四氢生物蝶呤通过蛋白激酶Cε途径对醋酸脱氧皮质酮—盐型高血压小鼠左室舒张功能的影响

发布时间：2018-05-09 12:30

本文选题：四氢生物蝶呤 + 醋酸脱氧皮质酮-盐型高血压小鼠　；参考：《兰州大学》2015年硕士论文

【摘要】：目的研究四氢生物蝶呤(BH4)对醋酸脱氧皮质酮(DOCA)-盐型高血压小鼠左心室舒张功能与蛋白激酶Cε(PKCε)表达的影响。以期为探索左室舒张性心功能不全的治疗提供实验依据。方法 90只10周龄雄性C57BL/6小鼠随机分成手术组(n=50)与假手术组(n=40)。手术组小鼠切除左侧肾脏后在颈部皮下埋入DOCA药片,术后以普通饲料及1.05%氯化钠(NaCl)溶液饲养,至术后14天,排除死亡小鼠6只后随机分为高血压组(n=22)与高血压+BH4组(n=22),并分别用生理盐水(每日每只0.1mL/10g)和2.5%的BH4溶液(每日每只0.1mL/10g)灌胃7天。假手术组小鼠仅游离左侧肾脏而不切除,并在颈部皮下埋入安慰剂药片,术后以普通饲料及常规不含盐净化水饲养,然后随机分成对照组(n=20)及对照+BH4组(n=20)。四个亚组于术后21天时,分别进行以下实验测定：1.鼠尾动脉压测定小鼠舒张期血压(DBP)及收缩期血压(SBP)(n=10)。2.应用彩色多普勒超声心动图二维成像与组织多普勒成像技术测定各组小鼠二尖瓣环舒张早期的运动速度(E’)、二尖瓣口舒张早期血流速度(E)及二尖瓣环舒张早期运动速度及二尖瓣环舒张晚期运动速度的比值(E'/A')等心脏的结构与功能参数(n=10)。3.血流动力学测定小鼠心率(HR)、Tau指数、左心室舒张末期的压力(LVEDP)、左心室收缩末期的压力(LVESP)、收缩末期压力-容积关系(ESPVR)、舒张末期压力-容积关系(EDPVR)等指标(n=6)。所有亚组均于术后22天时进行取材,所取组织均行以下实验测定：①酶联免疫吸附实验测定环磷酸鸟苷(cGMP)、超氧化物歧化酶(SOD)以及丙二醛(MDA)。②高效液相色谱法(HPLC)测定心肌组织中BH4及二氢生物蝶呤(BH2)的含量。③放免法测定血浆血管紧张素Ⅱ(AngⅡ)含量。④实时荧光定量聚合酶链反应(RT-PCR)测定心肌组织中α-SA、TGFβ1、PKCε及β-MHC的含量。⑤免疫组化测定血管内皮生长因子(VEGF)、受磷蛋白(PLB)、蛋白激酶C zeta (PKC-zeta)、肌浆／内质网钙ATP酶2(Serca2)、B细胞淋巴瘤／白血病-2(BCL-2)、磷酸化的受磷蛋白(Phospho-PLB)及Bcl-2相关X蛋白(Bax) 。⑥Western-blot实验测定内皮型一氧化氮合酶(eNOS)与PKCε的表达。结果①高血压组与对照组相比,SBP与DBP均增高(P0.05),高血压造模成功。高血压+BH4组与高血压组相比,血压差异无统计学意义(P0.05)。②高血压组与对照组相比,二尖瓣舒张早期血流速度及二尖瓣环舒张早期运动速度的比值(E/E')、EDPVR及Tau指数增高(均P0.05)③高血压+BH4组与高血压组相比,EDPVR及Tau指数减小(均P0.05)。④高血压组与对照组比较,SOD及NO减少(均P0.05),给予BH4干预后,SOD及NO增多(均P0.05)。⑤放免法结果显示,高血压组和对照组比较,AngⅡ的表达增加(P0.05),高血压+BH4组与高血压组相比,AngⅡ的表达减少(P0.05)。⑥T-PCR结果示高血压组与对照组相比,α-SA、PKCε及β-MHC的表达减少(P0.05),给予BH4干预后,α-SA、PKCε及β-MHC的表达增加(P0.05)。⑦免疫组化结果示高血压组与对照组相比,VEGF、PLB、Serca2及Bax表达增加,Phospho-PLB及BCL-2表达减少,高血压+BH4组与高血压组相比,VEGF、PLB、Serca2及Bax表达减少,Phospho-PLB及BCL-2表达增加,而对照+BH4组与对照组相比也出现了类似的结果。⑧Western-blot结果显示高血压组与对照组相比,PKCε的表达减少(P0.05),高血压+BH4组与高血压组相比,PKCε的表达增加(P0.05)。结论BH4无明显的降压作用,但可改善左室舒张功能不全,这可能与其通过PKCε途径降低体内氧化应激水平、提高NO表达等有关。
[Abstract]:Objective to study the effect of four hydrogen biotrexate (BH4) on the left ventricular diastolic function and the expression of protein kinase C e (PKC E) in mice with acetic acid deoxy corticosterone (DOCA) and salt type hypertension in order to provide experimental basis for the exploration of the treatment of left ventricular diastolic dysfunction. Methods 90 10 week old male C57BL/6 mice were randomly divided into surgical group (n=50) and artificial hand. The operation group (n=40). After resection of the left kidney of the operation group, the DOCA tablets were subcutaneously embedded in the neck. After the operation, the mice were fed with ordinary feed and 1.05% sodium chloride (NaCl) solution, and after 14 days after the operation, 6 rats were randomly divided into hypertension group (n=22) and hypertension +BH4 group (n=22), and the physiological saline (every 0.1mL/10g per day) and 2.5% BH4 solution were used respectively. The liquid (each 0.1mL/10g per day) was intragastric for 7 days. The sham operation group was only free left kidney in the sham operation group, and the placebo tablets were subcutaneously embedded in the neck. After the operation, the mice were fed with ordinary diet and conventional non salt purified water, and then randomly divided into the control group (n=20) and the control group +BH4 (n=20). The following experimental tests were carried out at 21 days after the operation. Determination of diastolic blood pressure (DBP) and systolic blood pressure (SBP) (n=10).2. using color Doppler echocardiography and tissue Doppler imaging in mice to determine the