【作者】 刘晓亮；

【导师】 赵彦艳；

【作者基本信息】 中国医科大学 ， 遗传学， 2010， 博士

【摘要】 目的细胞色素P450 4F2 (cytochrome P450 4F2, CYP4F2)通过ω-羟化作用将花生四烯酸(arachidonic acid, AA)代谢为20-羟基二十碳四烯酸(20-hydroxyeicosatetraenoic acid,20-HETE),是人类肾脏产生20-HETE最主要的酶。20-HETE通过调节血管和肾小管的活性而影响动脉血压：一方面,20-HETE收缩外周血管,并增强其它血管活性物质的缩血管效应而起促高血压作用；另一方面,20-HETE抑制肾脏近端小管和髓袢升支粗段对钠离子的重吸收而起抗高血压作用。本课题组前期对辽宁省西部高血压病高发区人群的关联研究表明,CYP4F2基因调控区功能性多态上调了该基因转录活性,增强了代谢产物20-HETE的生成,与高血压发病呈正相关。我们的结论与Ward等对白种人群报道的CYP4F2 V433M多态与20-HETE生成增多及血压升高呈正相关结论一致。然而,Gainer等则认为20-HETE的另一合酶CYP4A11 F434S多态性降低了转录活性,与高血压呈负相关。因此,建立高血压易感基因CYP4F2的转基因小鼠模型,将是从整体水平分析20-HETE对血压的调节作用及机制的重要手段。肾脏雄激素调节蛋白(kidney androgen-regulated protein, KAP)是小鼠肾脏近端小管表达的高丰度蛋白,与人CYP4F2在肾脏的表达部位基本一致。KAP启动子活性受雄激素、雌激素等多种激素的调节,其中以对雄激素的反应性最为明显。已有学者建立KAP-hAGT、KAP-LUC等转基因小鼠模型,并成功地实现了雄激素对转基因在小鼠肾脏的诱导表达。因此,选择KAP启动子构建CYP4F2转基因小鼠模型,旨在强调CYP4F2在小鼠肾脏的优势表达,兼顾20-HETE的双重血压调节作用,并为实现CYP4F2的雄激素诱导表达提供可行性。本研究通过建立CYP4F2转基因小鼠模型,从整体水平鉴定CYP4F2/20-HETE代谢通路对血压的调节作用,并进一步以该模型作为研究平台,借助雄激素诱导CYP4F2的表达及效应,为阐明CYP4F2/20-HETE对高血压的具体调节机制提供有力的条件。材料与方法一、实验材料：1、FVB/N小鼠2、人HEK293、HUVEC细胞系3、基因克隆及萤光素酶报告基因检测相关试剂4、Southern blot、Western blot及免疫组化相关试剂5、20-HET EELISA检测、醛固酮放免检测相关试剂6、Real-time PCR相关试剂7、ROS特异性荧光染料DHE及NO特异性荧光染料DAF-2 DA8、MDA及SOD检测相关试剂9、氧化应激基因mRNA芯片二、实验方法：1、构建pKAP-LUC、pKAP-CYP4F2/his载体,转染人肾脏细胞系HEK293,双萤光素酶检测系统检测LUC活性,以CYP4F2抗体和his标签抗体Western blot检测CYP4F2的表达,细胞水平判断KAP启动子活性。2、将pKAP-CYP4F2/his载体双酶切线性化,胶回收纯化转基因片段,显微注射至FVB/N小鼠受精卵,产生CYP4F2转基因Founder小鼠。提取鼠尾DNA,以CYP4F2特异性探针及引物,经Southern blot及PCR双重鉴定阳性小鼠,建立转基因系。3、提取小鼠肾脏等十余种组织的总蛋白并定量,his标签抗体Western blot检测CYP4F2的表达谱。以4%多聚甲醛固定肾脏等组织块,石蜡切片,免疫组化鉴定CYP4F2的表达定位。提取肾脏微粒体蛋白,his标签抗体Western blot检测CYP4F2在微粒体酶中的表达。4、将肾脏微粒体蛋白与AA底物、NADPH辅酶等成分孵育,建立体外的AA羟化反应体系,ELISA试剂盒检测20-HETE生成量。收集小鼠尿液,ELISA试剂盒检测尿20-HETE的水平。5、无创鼠尾血压仪测量转基因小鼠收缩压(SBP),并抽样以有创的颈动脉插管法评估前种方法的有效性。动态监测8-40周龄小鼠的血压变化情况。小鼠眶后静脉丛采血并分离血浆及血清,放射免疫法测量血浆醛固酮水平,干片法测量血清肌酐(CRE)、尿素氮(BUN),判断肾功能。6、对雌性转基因小鼠连续两周腹腔注射雄激素诱导剂5α-双氢睾酮(dihydrotestosterone, DHT), his标签抗体Western blot检测诱导后肾脏CYP4F2的表达情况。ELISA试剂盒检测诱导后微粒体对AA羟化孵育反应的20-HETE产量,并测量小鼠尿20-HETE的排泌水平。鼠尾动脉血压仪测量诱导后小鼠血压的变化。7、对雄性转基因小鼠实施去势术清除内源的雄激素分泌,再给与外源DHT诱导剂,分别检测肾脏CYP4F2的表达、代谢产物20-HETE及小鼠血压,并与诱导前加以比较。8、对小鼠实施DHT及20-HETE特异性抑制剂HET0016共给药,检测尿20-HETE及动脉血压,进一步确认20-HETE在DHT诱导的高血压中的作用。9、提取DHT诱导小鼠肾脏总RNA并反转录成cDNA, Real-time PCR检测DHT对小鼠内源性20-HETE合酶cyp4a12、cyp4a14、cyp4a10的表达调控。10、以DHT分别刺激人HEK293细胞0.5小时、24小时和48小时,CYP4F2抗体Western blot检测雄激素对人CYP4F2的诱导表达作用。11、检测转基因小鼠氧化应激相关指标：DHE染色法检测胸主动脉、肾脏冰冻切片活性氧自由基(ROS)、试剂盒检测血浆及肾组织匀浆液脂质过氧化标记物丙二醛(MDA)、试剂盒检测血浆及肾组织匀浆液总体超氧化物歧化酶(SOD)活力。12、以SOD类似物Tempol添加到饮水中抑制氧化应激,检测转基因小鼠SBP的变化,确立氧化应激对转基因小鼠高血压发生的重要作用。13、提取肾脏总RNA并反转录成cDNA,通过Real-time PCR仪进行氧化应激相关基因的mRNA芯片检测,筛查转基因与野生型小鼠之间差异表达的氧化应激基因。14、以芯片筛查的差异表达基因Nos2作为靶基因,对整个NOS家族的每个成员(iNOS、nNOS、eNOS),进行Western blot验证小鼠肾脏蛋白质水平的表达差异。15、体外培养人]HUVEC、HEK293细胞系并施加20-HETE刺激,DHE荧光染料检测ROS水平,分析20-HETE对细胞氧化应激的诱发作用。DAF-FM荧光染料检测NO水平,分析20-HETE对细胞整体NO水平的影响。