in retinal vasculature.MolVis.2004; 10(1):103-111.
14 Robson P, Stein P, Zhou B, et al.Inner cellmass-specific expression of a cell adhesion molecule(PECAM-1/CD31) in the mouse blasocyst.DevBiol.2001; 234(2):317-329.
15 Andreotti F, Porto I, Crea F, et al. Inflammatory gene polymorphisms and ischaemic heart disease:review of population association studieHeart.2002; 87(2):107-112.
16 DeLisser HM, Newman PJ, Albelda SM. Molecular and functional aspects of PECAM-1/CD31. Immunol Today.1994; 15(10):490-495.
17 Newman PJ. The biology of PECAM-1. J Clin Invest.1997; 100(11 Suppl):S25-29.
18 WnLzel K, Baunnannq Felix SB.The homogous combination of Leul25Val and Ser563Asn Polymophisms in the PECAM-1(CD31)gene are associated with early severe coronary heart disease.HumMut.1999; 14:545.
19 Hewett PW, Murray JC. Human microvessel endothelial cells:isolation, culture and characterization.In Vitro Cell Dev Biol Anim.1993; 29A(11):823-830.
20 Paul S, Newman DK, Newman P, et al.Platelet endothelial cell adhesion molecule-1 severe as aninhibitory receptor that modulates platelet responses to collagen.Blood.2001; 97:1727-1732.
21 Falati S, Patil S, Gibbins J, et al.Opposing effects of P-selection and PECAM-1 on arterial thrombus growth and stability in vivo. J Thromb Haemost.2003; 1(7):12-18.
22 Gibbins JM.Platelet adhesion signalling and the regulation of thrombus formation. J Cell Sci. 2004; 17(16):3415-3425.
23 Baumann CI, Bailey AS, Li W,et al. PECAM-1 is expressed on hematopoietic stem cells throughout ontogeny and identifies a population of erythroid progenitors. Blood.2004; 104(4):1010-6.
24 Woodfin A, Voisin MB, Nourshargh S. PECAM-1:a multi-functional molecule in inflammation and vascular biology. Arterioscler Thromb Vasc Biol.2007; 27(12):2514-23.
25 Garcia VE, Chuluyan HE. SLAM and CD31:signaling molecules involved in cytokine secretion during the development of innate and adaptive immune responses. Cytokine Growth Factor Rev.2007; 18(1-2):85-96.
26杨颖,罗晖.血小板内皮细胞粘附分子-1(PECAM-1)的研究与疾病.川北医学院学报,2008：23(6)：627-630.
27杨颖,程龙献.某些炎症因子基因多态性与冠心病关系的研究进展.心脏杂志,2006；18(6)：710-712.
28 Elrayess MA, Talmud PJ. Platelet endothelial cell adhesion molecule-1 (PECAM-1)& coronary heart diseaselndian J Med Res.2005; 121(2):77-9.
29 Wenzel K, Baumann G, Felix SB. The homozygous combination of Leu125Val and Ser563Asn polymorphisms in the PEC AMI (CD31) gene is associated with early severe coronary heart disease. Hum Mutat.1999; 14(6):545.
30 Gardemann A, Knapp A, Katz N,et al.No evidence for the CD31 C/G gene polymorphism as an independent risk factor of coronary heart disease.Thromb Haemost.2000; 83(4):629.
31 Auer J, Weber T, Berent R,et al. Genetic polymorphisms in cytokine and adhesion molecule genes in coronary artery disease. Am J Pharmacogenomics.2003; 3(5):317-28.
32宋福春,陈爱华,唐晓明,等.血小板内皮细胞粘附因子基因多态性与冠心病的关系.第一军医大学学报,2003；23(2)：156-158.
33 Fang L, Wei H, Chowdhury SH,et al. Association of Leu125Val polymorphism of platelet endothelial cell adhesion molecule-1 (PECAM-1) gene & soluble level of PECAM-1 with coronary artery disease in Asian Indians. Indian J Med Res.2005; 121(2):77-9.
34满晓,崔元孝,朱海英.急性脑梗塞患者血小板表达PECAM-1的动态变化和意义.山东医药,2008；48(22).
35 Wei YS, Lan Y, Liu YG,et al. Platelet-Endothelial Cell Adhesion Molecule-1 Gene Polymorphism and Its Soluble Level Are Associated with Ischemic Stroke.DNA Cell Biol. 2009 Feb 1.