| Part I：The isolation, culture, and identification of adiposetissue-derived adult stem cells and the detection onNeuregulin-1/ErbB pathwayObjective: To isolate, culture and identify rat adipose tissue-derived stem cells（ADSCs）, and detect the expression of NRG-1/ErbB signal pathways.Methods: Acquire ADSCs by digesting rat adipose tissue with collagenase type I andadherent method; Detect the expression of the cells’ surface antigens CD44, CD90,CD105and CD31by immunofluorescence, and detect the expression of CD29, CD45,CD90by flow cytometer; Take three generations of identified ADSCs, and detect theexpression of NRG-1/ErbB signal pathways by RT-PCR and ELISA.Results: The cultured rat ADSCs adhered to plastic in standard culture conditionsand grew rapidly. The immunofluorescence result indicated that CD44, CD90andCD105, all of which are surface antigens regarded as the characteristics ofmesenchymal stem cells, were expressed; while CD31, an endothelial-cells-associatedantigen, was not expressed. It could be further confirmed by flow cytometry resultthat the positive rates of CD29, CD90on ADSCs were above96%, while the negativemarker CD45was seldom expressed. Moreover, RT-PCR result showed that the ratADSCs expressed the genes of NRG-1，ErbB2，ErbB3and ErbB4. And, ELISA led tothe fact that ADSCs can secrete NRG-1, a kind of neuregulin.Conclusion: The cultured rat ADSCs expressed the surface antigens ofmesenchymal stem cells and the complete NRG-1/ErbB signal pathway.Part II：The effect of NRG-1/ErbB pathway intervention on thedifferentiation process from rat adipose tissue-derived stem cellsinto sinus node-like cellsObjective: To discuss the possibility of regulation and control on the differentiationfrom rat adipose tissue-derived stem cells (ADSCs) into sinus node-like cells or working-type cells, by intervening the NRG-1/ErbB pathway during certain period oftime when ADSCs differentiate into cardiomyocyte-like cells.Methods: Take three generations of identified ADSCs, add into the mediumcontaining10μ mol/L5-aza+10μg/L bFGF and wait for24hours, then co-culturethe cells with neonatal rat cardiomyocytes (indirect contact) on Transwell plates;Establish a rat ADSCs cardiomyocyte-like differentiation model. Design3groups:AG1478group (take out neonatal rat cardiomyocytes in5-11th days, discretely addAG1478, an ErbB receptor antagonist whose concentration is10μmol/L, every12hours); NRG-1group (take out neonatal rat cardiomyocytes in5-11th days, addexogenous NRG-1whose concentration is100μg/L); control group (add5-aza only,with other conditions being the same); use normal group as blank control group.After3weeks, detect the expression of NKx2.5, HCN4, TBX3and TBX2byRT-PCR, the expression of TBX3protein by Western bloting and the bias betweenthe action potentials in all the groups with patch clamps.Results:3weeks later, RT-PCR and immunofluorescence showed that ADSCsexpressed cardiac early transcription factor Nkx2.5and troponin I after treated with10μ mol/l5-azacytidine, while it was negative in blank control group. The inhibitionof Neuregulin-1/ErbB signaling (used AG1478) greatly enhanced the gene expressionof HCN4, TBX3, TBX2compared to the control group and NRG-1group（P＜0.05）.In addition, the expression of TBX3protein was also higher in AG1478group（P＜0.05）, and distinct nodal-type action potentials could be detected. In the other hand,the expression of Nkx2.5in NRG-1group was higher than the other two groups（P＜0.05）, and working-type action potentials can be detected.Conclusion: The expression of pacemaker related genes and proteins can be greatlyenhanced by intervening endogenous NRG-1/ErbB signal pathway in certain periodof time when rat ADSCs differentiate into cardiomyocytes. Moreover, addingexogenous NRG1can improve the expression of working-type related genes. Part III：The manufacture of biological pacemakers with Adiposetissue-derived stem cells transfected with hyperpolarization-activated cyclic nucleotide–gated channels-2geneObjective: To transfect rat ADSCs with the pacemaker gene of humanhyperpolarization-activated cyclic nucleotide–gated channels-2(hHCN2), andobserve its feasibility of functioning as pacemaker cells.Methods： First, take three generations of ADSCs identified already and transfectthem with hHCN2gene, and detected the expression of hHCN2gene and protein byquantitative real-time PCR (qRT-PCR)， Western blot analysis and immunof-luorescence, then use patch clamp techniques to measure the electrophysiologyproperties and record the inward currents. Second, establish a co-culture of thetransfected ADSCs and rat cadiomyocytes, and observe its influence on thespontaneous beating rate of cadiomyocytes.Results: The transfected ADSCs expressed an anticipated high level of hHCN2gene,and they also produced hyperpolarization-activated cation current. Furthermore, it canaccelerate the spontaneous beating rate of the ventricular myocytes（P＜0.05）whenco-cultured with rat ventricular myocytes.Conclusion: ADSCs transfected with hHCN2gene can differentiate into the kind ofpacemaker cells which possess pacemaker function, which can probably facilitate thefuture research of biological pacemakers. |
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