结果一、CYP4F2转基因小鼠模型的建立与鉴定1、转染pKAP-LUC载体的HEK293细胞LUC活性升高3倍,转染pKAP-CYP4F2/his载体的HEK293细胞CYP4F2表达上调2.6倍,说明KAP启动子能够驱动CYP4F2在肾脏细胞中表达。2、显微注射法建立CYP4F2转基因小鼠,Southern blot及PCR双重鉴定了三只转基因小鼠的Founder,经繁殖建立起三个转基因小鼠系：F0-6,F0-16和F0-56。3、三个转基因小鼠系的肾脏均有CYP4F2的表达,以F0-16表达最高。转基因小鼠十余种组织的表达谱表明：CYP4F2在雄鼠肾脏表达最明显,此外,在睾丸、附睾、子宫、卵巢等性激素反应性器官呈高表达。4、转基因小鼠肾脏的CYP4F2表达主要定位于近端小管；同野生型小鼠比较,转基因小鼠的肾脏微粒体中CYP4F2呈高水平表达,AA羟化活性增强,20-HETE生成量增多；且尿中CYP4F2功能性代谢产物20-HETE水平显著升高。5、转基因小鼠的SBP较野生型小鼠明显升高,且与20-HETE水平呈正相关。高血压的发生早于8周龄。转基因小鼠血浆醛固酮水平以及血清CRE、BUN水平与野生型小鼠无差异。二、雄激素对CYP4F2转基因小鼠的调节作用1、雌性转基因小鼠经DHT诱导后,肾脏CYP4F2的表达进一步上调,20-HETE生成增多,诱导后小鼠血压也随之进一步升高。2、雄性转基因小鼠去势后肾脏CYP4F2的表达下调,再给与外源DHT诱导后肾脏CYP4F2的表达明显回升,20-HETE和小鼠血压也同样随着小鼠体内雄激素水平的变化而先降低,后升高。3、HET0016与DHT联合给药后,消除了DHT对20-HETE和SBP的诱导作用,由此进一步确认20-HETE对雄激素依赖性高血压的促进作用。4、CYP4F2转基因小鼠肾脏内源性20-HETE合酶cyp4a12、cyp4a14和cyp4a10的表达比野生型小鼠明显降低,且对DHT的反应性也较野生型小鼠降低。DHT上调了cyp4a12表达、下调了cyp4a14的表达,而cyp4a10对DHT反应不明显。5、人肾脏细胞系HEK293经DHT刺激0.5小时、24小时和48小时后,CYP4F2的表达分别上调了1.4倍、2.3倍和2.5倍,说明人CYP4F2可能在肾脏受雄激素诱导表达。三、CYP4F2转基因小鼠的氧化应激反应1、转基因小鼠胸主动脉及肾脏的ROS水平明显高于野生型小鼠,血浆及肾组织匀浆液MDA水平也明显升高,而血浆及肾组织匀浆液总SOD活性下降,说明CYP4F2转基因小鼠处于氧化应激状态。2、抗氧化剂Tempol处理小鼠后,转基因小鼠SBP明显下降至接近野生型小鼠水平,说明氧化应激参与了CYP4F2转基因小鼠高血压的发生。3、小鼠肾脏组织氧化应激相关基因mRNA表达芯片共检测出差异表达基因23个(P<0.05)；其中P<0.01的差异表达基因12个4、对iNOS(芯片检测转基因小鼠肾脏表达上调105.55倍,P=0.0016)及其它NOS成员nNOS、eNOS的蛋白质水平的表达鉴定表明：野生型小鼠iNOS几乎不表达,转基因小鼠有较明显表达；nNOS表达下调。5、20-HETE刺激体外培养HUVEC和HEK293细胞系,细胞的ROS水平明显升高,诱发了细胞的氧化应激状态；而NO水平无明显变化。结论1、成功地建立了高血压易感基因CYP4F2的转基因小鼠模型,CYP4F2/20-HETE代谢通路功能性获得,引起转基因小鼠呈现慢性高血压表型。因此,从整体水平证明CYP4F2/20-HETE起促高血压作用。2、雄激素明显诱导了转基因小鼠肾脏CYP4F2的表达,放大了CYP4F2/20-HETE通路的效应,并引起雄激素依赖性高血压。雄激素对人肾脏细胞CYP4F2的诱导表达进一步提示CYP4F2/20-HETE通路可能是人类高血压性别差异的机制之一3、转基因小鼠氧化应激水平明显升高,抑制氧化应激明显改善了高血压表型,氧化应激相关基因存在较广泛的表达差异,提示氧化应激是CYP4F2转基因小鼠高血压发生的关键,参与了CYP4F2/20-HETE通路促高血压效应的机制。更多还原

【Abstract】 IntroductionCytochrome P450 4F2 (CYP4F2) encodes a major 20-hydroxyeicosatetraenoic acid (20-HETE) synthase in human kidney that converts arachidonic acid (AA) into 20-HETE throughω-hydroxylation.20-HETE participates in the development of hypertension by regulating vascular and renal tubular functions. On the one hand, 20-HETE promotes hypertension by vasoconstriction or by sensitizing the vascular smooth muscle cells to other vasoconstrictors. On the other hand,20-HETE attenuates hypertension by inhibiting sodium reabsorption in the renal proximal tubles and ascending thich limb of Henle’s Loop. Our group have rescently found that a functional haplotype of CYP4F2 with increased transcriptional activity was associated with elevated urinary 20-HETE and hypertension in Han Chinese residing in a hypertension-prevalent region of Liaoning Province. A similar result was reported that the variant V433M in CYP4F2 was associated with the increase of 20-HETE excretion and systolic blood pressure (SBP) in a