永生化骨骺干细胞株的建立及PTHrP亚基因对骨骺干细胞增殖和分化的影响
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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。
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.
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。
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.
引文
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1.Robinson D,Hasharoni A,Cohen N,et al.Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells.Clin Orthop,1999,(367Suppl): 163-175.
2.Kramer J,Bohrnsen F,Schlenke P,et al.Stem Cell-Derived Chondrocytes for Regenerative Medicine.Transplant Proc.2006;38(3): 762-765
3.Zelle S,Zantop T,Schanz S,Petersen W.Arthroscopic techniques for the fixation of a three-dimensional scaffold for autologous chondrocyte transplantation: structural properties in an in vitro model.Arthroscopy.2007;23(10): 1073-1078.
4．罗卓荆，靳小兵，杨柳，等．自体组织工程生长板治疗长骨生长板损伤的实验研究．中华小儿外科杂志，2003，24(6)：537-540．
5．焦强，卫小春．自体软骨细胞移植修复关节软骨缺损．中华骨科杂志，2004，24(3)：184-186
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1.Robinson D,Hasharoni A,Cohen N,et al.Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells.Clin Orthop,1999,(367Suppl): 163-175.
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1.Robinson D,Hasharoni A,Cohen N,et al.Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells.Clin Orthop,1999,(367 Suppl): 163-175.
2．游洪波，陈安民．藻酸盐水凝胶与前软骨干细胞构建组织化工程骺板。生物骨科材料与临床研究，2004，1(7)：1-5．
3.Van Donkelaar CC,Huiskes R.The PTHrP-Ihh feedback loop in the embryonic growth plate allows PTHrP to control hypertrophy and Ihh to regulate proliferation.Biomech Model Mechanobiol.2007,6(1-2): 55-62.
4． 曾晓峰，破骨细胞的分化及可溶性因子调节。医学研究生学报，2003，16(03)：215-218
5.N Loveridge.Bone: more than a stick.JAnim Sci.1999,77(2): 190-196.
6.Strid H,Care A,Jansson T,et,al.Parathyroid hormone-related peptide(38-94)amide stimulates ATP-dependent calcium transport in the Basal plasma membrane of the human syncytiotrophoblast.J Endocrinol.2002,175(2): 517-524.
7.Syed MA,Horwitz MJ,Tedesco MB,et,al.Parathyroid hormone-related protein(1-36)stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy.J Clin Endocrinol Metab.2001,86(4): 1525-1531.
8.Stewart AF,Cain RL,Burr DB,et,al.Six-month daily administration of parathyroid hormone and parathyroid hormone-related protein peptides to adult ovariectomized rats markedly enhances bone mass and biomechanical properties: a comparison of human parathyroid hormone 1-34,parathyroid hormone-related protein 1-36,and SDZ-parathyroid hormone 893.J Bone Miner Res.2000,15(8): 1517-1525.
9.Horwitz M J,Tedesco MB,Sereika SM,et,al.Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)2 vitamin D.J Bone Miner Res.2005,20(10): 1792-1803.
10.Ekstein J,Nasatzky E,Boyan BD,et,al.Growth-plate chondrocytes respond to 17beta-estradiol with sex-specific increases in IP3 and intracellular calcium ion signalling via a capacitative entry mechanism.Steroids.2005,70(11): 775-86.
11．薛松涛，施鑫。骨形态生成蛋白在成骨细胞分化机制中的研究进展。医学研究生学报，2007，20(05)：540-543
12.Cornish J,Callon KE,Lin C,et al.Stimulation of osteoblast proliferation by C-terminal fragments of parathyroid hormone-related protein.J Bone Miner Res,1999,14(6): 915-922
13.Mathe Z,Dupraz P,Rinsch C,et,al.Tetracycline-regulated expression of VEGF-A in beta cells induces angiogenesis: improvement of engraftment following transplantation.Cell Transplant.2006,15(7): 621-636.
1.Gossen M,Bujard H.Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.Proc Natl Acad Sci U S A.1992,15;89(12): 5547-5551.
2.Van Donkelaar CC,Huiskes R.The PTHrP-Ihh feedback loop in the embryonic growth plate allows PTHrP to control hypertrophy and Ihh to regulate proliferation.Biomech Model Mechanobiol,2007,6(1-2):55-62.
3.Horwitz M J,Tedesco MB,Sereika SM,et,al.Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)_2 vitamin D.J Bone Miner Res,2005,20(10): 1792-1803.
4.Lazarus JE,Hegde A,Andrade AC,et,al.Fibroblast growth factor expression in the postnatal growth plate.Bone,2007,40(3):577-586.
