| Objective:1. To establish the method of mouse bone marrow mesenchymal stem cells isolation and culture and confirm their biological features.2. To investigate the expression of glucagon-like peptide1receptor in mouse BM-MSCs and the effect of Exendin-4on BM-MSCs cell cycle.3. To construct and identify an adenoviral vector containing GLP-1R gene and investigate the expression of GLP-1R in BM-MSCs transfected by Ad. GLP-1R.4. To investigate the effect of Exendin-4on proliferation, apoptosis and differentiation capability into adipocytes and osteoblasts of Ad.GLP-1R-BM-MSCs.Methods:1. Mouse BM-MSCs were isolated by adherence culture method. Cell morphology was studied. BM-MSCs doubling time was determined at exponential stage of growth curve. Surface markers CD29, CD44, Sca-1, CD34and CD117expressions and positive rates were detected by flow cytometry. BM-MSCs were induced into adipocyte and osteoblast. Oil red O and Alizarin red staining were used to reveal the lipid droplets and calcium deposition.2. The expression of GLP-1R in BM-MSCs was examined by RT-PCR and Western blot. The effects of Exendin-4on cell cycle in BM-MSCs were evaluated by flow cytometry.3. Through the Ad.Max-system, the GLP-1R genes were cloned into pDC316to construct pDC316-GLP-1R, which was cotransfected with the rescue plasmid pBHGlox_E1,3Cre into293cells to obtain the recombinant adenovirus Ad.GLP-1R. The expression of GLP-1R in BM-MSCs transfected by Ad.GLP-1R was examined by RT-PCR, immunofluorescence, and Western blot, respectively.4. The effects of Exendin-4on cell growth, cell cycle and apoptosis of Ad.GLP-1R-BM-MSCs were evaluated by cell counting and flow cytometry. The effects of Exendin-4on Ad.GLP-1R-BM-MSCs differentiation into adipocytes and osteoblast were assessed by cell staining and real-time polymerase chain reaction.Results: 1. BM-MSCs were spheroid adherent monolayers. Doubling time was36.9-37.3hours. Positive rates of surface markers CD29, CD44, Sca-1, CD34and CD117were99.91%,90.59%,99.02%,97.75%and0.01%respectively. BM-MSCs could form lipid droplets and calcium deposition after inducing into adipocyte and osteoblast.2. Mouse BM-MSCs didn’t express the GLP-1R gene and protein natively. After stimulated by Exendin-4, compared with the control group, there were no significant differences in cell cycle between two groups (P>0.05).3. The recombinant adenoviral vector of GLP-1R was verified RT-PCR. And the tilter of the Ad.GLP-1R is1.26×1010TU/mL. When multiplicities of infection (MOI) were400, the infection efficiency was89.5%. BM-MSCs could express the GLP-1R gene and protein effectively after transfected by Ad.GLP-1R.4. After stimulated by Exendin-4, the cell growth curve of Ad.GLP-1R-BM-MSCs was significantly higher than the control group (P<0.05). Compared with the control group, G0/G1phase cells decreased significantly, G2/M phase cells was significantly higher, and proliferation index (S+G2/M) also increased significantly in the stimulation group (P<0.01). And the rate of apoptosis was decreased significantly (P<0.05). After inducing into adipocyte, Exendin-4could decrease lipid droplets and the expression of LPL, C/EBPβ and PPARy significantly (P<0.01). After inducing into osteoblast, Exendin-4could markedly increase calcium deposition (P<0.01) and OCN gene expression (P<0.05). The gene expression of col-1had increased trend, but there’s no significant difference in cbfa-1gene expression.Conclusion:1. Abundant mouse BM-MSCs homological in biological feature can be obtained after in vitro culture, and they have the differentiation potential of adult stem cells.2. Mouse BM-MSCs maybe don’t express the GLP-1R gene and protein natively.3. The recombinant adeno viral vector of GLP-1R was successfully constructed. BM-MSCs could be effectively transfected by adenovirus. BM-MSCs could express the GLP-1R gene and protein effectively after transfected by Ad.GLP-1R.4. Exendin-4/GLP-1R can promote BM-MSCs proliferation and cell differentiation into osteoblasts, and reduce BM-MSCs apoptosis and cell differentiation into adipocytes.
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