white cohort However, a different result showed that the variant F434S in CYP4A11, another human 20-HETE synthase gene, with reduced catalytic activity was associated with hypertension in white populations. Therefore, CYP4F2 transgenic approach would be a pivotal and irreplaceable strategy to clarify the correlation between 20-HETE and blood pressure at the level of whole organism.Kidney androgen-regulated protein (KAP) is one of the most abundant proteins expressed in mouse proximal tubule that resembles the expression locus of CYP4F2 in human kidney. KAP promoter activity is regulated by different hormones such as androgen and estrogen, among which androgen shows the strongest responsibility. Previous KAP-hAGT and KAP-LUC transgenic models have shown that KAP promoter could successfully regulate androgen-inducible transgene expression in the kidney. Herein, we constructed CYP4F2 transgenic mouse model utilizing KAP promoter to to highlight CYP4F2 expression in the kidney where the dual role of 20-HETE fulfils, and to better understand the mechanism of CYP4F2/20-HETE in blood pressure regulation by androgen induction.The present study developed a transgenic mouse model expressing CYP4F2 under the control of the heterologous KAP promoter to shed light on the impact of CYP4F2/20-HETE pathway on blood pressure at the level of whole organism. With androgen as an inducer of CYP4F2 expression, further studies based on this model would help to gain insight into the detailed mechanism of CYP4F2/20-HETE in blood pressure regulation.Materials and MethodsMaterials1.FVB/N mice2. Human cell line HEK293 and HUVEC3. Reagents for gene cloning and luciferase assay4. Reagents for Southern blot, Western blot and immunohistochemistry5. Reagents for 20-HETE ELISA and aldosterone RIA determination6. Reagents for Real-time PCR assay7. ROS-specific fluorescent dye DHE and NO-specific fluorescent dye DAF-FM8. Reagents for MDA and SOD activity assay9. Microarrays of oxidative stress gene mRNA expressionMethods 1. Kidney-derived HEK293 cells were transfected with constructed pKAP-LUC, pKAP-CYP4F2/his expression plasmid. Luciferase activity and CYP4F2 expression were determined to identify the promoter activity of KAP promoter in cultured cells.2. The linearized pKAP-CYP4F2/his was microinjected into FVB/N mouse one-cell fertilized embryoes to generate transgenic founders. Tail DNA was isolated for Southern blot and PCR identification of positive mice with probes and primers designed specific to CYP4F2.3. Total protein from various tissus including kidney was extracted and determined concentration. The expression profile of CYP4F2 was determined by Western blot using anti-his antiby. The 4% paraformaldehyde fixed kidney samples were embedded by paraffin