| Research background:Acute myocardial infarction,a severe illness,is characterized by consistent myocardial ischemia and hypoxia.The expression of receptor for advanced glycation end products(RAGE)soars while acute myocardial infarction happens,and inhibiting RAGE protein expression can attenuate myocardial injury.Currently,the delivery of small interfering RNA through virus or liposome exerting a drastic reduction in the expression of rage has been reported by large numbers of researches.Nevertheless,viral vector possesses cytotoxicity,immunogenicity and the shortcomings of triggering off inflammation and gene mutation,while the defects of liposome,such as low encapsulation efficiency,poor stability,rapid clearance rate in bloodstream,have been realized.As a biodegradable polysaccharide,that chitosan(CS)possesses low cytotoxicity,low immunogenicity,good biocompatibility,slow clearance rate in bloodstream and stable physical and chemical properties prevails among experts.Therefore,the transfection of CS/siRNA nanoparticles may become a way of high-efficient and low cytotoxic genetic treatment.Research objectives:(1)To explore the optimal transfection protocol of CS/siRNA nanoparticles towards primary myocardial cell in Sprague-Dawley(SD)rat;(2)To assure the cytotoxicity of CS/siRNA nanoparticles towards primary myocardial cell;(3)To explore the effects of CS/targeted rage siRNA towards primary myocardial cell in SD rat on rage expression and cell viability,which may supply reference to CS/siRNA research on myocardial ischemia and hypoxia animal model and drug therapies on clinical cardiological diseases.Research methods:(1)The preparation of CS/siRNA nanoparticles:for obtaining 1mg/ml CS solution,CS particles are dissolved in pH5.5 sodium acetate-acetic acid buffer.Then,while 20 μL 100μ mol/L siRNA solution is added to 1mL CS solution and the compound is blended by magnetic stirring for 2-3min,CS/siRNA nanoparticles is synthesized.The distribution of CS/siRNA particle size and Zeta potential in CS/siRNA are detected by laser particle size analyzer.(2)The isolation and culture of primary myocardial cell from 1-3day SD rat pups:hearts acquiring from twelve 1-3day SD rat pups are washed with PBS to eliminate blood.For obtaining ventricular cell,atrial tissue and artery tissue are clipped off and ventricular tissue is cut into small pieces of 1-2mm3 by scissors.After 0.25%trypsin enzymic digestion,primary myocardial cells are collected and then diluted with complete medium.Eventually,20ml cell suspension is incubated in 75 culture bottles for 100min,then cell suspension is collected and incubated in other culture plate.(3)The transfection method of CS/siRNA nanoparticles towards primary myocardial cell:CS/siRNA nanoparticles with distinct concentrations are transfected in primary myocardial cell at 37℃ and 21%O2.(4)The assurance of CS/siRNA nanoparticles’ optimal incubation time:the experiment is classified into 12 h incubation group and 24 h incubation group,and each group is incubated with distinct concentrations CS/siRNA nanoparticles respectively,whose myocardial cells viability is assessed by live cell imaging system and CCK8 after incubation.(5)The assurance of CS/cy5-NC-siRNA nanoparticles’ optimal transfection efficiency:12h incubation group,whose subgroups are incubated with distinct concentrations CS/cy5NC-siRNA nanoparticles respectively,is chosen for detection of the transfection efficiency of CS/cy5-NC-siRNA by live cell imaging system and laser scanning confocal microscope.(6)The establishment of myocardial cell hypoxia model:primary myocardial cell prepared for hypoxia is incubated with complete medium without glucose,then these cells are incubated in a tri-gas incubator at 3%O2 for different time.(7)The detection of myocardial cell viability:hypoxia incubation groups,which are incubated with CS/NC-siRNA nanoparticles with distinct concentrations including Omg/ml,20*10-3mg/ml and 24*10-3mg/ml at 3%O2 for distinct time,are detected by CCK8 for cell viability.