【作者】 彭庚；

【导师】 王东明；

【作者基本信息】 汕头大学 ， 心血管内科学， 2007， 硕士

【摘要】 背景急性冠脉综合征（Acute Coronary Syndrome，ACS）是冠心病的危重症，其发病存在明显的性别差异。即使考虑其他危险因素，男性性别仍是冠心病的独立危险因子，而雄激素的作用一直存在争议，是目前心血管领域的研究热点之一。研究证实血管内皮抗凝活性降低，血栓形成是急性冠脉综合征的主要特征，而我们的前期实验发现生理浓度睾酮（Testosterone，T）可通过雄激素受体（AndrogenReceptor，AR）促进人脐静脉内皮细胞（Human Umbilical Vein Endothelial Cell，HUVEC）合成、分泌组织因子途径抑制物（Tissue Factor Pathway Inhibitor，TFPI），增强内皮抗凝活性，但是其具体机制不明，本实验即是对该调节通路的进一步研究。目的观察生理浓度睾酮处理后，人脐静脉内皮细胞核转录因子（Nuclear Factor，NF）活性变化及对TFPI表达合成的影响，探讨生理浓度睾酮在转录因子（Transcription Factor，TF）环节调节TFPI表达的机制。方法（1）将体外培养的人脐带静脉内皮细胞（HUVEC）分为单纯培养基对照组、生理浓度睾酮组和雄激素受体拮抗剂（Flutamide，F）预处理组，应用转录因子活性芯片技术，分析三组人脐静脉内皮细胞核内转录因子活性的差异；（2）将体外培养的HUVEC细胞分为单纯培养基对照组（Control组）、不同浓度睾酮组(3×10^-9，3×10^-8，3×10^-6，3×10^-5mol／L Testosterone组：T_-9组，T_-8组，T_-6组，T_-5组)、Flutamide组（F组）、Flutamide预处理组(F+T_-8组)、TNF-α处理组（TNF-α组）、TNF-α预处理组(TNF-α+T_-8组)、PFT-α处理组（PFT-α组）、PFT-α预处理组(PFT-α+T_-8组)，ELISA法测各组TFPI蛋白水平，real-time RT-PCR检测各组TFPI mRNA水平。结果（1）睾酮作用后有5个转录因子的活性明显变化，其中活性明显上调的转录因子有3个，包括c-Myb、Myc和P53；显著下调的转录因子有2个，包括核因子κb（NF-κb）和SP1；Flutamide能有效消除睾酮的上述作用：（2）生理浓度睾酮(3×10^-8 mol／L)组TFPI蛋白与mRNA水平与对照组相比明显增加，而超生理浓度睾酮(3×10^-5 mol／L)组则明显低于对照组，Flutamide可消除上述作用；通过TNF-α上调NF-κb和SP1活性后，TFPI的表达明显受到抑制，生理浓度睾酮可抑制TNF-α的这种作用；而PFT-α增加TFPI的表达，与生理浓度睾酮共同作用后，TFPI的表达进一步增加。结论（1）生理浓度睾酮可通过雄激素受体使HUVEC核转录因子c-Myb、Myc-Max、P53的活性上调，同时使NF-κb和SP1的活性下调；（2）生理浓度睾酮可通过明显减弱转录因子NF-κb和SP1的活性，而促进HUVEC TFPI表达，发挥防止血栓形成的保护作用。更多还原

