血浆miRNAs表达特征作为高血压肝阳上亢证生物标记的相关性研究

To randomly select 200 hypertensive patients,100 hyperactivity and 100 non-hyperactivity of liver Yang ,another randomly selected 100 cases of healthy volunteers, and then randomly select 20 core pedigrees from each group. To investigate the risk factors of hypertension by using questionnaire and family survey methods. Blood pressure and heat rate rhythm will be detected by ambulatory blood pressure monitor(ABPM)and electrical cardiogram(ECG/EKG), ELISA and radioimmunoassay be used to detect the components of RAAS(AngII、aldosterone,etc ) ,chronic inflammatory state(CRP、IL-6、TNF-α),and the NO, ET, epinephrine and blood rheology, etc . Real-time fluorescence quantitative PCR detects the miRNAs(miRNA-17,31,33,122,126,146α,155,221,222,370,etc) associated with these physicochemical indexes and blood pressure regulation. The relationships between the expression of miRNAs and RAAS and the chronic inflammatory state components will be analyzed completely. Combined with the correlation analysis and linkage analysis, the relationship between the expression of miRNAs and hyperactivity of liver Yang of hypertension will be clear and determine the miRNAs of which is/are the genetic marker(s) of hypertension of hyperactivity of the liver-yang, and then provide experimental and theoretical bases of treatment of hypertension for Chinese medicine.

随机选择临床确诊的高血压患者，经中医辩证确诊为肝阳上亢证和非肝阳上亢证，各100例，另随机选取健康志愿者100例，并从中随机选取核心家系各20个。采用问卷和家系调查法，对所有研究对象进行高血压发病危险因素调查。动态血压、心电图、酶免或放免法检测患者血压特征、心律、RAAS（肾素、血管紧张素II等）和慢性炎症状态（CRP、TNF-α等）各组分及NO、ET、肾上腺素及血流变学等肝阳上亢证的客观化指标，实时荧光定量PCR检测与这些理化指标及血压调控相关的miRNAs（miRNA-17、31、33、122、126、146α、155、221、222、370等）的表达。全面分析miRNAs表达特征与RAAS及慢性炎症状态各组分的关系，结合群体关联和家系连锁分析，明确miRNAs与高血压肝阳上亢证之间的关系，确定高血压肝阳上亢证的miRNAs遗传标志，为肝阳上亢证形成的生物学机制研究奠定初步实验基础。

通过招募，结合纳入和排除标准，共纳入研究对象 300 例，根据数据完整性、溶血发生情况、循环 miRNAs 含量，最终非肝阳上亢证组纳入 95 例（男51例），其中家族遗传20（男17例）；肝阳上亢组 88例（男55例），其中家族遗传 20例（男17例）；正常血压人群 79例（男39例）。满足了每组最少 75 例合格的基本要求。miRNAs芯片检测结果：非肝阳上亢证组患者血清miRNAs表达与肝阳上亢组对比，有18个miRNAs上调超过2倍以上，下调16个；非肝阳上亢证组患者血清miRNAs表达与正常血压人群对比，有5个miRNAs上调超过2倍以上，下调22个；肝阳上亢证组患者血清miRNAs表达与正常血压人群对比，有25个miRNAs下调超过2倍以上。根据miRNA丰度和表达量，选取hsa-miR-16-RT，hsa-miR-16-AS，hsa-miR-483-3p-2-RT，hsa-miR-483-3p-2-AS等四个miRNAs最为目标miRNA，进行Q-PCR验证。，进一步的Q-PCR验证，发现miR-1304在非肝阳上亢人群表达明显高于正常血压人群。细胞因子：CRP，多巴胺，BK，ET-1，前列腺素，NA，醛固酮，肾素，AngII表达高血压人群（包括肝阳上亢和非肝阳上亢）显著高于正常血压人群。排除肾素、缓激肽、血管紧张素和醛固酮，其他细胞因子肝阳上亢证患者显著高于非肝阳上亢证患者。结论：高血压肝阳上亢患者发病较非肝阳上亢患者发病年龄早，且男性多于女性；不管何种证候特征，男性高血压发病与遗传关系较女性更为密切；肝阳上亢证患者血管内皮损伤明显、炎症突出、神经调节失常更加明显。