| Objective: To investigate the ability of differentiation of hAMSCs into hLFs in vitro by overexpressing Scx in hAMSCs via lentiviral vector.Methods: 1)The hAMSCs were separated through trypsin-enzyme digestion from the placenta and the phenotypic characteristics were tested by flow cytometry.Observation of organelles through transmission electron microscope(TEM)and the profileration ability was recorded by CCK-8 assay.After 21 days of culture in different induction medium,the alizarin red,toluidine blue and oil red O staining were used to evaluate the differentiation effect of the hAMSCs into osteocytes,chondrocytes and lipocytes like.2)Isolation and culture of hLFs by collagenase digestion and Cell morphology was observed by inverted phase contrast microscope.Compare collagenase digestion and in situ tissue isolation and culture.CCK-8 assay was used to detect cell proliferation and compared with hAMSCs.The expression of m RNA levels of hLFs of collagen type I,Tenomodulin,Mohawk and Scleraxis were detected and compared with hAMSCs by real-time fluorescence quantitative PCR.3)Lentivirus caontaining Scx/GFP or GFP were collected after the 293 T cells infected by plasmid.The concentration(TU/ml),and the MOI of the virus were determined as follows.Then,the cell lines that overexoress Scx/GFP or GFP were established and identified by RT-PCR and Western Blot.The expression of genes and proteins related to ligaments were detected,and cells transfeted with Scx/GFP or GFP were mixed with Matrigel to implant into the subcutaneous pockets of nude mice.Results: 1)A large number of stably proliferating hAMSCs could be obtained by enzymatic digestion and grow in fusiform and whirlpool-like.TEM showed cells are oval,clear structure;a small amount of microvilli on the surface of the cell,chromatin distribution is uniform,rich endoplasmic reticulum and arranged regular mitochondria in cytoplasm.Flow cytometry analysis showed that hAMSCs expressed mesenchymal stem cell phenotype.The Alizarin Red staining,Toluidine Blue staining and Oil red O staining showed positive results after 21 induction,separately.The inverted microscope showed that the primary hLFs were long fusiform adherent growth,the nuclei are round,and long fusiform colony growth after passage.CCK-8 showed that the curve of hl Fs was "S" type,but the proliferation ability was significantly lower than hAMSCs(P<0.05).RT-PCR showed the expression of m RNA levels of collagen type I,Tenomodulin,Mohawk and Scleraxis of hLFs were higher than hAMSCs(P<0.05).2)The PCR bands were 600 bp,detect of enzyme digestion products and bacterial,which accorded with the target strip size.The concentration of lentivirus we collected was 1.0×108 TU/ml,and the transfect rate was high when h AMCSs were modificated by 6.5?L virus solution and selected by 3.5?g/m L concentration of puromycin.RT-PCR showed the expression of m RNA levels of Scx in the treatment group was marked higher than the control group on days 3 and 7,respectively.Western Blot showed the expression of Scx in the treatment group was significantly higher than that of the control group(P<0.05).3)RT-PCR showed that modificated with Scx could significantly enhance the gene expressions of tenogenic-related molecules,including collagens ?,fibronectin,tenascin-C,matrix metalloproteinase-2(MMP-2),lysyl oxidase-1(LOX-1)and tenomodulin in all time-points(P<0.05),but the expression of collagen I in treatment group was highest on day 5(P<0.05),and showed no difference with hLFs group on day 7(P>0.05).Western Blot showed the secretion of proteins of collagens I,?,fibronectin and tenascin-C were increased in all groups overtime(P<0.05),and the expression of four ligament relative protiens in hLFs group were highest on days 3 and 7,respectively(P < 0.05).Immunofluorescence staining showed cobweb-like fusion of collagen I and fibronectin of treatment group on day 7 and the average fluorescence intensity was higher than the control(P<0.05).In addition,upon in vivo implantation with Matrigel subcutaneously in nude mice,hAMSCs modificated with Scx formed neoligament-like tissue,which revealed a histological feature of wave-like dense collagen fibres and cells aligned in parallel and no inflammatory reaction.By contrast,a disorganized connective tissue structure with randomly distributed cells was observed in the control group.Conclusion: 1)hAMSCs obtained by enzyme digestion and repeated adherence method has the characteristics of stem cell and multilineage differentiation.In vitro,the proliferation ability of hLFs is weak but the expression level of ligament related genes is higher than that of hAMSCs,and confirms that Scleraxis is the differential gene of hAMSCs and hLFs.2)Lentivirus containing Scx/GFP or GFP genes are successfully transfected into hAMSCs and unchanged the ability of cell proliferation,and the cell line of stable expression was established.3)After modificated with Scx lentiviral vector,hAMSCs are different into ligament fibroblasts directly with the signiciant increased expression levels of ligament related genes and proteins.And showed no obvious inflammatory reaction in vivo. |
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