本文選題：環(huán)磷酸腺苷　切入點(diǎn)：結(jié)締組織生長(zhǎng)因子　出處：《華中科技大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：第Ⅰ部分環(huán)磷酸腺苷抑制CTGF表達(dá)調(diào)控小鼠心肌梗死后心肌纖維化的作用觀察 摘要：目的觀察環(huán)磷酸腺苷(cAMP)對(duì)小鼠心肌梗死后結(jié)締組織生長(zhǎng)因子(CTGF)的調(diào)控作用以及對(duì)心肌纖維化的影響 方法72只SPF級(jí)C57BL雄性小鼠隨機(jī)分為假手術(shù)組(MS組)、心肌梗死組(MI組)及藥物干預(yù)組(MT組),每組各24只。MI組及MT組小鼠呼吸機(jī)輔助開(kāi)胸結(jié)扎左冠狀動(dòng)脈前降支(LAD)建立心肌梗死動(dòng)物疾病模型：MS組小鼠開(kāi)胸后不結(jié)扎,其余操作同MI組及MT組。術(shù)后1周開(kāi)始MT組腹腔注射環(huán)磷酸腺苷葡胺(MAC),連續(xù)給藥4周；同時(shí)MS組及MI組注射等量生理鹽水對(duì)照。術(shù)后第5周,各組隨機(jī)抽取10只小鼠行心臟超聲檢測(cè),測(cè)量心臟形態(tài)指標(biāo)及心功能指標(biāo)；超聲完畢后,立即開(kāi)胸收獲心臟標(biāo)本,行組織形態(tài)學(xué)HE染色、膠原纖維Masson染色及CTGF免疫組化染色。各組剩余小鼠按照5只、5只、4只隨機(jī)分成3組,收獲心臟標(biāo)本,分離左心室梗死區(qū)及非梗死區(qū),分別行羥脯氨酸含量測(cè)定、CTGF蛋白表達(dá)western blot檢測(cè)及CTGF mRNA、TGF-β1mRNA表達(dá)量RT-PCR檢測(cè). 結(jié)果超聲心動(dòng)圖提示MT組射血分?jǐn)?shù)(EF)為(19.83±3.95)%,左室短軸縮短率(FS)為(9.07±1.9)%,均高于MI組[EF(13.65±2.14)%,FS(6.22±1.03)%](P0.05)；左心室每搏輸出量(SV)MT組[(26.26±3.57)μL]優(yōu)于MI組[(18.82±2.71)μL](P0.05)；MT組左心室前、后壁厚度及內(nèi)徑與MI組之間無(wú)明顯差異(P0.05)。心臟Masson染色提示MT組膠原纖維向非梗死區(qū)心肌組織浸潤(rùn)減少；免疫組化CTGF陽(yáng)性表達(dá)較MI組減少。MT組梗死區(qū)羥脯氨酸含量[(1388.98±86.26)u g/mg]低于MI組[(1673.12±199.97) μ g/mg](P0.05)；非梗死區(qū)羥脯氨酸含量[(662.92±98.05)μ g/mg]低于MI組[(855.84±83.3)u g/mg)](P0.05)。Western blot檢測(cè)提示MT組CTGF蛋白表達(dá)量低于MI組(P0.05)。RT-PCR檢測(cè)提示MT組CTGF mRNA表達(dá)量低于MI組(P0.05);TGF-β1mRNA表達(dá)量MT組與MI組之間在梗死區(qū)與非梗死區(qū)均無(wú)明顯統(tǒng)計(jì)學(xué)差異(P0.05)。 結(jié)論環(huán)磷酸腺苷可以抑制CTGF的表達(dá),降低小鼠心肌梗死后膠原纖維的表達(dá),減輕梗死區(qū)膠原纖維向非梗死區(qū)心肌的浸潤(rùn)程度,改善心臟功能。 第Ⅱ部分激活cAMP信號(hào)通路調(diào)控小鼠心肌成纖維細(xì)胞結(jié)締組織生長(zhǎng)因子的表達(dá) 摘要：目的研究小鼠心肌成纖維細(xì)胞(MCFs)胞內(nèi)環(huán)磷酸腺苷(cAMP)濃度提高對(duì)結(jié)締組織生長(zhǎng)因子(CTGF)表達(dá)的影響及其信號(hào)通路。 方法采用1周齡內(nèi)C57BL乳鼠,獲取心臟后分離培養(yǎng)原代MCFs,第3代細(xì)胞用于實(shí)驗(yàn)。使用轉(zhuǎn)化生長(zhǎng)因子-β1(TGF-β1)培養(yǎng)細(xì)胞,快速上調(diào)MCFs對(duì)CTGF的表達(dá),模擬小鼠心肌梗死后CTGF高表達(dá)情況。使用腺苷酸環(huán)化酶激活劑Forskolin干預(yù)培養(yǎng)細(xì)胞后檢測(cè)MCFs表達(dá)CTGF的變化,同時(shí)檢測(cè)可能的信號(hào)分子蛋白激酶A(PKA)、p44/42絲裂素活化蛋白激酶(MAPK)及磷酸化p44/42MAPK表達(dá)的變化。分別給予PD98509及Rp-cAMPS抑制p44/42MAPK的磷酸化水平及PKA的表達(dá),檢測(cè)細(xì)胞PKA、p44/42MAPK、磷酸化p44/42MAPK表達(dá)的變化,并同時(shí)檢測(cè)細(xì)胞內(nèi)CTGF的表達(dá)。 