wax and sectioned for immunohistochemistry to localize the CYP4F2 expression. Renal microsomes were also extracted to test CYP4F2 expression by Western blot.4. The in vitro AA hydroxylation assay was assessed by incubating the AA substrate, NADPH coenzyme, etc. with the renal microsomes.20-HETE production was quantified by specific 20-HETE enzyme-linked immunosorbent assay (ELISA) kit. Mice urine was collected to measure the level of 20-HETE by ELISA kit.5. Blood pressure of transgenic mice was measured by noninvasive tail-cuff method as well as carotid catheter. Mice aging from 8 to 40 weeks were measured to record the time course of hypertension development. In addition, blood sample was collected from orbital sinus for plasma and serum separation. Plasma aldosterone was determined by radioimmunoassay (RIA), and serum creatinine (CRE) and blood urea nitrogen (BUN) were measured using CREA and BUN Slides.6. Female mice were i.p. injected with 5a-dihydrotestosterone (DHT) for consecutive 14 days. After DHT induction, renal CYP4F2 expression was examined by Western blot.20-HETE level was measured by ELISA in the urine as well as in the microsomal AA hydroxylation reaction mixture. Mouse blood pressure was measured by tail-cuff method. 7. Male mice underwent castration to clear endogenous androgens before exogenous DHT administration. The level of renal CYP4F2 expression,20-HETE and SBP was also measured accordingly.8. Mice furthermore underwent coadministration of either DHT with HET0016, the specific 20-HETE inhibitor, or DHT with lecithin vehicle as control. HET0016 was applied to confirm the role of 20-HETE in the development of androgen-induced hypertension.9. Total RNA from the DHT-induced mouse kidney was extracted and reverse-transcripted into cDNA. DHT regulation on mouse endogenouse 20-HETE synthase, cyp4a12, cyp4a14, and cyp4a10, was studied by Real-time PCR.10. Human HEK293 cells were treated with DHT for 0.5 hour,24 hours and 48 hours, respectively. CYP4F2 exptression was examined by Western blot analysis using anti-CYP4F2 antibody.11. The level of oxidative stress in mice was evaluated by relevant markers:the level of reactive oxygen species (ROS) was in situ examined in the frozen sectioned thoracic aorta and kidney; plasma and renal homogenate malondialdehyde (MDA) was evaluated as a marker of lipid peroxidation; plasma and renal homogenate total superoxide dismutase (SOD) was measured as a major antioxidative parameter.12. Mice were treated with Tempol, an SOD mimetic to relieve oxidative stress, and the SBP was measured by tail-cuff method.13. Total renal RNA was extracted and reverse-transcripted into cDNA, and the microarray expression analysis including 84 oxidative stress genes was preformed using