| Bradycardia arrhythmia seriously affects the quality of life of patients,and even life-threatening.The medical treatment of bradycardia arrhythmia is implantation of electrical pacemaker.Although it can relieve patients' symptoms,but there are still many deficiencies,such as limited battery life,lead fracture,infections,the lack of autonomic responses,electromagnet interference and so on,there is a serious safety hazard.Therefore,the construction of biological pacemakers have promise to be an alternative treatment strategies with bright prospects.Aiming at the basis providing of biological pacemaker in future,experiments were fulfilled in which brown adipose-derived stem cells(BADSCs)were tested whether they could differentiate into pacemaker-like cells or not after being transferred by adenoviruses piggybacking TBX18,thus lay the foundation for further optimization of biological pacemaker.Part 1 Morphological characteristics of brown and white adipose tissues and stem cell isolation and cultureObjective:To investigate the morphological characteristics of rat subcutaneous fat tissues between inguinal and interscapular region,culture and observe the morphology of stem cells.Methods:Interscapular and inguinal adipose tissues were obtained from Sprague Dawley(SD)rat(40-50g),and the general shape of two types of adipose tissue were observed.Morphological characteristics of both adipose tissues were examined by HE staining.BADSCs and white adipose-derived stem cells(WADSCs)were primary cultured with enzymatic digestion method,and observed their morphological changes.Results:From gross observation,rat interscapular adipose tissue was typical brown adipose tissue(BAT),rich in blood vessels,surface coated with a layer of white adipose tissue(WAT),while inguinal adipose tissue was WAT,with a small amount of blood vessels.HE staining showed that interscapular adipocytes contained several small lipid droplets,while inguinal adipocytes contained only a large lipid droplet which populated most of the cytoplasm.Primary BADSCs grew well in 15%FBS/DMEM,could spontaneously differentiate into beating myotube-like cells,while WADSCs presented myofibroblast-like phenotypes.Conclusions:(1)Interscapular adipose tissue is mainly BAT,while inguinal adipose tissue is mainly WAT.Two types of adipose tissue cells have obvious morphological differences.(2)BADSCs can differentiate into spontaneously beating myotube-like cells,but not WADSCs.Part 2 TBX18 can improve the efficiency of rabbit adipose tissue-derived mesenchymal stem cell differentiating into the pacemaker cells.Objective:To explore the effect of TBX18 on the differentiation of BADSCs into pacemaker-like cells.Methods:First of all,BADSCs were primary cultured with enzymatic digestion method.And transcription factors TBX18 and GFP,which were used to transfer BADSCs,were carried by constructed adenoviruses carrier.The expression of green fluorescent protein(GFP),cell growth and morphological change were observed under inverted fluorescence microscope and phase contrast microscope separately,and determined their optimum Multiplicity of Infection(MOI).After a week,the mRNA level of cardiomyocyte-specific markers sarcomeric a-actinin(a-SCA)and cardiac troponin T(cTnT)and pacemaker cell-specific gene HCN4 were measured by qRT-PCR.The expression levels of cTnI and HCN4 protein were assayed by Western blot and immunofluorescence assay(IFA).Results:The optimal MOI value of adenovirus transfection BADSCs was 50.After transfected by adenovirus,BADSCs changed significantly in morphology.The myotube-like cells were significantly increased,and showed spontaneous beating after 5-6 days.qRT-PCR demonstrated that the expression of a-SCA,cTnT,HCN4 mRNA were significantly higher in TBX18-BADSCs group compared to control groups(P<0.05).It was found that cTnI and HCN4 was expressed efficiently in Tbx 18 transfected cells as demonstrated by Western blot and IFA,which were statistically significant compared with control groups(P<0.05).Conclusion:The results suggested that TBX18 gene can induce BADSCs to differentiate into pacemaker-like cells,BADSCs can be used as seed cells of biological pacemaker. |
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