| Chapterâ… :Construction and transfection of eukaryotic expression vector for rat transforming growth factor-β1Objective To master the method of construction and transfection of eukaryotic expression vector for rat transforming growth factor-β1 and to study the possibility of gene transfection to adipose-derived stem cells (ADSCs)with this vector.Methods Recombining DNA techniques were applied to construct recombinant plasmid pcDNA3.1-TGF-β1.And this plasmid was verified by restriction endonuclease mapping and DNA sequencing;Then the plasmid with TGF-β1 gene or not was transfected into ADSCs by use of Lipofectamine 2000.After infection,transduced ADSCs were diluted and cultured with neomycin(G418).Selected clones and cultured in Dulbecco's modified eagle's medium supplemented with 10%new-born calf serum.Gene transfer efficiency compared on the basis of GFP expression was assessed by fluorescence microscopy,and TGF-β1 gene expression in ADSCs were analysed with RT-PCR and Western-blot.Results Digestion of the plasmid with double restriction endonuclease Xboâ… and Hindâ…¢showed about two specific electrophoretic strip(1.35bp and 5.4kb),and the sequence of the rat TGF-β1 gene in recombinant was concorded with that reported in Genbank.There were 80 percent of the cells which were transduced,and the expressions of mRNA and protein of TGF-β1 in ADSCs were discovered positively.Conclusion The eukaryotic expression vector for rat transforming growth factor-β1(i.e,pcDNA3.1-TGF-β1),which is able to transfect the ADSCs,can be constructed through genetic recombination. Chapterâ…¡:Chondrogenic potential of adipose-derived stem cells modified by TGF-β1 gene in vitroObjective To investigate the influence on chondrogenic potential of ADSCs after TGF-β1 gene transfection in vitro.Methods ADSCs from rat were isolated and cultivated.Recombinant plasmid pcDNA3.1-TGF-β1 was transferred into the 4thcells, then these cells were induced in a specific Dulbecco's modified Eagle's medium supplemented with 10%new-born calf serum,6.25μg/mL insulin, 6.25μg/mL transferrin,1×10-7mol/L Dexamethasone,and 50μg/mL ascorbic-2-phosphate.The ADSCs transfected with pcDNA3.1 or not were control;The growth and morphology of these cells were observed with insert microscope everyday;Toluidine blue staining,collagenâ…¡immunohistochemistry and aggrecan immunofluoresecence were used to evaluate the chondrogenesis of these cells at 14 days after they were cultivated in this specific Dulbecco's modified eagle's medium.The mRNA expressions of Col2al,Sox9 and TGF-β1 were detected by RTPCR at 0,14,28 days,and at the same time,Western-blot was employed to estimate the protein expressions of Col2al and Agcl. Results The growth of the ADSCs transfected by TGF-β1 gene increased slowly,and there were some cells lost their appearances in first 24 hours,then these cells proliferated significantly,and their appearances aslo changed.At 14 days,a positive staining for collagen typeâ…¡was investigated in these cells,so were for toluidine blue and aggrecan.But in control samples,the ADSCs maintain proliferation,and the outcome was negative for toluidine blue staining,collagenâ…¡immunohistochemistry and aggrecan immunofluoresecence.Using RT-PCR,we found that the gene expressions of mRNA for Col2al,Sox9 and TGF-β1 were up- regulation at 0,14,28 days after cultured in specific Dulbecco's modified Eagle's medium.At the same time,Western-blot indicated that the productions of Col2al protein and Agcl protein also increased.In contrast to the cells modified by gene,the results were not detected nearly in the control samples.The differences between experimental groups and control samples were predominance(P＜0.05).Conclusion ADSCs transfected by TGF-β1 gene can synthetize and secret the TGF-β1 protein,which will promote the chondogenesis of ADSCs.Chapterâ…¢:Heterotopic chondrogenesis of ADSCs modified by TGF-β1 gene in PLGA scaffolds in vivoObjective To explore the feasibility to construct the tissueengineered cartilage in vivo with ADSCs transfected by TGF-β1 gene in PLGA scaffolds.Methods After modified by TGF-β1 gene,ADSCs were loaded in PLGA and cultured in specific Dulbecco's modified Eagle's medium described as Chapterâ…¡for 24 hours.Then the complexes of cells and PLGA scaffolds were randomly implanted into the back subcutaneous pouches of nude mice.The morphologies of implants were evaluated approximately when they were in nude mice' s backs.The performances of the complexes were observed with the scanning electron microscope after 4 weeks of operation.The mouse were sacrificed and the implants were harvest at 12 weeks after transplantation,then all implants were fixed in formalin,dehydrated by alcohol with ascending concentrations, paraffin embedded,and cut into 5μm or 10μm slices.Some sections were stained with hematoxylin and eosin(H&E),toluidine blue and safranin O/fast Green for microscopy analyses of the regenerated cartilage,some sections were subsequently used for collagen typeâ…¡immunohistochemical staining.During the experiment,the mRNA expressions of Col2al,Sox9 and TGF-β1 were detected by RT-PCR at 4 weeks,8 weeks and 12 weeks after grafting.Results The nude mouse existed well and recovered ideally after operation.The morphology of implants in experimental groups kept their original sizes,their tenacities increased.While the implants in control groups could not maintain their shapes,they shrinked gradually, disappeared at last.By the use of scanning microscope,there were many spherical cells found in pores and on the surface of the scaffolds in both groups,but much more cells and extracellular matrices were observed in experimental groups at 4 weeks after transplantation.The histological analyses,which performed by hematoxylin eosin staining,toluidine blue staining and safranin O/fast Green staining,showed that the newly formed tissue in nude mouse had cartilage-like characteristics.A positive staining for collagen typeâ…¡was investigated in all experimental groups. RT-PCR indicated that the gene expressions of Col2al mRNA and Sox9 mRNA were up-regulation by degrees,and the level of mRNA gene expression for TGF-β1 at 8 weeks was higher compared to 4 weeks or 12 weeks.In spite of differences existed at each time,the variances of gene expressions of Col2al,Sox9 and TGF-β1 were not statistically significant (P＞0.05). Conclusion After combined with PLGA scaffolds,ADSCs transfected by TGF-β1 gene can construct heterotopic cartilage,and this cartilage will maintain their properties for a long time. |
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