【Abstract】 BackgroundSignificant difference in morbidity of acute coronary syndrome （ACS）, one of the critical coronary diseases, was found between males and females. Male gender was found to be an independent risk factor, despite other risk factors. But the role androgen played has been controversial, which becomes one interest of cardiovascμar research. It is confirmed that decreased vascμlar endothelial anticoagμlant activity and thrombosis development is the main feature of acute coronary syndrome. Our previous investigation demonstrated that physiological levels of testosterone had beneficial influence on hemostatic system to enhance the anticoagulant activity throμgh stimμlating the tissue factor pathway inhibitor （TFPI） levels secreted by the human umbilic vein endothial cells（HUVEC）, and the androgen receptor is involved in these processes. However, the specific mechanism, till now, is still not fμlly understood. The purpose of the present study, therefore, was to investigate the cellμlar mechanism in vitro.ObjectiveTo investigate the effects of testosterone at physiological concentration on the activities of nuclear transcription factors in HUVECs, further influence on TFPI expression. Then, study the cellμlar mechanism. Methods（1） HUVECs within 3-4 passages were cμltured in 25cm² flasks. The cells were incubated in the presence or absence of physiological testosterone (3×10^-8mol/L) for 12h. After the incubation, the nuclear proteins were isolated and then the transcription factor（TF） activity profile were examined using TransSignal Protein/DNA arrays according to the manufacture’s protocol. Then experiments were repeated with HUVECs preincubated in androgen receptor antagonist （flutamide 10μmol/L） for 3h.（2） HUVECs within 3-4 passages were divided into cμlture medium only group （control）, testosterone groups with different concentrations (T_-9 group: 3×10^-9 mol/L, T_-8 group: 3×10^-8 mol/L, T_-6 group: 3×10^-6 mol/L, T_-5 group:3×10^-5mol/L), Flutamide treat group （F group）, Flutamide pretreat group (F＋T_-8 group), TNF-αtreat group （TNF-αgroup）, TNF-αpretreat group (TNF-α＋T_-8 group), PFT-αtreat group （PFT-αgroup） and PFT-αpretreat group (PFT-α＋T_-8 group). Then, ELISA kit and Real-time RT-PCR were carried out to detect the TFPI antigen and mRNA levels in HUVECs.Reslμlts（1）Among a total of 54 screened TFs, 5 activity differential TFs significantly influenced by physiological levels of testosterone throμgh androgen receptor were found, of which 3 showed increased activity, including c-Myb, Myc-Max and P53, and 2 showed decreased activity including NF-κb and SP1.（2） Compared with control group, testosterone at physiological concentrations (3×10^-8 mol/L) stimμlated the secretion of TFPI significantly （antigen level: 2.925±0.054 VS. 2.725±0.044 ng/mL P＜0.01; mRNA level: 1.537±0.023 VS. 1.260±0.010 copies P＜0.01）. However, TFPI antigen levels markedly reduced at larger dose (3×10^-5mol/L) （antigen level: 2.649±0.029 VS. 2.725±0.044 ng/mLP=0.02; mRNA level: 1.253±0.012 VS. 1.260±0.010 copies P=0.49）. Flutamide attenuated physiological testosterone’s effects(antigen level: 2.732±0.063 VS. 2.925±0.054ng/mL P＜0.01; mRNA level: 1.273±0.015 VS. 1.537±0.023 copies, F＋T_-8VS.T_-8, P＜0.01). After the activity of NF-κb and SP1 had been activated by TNF-α, TFPI mRNA level significantly decreased than that of the control group（0.933±0.058 VS. 1.260±0.010 copies P＜0.01）. And physiological testosterone coμld inhibit the effects of TNF-αeffectly (1.163±0.015 VS. 0.933±0.058 copies, TNF-α＋T_-8 VS. TNF-α, P＜0.01,). However, PFT-α（P53 inhibitor） increased the express of TFPI notably （1.470±0.030 VS. 1.260±0.010 copies, PFT-αVS. control, P＜0.01）.Conclusion（1）Physiological levels of testosterone coμld up-regμlate the activities of c-Myb, Myc-Max and P53, and down regμlate activities of NF-κb and SP1 throμgh androgen receptor in HUVEC.（2）Testosterone with physiological concentrations may cast its effects on thrombosis prevention by stimμlating TFPI expression of HUVECs throμgh attenuating the inhibition for TFPI by transcription factor NF-κb and SP1, in which processes, the classical androgen receptor was closely involved.更多还原