高血压肝阳上亢证的分子机理研究

Study on Molecular Mechanism of Ganyang Shangkang Syndrome in Hypertension

目的:探讨高血压肝阳上亢证的分子机理。方法:应用高效液相色谱(HPLC)方法检测健康人、高血压病肝阳上亢证及肝肾阴虚证患者血浆去甲肾上腺素(NE)和肾上腺素(E)含量,并采用SouthernBlot方法分析上述3组间酪氨酸羟化酶(TH)基因多态性;用PCRSSCP方法分析高血压肝阳上亢证TH及单胺氧化酶A和B(MAOA,MAOB)基因相关微卫星多态性改变;同时,采用自发性高血压大鼠(SHR)加灌附子汤方法复制高血压肝阳上亢证动物模型,用免疫组织化学方法和原位杂交法分别测定该模型肾上腺组织TH蛋白质及mRNA表达水平。结果:高血压病肝阳上亢证患者血浆NE、E含量明显高于健康人组及肝肾阴虚组,其TH基因有显著扩增,且TH微卫星D11S4046中A1型明显增高;SHR肝阳上亢证模型肾上腺组织TH蛋白质及mRNA表达增强。结论:高血压肝阳上亢证具有TH基因显著扩增、THmRNA及蛋白质表达增强的特点。提示TH基因的过度表达可能是高血压肝阳上亢证的分子机理。

To investigate the molecular mechanism of Ganyang Shangkang (Liver Yang ascending, GYSK) syndrome in hypertension. Methods: The plasma norepinephrine (NE), epinephrine (E) contents in patients with GYSK syndrome or Ganshen Yinxu (Liver and Kidney Yin Deficiency, GSYX) syndrome who were suffered from hypertension, and normal controls were determined by high performance liquid chromatography electrochemical detector (HPLC ECD). The polymorphism of tyrosine hydroxylase (TH) gene in the three groups were analyzed by Southern Blot assay. The polymorphism of TH and MAOA, MAOB gene microsatellite in these groups were analyzed by polymerase chain reaction (PCR) SSCP; meanwhile, the GYSK model by administration of single TCM preparation, Radix Aconiti preparate (RAP) in spontaneous hypertension rats (SHR). The adrenal gland medulla were examined for their TH expression at the protein level after ABC immuno histochemical staining with the TH monoclonal antibody (McAb). The adrenal TH gene mRNA expression was demonstrated by in situ hybridization with the synthesized TH oligonucleic acid probe. The results of immunohistochemistry and in situ hybridization were analyzed with the image analysis system (IAS). Results: The plasma NE, E contents in patients with GYSK were more significantly increased than that in GSYX and normal controls. The TH gene amplified and the type A 1 TH microsatellite D11S4046 was increased more remarkably than other groups. The TH mRNA protein expression in their adrenal tissue of the single TCM preparation RAP induced GYSK model in SHR were elevated. Conclusions: The GYSK syndrome of hypertension possesses the character as amplified TH gene and increased TH mRNA and protein. It suggested that the hyperexpression of TH is probably the mechanism of GYSK syndrome in hypertension