| Objective:Bone marrow mesenchymal stem cells(BMSCs)are derived from mesoderm. BMSCs have the ability of differentiation into a variety of tissues.They can be isolated and cultured in vitro,and can differentiate into myocardial cells under some special environments.Induction of 5-azacytidine(5-aza)can cause demethylation of muscle-derived genetic locus,transcription activation and cells differentiation into muscle-derived cells.Fibroblast growth factor-2(FGF-2)is a kind of mitogen.It has the effect of promoting cell division and proliferation.It is reported that FGF-2 can protect heart by inducing myocardial hypertrophy and increasing survival rate of myocardial cells under anoxia condition.Ultrasound-mediated albumin microbubble destruction can promote exogenous gene transfer into genome of eukaryotic cells.In present study,our objectives are as follows:(1)to clone rat FGF-2 gene in vitro and construct its' eukaryotic expression vector.(2)to investigate the method of fractional cultivation of BMSCs and induce its differentiation into myocardial cells.(3)to investigate transfer of FGF-2 into BMSCs in vitro by ultrasound-mediated albumin microbubble destruction method and compare with the lipsome2000 method.Methods:(1)From Sprague-Dawley(SD)rats' bone marrow,BMSCs were isolated and cultured in vitro successfully,their growth characteristic and phenotypes were observed. Nucleus,endochylema and cell morphology of BMSCs were observed with HE staining. Growth curves of induced and non-induced BMSCs were detected by MTT method. BMSCs were induced for 24hr by 5-aza(10μmol/L)during primary culture in generation 4 and 7.The phenotypes of BMSCs induced or not were identified by flow cytometry, immunofluorescence and reverse transcription-polymerase chain raction(RT-PCR).(2) Ventricular myocardial cells were isolated and cultured from neonate SD rat,Growth characteristic and phenos were observed.(3)Ventricular myocardial cells were collected, Total mRNA were extracted through Trizol method.The complementary gene encoding rat FGF-2 was amplified by RT-PCR,and then the target gene fragment was inserted into vector PIRES2-EGFP after being digested with EcoR I and BamH I.Eukaryotic expression vectors was constructed at last.(4)Intensity and time of ultrasound were regulated to confirm the best parameters.The plasmid DNA was mixed gently with microbubbles,and then the mixture were transfered into BMSCs by ultrasound-mediated albumin microbubble destruction method.The green fluorescence was observed under inverted phase contrast Microscope after 24hr,It shows that Green Fluorescence Protein(GFP)was expressed by transfered BMSCs.(5)Lipsome2000 could promote FGF-2 transfer into BMSCs at some does.(6)Cell activity was detected by trypan blue staining after different ultrasound intensity and time.(7)Expression of EGFP was detected by flow cytometry after transferring BMSCs with FGF-2 24hr later.Results:(1)BMSCs were derived from SD rat bone marrow,grew adhering to the wall in shuttle shape,part of hematopoietic cells floated in substratum.MTT growth curve indicated that cells came into exponential phase of growth on the second day after inoculation and proliferated actively.Observed under contrast phase microscope,BMSC extended,axle protuberance,in shuttle shape,two or more circular nucleuses,abundant karyoplasms.Cell density increased,connected with each other.After the cells were cultured about seven days,cells-could spread the whole bottom,and expanded rapidly. After the cells were cultured about twelve days,BMSCs grew torpidity and came into platform phase.(2)Immunofluorescence and flow cytometer test indicated that BMSCs highly expressed CD44(99.3%),CD54(97.7%),CD90(99.4%),CD71(98.2%),CD106(25.8%) correspondence lower,and lowly expressed CD45(8.9%),CD31(15.9%).After passage, BMSCs was in uniform shape,grew rapidly and spread the whole bottom in 3 to 4 days. Contact inhibition was obvious because of high cell inoculum density.Cell morphology of 10 generation BMSCs in HE staining is shuttle shape,axle protuberance,abundant cytoplasm and blue circular nucleus.(3)Induced by 5-aza for 5 days,BMSCs size was getting bigger and rounder. Observed under contrast phase microscope,cells were localized in areas with high cell density in different size with abundant cytoplasm and blue circular nucleus.Cells proliferated and connected with each other tightly.Immunofluorescence staining indicated BMSCs induced by 5-aza could express MHC-β,connexin43,desmin and actinin-a.While part of induced cells could also express vascular endothelial cell specific moleculars (Flk-1/VEGFR2/KDR).RT-PCR test indicated that induced BMSCs could express myocardial cell early development gene NKX2.5 and GATA4.(4)Ventrieular myocardial cells which were isolated from neonate rats heart grew in cluster and overlapped into compact cell masses.Cultured for about 10 days,conglobate myocardial cells under contrast phase microscope show rhythmic contraction,which continued for half a month.(5)The FGF-2 gene was amplificated from ventricular myocardial cells of neonate rat by RT-PCR,and then was inserted into eukaryon expression vector PIRES2-EGFP after being digested with EcoR I and BamH I to construct the plasmid vector PIRES2-EGFP-FGF-2.(6)FGF-2 gene was transferred into BMSCs induced by ultrasound-mediated albumin microbubble destruction method.BMSCs could express EGFP observed under fluorescence microscope.FGF-2 gene were transferred into BMSCs mediated by Lipsome2000,after 24hr,BMSCs could express EGFP observed under fluorescence microscope.It showed that FGF-2 has been tansferred into BMSCs.(7)After different intensity and time of ultrasound,BMSCs activity was different;it indicated that it was salty within 0.75W/cm~2,32see extent.(8)Expression of EGFP was detected by flow cytometry after transferring BMSCs with FGF-2 24hr.In liposome group, expression efficiency was(49.35±9.34)%,compared with(30.42±4.58)%in ultrasound group,P＜0.05.It showed that transfection efficiency in liposome group was higher than in ultrasound group. Conclusions:(1)Ventricular myocardial cells were isolated from neonate Sprague-Dawley (SD)rats heart successfully.The antigen of ventricular myocardial cells tested by immunofluorescence.(2)Ventricular myocardial cells were collected;Total mRNA was extracted by Trizol method.The complementary gene encoding rat FGF-2 was amplified by RT-PCR,and then the target gene fragment was inserted into vector PIRES2-EGFE Eukaryotic expression vectors was constructed successfully.(3)BMSCs could differentiate into myocardial cell which were induced by 5-aza.(4)Cell activity could be influenced by different intensity and time of ultrasound.(5)Both lipsome and Ultrasound-mediated albumin microbubble destruction could improve FGF-2 transferring into BMSCs.(6)Ultrasound-mediated albumin microbubble destruction could promote FGF-2 transfer into BMSCs.It is a new method for transferring with non-virus vector,but transfer efficiency should be increased...
|
PDF Full Text Request