Compere With The Specific MicroRNAs Expression In Rat C-Kit~+ CSCs,BMSCs And ESCs

Objective:Compare with the specific MicroRNAs expression in rat C-Kit+ cardiac stem cells(CSCs),rat embryonic stem cells(ESCs)and rat bone marrow mesenchymal stem cells(BMSCs)by MicroRNAs gene chip method,and explore the MicroRNAs expression profile differences and the origins of CSCs,and search for CSCs specific MicroRNAs.Method:1.Primary CSCs cultures:Neonatal Kunming mice(<1 week)were sacrificed by dislocating,sterilized chest skin and Operated by thoracotomy,pericardial tissue was removed,isolated myocardial tissue sliced into small clumps about 1mm3 pieces,washed with cold phosphate-buffered solution(PBS),and digested for 2 minutes at 37? with 0.25%trypsin and 0.1%collagenase ?.The supernatant were discarded,and the remaining tissue fragments,washed with fresh complete explant medium(CEM)and were cultured as explants in CEM at 37? and 5%CO2,and were replaced the culture medium periodically 2.FCM sorting for C-Kit+ CSCs:In 14±2days,the CSCs were differentiating and proliferating,and the confluent cells were up to 40%?50%,heart tissue fragments was washed with phosphate-buffered solution for 2 times,and was digested gentle differential for 5 minute at 37? with 0.25%trypsin,trypsin was neutralized with equivalent volume CEM,then centrifuged(1000r/min,5min),the supernatant was abnegated,substrate was added PerCP-CyTM5.5 Rat Anti-Mouse CD117 antibody and lml PBS,incubated for 20?30 minute at 4?,centrifuged(1000r/min,5min),supernatant was abandoned and centrifuged repeatedly,substrate was washed with PBS 2 times for excessing antibody,substrate was mixed with 1ml PBS,and was referred to Luzhou Medical Center Laboratory.3.Recovering and proliferation the rat ESCs and BMSCs 4.To detect the rat MicroRNAs expression of C-Kit+ CSCs,ESCs and BMSCs by MicroRNAs gene chip:C-Kit + CSCs,ESCs and BMSCs were extracted total RNA respectively,and inspected quality of total RNA,MicroRNAs was made for the MicroRNAs microarray chip,after scanning fluorescence image data and was filtered out the specific RNA MicroRNAs.Result:1.To get a relatively large number of small,round,bright form CSCs by Myocardial tissue culture method,myocardial tissue have begun to grow CSCs in 4 ± 1days,cells were plated on the stacked on top of fibroblasts,cells are shape of the"teardrop" or "sunflower",the cells edge are rounded and smooth,translucent good,nucleus cannot be observed under the High-frequency microscope;the small,round,bright cell number have grown rapidly in 10±1 days,the confluent cells have being up to 40%to 50%in 14 ± 2days;2.The tissue as myocardium of CSCs differentiation have potential to beat;3.FCM sorting C-Kit+CSCs positive rate is 8.4%± 0.6%,C-Kit+CSCs is small,round,bright morphology in microscope,cell edges are rounded and smooth,good transparency,the nucleus cannot be observed at high magnification,there are different degrees of brightness of the fluorescence microscope chromogenic;4.1n the pure CEM environment,C-Kit+ CSCs maintain a small,round,bright morphology;added the inducing factors,C-Kit+ CSCs started to grow " pseudopodia " and some began to increase;5.Compare with C-Kit+CSCs and ESCs,there are 5 MicroRNAs up-regulation,and 21 MicroRNAs down regulated;Compare with C-Kit+ CSCs and BMSCs:there are 34 MicroRNAs up-regulation,and 27 MicroRNAs down-regulation;compare with ESCs and BMSCs:there are 64 MicroRNAs up-regulation,and 20 MicroRNAs down-regulation.Conclusions:1.Myocardial tissue adherent culture method can harvest C-Kit+ CSCs stably;2.CSCs have the potential to differentiate into cardiomyocytes;3.C-Kit can be used as a screening criterion of CSCs;4.FCM sorting C-Kit+CSCs positive rate is 8.4%± 0.6%;5.FCM sorting:The C-Kit+ CSCs is "sleep" state in the pure CEM environment,and the C-Kit+ CSCs maintain good activity with proliferation and differentiation potential in the inducing factors;6.Primary CSCs medium contain the factors of inducing C-Kit+ CSCs proliferation and differentiation;7.Compare with C-Kit+ CSCs,ESCs and BMSCs,the 1.5-fold up and down regulated MicroRNAs that are some new discovering,they may have a role in the regulation of cardiac stem cell differentiation and proliferation,it is worth to study;8.The CSCs is more closely ESCs than BMSCs in the origin of species,and we do not rule out that CSCs come from other origin.