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Research on CYP4F2/20-HETE Pathway in CYP4F2 Transgenic Mouse Model

Author: LiuXiaoLiang
Tutor: ZhaoYanYan
School: China Medical University
Course: Genetics
Keywords: CYP4F2 20-HETE Transgenic Hypertension Kidney Androgen Oxidative Stress
CLC: R-332
Type: PhD thesis
Year: 2010
Downloads: 248
Quote: 0
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Abstract


Objective cytochrome P450 4F2 (cytochrome P450 4F2, CYP4F2) ω-hydroxylation of the arachidonic acid metabolism of 20 - hydroxy-eicosatetraenoic acid (arachidonic acid, AA) (20-hydroxyeicosatetraenoic acid ,20-HETE) , human kidneys produce 20-HETE main enzyme. 20-HETE affecting arterial blood pressure by regulating the activity of the vascular and tubular: on the one hand ,20-HETE contraction of peripheral vascular and enhance other vasoactive substances vasoconstrictor effect of the sky to promote the hypertensive effects; other hand ,20-HETE Inhibition of renal proximal tubule and medullary thick ascending limb segment re-absorption of sodium ions into the sky antihypertensive effect. The high incidence populations of western Liaoning Province, hypertension associated with pre-study in our group of CYP4F2 gene regulatory region functional polymorphism raised the transcriptional activity of the gene, enhances the generation of 20-HETE metabolites showed a positive correlation with hypertension . Consistent with our conclusions and Ward CYP4F2 V433M polymorphic Caucasian reported an increase in the formation of 20-HETE and blood pressure was positively correlated conclusions. However, Gainer that another of 20-HETE synthase CYP4A11 F434S polymorphism reduces the transcriptional activity was negatively correlated with hypertension. Therefore, the establishment of a transgenic mouse model of hypertension susceptibility gene CYP4F2, the analysis from the overall level of 20-HETE important role and mechanism of blood pressure regulation means. Kidney androgen-regulated protein (kidney androgen-regulated protein, KAP) is a high abundance protein expression in mouse kidney proximal tubule, consistent with the people CYP4F2 expression in kidney parts. KAP promoter activity is regulated by androgen, estrogen and other hormones, the most obvious of which the reactivity of androgen. Some scholars establish KAP-hAGT the KAP-LUC transgenic mouse model, and successfully achieve androgen-induced expression of transgenic mouse kidney. Therefore, the choice the KAP promoter constructs CYP4F2 transgenic mouse model, seeks to highlight the CYP4F2 expression in mouse kidney advantage, taking into account the 20-HETE dual regulation of blood pressure, and to achieve the CYP4F2 the androgen induced expression feasibility. In this study, through the establishment of CYP4F2 transgenic mouse model, from the overall level the identification CYP4F2/20-HETE metabolic pathway of blood pressure regulation, and further to the model as a research platform, with androgen-induced CYP4F2 expression and effect, to elucidate of CYP4F2 / 20-HETE specific regulation mechanism of hypertension strong conditions. MATERIALS AND METHODS Materials: 1, FVB / N mice, human HEK293 HUVEC cell line 3 gene and luciferase reporter gene assay reagents, Southern blot analysis, Western blot and immunohistochemistry of Reagents 5,20-HET EELISA detection, aldosterone radioimmunoassay Reagents Real-time PCR reagents ROS-specific fluorescent dye DHE NO-specific fluorescent dye DAF-2 DA8, MDA and SOD detection reagents 9, oxidative stimulated gene mRNA chip two experimental methods: 1, build pKAP-LUC, pKAP-CYP4F2/his vector transfected human kidney cell line HEK293, dual luciferase detection system LUC activity to CYP4F2 antibody and his tag antibody Western blot to detect the expression of CYP4F2 cellular level to determine the KAP promoter activity. 2 digested pKAP-CYP4F2/his carrier linear glue purified transgenic fragment microinjection of fertilized eggs to FVB / N mice, genetically modified produce CYP4F2 Founder mice. Extracted tail DNA CYP4F2 specific probes and primers, Southern blot and PCR identification of double transgenic mice to establish transgenic lines. 