本文關(guān)鍵詞：β3-AR對(duì)心肌細(xì)胞凋亡的影響及介導(dǎo)機(jī)制研究　出處：《石河子大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 心肌細(xì)胞 β3腎上腺素能受體 細(xì)胞凋亡 Akt p38MAPK

【摘要】：目的:采用去甲腎上腺素(noradrenaline,NE)誘導(dǎo)心肌細(xì)胞凋亡,分別應(yīng)用β3-AR激動(dòng)劑及阻滯劑進(jìn)行干預(yù),觀察β3-AR對(duì)心肌細(xì)胞凋亡的影響,探討β3-AR與PI3K/Akt和p38MAPK信號(hào)通路對(duì)心肌細(xì)胞凋亡的作用,為β3-AR的信號(hào)通路研究提出新的理論。方法:使用優(yōu)化的Ⅱ型膠原酶和差速貼壁法分離培養(yǎng)心肌細(xì)胞,分別用總蛋白法、超速離心法、試劑盒法提取心肌細(xì)胞β3-AR膜蛋白。BCA法進(jìn)行蛋白定量,Western blot法檢測(cè)蛋白樣品中β3-AR提取蛋白的特異性。實(shí)驗(yàn)分組:正常對(duì)照組;NE誘導(dǎo)組:NE培養(yǎng)48 h;BRL組:β3-AR激動(dòng)劑(BRL37344)干預(yù)+NE培養(yǎng)48 h;SR組:β3-AR阻滯劑(SR59230A)干預(yù)+NE培養(yǎng)48 h。TUNEL法及流式細(xì)胞儀雙染法檢測(cè)心肌細(xì)胞凋亡率;Western blot檢測(cè)各組凋亡相關(guān)因子Bcl-2、Bax和caspases-3蛋白的表達(dá);Western blot和q RT-PCR檢測(cè)各組PI3K/Akt和p38MAPK信號(hào)通路蛋白和m RNA的表達(dá)。結(jié)果:1.優(yōu)化心肌細(xì)胞的培養(yǎng)方法可獲得產(chǎn)量大、濃度高的細(xì)胞,能滿足后續(xù)實(shí)驗(yàn);2.與其他組比較,試劑盒法提取蛋白的濃度最高,差異均有統(tǒng)計(jì)學(xué)意義(P0.05);Western blot檢測(cè)結(jié)果示:與其他組比較,試劑盒提取法所得β3-AR膜蛋白含最高,差異均有統(tǒng)計(jì)學(xué)意義(P0.05);3.TUNEL法及流式細(xì)胞儀檢測(cè)心肌細(xì)胞凋亡率:BRL組細(xì)胞凋亡率明顯增高,與NE誘導(dǎo)組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05);4.Western blot檢測(cè)結(jié)果示:BRL組caspases-3和Bax/Bcl-2比值增高,與NE誘導(dǎo)組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05);5.Western blot檢測(cè)結(jié)果示:BRL組磷酸化的p38MAPK蛋白表達(dá)增高,與NE誘導(dǎo)組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05);SR組磷酸化的Akt蛋白表達(dá)增高,與NE誘導(dǎo)組比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.試劑盒提取法可有效提高β3-AR膜蛋白的濃度和特異性;2.β3-AR可促進(jìn)體外培養(yǎng)NE誘導(dǎo)的心肌細(xì)胞凋亡;3.β3-AR過度激活可通過下調(diào)PI3K/Akt信號(hào)和上調(diào)p38MAPK信號(hào)通路促進(jìn)心肌細(xì)胞凋亡。
[Abstract]:Objective: using norepinephrine (noradrenaline, NE) were used to induce apoptosis of myocardial cells, beta 3-AR agonists and antagonists intervention, to observe the effect of beta 3-AR on myocardial cell apoptosis, explore the beta 3-AR and PI3K/Akt and p38MAPK signal pathway on apoptosis of myocardial cells with new theory for the study of beta 3-AR signal pathway. Methods: using the optimized type II collagenase and differential adherent cultured myocardial cells, respectively, with the total protein, ultracentrifugation, protein quantitative extraction of myocardial cell beta 3-AR membrane protein.BCA kit method, specific protein beta 3-AR extraction protein samples Western blot method in the experimental groups. Normal control group; NE group: NE culture induced by 48 h; group BRL: 3-AR beta agonists (BRL37344) intervention +NE cultured for 48 h; group SR: 3-AR beta blockers (SR59230A) intervention +NE 48 h.TUNEL culture method and flow cytometry double staining The apoptosis rate of myocardial cells; apoptosis related factor blot detection of Western Bcl-2, the expression of Bax and caspases-3 protein expression of Western and Q; blot RT-PCR was used to detect PI3K/Akt and p38MAPK signal pathway of M protein and RNA. Results: 1. optimized culture of myocardial cells can be obtained from the large amount, high concentration of cells, can satisfy the follow-up experiment 2.; compared with the other groups, the kit method for extracting protein was the highest, the differences were statistically significant (P0.05); Western blot assay results showed that: compared with the other groups, extraction kit method the beta 3-AR membrane protein containing the highest, the differences were statistically significant (P0.05); 3.TUNEL method and flow detection of myocardial cell apoptosis type cell rate: the apoptosis rate in BRL group increased significantly, compared with the induction of NE, the difference was statistically significant (P0.05); 4.Western blot assay results showed: in group BRL, caspases-3 and Bax/Bcl-2 ratio increased, and NE induced group Comparison, the difference was statistically significant (P0.05); 5.Western blot assay results showed: in BRL group the expression of phosphorylated p38MAPK protein increased, compared with the induction of NE, the difference was statistically significant (P0.05); SR group the expression of phosphorylated Akt protein increased, compared with the induction of NE, the difference was statistically significant (P0.05) conclusion: 1.. Extraction Kit method can effectively improve the beta 3-AR membrane protein concentration and specificity; 2. beta 3-AR can promote cultured myocardial cell apoptosis induced by NE in vitro; 3. beta excessive activation of 3-AR can downregulate the PI3K/Akt signal and the up regulation of p38MAPK signaling pathway to promote myocardial cell apoptosis.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R54