early diastolic velocity of mitral annulus (E '), early diastolic velocity of two apical mouth (E) and early diastolic transport of mitral annulus Cardiac structural and functional parameters (E'/A') and other cardiac structural and functional parameters (n=10).3. hemodynamics, the heart rate (HR), Tau index, left ventricular end diastolic pressure (LVEDP), left ventricular end systolic pressure (LVESP), end systolic pressure volume relation (ESPVR), end diastolic pressure volume close (n=10) System (EDPVR) and other indexes (n=6). All subgroups were collected at 22 days after the operation. The tissues taken were determined as follows: (1) enzyme-linked immunosorbent assay was used to determine cyclophosphate (cGMP), superoxide dismutase (SOD) and malondialdehyde (MDA). (2) high performance liquid chromatography (HPLC) was used to determine the content of BH4 in myocardial tissue and the content of two methotrexate (BH2) in myocardial tissue The content of plasma angiotensin II (Ang II) was measured by radioimmunoassay. (4) real-time fluorescence quantitative polymerase chain reaction (RT-PCR) was used to determine the content of alpha -SA, TGF beta 1, PKC E and beta -MHC in myocardial tissue. (5) immunohistochemical determination of vascular endothelial growth factor (VEGF), phosphotin (PLB), protein kinase C zeta (PKC-zeta), sarcoplasmic / endoplasmic reticulum calcium ATP enzyme 2 A2), B cell lymphoma / leukemia -2 (BCL-2), phosphorylated phosphoprotein (Phospho-PLB) and Bcl-2 related X protein (Bax). 6. Western-blot experiment was used to determine the expression of endothelial nitric oxide synthase (eNOS) and PKC epsilon. Results (1) the hypertension group was higher than the control group. Compared with the pressure group, the blood pressure difference was not statistically significant (P0.05). Compared with the control group, the ratio of the early diastolic velocity of mitral valvular and the early diastolic velocity of mitral annulus (E/E'), EDPVR and Tau index increased (P0.05). The EDPVR and Tau index decreased (all P0.05) in hypertensive group +BH4 (all P0.05). (4) hypertension group and hypertension group Compared with the control group, SOD and NO decreased (P0.05), and the prognosis of BH4 was given, SOD and NO increased (P0.05). 5. The result of radioimmunoassay showed that the expression of Ang II increased (P0.05) in the hypertension group and the control group (P0.05), and the expression of Ang II in the +BH4 group of hypertension was less than that of the hypertension group (P0.05). The expression of beta -MHC decreased (P0.05), the expression of alpha -SA, PKC E and beta -MHC increased (P0.05). (P0.05). Immunohistochemistry results showed that the expression of VEGF, PLB, Serca2 and Bax was increased compared with the control group. The expression of CL-2 increased, while the control group had similar results compared with the control group. The Western-blot results showed that the expression of PKC e decreased (P0.05) compared with the control group (P0.05). The expression of PKC epsilon in the hypertensive group +BH4 and the hypertension group increased (P0.05). Conclusion BH4 has no significant antihypertensive effect, but it can improve the left ventricular diastolic function. All this may be related to lowering the level of oxidative stress and enhancing the expression of NO through PKC pathway.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R544.1

【共引文献】