本文選題：SWNTs + Caspse��； 參考：《中國(guó)人民解放軍醫(yī)學(xué)院》2015年博士論文

【摘要】：目的：(一)通過(guò)體外及體內(nèi)研究驗(yàn)證基于單壁碳納米管(SWNTs)的siRNA載體沉默Caspase 3對(duì)急性梗死(AMI)早期細(xì)胞凋亡及心功能的影響；(二)評(píng)估納米金顆粒(AuNP)/膠原(Co1)復(fù)合材料對(duì)新生大鼠心肌細(xì)胞(NRVM)閏盤(pán)(ID)組裝及功能成熟的影響,并探討AuNPs調(diào)控ID組裝的分子機(jī)制。方法：(一)構(gòu)建SWNTs與siRNA復(fù)合載體F-CNT-siCas3,通過(guò)western blotting、RT-PCR等評(píng)估其對(duì)體外培養(yǎng)NRVM目的基因表達(dá)的影響；通過(guò)超聲,Tunel染色等評(píng)估其對(duì)AMI大鼠早期凋亡、心功能的影響；(二)采用原子力顯微鏡(AFM)、透射電鏡(TEM)、掃描電鏡(SEM)、電化學(xué)工作站等表征AuNP/Col復(fù)合材料特性；采用live-dead、阿爾瑪藍(lán)染色等評(píng)估復(fù)合材料的生物相容性；從形態(tài)學(xué)、分子生物學(xué)及功能上分別評(píng)估各組心肌細(xì)胞ID相關(guān)蛋白在不同時(shí)間點(diǎn)(3d、7d、14d)的表達(dá)、分布及組裝情況；采用免疫熒光染色、western blotting、RT-PCR等方法探究β1-整合素信號(hào)通路在AuNPs調(diào)控ID成熟及功能發(fā)揮中的作用。結(jié)果：(一)western blotting、RT-PC R結(jié)果表明與對(duì)照組相比,F-CNT-siCas3降低了NRVM的Caspase3表達(dá)(P0.05)；超聲,Tunel染色等結(jié)果表明F-CNT-siCas3可減輕AMI大鼠早期細(xì)胞凋亡,改善心功能(P0.05)；(二)SEM檢測(cè)結(jié)果表明低濃度AuNP在Col溶液中分散更為均勻；AFM檢測(cè)表明材料的粗糙度隨AuNPs濃度的增加而增加；電化學(xué)工作站檢測(cè)顯示AuNPs農(nóng)度與復(fù)合基質(zhì)的電阻抗呈負(fù)相關(guān)；Live/Dead染色及阿爾瑪藍(lán)染色結(jié)果提示：當(dāng)AuNPs農(nóng)度為0.1mmol/L時(shí)材料對(duì)細(xì)胞活力的影響最小且有較好的促細(xì)胞增殖作用；TEM、及western blotting結(jié)果表明：AuNP/Col組NRVM細(xì)胞在各時(shí)間點(diǎn)的伸展、肌節(jié)發(fā)育情況均明顯優(yōu)于Co1組,且AuNPs可顯著增強(qiáng)ID相關(guān)蛋白(Cx43、PKP2、PG和N-cadherin)的表達(dá)(P0.05),尤其加速縫隙連接蛋白Cx43蛋白的極化；鈣瞬變結(jié)果顯示AuNPs/Col組Ca2+的釋放節(jié)律性更顯著(P0.05),并形成更大幅度的一致性收縮；p1-整合素阻斷實(shí)驗(yàn)表明p1-整合素抗體同時(shí)阻斷了縫隙連接蛋白Cx43和力學(xué)連接蛋白N-cadherin的表達(dá)(P0.05)。western blotting檢測(cè)兩組細(xì)胞3、7和14d時(shí)的p-FAK、Src和ILK的蛋白表達(dá)情況,結(jié)果表明實(shí)驗(yàn)組ILK在各個(gè)時(shí)間點(diǎn)的表達(dá)量均顯著高于對(duì)照組(P0.05)。進(jìn)一步通過(guò)Western blotting檢測(cè)兩組心肌細(xì)胞不同時(shí)間點(diǎn)p-AKT等蛋白表達(dá)情況,結(jié)果表明實(shí)驗(yàn)組p-AKT在各個(gè)時(shí)間點(diǎn)的表達(dá)量均顯著高于對(duì)照組(P0.05),而其余蛋白無(wú)明顯變化(P0.05)；p-AKT信號(hào)通路阻斷實(shí)驗(yàn)顯示,與對(duì)照組相比,呈線性斑塊分布于心肌細(xì)胞間區(qū)域的Cx43蛋白顯著減少。相反,添加β-catenin的信號(hào)阻斷劑IWP-2后,未抑制Cx43蛋白的表達(dá)及分布。而添加這兩種阻斷劑對(duì)心肌細(xì)胞間N-cadherin表達(dá)無(wú)影響。結(jié)論：(一)基于SWNTs的F-CNT-siCas3載體有效沉默了Caspase 3表達(dá)，并可減少AMI大鼠早期細(xì)胞凋亡,改善心功能,為CNTs用于心肌基因治療提供了新的思路；(二)AuNP/Col復(fù)合基質(zhì)具有良好的生物相容性,且基于AuNPs的膠原復(fù)合基質(zhì)可促進(jìn)NRVM中ID的形成、組裝和功能成熟。β1-整合素／ILK/p-AKT信號(hào)通路在AuNPs促進(jìn)ID縫隙連接相關(guān)蛋白Cx43表達(dá)方面起到了重要作用,此信號(hào)通路的激活加速了縫隙連接的形成。本研究為AuNPs用于心肌組織工程研究提供了重要的分子機(jī)制理論。
[Abstract]:Objective : To investigate the effect of Caspase 3 on early apoptosis and cardiac function in acute myocardial infarction ( AMI ) by means of in vitro and in vivo studies .
( 2 ) To evaluate the influence of nano - gold particle ( AuNP ) / collagen ( Co1 ) composite on the assembly and functional maturation of the intercalated disk ( ID ) of neonatal rat cardiomyocytes ( NRVM ) , and to explore the molecular mechanism of the self - regulation ID assembly .
