The Study Of The Effect And Mechanism Of The NLRP3 Inflammasome In Cardiac Fibroblasts On The Neonatal Mice Cardiomyocytes' In Vitro Proliferation

Many heart disease such as cardiomyopathy,coronary heart disease and rheumatic heart disease,which caused by various reasons,has the main common pathological character: lack functional cardiomyocytes.The research on cardiac regeneration and myocardial repairation of injury,the proliferation characteristics of mammal myocardial cells and the initiation of inflammation,especially the influence of NLRP3 inflammasome on proliferation and repairation of myocardial tissue become hot topics.In-vitro co-culture system of cardiomyocytes and cardiac fibroblasts in neonatal C57BL/6 mice were used to research the proliferation characteristics of cardiomyocytes of mice at different day-age.Inflammation model was established to explore the effect of NLRP3 inflammasome on the proliferation of neonatal C57BL/6 cardiomyocytes.This research has important significance on evolution,development and regenerative biology,simultaneously has definite guidance for the clinic treatment of infant heart disease or adolescent patients with myocarditis caused by inflammation.1.The establishment of a co-culture system of cardiac cells in neonatal C57BL/6 mice and its application in the study of cardiomyocytes proliferationObjective: To establish in vitro culture and co-culture system of cardiomyocytes and cardiac fibroblasts in neonatal C57 BL/6 mice,and to research the proliferation characteristics of neonatal cardiomyocytes.Methods(1)Myocardial cells of neonatal C57 BL/6 mice at different day-age(at postnatal 1 d,3 d,5 d,7 d,9 d)were isolated by mixed enzymes(composed with 0.08% trypsinase and 0.1% type II collagenase),then collected and purified by the methods of different speed adherence and chemical inhibitor;Trypan Blue was used to detect the cell livability.(2)Cardiomyocytes and fibroblasts were inoculated for in vitro culture and co-culture.(3)The growth situation and the basic change of cell shape of cardiomyocytes and fibroblasts in single cultivation and co-culture system were observed by inverted phase contrast microscope;Cardiomyocytes were assayed by ?-sarcomeric actin and fibroblasts by vimentin,to authenticate the purity of cardiomyocytes and fibroblasts;the vitality of cardiomyocytes was measured with the number of beats per minute.(4)Proliferation vitality of fibroblasts and cardiomyocytes in the two systems were measured by the method of MTT.(5)The expression of cell cycle regulation factors Cyclin D1 and CDK4,cardiac differentiation related factors GATA4 and Nkx2.5,and FGF1 were detected through the Western Blotting method.Results(1)The mixed enzymes could extract much more cells with high livability.In primary cell culture alone,fibroblasts were adhered 1.5 hours later,and cardiomyocytes,12 hours later.(2)The positive expression rate of cardiac ?-sarcomeric actin in cardiomyocytes was more than 93 %,and the vimentinin fibroblasts 95 %,and the vitality of cardiomyocytes was good.(3)In co-cultured system,several cardiomyocytes were gathered as clusters,there was spontaneous beating phenomenon of part cardiomyocytes clusters,while the ones cultured alone became fusiform after 24 hours.The pseudopodiums of the co-cultured cardiomyocytes were extended more quickly,the crosslinking speed was faster,and the time of cardiomyocytes' beat reaching synchronization was earlier than the ones cultured alone.(4)The cell number and volume for cardiomyocytes were significantly changed for the cell got from the mice born within 5 days.Co-cultured cardiomyocytes grew faster and had relatively higher proliferation activity than the ones cultured alone.(5)The expression of Cyclin D1 and CDK4 of cardiomyocytes isolated from the mice born within 5 days was higher than those born after 5 days,the co-cultured cardiomyocytes was higher than the cultured alone,and the expression of GATA4?Nkx2.5 and FGF1 presented an increasing trend with the increase of age.Conclusions(1)Cardiomyocytes and cardiac fibroblasts could be effectively separated via the method of different speed adherence combined with chemical inhibitor,the purity of cardiomyocytes was high,and the vitality was fine.(2)Myocardial cells of neonatal C57 mice at different day-age can grow well in this in vitro co-culture system.The in vitro primary cell culture and co-culture method of neonatal cardiac cells was effective and feasible.(3)Cardiomyocytes isolated from the mice born within 5 days have the ability of proliferation,while the proliferation ability of cardiomyocytes isolated from the mice born after 5 days is weak or even disappeared.2.Research of the effect mechanism of the NLRP3 inflammasome in fibroblasts to the cardiomyocytes' in vitro proliferationObjective: To research the effect of NLRP3 inflammasome in cardiac fibroblasts on the proliferation of neonatal C57BL/6 cardiomyocytes.Methods(1)Cardiomyocytes and cardiac fibroblasts of neonatal wild and Nlrp3-/-mice born within 24 h,were isolated by mixed enzymes,then collected and purified by the methods of different speed adherence and chemical inhibitor.(2)Cells were inoculated to tissue culture plate for in vitro single cultivation and co-culture: wild fibroblasts in single cultivation,co-cultured wild cardiomyocytes and wild fibroblasts,co-cultured wild cardiomyocytes and Nlrp3-/-fibroblasts,and Nlrp3-/-cardiomyocytes in single cultivation.Cells were cultured for 24 h with normal medium,then pre-treated cells with LPS(10ng/m L)for 24 h,after that,further incubated the cells with extracellular ATP(3 m M)for 1 h.Synchronously,the control groups were disposed with normal medium.(3)The growth situation and the basic change of cells in single cultivation and co-culture system were observed by inverted phase contrast microscope.Proliferation vitality of fibroblasts and cardiomyocytes in the two systems were measured by the method of MTT.Cells pellets were collected to detect the protein expression of NLRP3,ASC,Caspase-1,CDK4,Cyclin D1,Nkx2.5 and GATA4 via the Western Blot method,and for the quantification of m RNA level of Nlrp3,Caspase-1,ASC,Cdk4,Cyclin D1,Nkx2.5 and Gata4 via the method of real-time RT-PCR.Cell culture supernatant was collected for the measurement of IL-1? via ELISA.Results(1)For both of the wild and Nlrp3-/-cells,compared with the cardiomyocytes in single cultivation of the control group,the vitality of cardiomyocytes of the experimental group were decreased and the number of cells was slightly reduced.However,the wild co-cultured cardiomyocytes were severely damaged and the number was significantly reduced than the Nlrp3-/-co-cultured cardiomyocytes.(2)The Western Blot results showed that: compared with the control group,the expression of NLRP3,ASC and Caspase-1 of the experimental group were increased,while,the protein level of CDK4,Cyclin D1,Nkx2.5,and GATA4 were decreased,the trend of the quantification of m RNA level performed using real-time RT-PCR was consistent with the protein level.The IL-1? measured by ELISA was significantly higher in experimental group compared with the control group.Conclusions(1)The myocardiac NLRP3 inflammasome can be activated by the inflammatory stimulation.(2)The increasing expression of NLRP3 in fibroblasts may cause the inhibition of the proliferation and differentiation of cardiomyocytes.