Real-time PCR.14. Nos2, one of the differentially expressed genes screened by microarray analysis, was chosen as candidate for detailed analysis. The renal expression of iNOS, nNOS, and eNOS was further confirmed by Western blot.15. HUVEC and HEK293 cells were cultured and stimulated by 20-HETE. The level of oxidative stress was studied by ROS-specific fluorescent dye DHE, and the level of NO was determined by the fluorescent dye DAF-FM.ResultsⅠ. Generation and identification of CYP4F2 transgenic mouse model1. Kidney-derived HEK293 cells transfected with pKAP-LUC showed increased luciferase activity by 3-fold over control, and cells with pKAP-CYP4F2/his plasmid expressed CYP4F2 of 2.6-fold higher than control.2. Three CYP4F2 transgenic founders, generated by microinjection, were identified by Southern blot and PCR Therefore, three transgenic lines were established after breeding, namely F0-6, F0-16, and F0-56.3. Renal CYP4F2 expression was the highest in line F0-16, and was also positive in the other two transgenic lines. The expression profile demonstrated that:CYP4F2 was expressed at the highest level in male kidney; in addition, the expression was also high in sexual-hormone responsive organs such as testis, epididymis, uterus, and ovary.4. In CYP4F2 transgenic mice, renal CYP4F2 mainly localized in the proximal tubules, and CYP4F2 expression and AA hydroxylation were increased in the renal microsomes compared with wild-type controls. Transgenic mice also had significantly higher level of urinary 20-HETE excretion than wild-type mice.5. The systolic blood pressure (SBP) in transgenic mice was obviously elevated than in wild-type control, and was in positive correlation with urinary 20-HETE excretion. Hypertension developed in early lives of transgenic mice. Plasma aldosterone, serum CRE and BUN were not different between transgenic mice and wild-type controls.Ⅱ. Androgen effect on CYP4F2 transgenic mouse model1. Renal CYP4F2 expression was further up-regulated after DHT induction in female transgenic mice. Consequently,20-HETE production was enhanced. The blood pressure in transgenic mice was furthermore elevated after induction.2. Male transgenic mice showed decreased renal CYP4F2 expression after castration, and exhibited dramaticly increased expression after exogenous DHT induction.20-HETE excretion and SBP accompanied the changes in androgen level, i.e., first declined, and then elevated.3. Coadministration of DHT and 20-HETE specific inhibitor HET0016 negated DHT induced 20-HETE excretion and hypertension. This further confirmed the contributive role of 20-HETE in the development of androgen-induced hypertension.4. All the three isoforms of mouse endogenouse 20-HETE synthases were greatly depressed and were less sensitive