3, extract the total protein of the mouse kidney and more than 10 kinds of organizations and quantify the spectrum of the CYP4F2 the expression of his tag antibody detected by Western blot. 4% paraformaldehyde kidney tissue blocks, paraffin sections, identified by immunohistochemistry CYP4F2 Expression and localization. Extraction of kidney microsomal protein, his tag antibody detected by Western blot CYP4F2 the microsomal enzyme expression. AA 4 kidney microsomal protein AA substrate, NADPH coenzyme ingredients incubated build vitro hydroxylation system, ELISA kits to detect the formation of 20-HETE. Collection of mouse urine ELISA kit detects urine levels of 20-HETE. 5, no the invasive rat tail blood pressure measured transgenic mice systolic blood pressure (SBP), and sampling to the effectiveness of the carotid artery intubation invasive assessment of the former method. Dynamic monitoring of changes in blood pressure of 8-40 week old mice. Mouse orbital venous plexus blood collection and separation of plasma and serum, radioimmunoassay measurement of plasma aldosterone levels the dry chip measurement of serum creatinine (CRE), blood urea nitrogen (BUN), to determine kidney function. 6, intraperitoneal injection androgen inducer 5α-DHT (dihydrotestosterone, DHT), his tag antibody detected by Western blot induced kidney the CYP4F2 the expression of female transgenic mice for two consecutive weeks. ELISA kits induced microsomal production of 20-HETE AA hydroxylation incubation reaction and measuring mouse urinary excretion of 20-HETE level. Tail arterial blood pressure was measured after the induction of changes in blood pressure in mice. 7 implementation of the castration of male transgenic mice remove endogenous androgen secretion, give DHT with exogenous inducer, respectively, to detect kidney the CYP4F2 the expression, metabolite 20-HETE and mouse blood pressure before induction be comparison. 8, mice implementation of DHT and 20-HETE specific inhibitor HET0016 of total administration, urinary 20-HETE and arterial blood pressure is detected, further confirm the role of 20-HETE in the DHT-induced hypertension. 9, extract the the DHT induced mouse kidney total RNA was reverse-transcribed into cDNA, Real-time PCR detection DHT mouse endogenous 20-HETE synthase cyp4a12 cyp4a14, cyp4a10 of expression and regulation. 10, to DHT were stimulated human HEK293 cells 0.5 hours, 24 hours and 48 hours, CYP4F2 antibody Western blot analysis of androgen role in the induction of the expression of the human CYP4F2. 11: the DHE staining thoracic aorta, kidney frozen sections of reactive oxygen radicals (ROS), kit test plasma and renal tissue homogenate lipid oxidation markers malondialdehyde to detect transgenic mice oxidative stress indicators ( MDA), vitality kit detects plasma and renal tissue homogenate overall superoxide dismutase (SOD). 12, SOD analogs Tempol added to the drinking water in the inhibition of oxidative stress, was detected by the change of the gene in mice SBP, established the important role of oxidative stress in transgenic mice hypertension. 13, extracted kidney total RNA and reverse transcribed into cDNA by Real-time PCR instrument oxidative stress-related gene mRNA microarray screening transferred oxidative stress genes differentially expressed between the gene and wild-type mice. 14 gene expression differences chip screening Nos2 as the target gene, each member of the entire family of NOS (iNOS, nNOS, eNOS), by Western blot to verify protein expression differences in mouse kidney. 15, cultured in vitro human] HUVEC, HEK293 cell lines and 20-HETE stimulation is applied DHE fluorescent dye detection ROS level of 20-HETE cellular oxidative stress induced. DAF-FM fluorescent dye detection of NO level, analysis of the impact of 20-HETE on the level of the whole cell NO. Results, CYP4F2 turn Establishment and identification of the gene mouse model, HEK293 cells transfected pKAP-LUC vector LUC activity increased by 3-fold, HEK293 cells transfected pKAP-CYP4F2/his carrier CYP4F2 expression up 2.6 times, indicating that the KAP start the sub drive CYP4F2 expression in kidney cells. The microinjection establish CYP4F2 transgenic mice, Southern blot and PCR identified three transgenic mice Founder dual breeding established three transgenic mouse lines: F0-6, F0-16 and F0-56. 3, three transfected gene mouse lines kidneys were the CYP4F2 the expression, the highest expression of F0-16. Transgenic mice expression profiles of more than 10 kinds of organizations: CYP4F2 expression in male rats kidney is the most obvious, was highly expressed in the testis, epididymis, uterus, ovaries, and sex hormone reaction sexual organs. 