(8)The inhibition of rage expression:hypoxia incubation groups are incubated with CS/targeted rage siRNA nanoparticles with distinct concentrations including Omg/ml,20*10-3mg/ml and 24*10-3mg/ml at 3%O2.Then,the expression level of rage’s mRNA is assessed by qPCR,the expression level of RAGE protein is detected by Western Blot,and each groups’ cell viability is measured by CCK8 after incubation.Research results:(1)The particle size of CS/siRNA nanoparticles with N/P ratios 70:1 is 370.50 nm,and its Zeta potential is 28.17 mV,which is synthesized by magnetic stirring and detected by laser particle size analyzer.(2)According to live cell imaging system,compared with 24h incubation group,12h incubation group possesses more definite and complete cardiomyocytes morphology,whose cell nucleus and the state of cell division are more manifest.(3)There were no statistically significant differences between 0 mg/mL group and other groups on the cell viability in 12 h incubation group(P>0.05),while the cell viability of 0 mg/mL group is higher than those of the other groups in 24 h incubation group(P<0.05),and meanwhile,the cell viabilities of each groups in 12 h incubation group are distinctly superior to those of 24 h incubation group’s corresponding groups,except 0 mg/mL group(P<0.01).(4)According to live cell imaging system,each groups possess more efficient transfections in 24 h after the administration of CS/siRNA compared with corresponding groups in 12h(P<0.0001),yet there are no significant differences between 20*10-3 mg/mL group and other groups on transfection efficiency in 24 h after the administration of CS/siRNA(P>0.05),and after the administration of CS/siRNA in 48h,the transfection efficiency of 20*10-3mg/mL group is not preferable to 24*10-3 mg/mL group(P>0.05),which possesses more efficient transfection than other groups(P<0.0001).(5)The detection of laser scanning confocal microscope manifests that both 20*10-3 mg/mL group and 24*10-3 mg/mL group can be transfected by CS/siRNA perfectly after administration in 24 h,and also the transfection efficiency is similar between them without significant statistical difference(P>0.05).(6)Compared with normal incubation group,the cell viabilities of 0 mg/ml group,20*10-3mg/mL group and 24*10-3 mg/mL group,which are cultured at 3%O2 for 2h,4h and 6h respectively,are no significant statistical differences(P>0.05).Nonetheless,the cell viabilities of 0 mg/ml group,20*10-3mg/mL group and 24*10-3 mg/mL group,which are cultured at 3%O2 for 8h,are inferior to normal incubation group(P<0.05).(7)The expression level of rage’s mRNA(4.0623±0.2230)and RAGE protein(1.2115±0.1013)in Omg/ml group are obviously higher than normal incubation group[mRNA expression level(1.0000±0.0681)and RAGE protein expression level(0.8236±0.0374)](P<0.0001),the expression level of rage’s mRNA and RAGE protein in 20*10-3mg/mL group[mRNA expression level(0.0492±0.0050)and RAGE protein expression level(0.4227±0.1031)]and 24*10-3 mg/mL group[mRNA expression level(0.0408±0.0086)and RAGE protein expression level(0.1692±0.0261)]are distinctly lower than normal incubation group(P<0.0001).(8)The cell viability of Omg/ml group(0.7756±0.0325)is inferior to normal incubation group(0.9747±0.0997).The cell viability of 20*10-3mg/mL group(0.8828±0.0543)and 24*10-3mg/mL group(0.8931±0.0369)is a little inferior to normal incubation group,but there are no significant statistical differences(P>0.05).Research conclusions:(1)Besides being suitable for the transfection of primary myocardial cells,20*10-3mg/mL and 24*10-3mg/mL CS/siRNA solution with N/P ratios 70:1 possess more efficient transfection and less toxicity towards primary myocardial cells after incubation in 12h;(2)The transfection of CS/targeted rage siRNA towards primary myocardial cell can efficiently inhibit the expression of RAGE protein;(3)The inhibition of the expression of RAGE protein can attenuate myocardial cell injury induced by hypoxia,which supplies theoretical basis to the development of genetic therapies on myocardial remodeling after myocardial infarction. |
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