5．游洪波，陈安民，程浩．免疫磁性细胞分选技术分离纯化新生大鼠骨骺前软骨干细胞．中华创伤杂志，2004，20(10)：606-608．
6.Syed MA,Horwitz M J,Tedesco MB,et,al.Parathyroid hormone-related protein(1-36)stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy.J Clin Endocrinol Metab,2001,86(4): 1525-1531.
7.Stewart AF,Cain RL,Burr DB,et,al.Six-month daily administration of parathyroid hormone and parathyroid hormone-related protein peptides to adult ovariectomized rats markedly enhances bone mass and biomechanical properties: a comparison of human parathyroid hormone 1-34,parathyroid hormone-related protein 1-36,and SDZ-parathyroid hormone 893.J Bone Miner Res,2000,15(8): 1517-1525.
8.Strid H,Care A,Jansson T,et,al.Parathyroid hormone-related peptide(38-94)amide stimulates ATP-dependent calcium transport in the Basal plasma membrane of the human syncytiotrophoblast. J Endocrinol, 2002,175(2):517-524.
9. Mathe Z, Dupraz P, Rinsch C, et,al. Tetracycline-regulated expression of VEGF-A in beta cells induces angiogenesis: improvement of engraftment following transplantation. Cell Transplant, 2006,15(7):621-636.
1. Van Donkelaar CC, Huiskes R. The PTHrP-Ihh feedback loop in the embryonic growth plate allows PTHrP to control hypertrophy and Ihh to regulate proliferation. Biomech Model Mechanobiol. 2007;6(1-2):55-62.
2. Horwitz MJ, Tedesco MB, Sereika SM, et, al. Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)_2 vitamin D. J Bone Miner Res, 2005, 20(10):1792-1803.
3. Kuo AC, Rodrigo JJ, Reddi AH, et al. Microfracture and bone morphogenetic protein 7 (BMP-7) synergistically stimulate articular cartilage repair.Osteoarthritis Cartilage. 2006,14(11): 1126-1135.
4. Han F, Adams CS, Tao Z, et al. Transforming growth factor-betal (TGF-betal) regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression. J Cell Biochem. 2005; 95(4):750-762.
5. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA. 1992, 15; 89(12): 5547-5551.
6. Mathe Z, Dupraz P, Rinsch C, et,al. Tetracycline-regulated expression of VEGF-A in beta cells induces angiogenesis: improvement of engraftment following transplantation. Cell Transplant, 2006, 15(7):621-636.
7. Syed MA, Horwitz MJ, Tedesco MB, et, al. Parathyroid hormone-related protein(1-36) stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy. J Clin Endocrinol Metab, 2001, 86(4): 1525-1531.
8. Stewart AF, Cain RL, Burr DB, et, al. Six-month daily administration of parathyroid hormone and parathyroid hormone-related protein peptides to adult ovariectomized rats markedly enhances bone mass and biomechanical properties: a comparison of human parathyroid hormone 1-34, parathyroid hormone-related protein 1-36, and SDZ- parathyroid hormone 893. J Bone Miner Res, 2000,15(8): 1517-1525.
9. Strid H, Care A, Jansson T, et,al. Parathyroid hormone-related peptide (38-94) amide stimulates ATP-dependent calcium transport in the Basal plasma membrane of the human syncytiotrophoblast. J Endocrinol, 2002,175(2):517-524.
10. Cornish J, Callon KE, Lin C, et al. Stimulation of osteoblast proliferation by C-terminal fragments of parathyroid hormone-related protein. J Bone Miner Res, 1999, 14(6):915-922
11. Brouwers JE, van Donkelaar CC, Sengers BG, et al. Can the growth factors PTHrP, Ihh and VEGF, together regulate the development of a long bone?. Biomech, 2006, 39(15):2774-2782.