4, transgenic mice kidneys of CYP4F2 expression mainly located in the proximal tubule; compared with wild-type mice, the transgenic mice kidney microsomal CYP4F2 was expressed at high levels, AA hydroxylation active for enhanced ,20-HETE increased generation; CYP4F2 functional metabolite 20-HETE levels in urine were significantly increased. 5, the SBP transgenic mice than in wild-type mice was significantly higher, and was positively correlated with the level of 20-HETE. The incidence of hypertension as early as 8 weeks of age. Transgenic mouse plasma aldosterone levels and serum CRE, BUN levels of wild-type mice no difference. Second, the regulatory role of androgen CYP4F2 transgenic mice, female transgenic mice after DHT induced the kidney the CYP4F2 the expression further raised ,20-HETE generation increased, induced in mice after blood pressure also will be increased further. 2 male transgenic mice the kidney the CYP4F2 the expression down after castration, give exogenous DHT induced expression of renal CYP4F2 rebounded significantly ,20-HETE and mouse blood pressure also as mice androgen levels change first decreased and then increased. HET0016 co-administered with DHT eliminate DHT induced 20-HETE and SBP, confirming the role of 20-HETE androgen-dependent hypertension. 4, CYP4F2 transfer of 20-HETE synthase gene mouse kidney endogenous cyp4a12 the expression of cyp4a14 and cyp4a10 significantly lower than in wild-type mice, and the reaction of DHT also reduced compared with the wild-type mice. DHT raised cyp4a12 expression, to cut the cyp4a14 expression, while cyp4a10 no obvious reaction to DHT. 5, the human kidney cell line HEK293 DHT stimulation 0.5 hours, 24 hours and 48 hours after the expression of of CYP4F2 are raised 1.4 times, 2.3 times and 2.5 times, respectively, described human CYP4F2 may androgen induced expression in the kidney. Of CYP4F2 transgenic mice, oxidative stress reaction, the ROS level of the thoracic aorta and kidney of transgenic mice was significantly higher than that in wild-type mice, plasma and renal tissue homogenate MDA levels were also significantly higher, and plasma and renal tissue homogenate SOD activity decreased the CYP4F2 state of oxidative stress in transgenic mice. 2, antioxidants the Tempol treated mice, turn transgenic mice SBP significantly decreased to close to the level of wild-type mice, indicating oxidative stress in a of CYP4F2 transgenic mouse hypertension occurs. Mouse kidney tissue oxidative stress-related gene mRNA expression chip were detected differentially expressed gene 23 (P lt; 0.05); P lt; 0.01 difference in expression of genes 12 and iNOS (microarray transgenic mice the kidney upregulation 105.55, P = 0.0016), and other NOS members of nNOS protein levels of eNOS expression was identified: the wild-type mice iNOS almost no expression, a more visible expression in transgenic mice; nNOS downregulation. 5,20-HETE stimulation in vitro cultured HUVEC and HEK293 cell lines, cell ROS levels were significantly increased induced oxidative stress status of cells; while no significant change in the level of NO. Conclusion 1, successfully established a transgenic mouse model of hypertension susceptibility gene CYP4F2 functionality can be obtained CYP4F2/20-HETE metabolic pathway, caused by the transgenic mice showed chronic hypertension phenotype. Therefore, from the overall level to prove that to promote hypertension CYP4F2/20-HETE since. 2, androgen significantly induced the expression of the transgenic mouse kidney CYP4F2 effect amplified CYP4F2/20-HETE passage, and cause androgen-dependent hypertension. Androgen-inducible expression of human kidney cells CYP4F2 further prompted CYP4F2/20-HETE pathway may be one of the mechanisms of human hypertension gender differences, significantly increased level of oxidative stress in transgenic mice, inhibition of oxidative stress significantly improved high blood pressure phenotypes, oxidative stress related genes exist broader expression differences, suggesting that oxidative stress is the CYP4F2 turn the key gene in mice of hypertension, involved CYP4F2/20-HETE pathway pro-hypertensive effect mechanism.

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