An Experimental Study On Directing Chondrogenetic Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells By Sox9 Gene Modification In Vitro

Objective.The purpose of this study was to construct a lentiviral vector containing Sox9 gene and transfect into mesenchymal stem cells derived from human umbilical cord (hUC-MSCs) in vitro,to observe whether the biomedical behaviours of hUC-MSCs were affected by transfecting with Sox9 gene and the capacity of hUC-MSCs modified with Sox9 under the condition of monolayer culture. It will facilitated the following development of gene and cell therapy in treating diseases of cartilage injure and degeneration.Methods.Human Sox9 gene coding region fragment was obtained using PCR method and then cloned it into plasmid of Pwpxl-MOD to product Pwpxl-MOD/Sox9 , The correct Sox9 gene was confirmed by sequencing and restriction analysis.The plasmid of Pwpxl-MOD/Sox9 vector was co-transfected together with lentivirus-packaging plasmid pRsv-REV,pMDlg-pRRE,pMD2G into 293T cells to obtain recombinant virus containing Sox9 gene. MSCs were harvested from human umbilical cord stromas using the methods of enzymatic digestion and phenotypic characteristic of hUC-MSCs was identified by Flow Cytometry. In order to confirm the multipotent of hUC-MSCs,the cells were induced differentiation toward adipose and osteogenesis. The packaging lentivirus vectors transfected hUC-MSCs in vitro, morphologic change and expression of green fluorescent protein (GFP) was observed by flourescence phase contrast microscope. The efficiency of transfection was investigated by fluorescence microscopy 48 h after transduction. Reverse Transcriptase Polymerase Chain Reaction(RT-PCR) and Western-blot was used to confirm expression of Sox9 in hUC-MSCs.The cellular proliferation capacity of MSCs was detected by MTT assay.hUC-MSCs modified with Sox9 grown on the high density monolayer culture without addition of any exogenous growth factors for 21 days.The cartilage-specific gene collagenⅡand Aggrecan were detected by RT-PCR, Western-blot,immunohistochemisty and immunofluorescence staining and accumulation of sulfated glycosaminoglycans was detected by Alcian Blue staining.Results. The sequencing and restriction analysis showed that Sox9 gene fragment was correctly connected and was cloned into plasmid Pwpxl-MOD vector, and the titer of virus was 6.0×10~7TU/ml.The hUC-MSCs isolated from human umbilical cord stromas exhibited fibroblastic morphology and they were positive for CD29(95.9%),CD44(96.5%)and negtive for hematopoietic stem cells surface markers CD34(3.0%) ,CD45(2.6%).At days 21 hUC-MSCs induced differentiation toward adipose and osteogenesis, oil red O,ALP and von kossa staining were all intensely positive which indicated multilineage potential of hUC-MSCs.The intensely expression of GFP was observed via flourescence microscope and the efficiency of transfection of cells transfected with Lenti-GFP-Sox9 or Lenti-GFP was more than 90% respectively. RT-PCR, Western blot have detected Sox9 gene was overexpressed in hUC-MSCs(P<0.001). Cells growth curve showed the proliferation of hUC-MSCs has no significant effect after Sox9 gene transfection(P>0.05).In vitro high density monolayer culture of these Sox9 transfected hUC-MSCs demonstrated that spontaneous cell aggregation were formed at day 14 of culture and subsequently generate large cartilage nodules.However,there was no the phenomenon of cell aggregation which occured in the cells trasducted by Lenti-GFP vectors or untreated. The expression of collagenⅡa nd agrrecan in Sox9 transducted cells was dectected by RT-PCR, Western-blot and, the abundant accumulation of cartilage-specific extracellular matrix in the cartilage nodules were detected by immunohistochemistry assay and alcian blue staining.Conclusion.1.Lentiviral vector carrying Sox9 gene has been successfully constructed.2.MSCs obtained from human umbilical cord stromas display multilineage potential and they are amenable to modify by exogenous gene;3. hUC-MSCs modified with Sox9 grown on the high density monolayer culture without the stimulation of exogenous growth factors could also been induced differentiation along with chondrogenetic pathway. These results indicated that Sox9 gene plays a important role in regulating chondrogenic differentiation of MSCs.