纳米材料作为心肌细胞siRNA递送载体及对闰盘组装发育影响的研究

发布时间：2018-05-12 10:30

  本文选题：SWNTs + Caspse　； 参考：《中国人民解放军医学院》2015年博士论文

【摘要】：目的：(一)通过体外及体内研究验证基于单壁碳纳米管(SWNTs)的siRNA载体沉默Caspase 3对急性梗死(AMI)早期细胞凋亡及心功能的影响；(二)评估纳米金颗粒(AuNP)/胶原(Co1)复合材料对新生大鼠心肌细胞(NRVM)闰盘(ID)组装及功能成熟的影响,并探讨AuNPs调控ID组装的分子机制。方法：(一)构建SWNTs与siRNA复合载体F-CNT-siCas3,通过western blotting、RT-PCR等评估其对体外培养NRVM目的基因表达的影响；通过超声,Tunel染色等评估其对AMI大鼠早期凋亡、心功能的影响；(二)采用原子力显微镜(AFM)、透射电镜(TEM)、扫描电镜(SEM)、电化学工作站等表征AuNP/Col复合材料特性；采用live-dead、阿尔玛蓝染色等评估复合材料的生物相容性；从形态学、分子生物学及功能上分别评估各组心肌细胞ID相关蛋白在不同时间点(3d、7d、14d)的表达、分布及组装情况；采用免疫荧光染色、western blotting、RT-PCR等方法探究β1-整合素信号通路在AuNPs调控ID成熟及功能发挥中的作用。结果：(一)western blotting、RT-PC R结果表明与对照组相比,F-CNT-siCas3降低了NRVM的Caspase3表达(P0.05)；超声,Tunel染色等结果表明F-CNT-siCas3可减轻AMI大鼠早期细胞凋亡,改善心功能(P0.05)；(二)SEM检测结果表明低浓度AuNP在Col溶液中分散更为均匀；AFM检测表明材料的粗糙度随AuNPs浓度的增加而增加；电化学工作站检测显示AuNPs农度与复合基质的电阻抗呈负相关；Live/Dead染色及阿尔玛蓝染色结果提示：当AuNPs农度为0.1mmol/L时材料对细胞活力的影响最小且有较好的促细胞增殖作用；TEM、及western blotting结果表明：AuNP/Col组NRVM细胞在各时间点的伸展、肌节发育情况均明显优于Co1组,且AuNPs可显著增强ID相关蛋白(Cx43、PKP2、PG和N-cadherin)的表达(P0.05),尤其加速缝隙连接蛋白Cx43蛋白的极化；钙瞬变结果显示AuNPs/Col组Ca2+的释放节律性更显著(P0.05),并形成更大幅度的一致性收缩；p1-整合素阻断实验表明p1-整合素抗体同时阻断了缝隙连接蛋白Cx43和力学连接蛋白N-cadherin的表达(P0.05)。western blotting检测两组细胞3、7和14d时的p-FAK、Src和ILK的蛋白表达情况,结果表明实验组ILK在各个时间点的表达量均显著高于对照组(P0.05)。进一步通过Western blotting检测两组心肌细胞不同时间点p-AKT等蛋白表达情况,结果表明实验组p-AKT在各个时间点的表达量均显著高于对照组(P0.05),而其余蛋白无明显变化(P0.05)；p-AKT信号通路阻断实验显示,与对照组相比,呈线性斑块分布于心肌细胞间区域的Cx43蛋白显著减少。相反,添加β-catenin的信号阻断剂IWP-2后,未抑制Cx43蛋白的表达及分布。而添加这两种阻断剂对心肌细胞间N-cadherin表达无影响。结论：(一)基于SWNTs的F-CNT-siCas3载体有效沉默了Caspase 3表达，并可减少AMI大鼠早期细胞凋亡,改善心功能,为CNTs用于心肌基因治疗提供了新的思路；(二)AuNP/Col复合基质具有良好的生物相容性,且基于AuNPs的胶原复合基质可促进NRVM中ID的形成、组装和功能成熟。β1-整合素／ILK/p-AKT信号通路在AuNPs促进ID缝隙连接相关蛋白Cx43表达方面起到了重要作用,此信号通路的激活加速了缝隙连接的形成。本研究为AuNPs用于心肌组织工程研究提供了重要的分子机制理论。
[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 .

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R542.22

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