本文選題：四氫生物蝶呤 + 醋酸脫氧皮質酮-鹽型高血壓小鼠��； 參考：《蘭州大學》2015年碩士論文

【摘要】：目的研究四氫生物蝶呤(BH4)對醋酸脫氧皮質酮(DOCA)-鹽型高血壓小鼠左心室舒張功能與蛋白激酶Cε(PKCε)表達的影響。以期為探索左室舒張性心功能不全的治療提供實驗依據。方法 90只10周齡雄性C57BL/6小鼠隨機分成手術組(n=50)與假手術組(n=40)。手術組小鼠切除左側腎臟后在頸部皮下埋入DOCA藥片,術后以普通飼料及1.05%氯化鈉(NaCl)溶液飼養(yǎng),至術后14天,排除死亡小鼠6只后隨機分為高血壓組(n=22)與高血壓+BH4組(n=22),并分別用生理鹽水(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。假手術組小鼠僅游離左側腎臟而不切除,并在頸部皮下埋入安慰劑藥片,術后以普通飼料及常規(guī)不含鹽凈化水飼養(yǎng),然后隨機分成對照組(n=20)及對照+BH4組(n=20)。四個亞組于術后21天時,分別進行以下實驗測定：1.鼠尾動脈壓測定小鼠舒張期血壓(DBP)及收縮期血壓(SBP)(n=10)。2.應用彩色多普勒超聲心動圖二維成像與組織多普勒成像技術測定各組小鼠二尖瓣環(huán)舒張早期的運動速度(E’)、二尖瓣口舒張早期血流速度(E)及二尖瓣環(huán)舒張早期運動速度及二尖瓣環(huán)舒張晚期運動速度的比值(E'/A')等心臟的結構與功能參數(n=10)。3.血流動力學測定小鼠心率(HR)、Tau指數、左心室舒張末期的壓力(LVEDP)、左心室收縮末期的壓力(LVESP)、收縮末期壓力-容積關系(ESPVR)、舒張末期壓力-容積關系(EDPVR)等指標(n=6)。所有亞組均于術后22天時進行取材,所取組織均行以下實驗測定：①酶聯(lián)免疫吸附實驗測定環(huán)磷酸鳥苷(cGMP)、超氧化物歧化酶(SOD)以及丙二醛(MDA)。②高效液相色譜法(HPLC)測定心肌組織中BH4及二氫生物蝶呤(BH2)的含量。③放免法測定血漿血管緊張素Ⅱ(AngⅡ)含量。④實時熒光定量聚合酶鏈反應(RT-PCR)測定心肌組織中α-SA、TGFβ1、PKCε及β-MHC的含量。⑤免疫組化測定血管內皮生長因子(VEGF)、受磷蛋白(PLB)、蛋白激酶C zeta (PKC-zeta)、肌漿／內質網鈣ATP酶2(Serca2)、B細胞淋巴瘤／白血病-2(BCL-2)、磷酸化的受磷蛋白(Phospho-PLB)及Bcl-2相關X蛋白(Bax) 。⑥Western-blot實驗測定內皮型一氧化氮合酶(eNOS)與PKCε的表達。結果①高血壓組與對照組相比,SBP與DBP均增高(P0.05),高血壓造模成功。高血壓+BH4組與高血壓組相比,血壓差異無統(tǒng)計學意義(P0.05)。②高血壓組與對照組相比,二尖瓣舒張早期血流速度及二尖瓣環(huán)舒張早期運動速度的比值(E/E')、EDPVR及Tau指數增高(均P0.05)③高血壓+BH4組與高血壓組相比,EDPVR及Tau指數減小(均P0.05)。④高血壓組與對照組比較,SOD及NO減少(均P0.05),給予BH4干預后,SOD及NO增多(均P0.05)。⑤放免法結果顯示,高血壓組和對照組比較,AngⅡ的表達增加(P0.05),高血壓+BH4組與高血壓組相比,AngⅡ的表達減少(P0.05)。⑥T-PCR結果示高血壓組與對照組相比,α-SA、PKCε及β-MHC的表達減少(P0.05),給予BH4干預后,α-SA、PKCε及β-MHC的表達增加(P0.05)。⑦免疫組化結果示高血壓組與對照組相比,VEGF、PLB、Serca2及Bax表達增加,Phospho-PLB及BCL-2表達減少,高血壓+BH4組與高血壓組相比,VEGF、PLB、Serca2及Bax表達減少,Phospho-PLB及BCL-2表達增加,而對照+BH4組與對照組相比也出現(xiàn)了類似的結果。⑧Western-blot結果顯示高血壓組與對照組相比,PKCε的表達減少(P0.05),高血壓+BH4組與高血壓組相比,PKCε的表達增加(P0.05)。結論BH4無明顯的降壓作用,但可改善左室舒張功能不全,這可能與其通過PKCε途徑降低體內氧化應激水平、提高NO表達等有關。
[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.

【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R544.1

【共引文獻】

相關期刊論文 前1條

1 田穎;張書余;朱衛(wèi)浩;羅斌;王寶鑒;;熱浪對ApoE基因缺陷小鼠超氧化物歧化酶的影響[J];科學技術與工程;2013年35期

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