| Objective:The rat bone marrow mesenchymal stem cells(BMSCs)and endothelial progenitor cells(EPCs) were isolated and cultured in vitro.We will construct the engineered myocardium-like tissue based on rat BMSCs and acellular bovine pericardium. At the same time, endothelial progenitor cells at the different ratio will be coimplanted in the scaffolds in order to build the vascular networks of the tissue engineered myocardium. Then the newborned myocardium will be transplanted into immunodeficient mice. We will explore the possibilities of constructing vascularization network of the engineered myocardium based on bone mesenchymal stem cells as seeding cells and the effectiveness of the function of the new blood vessels network,and the suitable ratio of EPCs in vitro.Methods:Part one: The rat bone marrow mesenchymal stem cells were isolated and cultured. The identifications were made for the separation fo cells by FACS.Bone marrow mesenchymal stem cells were induced into myocardial cells by Ang II, and the control group without any inducer. MTT assay was used to detect the absorbance values at days 1, 3, 5, 7 to calculate the cell proliferation inhibition rate in each group. Expression of cardiac-specific protein of c Tn T and β-MHC in four induced groups and the control groups was detected by immuno?uorescence staining at 1 and 4 weeks after induction. The cell conversion rates were calculated.m RNA expression of cardiac-transcription-factor GATA-4 and NKx2.5 in the experimetal groups was measured by real-time PCR at 4 weeks after induction.Part two: The isolation, culture and identification of Endothelial progenitor cells from the rat bone marrow.The cell phenotype were detected by immunofluorescence staining to identify the EPCs.The bovine pericardiums were managed by the detergent-enzyme digestion method.And acellular bovine pericardiums were observed by HE staining and SEM. The efficiency of acellular was calculated.Bone marrow mesenchymal stem cells induced by Ang II for 4 weeks and the Passage 1 endothelial progenitor cells were seeded into the biological scaffold materials of acellular bovine pericardium in virto.we can observed the cells on the the biological scaffold materials through the mi croscope and SEM.Construction of the engineered myocardium-like tissue based on rat BMSCs and acellular bovine pericardium. Then the cells/scaffolds we build in vitro were transplanted in the back of the nude mice. In the 1th and 4th week after implanting, the samples were harvested from the mice. the cardiac-specific protein and microvasular were detected by HE staining. Observe vascular network formation in the engineering myocardial.Results:Part one: The formation of colony of primary cells can be observed in 5-6 d.the morphology of cells was long spindle and polygon. Then cell colony increased rapidly and the morphology was spiral arrangement. Monolayer cells can be obsevered on 12-14 d. Results of FACS: the expression of CD29 is positive; the expression of CD45 is negative.The cell proliferation curves were similar among each group. By comparsion of inhibition of cell proliferation rate, the 0.05umol/L group began to appear negative and statistically significant compared with other groups in the 5th day. The cell proliferation inhibition rate of 0.1μmol/L group is significantly lower than 0.2 or 0.5μmol/L groups on the 5th and 7th day. c Tn T and β-MHC were detected in BMSCs of each induced group, the control group was negative. the group of 0.5μmol/L protein conversion rate is higher than that of 0.05μmol/L after induced 1 week. When induced culture 4 weeks,the protein conversion rate in the 0.1umol/L is similar with 0.2μmol/L.The early cardiac transcription factor GATA-4, NKx2.5 was detected by RT-PCR in the cells cultured at four weeks among the each induced group. The control group is negative.Part two: EPCs from rats were isolated, cultured and purified by the density gradient centrifugation.The EPCs exhibited small spindle shape at 7th day. The cellular morphology like paving-stone can be observed in the passage 1 EPCs.The protein of v WF、FLK-1、CD34 and CD133 were detected by immunofluorescence staining.The acellular bovine pericardiums were steriliazed and covered in the DMEM. The rate of acelluar in the bovine pericardium can reach 100%. The surface of the ultrastructure of acellular bovine pericardiums were detected by SEM.There was no cell remaining on the surface of the acellular bovine pericardiums, and 2um-aperture were observed in the acellular bovine pericardiums.The cells of three groups can ahere the scaffold material of acellular bovine pericardiums.The cells gradually increased with the extension of incubation time. We can see the cells on the surface of the acellular bovine pericardiums by SEM. The construction of the engineered myocardium-like tissue with the vascular networks in vivo. There was no acute inflammation in the surrounding tissue of both types of implants. The samples combined with surrounding soft tissue were harvested from the mice in the 1th and 4th week after implanting. Blood vessel and microvessel were observed by HE staining. The ratio of BMSCs/EPCs was effect the formation of microvessel. The best ratio is 80:20.Conclusions:(1) Rat bone marrow stem cells and endothelial progenitor cells can be isolated and cultured in vitro.(2) BMSCs can express the cardiac-specific protein c Tn T,β-MHC and transcription factor GATA-4 and NKx2.5 by AngⅡ induced. The suitable concentration of AngⅡ is 0.1μmol/L.(3) The bovine pericardium processed through four steps of detergent-enzyme acelluar methods. The rate of acelluar in the bovine pericardium can reach 100%. Acellular bovine pericardium was a good scaffold material of myocardial tissue engineering. The acellular bovine pericardium can provide a good condtion for the adhesion and cell proliferation of BMSCs and EPCs.(4) BMSCs/EPCs are ideal seed cells building myocardial/vascular engineering tissue, which can construcr tissue-engineered cardiac muscle via the scaffolds, and confirmed that BMSCs/EPCs can promote angiogenesis. |
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