結(jié)果TGF-β1可以快速上調(diào)MCFs對(duì)CTGF的表達(dá)。Forskolin干預(yù)培養(yǎng)MCFs后,細(xì)胞內(nèi)cAMP濃度明顯提高、細(xì)胞活力下降；同時(shí)細(xì)胞中CTGF mRNA及蛋白表達(dá)均下降；其可能的信號(hào)分子PKA表達(dá)上升,磷酸化p44/42MAPK水平表達(dá)下降而p44/42MAPK總蛋白無(wú)明顯改變。MCFs給予PD98509抑制p44/42MAPK磷酸化水平后,PKA表達(dá)上升,同時(shí)細(xì)胞中CTGF mRNA及蛋白的表達(dá)下調(diào)：給予Rp-cAMP抑制PKA活化后,p44/42MAPK磷酸化水平升高,細(xì)胞中CTGF mRNA及蛋白表達(dá)出現(xiàn)上調(diào)。 結(jié)論Forskolin可以通過(guò)提高M(jìn)CFs胞內(nèi)cAMP濃度抑制MCFs對(duì)CTGF的表達(dá)。其作用信號(hào)通路為高濃度cAMP上調(diào)PKA表達(dá),降低下游p44/42MAPK磷酸化水平,抑制CTGF的表達(dá)。
[Abstract]:The effect of cyclic adenosine cyclic adenosine on CTGF expression in regulating myocardial fibrosis after myocardial infarction in mice
Abstract: Objective To observe the effect of cAMP on the regulation of connective tissue growth factor (CTGF) after myocardial infarction in mice and the effect on myocardial fibrosis
Methods 72 SPF male C57BL mice were randomly divided into sham operation group (group MS), myocardial infarction group (MI group) and drug intervention group (MT group), each group had 24 rats in group.MI and MT group were ventilator assisted ligation of left anterior descending coronary artery (LAD) to establish the model of animal myocardial disease infarction in MS group after thoracotomy without ligation, the same with the MI group and MT group. MT group received intraperitoneal injection of meglumine Cyclicadenylate started 1 weeks after operation (MAC), administered continuously for 4 weeks; at the same time, MS group and MI group were injected with normal saline. After fifth weeks, echocardiography 10 mice for each group were randomly selected, measuring heart shape index and heart function index; ultrasound after thoracotomy, immediately harvested heart specimens for histological HE staining, collagen Masson staining and CTGF immunohistochemical staining. All remaining mice according to 5, 5, 4 were randomly divided into 3 groups, the harvest of heart were left The content of hydroxyproline was measured, the expression of CTGF protein was detected by Western blot and CTGF mRNA, and the expression of TGF- beta 1mRNA was detected in the ventricular infarction area and non infarct area.
緇撴灉瓚呭０蹇?jī)鍔ㄥ浘鎻惤C篗T緇勫皠琛，

本文編號(hào)：1631230