Construction Of Immortorlized Precartilaginous Stem Cell Strain And Effects Of PTHrP Sub-gene On Proliferation And Differentiation In Precartilagenous Stem Cells

Objective To investigate the method of separating,cultivating and identifyingprecartilaginous stem cells (PCSCs) and establish immortalized precartilaginous stemcell(IPCSC) strain which provides stable cell resource for cell-transplantation and genetherapies.To establish doxycyline-controlled PCSCs highly expressing aline genes.Tostudy the modulating effects of PTHrP(1-36),PTHrP(38-94) and PTHrP(107-139) oncultured precartilagenous stem cells in vitro and the relationship between PTHrP(107-139)and other regulating factors.Methods Immunomagnetic separation was used to segregate PCSCs labeled withfibroblast growth factor receptor-3(FGFR-3).Plasmids pCMVSV40T/PUR containing thesimian virus 40 large T antigen gene (SV40Tag) were transfected into the PCSCs byelectroporation method.Colonies were isolated by puromycin selection and amplified tocell strain which was named immortalized precartilaginous stem cell(IPCSC).The totalRNA was extracted from precartilaginous stem cells.Sub-gene PTHrP(1-36),PTHrP(38-94)and PTHrP(107-139) were obtained by RT-PCR method.Then the sub-genes werecombined with plasmids pTRE-2Hyg to construct recombinant eukaryotic responsiveplasmids pTRE-PTHrP(1-36),pTRE-PTHrP(38-94) and pTRE-PTHrP(107-139).PlasmidspTet-on was transfected into PCSCs with Lipoinfectamin^TM 2000 and then the stablytransfected positive clones were screened by G 418.Doxycycline was added into themedium of monoclonal cells which were transiently transfected with plasmidpTRE-2Hyg-Luc.The expression activitity of luciferase was detected to screen the doxycycline-controlled PCSCs highly expressing alien genes.Plasmids pTRE-PTHrP(1-36),pTRE-PTHrP(38-94) and pTRE-PTHrP(107-139) were transfected by lipofectamine 2000to pTet-on precartilagenous stem cells highly expressing alien genes.RT-PCR method wasused to detect the expression of PCNA for each group.Plasmids pTRE-PTHrP(107-139)were transfected into pTet-on precartilagenous stem cells which were induced byDoxycycline with different concentration respectively.Then the changes of expression ofgenes such as Colâ…¡,Colâ…©,Ihh,Ptc,Sox-9 and Bmp6 were examined by semiquant-itative RT-PCR.Results The results of immunocytochemistry confirmed that the rat precartilaginousstem cells was obtained and purified by micro-segregation and immunomagnetictechnology.The PCSCs stablely transfected with SV40Tag were detected in transfectedcells after stable transfection.The transfected cells were amplified to immortalized cellstrain maintained for more than 30 passages,named immortalized precartilaginous stemcells(IPCSCs).Double enzyme digestion analysis and sequencing showed that the targetsub-genes PTHrP(1-36),PTHrP(38-94) and PTHrP(107-139) were cloned into recombinantplasmids pTRE-PTHrP(1-36),pTRE-PTHrP(38-94) and pTRE-PTHrP(107-139).Theexpression activitity of luciferase doxycycline-controlled PCSCs transfected with plasmidpTet-on was 50 times more than that of the non-induced cell line.Compared with the grouptransfected with pTRE-PTHrP(1-36) or pTRE-PTHrP(38-94),the group transfected withpTRE-PTHrP(107-139) expressed more PCNA.For the group transfected withpTRE-PTHrP(107-139),the more doxycycline,the more Colâ…¡and Sox9.Otherwise;themore doxycycline,the less of Ptc,Colâ…©and BMP6.There was no significant differencein each group with different concentration of doxycycline for the expression of Ihh.Conclusions Rat PCSCs can be purified accurately in vitro.Immortalizedprecartilaginous stem cell strain was established successfully after SV40Tag wastransfected into the PCSCs.The eukaryotic responsive plasmids pTRE-PTHrP(1-36),pTRE-PTHrP(38-94) and pTRE-PTHrP(107-139) containing PTHrP(1-36),PTHrP(38-94), PTHrP(107-139) respectively were successfully constructed.After being screening,thePCSCs transfected with plasmid pTet-on highly expressed alien genes.As parts of PTHrP,PTHrP(107-139) may promote proliferation and inhibit differentiation for precartilagenousstem cells by regulating the expression of Sox-9,Ptc and BMP6.However,PTHrP(1-36) orPTHrP(38-94) was useless in cell proliferation.And what's more,pTet-on system canmodulate the expression of alien genes effectively.