【作者】 张树威；

【导师】 陈安民；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 目的体外分离、培养、鉴定骨骺干细胞并诱导其永生化，建立高水平诱导、低背景表达的骨骺干细胞株，研究甲状旁腺相关蛋白亚基因PTHrP(1-36)、PTHrP(38-94)和PTHrP(107-139)对体外培养的骨骺干细胞的调控作用以及PTHrP(107-139)与其他调控因子的相互影响。方法采用显微取材和免疫磁性分选技术分离纯化具有成纤维生长因子受体-3(fibroblast growth factor receptor-3，FGFR-3)特异性表面标志的骨骺干细胞。利用电穿孔转染技术将含有猿肾病毒40大T抗原基因(SV40Tag)的质粒pCMVSV40T／PUR转染骨骺干细胞，经嘌呤霉素筛选，抗性克隆扩大培养获得永生化骨骺干细胞。提取骨骺干细胞的总RNA，以RT-PCR方法获得带有酶切位点的PTHrP(1-36)、PTHrP(38-94)和PTHrP(107-139)基因片段，扩增的DNA片段分别与载体pTRE-2Hyg双酶切后连接，转化扩增后对重组质粒进行提取和酶切、测序鉴定。用脂质体介导的基因转染技术将pTet-on质粒转染于骨骺干细胞，G418筛选得到稳定克隆，再瞬时转染pTRE-2Hyg-Luc质粒并筛选出高水平诱导、低背景表达的pTet-on骨骺干细胞系。用脂质体介导的基因转染技术将将质粒pTRE-PTHrP(1-36)、pTRE-PTHrP(38-94)和pTRE-PTHrP(107-139)分别转染高水平诱导、低背景表达的-pTet-on骨骺干细胞株，放入诱导分化培养基中进行培养，加入强力霉素进行诱导，以RT-PCR的方法检测PCNA基因的表达变化。应用RT-PCR的方法检测不同浓度的强力霉素下质粒pTRE-PTHrP(107-139)转染组的ColⅡ、Col X、Ihh、Ptc、Sox9、BMP6基因的表达变化。结果经免疫细胞化学法证实所取材分离并纯化的细胞为骨骺干细胞。SV40Tag稳定转染入骨骺干细胞经扩大培养，命名为永生化骨骺干细胞。从骨骺干细胞中克隆出的PTHrP亚克隆基因PTHrP(1-36)、PTHrP(38-94)和PTHrP(107-139)经酶切图谱分析和DNA序列测定证实已经插入重组质粒pTRE-PTHrP(1-36)、pTRE-PTHrP(38-94)和pTRE-PTHrP(107-139)。pTet-on质粒转染于骨骺干细胞，并瞬时转染pTRE-2Hyg-Luc质粒获得1株诱导后荧光素酶的表达活性增加50倍的pTet-on骨骺干细胞株。强力霉素诱导的质粒pTRE-PTHrP(107-139)转染组的PCNA表达明显高于质粒pTRE-PTHrP(1-36)转染组和质粒pTRE-PTHrP(38-94)转染组；在强力霉素诱导的质粒pTRE-PTHrP(107-139)转染组中ColⅡ、Sox-9表达明显增强，Ihh表达未见明显变化，而Ptc、Col X、BMP6表达明显降低。而且其表达量与强力霉素存在剂量依赖性。结论运用显微取材和免疫纯化技术可以成功分离纯化骨骺干细胞。经SV40Tag转染构建了永生化的骨骺干细胞株。成功克隆出PTHrP亚克隆基因PTHrP(1-36)、PTHrP(38-94)和PTHrP(107-139)并构建了反应质粒pTRE-PTHrP(1-36)、pTRE-PTHrP(38-94)和pTRE-PTHrP(107-139)。筛选得到的pTet-on骨骺干细胞株受强力霉素诱导后能够低背景、高水平表达。PTHrP(107-139)可能是通过调控Sox-9、Ptc、BMP6的表达来促进骨骺干细胞增殖并抑制其分化的，而PTHrP(1-36)、PTHrP(38-94)对PCSCs的增殖并无明显作用。pTet-on质粒系统在PCSCs能有效的调控外来基因的表达。更多还原

【Abstract】 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 ColⅡ,ColⅩ,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 ColⅡand Sox9.Otherwise;themore doxycycline,the less of Ptc,ColⅩand 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.更多还原