Studies On Construction And Identification Of The Immortorlized Precartilaginous Stem Cell

This paper is to establish the methods for separation and culture the subpopulations chondrocyte of rat tibiofibula epiphysis growth plate, and to investigate their biological features. Then this ressarch cloned the parathroid hormone-related peptide gene from proliferating zone cell and constructed its eukaryotic expression vector by gene engineering technology. After established the immortalized precartilaginous stem cell strain, this project could provide stable cell resource for cell-transplantation and gene therapies. The 31P-NMR and the ion concentration of the IPSCs were investigated by using MR spectroscopy. The compatibility between IPSCs and nanoHA/PDLLA was evaluated through combination culture. Thereby this paper could provide experimental bases for studying the regulation of chondrocyte proliferation and differentiation and for repairing of cartilage using tissue-engineering materials.The chondrocyte of rat tibiofibula epiphysis growth plate was obtained by microdissection and digestion. Subpopulations of these chondrocytes were separated by discontinues percoll gradient and were cultured in monolayer. Morphological changes of the serial passage of subpopulations and the cell growth kinectics were observed. The ultramicro structures were observed by electron microscope. The cellular FGFR-3, collagen type II and type X expression were detected by histochemistry and immunocytochemistry. The total RNA was extracted from the proliferating zone cells and the full length cDNA encoding PTHrp gene was obtained by RT-PCR method. Product of PCR, amplified by transformed into E-coli DH5α, was inserted into the eukaryotic expression vector pEGFP-IRES2 after digestion and ligation by using restriction endonucleases and ligase. Recombinated plasmid pEGFP-IRES2-PTHrp containing the PTHrp gene was transfected into the primarily cultured precartilaginous stem cell(PSC) of newborn rat by using lipofectin transfection method. Colonies were isolated by G418 selection and expanded to immortalized cell strains. FGFR-3, collagen type II and type X antibodies were used to identify the cultured cells and to investigate the capability of differentiation of the transfected cells. The expression of PTHrp in expanded cells was identified by RT-PCR, Southern blot, western blot and immunocytochemistry method. IPSCs were resuspension by tris-buffer prepared in D2O. 31P-NMR spectroscopy and Mg2+ concentration were carried by using high magnetic field spectrometer inova 600. At last, cells were seeded on the tissue-engineering material nanoHA/PDLLA, and were observed under inverted microscope and environmental scanning electron microscope and recorded. The number of the cell adhered on the materials was calculated 7days after the culture.There were four subpopulations in the tibiofibula epiphysis growth plate, were more than 96% viable cells in the obtained subpopulations. The morphology of primary cultured subpopulations was fusiform shape or polygon. In this experiment, the eighth passage cell was still maintained and showed polygonal morphology. The index of duplicating day increased in the preceding fourth passage cell and decreased afterwards. There was more than 95 % cells expressed collagen type II in C4 but type X in C1. As the passagenumber increasing, the ratio of these collagens expression and dropped abruptly. The size was smaller in C4, but the density smaller in C1. FGFR-3 was expressed only in the cell membrane of C4. Both the content and the purity quotient of total RNA were qualified. The correct PCR production was obtained and it was confirmed that PTPrp gene was inserted into the eukaryotic expression vector correctly by using digestion identification and sequencing. One anti-G418 cell clone was obtained, which was confirmed as FGFR-3 positive PSC with the capability of proliferation after transfection. mRNA and protein of PTHrp were expressed in transfected cells after stable transfection. The transfected cells were expanded to immortalized cell strains maintained for more than 50 passages, named as immortalized precartilaginous stem cell (IPSCs). IPSCs were elliptic or triangular cells with two or three short axons. The population doubling time of IPSC was 23.52 h. Subculture, freezing and recovering had no effect on cellular shape and proliferation. There were five resonance peaks in the 31P-NMR Spectra of IPSCs, as PCr, ATP and Pi, et al. Intracellular free Mg2+ was 0.326mmol/L. IPSCs were attached to nanoHA/PDLLA and grew favorably on surface of it.The separation and culture methods adopted in this study can obtain high pure and viable subpopulations of epiphysis growth plate. The preceding four passage cells maintains their in vivo phenotype, they are ideal experimental materials for studying the regulation of proliferation and differentiation of chondrocyte and tissue engineering. The PThrp gene was cloned accurately from proliferating zone cell. The recombinant eukaryotic expression vector pEGFP-IRES2-PTHrp is successfully constructed, which may be a promising for studying the biological function of the PTHrp gene and its role in chondrocyte differentiation and bone formation. Transfection PTHrp gene could immortalize precartilaginous stem cells. The establishment of FGFR-3 positive IPSC line may provide stable cell resource for the basic researches and cell-transplantation therapies with PSC. The metabolism and internal environment of IPSCs were normal and stable. There was a good compatibility between IPSCs and bone tissue-engineering material nanoHA/PDLLA. The proliferation and differentiation of IPSCs adhered on materials were normal. This material is a safe tissue-engineering material, IPSCs and this material complex could be substitute for bone graft.