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉小娟;朱正炎;沈彬;張紅衛(wèi);牛翠;劉燕;呂建華;張建國(guó);唐立榮;;系統(tǒng)化管理模式對(duì)慢性心力衰竭患者生活質(zhì)量的影響[J];中西醫(yī)結(jié)合心腦血管病雜志;2015年02期

2 張健;張宇輝;;中國(guó)心力衰竭診斷和治療指南2014[J];中華心血管病雜志;2014年02期

3 孫路路;呂蓉;梁濤;季詩(shī)明;康曉鳳;郭金玉;張健;;心力衰竭患者出院后1年內(nèi)預(yù)后狀況及影響因素分析[J];中國(guó)循環(huán)雜志;2013年02期

4 邢作英;王永霞;朱明軍;;慢性心力衰竭流行病學(xué)研究現(xiàn)狀及其病因[J];中華實(shí)用診斷與治療雜志;2012年10期

5 鄧新桃;石桂良;王如興;趙建祥;鄭金國(guó);鮑穎芳;;B型利鈉肽水平對(duì)慢性心力衰竭患者預(yù)后的影響[J];中華心血管病雜志;2012年06期

6 秦曉毅;盧新政;;2010年NICE慢性心力衰竭診治指南更新的解讀[J];心血管病學(xué)進(jìn)展;2011年04期

7 周斌;劉曉東;王鳳鵑;劉珂;陳溥言;;PK-15細(xì)胞膜蛋白提取方法的比較及豬瘟病毒膜表面受體的初步鑒定[J];畜牧與獸醫(yī);2011年05期

8 陳晶瑜;郭寶峰;何付麗;曲春鶴;尹克鑫;楊洪達(dá);趙長(zhǎng)山;;適合雙向電泳的植物全蛋白提取方法比較[J];中國(guó)農(nóng)學(xué)通報(bào);2010年23期

9 鄭猛;符永恒;肖定璋;劉曉穎;梁家亮;;b3AR通過cGMP-PKG-p38通路調(diào)控心肌MIF發(fā)揮抗凋亡作用[J];中國(guó)病理生理雜志;2010年10期

10 孔一慧;劉玉冰;張莉;邵群;李悅;;心力衰竭大鼠心肌β_3腎上腺素受體表達(dá)及其對(duì)鈣離子相關(guān)調(diào)節(jié)蛋白的作用[J];中國(guó)老年學(xué)雜志;2010年15期

相關(guān)博士學(xué)位論文 前1條

1 李振魁;β腎上腺素受體與心力衰竭關(guān)系的研究[D];第三軍醫(yī)大學(xué);2004年

，

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