Study Of Nanomaterials Used As Myocardial Cells Sirna Delivery Vehicle And Its Effects On Intercalated Disc Assembly And Development

Objective:(a) To examine the effects of Caspase 3 silence by siRNA carrier based on SWNTs on apoptosis and heart function in early AMI.(b) To assess effects of AuNP/Col composites on the assembly and functional maturation of ID of NRVMMethods:(a)To construct F-CNT-siCas3 composite of SWNTs and siRNA,further to assess its impact on gene expression in cultured NRVM by western blotting, RT-PCR etc.;To assess its effects on the apoptosis and cardiac function in early AMI by ultrasound and Tunel staining; (b) To characterize the properties of AuNP/Col composite material by using AFM, TEM, SEM and electrochemical workstation;To assess the biocompatibility of this composite materials by live-dead and Alma Blue staining;To test the expression,distribution and assembly of the proteins associated to ID in each group at different time points (3d,7d,14d) from the level of morphological, molecular biology and electrophysiology function respectively;To explore the role of β1-integrin signaling pathway in regulating maturation of ID by AuNPs through immunofluorescence staining, western blotting, RT-PCR and so on.Results:(a) western blotting, RT-PCR results showed that compared with the control group, F-CNT-siCas3 reduced Caspase 3 expression of NRVM (P<0.05); Ultrasound, Tunel staining results demonstrated that F-CNT-siCas3 alleviated apoptosis and improved heart function in early available AMI rats (P<0.05); (b) SEM examination results showed that different concentrations of AuNPs in Col solution can be uniformly dispersed; AFM analysis showed that roughness increases with the concentration of the material AuNPs increase; Electrochemical workstation detection display electrical impedance of composite substrate was negatively correlated to the concentration ofAuNPs; Live/Dead and Alma Blue staining results showed:When AuNPs concentration was 0.1mmol/L,the substrate material excert least impact on cell viability and better effect to promote cell proliferation;TEM, and western blotting results showed that:In AuNP/Col group,the extended situation, development sarcomere of NRVM cells at different time points were better than Col group, and AuNPs can significantly enhance the ID-related protein (Cx43, PKP2, PG, and N-cadherin) expression (P<0.05), especially accelerated polarization of Connexin43; Calcium transients showed Ca2+ release rhythm in AuNP/Col group was more obvious;β1-integrin blocking experiments showed that (31-integrin antibody blocked the expression of Cx43 and mechanical connection of N-cadherin protein (P<0.05). western blotting were detected at time point of 3,7 and 14d,it is demonstrated that ILK expression levels were significantly higher (P<0.05) compare to Col group.Western blotting results further showed that at different time points, such as p-AKT protein expression was significantly higher (P<0.05) than Col group.The immunofluorescence staining results showed that blocking of the p-AKT signaling pathway could significantly reduce a linear plaques distributed in myocardial cells of Cx43 area compared with the other groups,Instead,add the P-catenin signal blocker IWP-2 could not inhibit the expression and distribution of Cx43 protein.N-cadherin expression in all groups have no significant changes in myocardial cells After adding these two blockers.Conclusion:(a) F-CNT-siCas3 carrier based on SWNTs effectively silence the expression of Caspase 3,reduce apoptotic and improve heart function in early AMI rats, which may provide a way for cardiac gene therapy; (b) AuNPs/Col composite biocompatible matrix can promote the formation,assembly and functional maturation of ID in NRVM.β1-integrin/ILK/p-AKT signaling pathway play an important role in AuNPs promoting gap junction protein Cx43 expression and accelerating the formation of gap junctions,which offerd an important theoretical molecular mechanisms on the applying of AuNPs to cardiac tissue engineering research.