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

【摘要】：目的研究四氫生物蝶呤(BH4)對(duì)醋酸脫氧皮質(zhì)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(dá)的影響。以期為探索左室舒張性心功能不全的治療提供實(shí)驗(yàn)依據(jù)。方法 90只10周齡雄性C57BL/6小鼠隨機(jī)分成手術(shù)組(n=50)與假手術(shù)組(n=40)。手術(shù)組小鼠切除左側(cè)腎臟后在頸部皮下埋入DOCA藥片,術(shù)后以普通飼料及1.05%氯化鈉(NaCl)溶液飼養(yǎng),至術(shù)后14天,排除死亡小鼠6只后隨機(jī)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(shù)組小鼠僅游離左側(cè)腎臟而不切除,并在頸部皮下埋入安慰劑藥片,術(shù)后以普通飼料及常規(guī)不含鹽凈化水飼養(yǎng),然后隨機(jī)分成對(duì)照組(n=20)及對(duì)照+BH4組(n=20)。四個(gè)亞組于術(shù)后21天時(shí),分別進(jìn)行以下實(shí)驗(yàn)測(cè)定：1.鼠尾動(dòng)脈壓測(cè)定小鼠舒張期血壓(DBP)及收縮期血壓(SBP)(n=10)。2.應(yīng)用彩色多普勒超聲心動(dòng)圖二維成像與組織多普勒成像技術(shù)測(cè)定各組小鼠二尖瓣環(huán)舒張?jiān)缙诘倪\(yùn)動(dòng)速度(E’)、二尖瓣口舒張?jiān)缙谘魉俣?E)及二尖瓣環(huán)舒張?jiān)缙谶\(yùn)動(dòng)速度及二尖瓣環(huán)舒張晚期運(yùn)動(dòng)速度的比值(E'/A')等心臟的結(jié)構(gòu)與功能參數(shù)(n=10)。3.血流動(dòng)力學(xué)測(cè)定小鼠心率(HR)、Tau指數(shù)、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(guān)系(ESPVR)、舒張末期壓力-容積關(guān)系(EDPVR)等指標(biāo)(n=6)。所有亞組均于術(shù)后22天時(shí)進(jìn)行取材,所取組織均行以下實(shí)驗(yàn)測(cè)定：①酶聯(lián)免疫吸附實(shí)驗(yàn)測(cè)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(cè)定心肌組織中BH4及二氫生物蝶呤(BH2)的含量。③放免法測(cè)定血漿血管緊張素Ⅱ(AngⅡ)含量。④實(shí)時(shí)熒光定量聚合酶鏈反應(yīng)(RT-PCR)測(cè)定心肌組織中α-SA、TGFβ1、PKCε及β-MHC的含量。⑤免疫組化測(cè)定血管內(nèi)皮生長(zhǎng)因子(VEGF)、受磷蛋白(PLB)、蛋白激酶C zeta (PKC-zeta)、肌漿／內(nèi)質(zhì)網(wǎng)鈣ATP酶2(Serca2)、B細(xì)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(guān)X蛋白(Bax) 。⑥Western-blot實(shí)驗(yàn)測(cè)定內(nèi)皮型一氧化氮合酶(eNOS)與PKCε的表達(dá)。結(jié)果①高血壓組與對(duì)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(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。②高血壓組與對(duì)照組相比,二尖瓣舒張?jiān)缙谘魉俣燃岸獍戥h(huán)舒張?jiān)缙谶\(yùn)動(dòng)速度的比值(E/E')、EDPVR及Tau指數(shù)增高(均P0.05)③高血壓+BH4組與高血壓組相比,EDPVR及Tau指數(shù)減小(均P0.05)。④高血壓組與對(duì)照組比較,SOD及NO減少(均P0.05),給予BH4干預(yù)后,SOD及NO增多(均P0.05)。⑤放免法結(jié)果顯示,高血壓組和對(duì)照組比較,AngⅡ的表達(dá)增加(P0.05),高血壓+BH4組與高血壓組相比,AngⅡ的表達(dá)減少(P0.05)。⑥T-PCR結(jié)果示高血壓組與對(duì)照組相比,α-SA、PKCε及β-MHC的表達(dá)減少(P0.05),給予BH4干預(yù)后,α-SA、PKCε及β-MHC的表達(dá)增加(P0.05)。⑦免疫組化結(jié)果示高血壓組與對(duì)照組相比,VEGF、PLB、Serca2及Bax表達(dá)增加,Phospho-PLB及BCL-2表達(dá)減少,高血壓+BH4組與高血壓組相比,VEGF、PLB、Serca2及Bax表達(dá)減少,Phospho-PLB及BCL-2表達(dá)增加,而對(duì)照+BH4組與對(duì)照組相比也出現(xiàn)了類似的結(jié)果。⑧Western-blot結(jié)果顯示高血壓組與對(duì)照組相比,PKCε的表達(dá)減少(P0.05),高血壓+BH4組與高血壓組相比,PKCε的表達(dá)增加(P0.05)。結(jié)論BH4無(wú)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(guò)PKCε途徑降低體內(nèi)氧化應(yīng)激水平、提高NO表達(dá)等有關(guān)。
[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(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R544.1

【共引文獻(xiàn)】

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

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本文編號(hào)：1866020