12. Kawakami Y, Tsuda M, Tskahashi S, et al. Transcriptional coactivator PGC- lalpha regulates chondrogenesis via with SOX9. Proc Natl Acad Sci USA , 2005, 102(7): 2414-2419
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3.Robinson D,Hasharoni A,Cohen N,et al.Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells.Clin Orthop,1999;367(Suppl):S 163-S 175
4． 游洪波，陈安民，程浩．免疫磁性细胞分选技术分离纯化新生大鼠骨骺前软骨干细胞．中华创伤杂志， 2004；20(10)：606-608
5． 游洪波，陈安民，程浩．新生大鼠骨骺前软骨干细胞培养及细胞库建立．中国修复重建外科杂志，2005；19(3)：238-240
6．黄仕龙，陈安民，郭风劲，等．鼠骨骺增殖区细胞的分离鉴定和克隆构建PTHrp基因真核表达载体．中国矫形外科杂志，2005；13(19)：1489-1491
7.Li CF,Hughes-Fulford M.Fibroblast growth factor-2 is an immediate-early gene induced by mechanical stress in osteogenic cells.J Bone Miner Res,2006;21(6): 946-955
8.Deng C,Wynshaw-Boris A,Zhou F,et al.Fibroblast growth factor receptor 3 is a negative regulator of bone growth.Cell,1996;84(6):911-921
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20．张衣北，陈安民，郭风劲，等．Notchl信号系统对骨骺干细胞增殖与分化调控作用的初步观察．中华医学杂志，2005；85(48)：3430-3434
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25.Wattanasirichaigoon D,Charoenpipop D.Thanatophoric dysplasia: roentgenographic findings and detection of a de novo mutation of FGFR3 gene in a Thai patient.J Med Assoc Thai,2006;89(8): 1287-1292
26.Bellus GA,Spector EB,Speiser PW,et al.Distinct missense mutations of the FGFR3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype.Am J Hum Genet,2000;67(6): 1411-1421
27.Kinkl N,Ruiz J,Vecino E,et al.Possible involvement of a fibroblast growth factor 9(FGF9)-FGF receptor-3-mediated pathway in adult pig retinal ganglion cell survival in vitro.Mol Cell Neurosci,2003;23(1):39-53
28.Govindarajan V,Overbeek PA.FGF9 can induce endochondral ossification in cranial mesenchyme.BMC Dev Biol,2006;6:7
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30．游洪波，陈安民．藻酸盐水凝胶与前软骨干细胞构建组织化工程骺板．生物骨科材料与临床研究， 2004；1(6)：1-5
31.Ibrahimi OA,Yeh BK,Eliseenkova AV,et al.Analysis of mutations in fibroblast growth factor(FGF)and a pathogenic mutation in FGF receptor(FGFR)provides direct evidence for the symmetric two-end model for FGFR dimerization.Mol Cell Biol，2005；25(2)：671-684
32．郭风劲，陈安民，黄仕龙．免疫分选软骨前体细胞并诱导永生化的研究．中华实验外科杂志，2007；24(2)：226-228
2.Zelle S,Zantop T,Schanz S,Petersen W.Arthroscopic techniques for the fixation of a three-dimensional scaffold for autologous chondrocyte transplantation: structural properties in an in vitro model.Arthroscopy.2007;23(10): 1073-1078.
3．罗卓荆，靳小兵，杨柳，等．自体组织工程生长板治疗长骨生长板损伤的实验研究．中华小儿外科杂志，2003，24(6)：537-540．
4．焦强，卫小春．自体软骨细胞移植修复关节软骨缺损．中华骨科杂志，2004,24(3)：184-186．
5.Robinson D,Hasharoni A,Cohen N,et al.Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells.Clin Orthop,1999,(367Suppl): 163-175.
6．张衣北，陈安民，郭风劲。骨骺干细胞的体外培养以及生长特性观察。中国康复杂志，2006，2(1)：79-82．
7.Van Donkelaar CC,Huiskes R.The PTHrP-Ihh feedback loop in the embryonic growth plate allows PTHrP to control hypertrophy and Ihh.to regulate proliferation.Biomech Model Mechanobiol.2007;6(1-2):55-62.
8.Horwitz MJ,Tedesco MB,Sereika SM,et,al.Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)_2 vitamin D.J Bone Miner Res,2005,20(10): 1792-1803.
9.Kuo AC,Rodrigo J J,Reddi AH,et al.Microfracture and bone morphogenetic protein 7(BMP-7)synergistically stimulate articular cartilage repair.Osteoarthritis Cartilage.2006,14(11): 1126-1135.
10.Han F,Adams CS,Tao Z,et al.Transforming growth factor-beta1(TGF-betal)regulates ATDC5 chondrogenic differentiation and fibronectin isoform expression.J Cell Biochem.2005;95(4):750-762.
11. Syed MA, Horwitz MJ, Tedesco MB, et, al. Parathyroid hormone-related protein(l-36) stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy. J Clin Endocrinol Metab. 2001,86(4): 1525-1531.
12. Stewart AF, Cain RL, Burr DB, et, al. Six-month daily administration of parathyroid hormone and parathyroid hormone-related protein peptides to adult ovariectomized rats markedly enhances bone mass and biomechanical properties: a comparison of human parathyroid hormone 1-34, parathyroid hormone-related protein 1-36, and SDZ-parathyroid hormone 893. J Bone Miner Res, 2000,15(8):1517-1525.
