The New Nano-system Build And Its Role In The Proliferation And Apoptosis Of Pulmonary Artery Smooth Muscle Cell

Hypoxic pulmonary arterial hypertension (HPAH) is an important pathophysiologicalbasis of clinical pulmonary diseases, including the development of chronic obstructivepulmonary disease to the pulmonary heart disease, so the prevention and treatment of theHPAH has important clinical significance. Pulmonary arterial hypertension is characterizedby vascular remodeling, which involves the lack of pulmonary arterioles and the excessiveproliferation of endothelial and smooth muscle cells. Of them, the abnormal proliferation ofthe pulmonary arterial smooth muscle cells is important in the process of pulmonaryvascular remodeling. Thus, study how to prevent and reverse the abnormal pulmonaryartery smooth muscle cells proliferation in specific pathological conditions is the key todetermine the pathogenesis and treatment of the pulmonary arterial hypertension.Recent years, more and more people getting their attention to gene therapy. Genetherapy is to inject the normal gene or the therapeutic gene into target cells, therebycorrecting the gene defects or playing a therapeutic effect to treat diseases. Nowadays, wemainly transfect the host with small interfering RNA (siRNA) or antisense oligonucleotide(ASON) to do the gene targeted therapy. However, how to make them safely and effectivelytransport inside the cells, or release steadily is one of the challenges. With the developmentof the nano-technology, it is a good choice to use the nano-system as a carrier to solve theproblem of the degradation of the ASON and siRNA inside cells, and it can also promotethe intake of ASON and siRNA. The meaning of our study is to synthetize a novel hybridnano-system composed of PH-responsive hydroformylation of cyclodextrins(α-cyclodextrin, αCD) and Low molecular weight polyethylene imine PEI1800NPs.Furthermore, we packed Bcl-xl ASON/mTOR siRNA with nano-system Ac-αCD NPs,researched the safety and effectiveness, and analyzed the influence of the nano-system onnormoxia or hypoxia-induced proliferation and apoptosis of the pulmonary arterial smoothmuscle cells. To conclude, our study will offer new theoretical basis and strategy for the prevention and treatment of the diseases such as pulmonary arterial hypertension,pulmonary vascular remodeling and so forth.ObjectTo build new nano-system, Ac-αCD NPs wrapped up with Bcl-xl ASON or mTORsiRNA, researching the safety and effectiveness of the nano-system, and analyzing theinfluence of the system on the proliferation and apoptosis of PASMCs.Methods1. Preparation and characteristics of the new nano-system Ac-αCD packaged withASON⑴Packaging Ac-αCD with ASON by using the improved double nano emulsionprocess.⑵Using scanning electron microscopy, transmission electron microscopy anddynamic light scattering to observe the morphology and distribution of nanometer particles,as well as the hydrolysis of nanometer particles in the buffer.2. The influence of the nano-system Ac-αCD packaged with ASON on theproliferation and apoptosis of RPASMCsCulturing the rat pulmonary arterial smooth muscle cells (RPASMCs) in vitro, andtreat RPASMCs transfected nano-system Ac-αCD packaged with ASON for48h in normalcondition. Using laser confocal microscopy to observe the intake of Ac-αCD packaged withASON by RPASMCs, RT-PCR and Western blot to detect the mRNA and proteinexpression of Bcl-xl, MTT to determine the proliferation of RPAMCs, and flow cytometryto determine the apoptosis of the RPASMCs.3. The influence of the nano-system Ac-αCD packaged with mTOR siRNA on theproliferation and apoptosis of hypoxia-treated RPASMCsCulturing RPASMCs in vitro and transfecting RPASMCs respectively with Ac-αCDNPs, PLGA NPs, PEI1800NPs, PEI25000NPs and Lipofectamine2k, which are allbringed with mTOR siRNA. Using MTT technology to detect the impact of differentvectors on toxicity of RPASMCs, laser confocal microscopy to observe the intake ofAc-αCD packaged with different concentration of mTOR siRNA by RPASMCs, also theintake of