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功能磁性纳米颗粒在原发性高血压易感性研究中的应用
Application of Functional Magnetic Nanoparticles in the Study of Essentional Hypertension Susceptibility
【作者】 刘丽赏；
【作者基本信息】 湖南工业大学 ， 生物医学工程， 2010， 硕士
【摘要】 研究复杂疾病背后的遗传作用有助于有效药物的开发。原发性高血压是最常见的心血管疾病,其发病率逐年上升,是遗传因素与环境因素综合作用的结果,原发性高血压呈家族聚集现象进一步说明遗传基因的存在。而肾素-血管紧张素系统（Renin-angitensin System,RAS）基因群中血管紧张素原（Angiotensinogen AGT）基因与原发性高血压（Essential Hypertension EH）相关性的研究成为人们关注的焦点。为了进一步揭示AGT基因与原发性高血压的相关性,我们对AGT基因两个多态位点:M235T位点和A-6G位点与湖南地区原发性高血压的易感相关性进行了研究。我们制备了平均粒径为30 nm的γ-Fe2O3颗粒,颗粒的饱和磁化强度为48.4 emu/g,具有超顺磁性,并对其进行了链霉亲和素修饰,实验证明链霉亲和素修饰的γ-Fe2O3磁性颗粒具有很好的生物活性,可以进一步用于生物检测。利用生物素与链霉亲和素系统的超强特异结合作用,将链霉亲和素修饰的γ-Fe2O3磁性颗粒应用于下一步SNP分型检测中。本文中,我们收集了湖南地区243例原发性高血压样本,247例正常对照样本,并对血管紧张素原基因中的两个最受关注的位点M235T位点和A-6G位点进行易感性研究。首先扩增生物素标记的PCR产物,将其固定于链霉亲和素标记的磁性纳米颗粒上,然后与双色荧光标记（Cy3和Cy5）的等位基因特异性探针进行杂交;最后通过扫描以获得样本的基因型。利用该方法,我们成功地对来自243个高血压及正常样本的2个位点进行了分型,对于纯合型样本,其正错配信号强度比均大于3,杂合型样本其Cy3:Cy5信号强度比介于0.64和1.7之间,分型方法经测序验证。通过用SPSS 13.0分析软件对分型结果进行统计学分析,发现原发性高血压病例组血管紧张素原基因M235T位点CC,CT,TT基因型频率与正常对照组比较差异有显著性意义（χ2=5.532,P=0.063）;C,T等位基因频率与正常对照组比较差异有显著性意义（χ2=5.745,P=0.017）。提示M235T位点中T等位基因是湖南地区人口原发性高血压的危险因素。原发性高血压病例组血管紧张素原基因A-6G位点AA,AG,GG基因型频率与正常对照组比较差异无显著性意义（χ2=1.701,P=0.427）;A,G等位基因频率与正常对照组比较差异无显著性意义（χ2=1.436,P=0.231）。提示AGT基因A-6G多态不是湖南地区人口原发性高血压发生的危险因素。
【Abstract】 The study of genetic effect of complex diseases benefits to the exploitation of effective medicines. Essential hypertension （EH） is a common kind cardiovascular disease, with growing up morbidity. EH is a result of multiple reactions of genetics and environment. The symptom of family EH certified the existence of genetic factor in a further step. The susceptibility between angiotensinogen gene of rennin-angitensin system gene group and EH has become a hot topic.In order to show the susceptibility of AGT and EH, the relationship between EH and two loci M235T and A-6G located in angiotensinogen gene was studied.In this work, superparamagneticγ-Fe2O3 magnetic nanoparticles with average size of 30 nm were prepared, with magnetic saturation of 48.4 emu/g. The particles functionalized with streptavidin were proved to be bio-active and could be used in biology test. Utilizing the super specific bonding effect of biotin-streptavidin system, streptavidin modifiedγ-Fe2O3 nanoparticles were used in SNP detection. 243 EH samples and 247 healthy samples in Hunan area were collected. The SNP of two loci, M235T and A-6G located in angiotensinogen gene, were detected. PCR products were modified on the streptavidin-γ-Fe2O3 nanoparticles, then hybridized with two probes labeled with two fluorescence colors: Cy5 and Cy3. The hybridization results were scanned with a GenePix 4100 scanner. When the sample is homozygous, the ratio of Cy3 to Cy5 is higher than 3. When the sample is heterozygous, the ratio is between 0.64 and 1.7. This SNP typying method had been proved to work well.After statistic analysis using SPSS 13.0 software, the difference of gene type CC, CT, TT ratio on M235T locus between the sick group of EH and the healthy group was found significant （χ2=5.532,P=0.063）. And so was the difference between the allele gene C, T （χ2=5.745,P=0.017）. The morbidity of individuals with mutation type TT was found greater than the others. There may be some correlation of the mutation type TT with essential hypertension in people of Hunan area. The difference of gene type AA, AG, GG ratio on A-6G site between the sick group of the EH and healthy group was found not significant （χ2=1.701,P=0.427）. And neither was the difference between the allele gene C, T （χ2=1.436,P=0.231）. The SNP of A-6G locus may not be a dangerous factor of essential hypertension in people of Hunan area.