13. Strid H, Care A, Jansson T, et,al. Parathyroid hormone-related peptide (38-94) amide stimulates ATP-dependent calcium transport in the Basal plasma membrane of the human syncytiotrophoblast. J Endocrinol. 2002,175(2): 517-524.
14. Cornish J, Callon KE, Lin C, et al. Stimulation of osteoblast proliferation by C-terminal fragments of parathyroid hormone-related protein. J Bone Miner Res, 1999, 14(6):915-922.
15. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA. 1992, 15; 89(12):5547-5551.
16. Mathe Z, Dupraz P, Rinsch C, et,al. Tetracycline-regulated expression of VEGF-A in beta cells induces angiogenesis: improvement of engraftment following transplantation. Cell Transplant, 2006, 15(7):621-636.
1.Robinson D,Hasharoni A,Cohen N,et al.Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells.Clin Orthop,1999,(367Suppl): 163-175.
2.Kramer J,Bohrnsen F,Schlenke P,et al.Stem Cell-Derived Chondrocytes for Regenerative Medicine.Transplant Proc.2006;38(3): 762-765
3.Zelle S,Zantop T,Schanz S,Petersen W.Arthroscopic techniques for the fixation of a three-dimensional scaffold for autologous chondrocyte transplantation: structural properties in an in vitro model.Arthroscopy.2007;23(10): 1073-1078.
4．罗卓荆，靳小兵，杨柳，等．自体组织工程生长板治疗长骨生长板损伤的实验研究．中华小儿外科杂志，2003，24(6)：537-540．
5．焦强，卫小春．自体软骨细胞移植修复关节软骨缺损．中华骨科杂志，2004，24(3)：184-186
6.Li CF,Hughes-Fulford M.Fibroblast growth factor-2 is an immediate-early gene induced by mechanical stress in osteogenic cells.J Bone Miner Res,2006; 21(6):946-955
7.Deng C,Wynshaw-Boris A,Zhou F,et al.Fibroblast growth factor receptor 3 is a negative regulator of bone growth.Cell,1996;84(6):911-921
1.Robinson D,Hasharoni A,Cohen N,et al.Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells.Clin Orthop,1999,(367Suppl): 163-175.
2． 游洪波，陈安民。藻酸盐水凝胶与前软骨干细胞构建组织化工程骺板。生物骨科材料与临床研究，2004，1(7)：1-5．
3． 张衣北，陈安民，郭风劲。骨骺干细胞的体外培养以及生长特性观察。中国康复杂志，2006，2(1)：79-82．
4． 黄仕龙，陈安民，郭风劲，张衣北。鼠骨骺增殖区细胞的的分离鉴定和克隆构建PFHrP基因真核表达载体。中国矫形外科杂志，2005，13(19)：1489-91．
5． 游洪波，陈安民，程浩。免疫磁性细胞分选技术分离纯化新生大鼠骨骺前软骨干细胞。中华创伤杂志，2004，20(10)：606-608．
6.Nakanishi A,Nakamura A,Liddington R,et al.Identification of amino acid residues within simian virus 40 capsid proteins Vpl,Vp2,and Vp3 that are required for their interaction and for viral infection.J Virol.2006;80(18): 8891-8.
7.May T,Wirth D,Hauser H,et al.Transcriptionally regulated immortalization overcomes side effects of temperature-sensitive SV40 large T antigen.Biochem Biophys Res Commun.2005;327(3): 734-41.
8.Gai D,Li D,Finkielstein CV,et al.Insights into the oligomeric states,conformational changes,and helicase activities of SV40 large tumor antigen.J Biol Chem,2004,279: 38952-38959.
1.Robinson D,Hasharoni A,Cohen N,et al.Fibroblast growth factor receptor-3 as a marker for precartilaginous stem cells.Clin Orthop,1999,(367 Suppl): 163-175.
2．游洪波，陈安民．藻酸盐水凝胶与前软骨干细胞构建组织化工程骺板。生物骨科材料与临床研究，2004，1(7)：1-5．
3.Van Donkelaar CC,Huiskes R.The PTHrP-Ihh feedback loop in the embryonic growth plate allows PTHrP to control hypertrophy and Ihh to regulate proliferation.Biomech Model Mechanobiol.2007,6(1-2): 55-62.
4． 曾晓峰，破骨细胞的分化及可溶性因子调节。医学研究生学报，2003，16(03)：215-218
5.N Loveridge.Bone: more than a stick.JAnim Sci.1999,77(2): 190-196.