distinct vectors packaged with the same mTOR siRNA by RPASMCs, flowcytometry to determine the transfection efficiency of distinct vectors packaged with mTOR siRNA, MTT to detect the inhibit effect of different vectors packaged with mTOR siRNAon the proliferation of RPASMCs, RT-PCR, Western blot,3H-TdR and flow cytometry todetermine the mRNA and protein expression of mTOR induced by RPASMCs treated48hin hypoxic condition with different vectors packaged with mTOR siRNA, and the influenceof mTOR siRNA and p-mTOR on the proliferation and apoptosis of RPASMCs.Results1. Successfully construct new PH-responsive hydroformylation of cyclodextrinsnano-system with excellent cell compatibility. Using scanning electron microscopy,transmission electron microscopy and dynamic light scattering, spherical nanoparticles withclear boundaries were prepared according to the formula, hybrid NPs release ASON at pH5.0is much more easier to hydrolyze in PBS buffer2. Observation from laser confocal microscopy demonst rated that cells treated withASON-NPs showed a large number of granular red fluorescent substances, no suchsubstances were observed in control and NPs group, the mRNA and protein expression ofBcl-xl in ASON-NPs group obviously lower than control and NPs group (P<0.05). Theinhibition ratio of ASON-NPs, NPs and control group were53.61±3.02%，6.30±1.90%and1.40±0.62%, while apoptosis ratio were53.04±2.09%，10.98±2.03%，2.19±0.11%.The apoptosis ratio of ASON-NPs group obviously higher than the control group (P<0.01)and the NPs group (P<0.01).3. After RPASMCs were treated with Ac-αCD NPs, PLGA NPs, PEI1800NPs, PEI25000NPs and Lipofectamine2k, with the same concentration, toxicity of Ac-αCD NPswas in a minimum. After RPASMCs were transfected with Ac-αCD NPs packaged withmTOR siRNA, with the concentration of mTOR siRNA increased, intensity of redfluorescence in RPASMCs gradually enhanced, of which50,100,200pmol/ml groupobviously higher than25pmol/ml group (P<0.05). Results of Semi-quantitative andquantitative demonstrated that the transfection effeciency of the Ac-αCD NPs group wasobviously higher than the PEI1800NPs, PEI25000NPs and Lipofectamine2k group(P<0.05). The inhibition ratio of Ac-αCD NPs/PEI NPs, PLGA NPs, PEI1800NPs, PEI25000NPs and Lipofectamine2k group were49.26±2.22%,42.9±2.24%,35.79±1.52%,34.76±1.68%and30.66±1.55%. The inhibition ratio of Ac-αCD NPs group packagedwith mTOR siRNA was obviously higher than other vectors (P<0.05). After RPASMCs were treated in hypoxic condition for24h,48h and96h, the mRNA and Phosphorylationexpression of mTOR were all increased in24h (P<0.05)，48h expressed the most (P<0.01).The proliferation ratio of RPASMCs treated in hypoxic condition for12h,24h,48h and96hwas obviously higher than24h (P<0.05). RPASMCs transfected with different vectors(Ac-αCD NPs, PLGA NPs, PEI1800NPs, PEI25000NPs or Lipofectamine2k) packagedwith mTOR siRNA for48h, the mRNA and Phosphorylation expression of mTOR inAc-αCD NPs group was obviously lower than other vectors treated group (P<0.05).RPASMCs transfected with different vectors packaged with mTOR siRNA for48h, theproliferation ratio of the Ac-αCD/PEI NPs group was obviously lower than other vectorstreated group (P<0.05) and the apoptosis ratio of Ac-αCD NPs group was obviously higherthan other vectors treated group, while the proliferation ratio was obviously higher thanother vectors treated group (P<0.05).Conclusions1. Successfully constructed new nano-system Ac-αCD NPs packaged with Bcl-xlASON/mTOR siRNA, while it is much more easier to hydrolyze in pH5.0PBS buffer.2. The Ac-αCD NPs packaged with Bcl-xl ASON can effectively transfect RPASMCs,Bcl-xl ASON obviously inhibited the proliferation of RPASMCs and promote the apoptosisof RPASMCs.3. The Ac-αCD NPs packaged with mTOR siRNA can effectively transfect RPASMCs,with low level of toxicity and high efficiency of transfection, which induces mTOR siRNArelease effectively and plays a perfect biological effect, therefore inducing the apoptosis ofRPASMCs in hypoxic condition and obviously inhibiting the proliferation of RPASMCs.