| Objective To establish immortalized precartilaginous stem cells (IPSCs) from which we clone the Sox9 gene and construct its eukaryotic expression vector. Then we transfect the plasmid into the bone marrow stromal cells (MSCs). We study the possibility of MSCs differentiating to PSCs and remaining in the PSCs state for the further related research on the differentiation mechanism and clinic application of precartilaginous stem cells.Methods pCMVSV40T/PUR was transfected into the primary cultured PSCs isolated by immuniomagnetic beads select system. Colonies were isolated by puromycin selection and expanded by many passages. Investigate the capability of differentiation of the transfected cells. The expression of SV40Tag in expanded cell lines was identified by immunocytochemistry method and RT-PCR. The total RNA was extracted from immortalized PSCs (IPSCs) after identification and the Sox9 gene was obtained by RT-PCR and be inserted into pGEM-T Easy cloning vector. After the sequencing was confirmed, the gene was subcloned to pEGFP-IRES2 to construct recombinant eukaryotic expression vector pEGFP-IRES2. After that, we transfected the plasmid into MSCs by liposome. We observed the transfection efficiency and expression of Sox9 gene and Sox9 protein, then we detected the transfected cell's expression of FGFR-3 and expression of collagen type II and collagen type X after 12 days after transfection. Detecting the grouping of cells by flow cytometry was applied. Proliferation was determined by MTT.Results A particular anti-puromycin cell clone was acquired, which was confirmed as fibroblast growth factor receptor-3(FGFR-3) positive PSCs. The total RNA were isolated from the positive cell clones, and a 588 bp fragment, which was specific for the SV40T antigene gene, was amplified. The transfected cells were expanded to immortalized cell strain, named as immortalized precartilaginous stem cells(IPSCs). The population doubling time of IPSCs was 22.98±2.77 h, no significant effect of subculture, freezing and recovering had been found. The total RNA extracted from IPSCs through electrophoresis can get 28s,18s,3s three bands. The OD value is 0.2635 and the A260/A280 is 1.8741 which shows that the total RNA extracted has a good integrity and purity, and accord with the request of RT-PCR.The PCR product through electrophoresis can get a 2000bp specificity electrophoretic band which is accord with anticipation. Enzyme digestion analysis and DNA sequencing showed that the target gene had been cloned into recombinant plasmids。The Sox9 recombinant plasmids were constructed successfully. Observing under fluorescence microscope confirmed that the Sox9 gene has transfected the bone marrow stromal stem cells successfully. The transfection efficiency is about 50%. The transfected cells expressing Sox9 gene and protein Sox9 stably. Immunohistochemistry and Western blot tests showed that the transfected cells express of FGFR-3 but notⅡtype of collagen and X type of collagen. Flow cytometry revealed:The majority of cells is in the first quadrant 1 after transfection from 1 day to 40 days, which hint FGFR-3 positive and the cells are PSCs, whose proliferation activity was similar to precartiliaginous stem cells (PSCs) in cells proliferation curve derived from MTT.Conclusion Precartilaginous stem cells could be isolated from neonatal SD rats, cultured in vitro, and immortalized through the transfection of pCMVSV40T/PUR.The Sox9 gene was cloned successfully from IPSCs. The eukaryotic expression plasmid containing Sox9 gene was successfully constructed. The bone marrow stromal stem cells were transfected by Sox9 gene eukaryotic expression vector successfully. The MSCs transfected by Sox9 gene eukaryotic expression vector differentiate to PSCs and have the characteristics of PSCs.Which may be a promising solid foundation for artificially controlling the differentiation of PSCs and regeneration of epiphyseal,autologous stem cell transplantation.
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