to DHT in the kidney of CYP4F2 transgenic mice compared to wild-type controls. The mRNA expression of cyp4a12 was up-regulated by androgen, and cyp4al4 was down-regulated by androgen. Nontheless, cyp4a10 responded to androgen with no statistical significance.5. Human HEK293 cells incubated with DHT for 0.5 hour,24 hours and 48 hours, showed increased CYP4F2 exptression of 1.4-fold,2.3-fold and 2.5-fold over control, respectively, indicating a possibility that CYP4F2 was androgen-dependent in human kidney.Ⅲ. Oxidative stress of CYP4F2 transgenic mouse model1. The level of ROS in the frozen sectioned thoracic aorta and kidney was obviously higher in CYP4F2 transgenic mice than in control. Meanwhile, plasma and renal homogenate MDA was elevated, whereas total SOD activity was decreased in transgenic mice.2. Transgenic mice administered with Tempol significantly lessened the degree of hypertensive phenotype to nearly normal level, indicating a key role of oxidative stress in hypertension pathogenesis.3. Microarray expression analysis of oxidative stress genes revealed 23 differentially expressed genes at the significance level of P＜0.05, and 12 genes at the significance level of P＜0.01. 4. After gene screening, iNOS, as well as nNOS and eNOS, were chosen as candidate for further analysis. Their expressions in mice kidney were confirmed by Western blot, displaying the increased iNOS and the decreased nNOS in CYP4F2 transgenic mice compared to wild-type controls.5. HEK293 cells stimulated with 20-HETE showed significantly higher level of ROS determined by DHE fluorescent dye. There is no obvious change of NO with 20-HETE as stimulus using DAF-FM fluorescent dye.Conclusions1. In present study, we successfully established a novel CYP4F2 transgenic mouse model. Transgenic mice with gain-in-function of CYP4F2/20-HETE metabolic pathway showed chronic hypertensive phenotype. Therefore, we provided evidence that the CYP4F2/20-HETE played a pro-hypertensive role at the level of whole organism.2. Androgen strongly induced the renal CYP4F2 expression and magnified the bioeffect of CYP4F2/20-HETE pathway, which thereby caused androgen-responsive hypertension. In addition, the androgen-inducibility of CYP4F2 in human kidney cells further indicated a possible mechanism of CYP4F2/20-HETE underlying gender-specific differences of human hypertension.3. CYP4F2 transgenic mice suffered severe oxidative stress, and anti-oxidative treatment ameliorated the hypertensive phenotype. In addition, the expression of oxidative stress genes extensively varied between transgenic and wild-type mice, suggesting oxidative stress was the key point in the pathogenesis of hypertension, and was involed in the pro-hypertensive mechanism of CYP4F2/20-HETE pathway.更多还原