6.Strid H,Care A,Jansson T,et,al.Parathyroid hormone-related peptide(38-94)amide stimulates ATP-dependent calcium transport in the Basal plasma membrane of the human syncytiotrophoblast.J Endocrinol.2002,175(2): 517-524.
7.Syed MA,Horwitz MJ,Tedesco MB,et,al.Parathyroid hormone-related protein(1-36)stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy.J Clin Endocrinol Metab.2001,86(4): 1525-1531.
8.Stewart AF,Cain RL,Burr DB,et,al.Six-month daily administration of parathyroid hormone and parathyroid hormone-related protein peptides to adult ovariectomized rats markedly enhances bone mass and biomechanical properties: a comparison of human parathyroid hormone 1-34,parathyroid hormone-related protein 1-36,and SDZ-parathyroid hormone 893.J Bone Miner Res.2000,15(8): 1517-1525.
9.Horwitz M J,Tedesco MB,Sereika SM,et,al.Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)2 vitamin D.J Bone Miner Res.2005,20(10): 1792-1803.
10.Ekstein J,Nasatzky E,Boyan BD,et,al.Growth-plate chondrocytes respond to 17beta-estradiol with sex-specific increases in IP3 and intracellular calcium ion signalling via a capacitative entry mechanism.Steroids.2005,70(11): 775-86.
11．薛松涛，施鑫。骨形态生成蛋白在成骨细胞分化机制中的研究进展。医学研究生学报，2007，20(05)：540-543
12.Cornish J,Callon KE,Lin C,et al.Stimulation of osteoblast proliferation by C-terminal fragments of parathyroid hormone-related protein.J Bone Miner Res,1999,14(6): 915-922
13.Mathe Z,Dupraz P,Rinsch C,et,al.Tetracycline-regulated expression of VEGF-A in beta cells induces angiogenesis: improvement of engraftment following transplantation.Cell Transplant.2006,15(7): 621-636.
1.Gossen M,Bujard H.Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.Proc Natl Acad Sci U S A.1992,15;89(12): 5547-5551.
2.Van Donkelaar CC,Huiskes R.The PTHrP-Ihh feedback loop in the embryonic growth plate allows PTHrP to control hypertrophy and Ihh to regulate proliferation.Biomech Model Mechanobiol,2007,6(1-2):55-62.
3.Horwitz M J,Tedesco MB,Sereika SM,et,al.Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)_2 vitamin D.J Bone Miner Res,2005,20(10): 1792-1803.
4.Lazarus JE,Hegde A,Andrade AC,et,al.Fibroblast growth factor expression in the postnatal growth plate.Bone,2007,40(3):577-586.
5．游洪波，陈安民，程浩．免疫磁性细胞分选技术分离纯化新生大鼠骨骺前软骨干细胞．中华创伤杂志，2004，20(10)：606-608．
6.Syed MA,Horwitz M J,Tedesco MB,et,al.Parathyroid hormone-related protein(1-36)stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy.J Clin Endocrinol Metab,2001,86(4): 1525-1531.
7.Stewart AF,Cain RL,Burr DB,et,al.Six-month daily administration of parathyroid hormone and parathyroid hormone-related protein peptides to adult ovariectomized rats markedly enhances bone mass and biomechanical properties: a comparison of human parathyroid hormone 1-34,parathyroid hormone-related protein 1-36,and SDZ-parathyroid hormone 893.J Bone Miner Res,2000,15(8): 1517-1525.
8.Strid H,Care A,Jansson T,et,al.Parathyroid hormone-related peptide(38-94)amide stimulates ATP-dependent calcium transport in the Basal plasma membrane of the human syncytiotrophoblast. J Endocrinol, 2002,175(2):517-524.
9. Mathe Z, Dupraz P, Rinsch C, et,al. Tetracycline-regulated expression of VEGF-A in beta cells induces angiogenesis: improvement of engraftment following transplantation. Cell Transplant, 2006,15(7):621-636.
1. Van Donkelaar CC, Huiskes R. The PTHrP-Ihh feedback loop in the embryonic growth plate allows PTHrP to control hypertrophy and Ihh to regulate proliferation. Biomech Model Mechanobiol. 2007;6(1-2):55-62.
2. Horwitz MJ, Tedesco MB, Sereika SM, et, al. Continuous PTH and PTHrP infusion causes suppression of bone formation and discordant effects on 1,25(OH)_2 vitamin D. J Bone Miner Res, 2005, 20(10):1792-1803.
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