The effects of ultrasound and Tunel staining on early apoptosis and cardiac function in AMI rats were assessed .
( 2 ) The properties of AuNP / Col composites were characterized by atomic force microscopy ( AFM ) , transmission electron microscope ( TEM ) , scanning electron microscope ( SEM ) and electrochemical workstation .
the biocompatibility of the composite material is evaluated by adopting live - dead , Alpha - blue staining and the like ;
The expression , distribution and assembly of myocardial cell ID - related protein in different time points ( 3d , 7d , 14d ) were assessed from morphology , molecular biology and function .
The results showed that F - CNT - siCas3 reduced the expression of Caspase3 in NRVM compared with the control group ( P0.05 ) .
The results showed that F - CNT - siCas3 could alleviate the early apoptosis of AMI rats and improve cardiac function ( P0.05 ) .
( 2 ) The results of SEM showed that the concentration of AuNP was more uniform in Col solution .
The results show that the roughness of the material increases with the increase of AuSn concentration .
The electrochemical workstation showed negative correlation with the electrical impedance of the composite substrate .
The results suggested that the effect of material on cell viability was minimal and had a better effect on cell proliferation when the yield was 0.1 mmol / L .
The results of TEM and western blotting showed that in AuNP / Col group NRVM cells were obviously superior to Co1 group at different time points , and AuxAcan significantly enhance the expression of Cx43 , PKP2 , PG and N - cadherin ( P0.05 ) , especially to accelerate the polarization of Cx43 protein in the gap junction protein .
The results showed that the release rhythm of Ca 2 + was more significant ( P0.05 ) .
The results showed that the expression level of p - fos protein in the experimental group was significantly higher than that in the control group ( P0.05 ) . The results showed that the expression of p - cadherin at various time points was significantly higher than that of the control group ( P0.05 ) .
Conclusion : ( 1 ) The expression and distribution of Cx43 protein were not inhibited by the addition of 尾 - catenin signaling blocker IWP - 2 .
( 2 ) The AuNP / Col composite matrix has good biocompatibility , and the formation , assembly and functional maturation of the ID in NRVM can be promoted by the collagen composite matrix . The activation of the signal pathway accelerates the formation of the gap junction . The present study provides important molecular mechanism theory for the research of myocardial tissue engineering .

【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R542.22

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