摘要
背景:甲状旁腺素相关蛋白-印第安刺猬蛋白两者相互作用调节着骨骺区软骨的分化成熟及增殖,促使骨骺区细胞处于一种相对稳定的状态。目的:分析甲状旁腺相关蛋白亚基因PTHrP(1-36)、PTHrP(38-94)和PTHrP(107-139)对骨骺干细胞的调控作用以及PTHrP(107-139)与其他调控因子的影响。方法:用脂质体介导的基因转染技术将质粒pTRE-PTHrP(1-36)、pTRE-PTHrP(38-94)和pTRE-PTHrP(107-139)分别转染pTet-on骨骺干细胞株,放入诱导分化培养基中进行培养,加入1mg/L的强力霉素进行诱导,以RT-PCR的方法检测增殖细胞核抗原基因的表达变化。再次将质粒pTRE-PTHrP(107-139)转染pTet-on骨骺干细胞株,加入不同浓度的强力霉素进行诱导,应用RT-PCR的方法检测Ⅱ型胶原、X型胶原、印第安刺猬蛋白、Ptc、Sox9、骨形态发生蛋白6基因的表达变化。结果与结论:强力霉素诱导的质粒pTRE-PTHrP(107-139)转染组的增殖细胞核抗原表达明显高于质粒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Ⅱ型胶原、Sox-9表达明显增强,Ihh表达未见明显变化,而Ptc、X型胶原、骨形态发生蛋白6表达明显降低。而且其表达量与强力霉素存在剂量依赖性。提示PTHrP(107-139)可能是通过调控Sox-9、Ptc、骨形态发生蛋白6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)对前癌干细胞的增殖并无明显作用。pTet-on质粒系统在前癌干细胞能有效的调控外来基因的表达。
BACKGROUND： The interaction between parathyroid hormone-related protein and Indian hedgehog protein both can regulate the differentiation, maturation and proliferation of cartilage in epiphyseal zone, which can help the cells in the epiphyseal stay in the relatively stable state. OBJECTIVE： To analyze the regulatory effects of parathyroid hormone-related protein sub-gene （1-36）, （38-94） and （107-139） on precartilaginous stem cells and the relationship between parathyroid hormone-related protein （107-139） and other regulating factors. METHODS： The pTRE-parathyroid hormone-related protein （1-36）, pTRE-parathyroid hormone-related protein P（38-94） and pTRE-parathyroid hormone-related protein （107-139） were transfected into pTet-on precartilagenous stem cells by liposome transfection method. The cells were cultured by differentiation-induced medium and the Tet-on was induced by 1 mg/L doxycycline respectively. Then the changes of expression of proliferating cell nuclear antigen gene were examined by reverse transcription-PCR. Plasmids pTRE-parathyroid hormone-related protein （107-139） were transfected into pTet-on precartilagenous stem cells which were induced by doxycycline with different concentrations respectively. Then the changes of expression of genes such as collagen type Ⅱ, collagen type Ⅹ, Indian hedgehog protein, Ptc, Sox-9 and bone morphogenetic protein 6 were examined by semiquantitative reverse transcription-PCR. RESULTS AND CONCLUSION： Compared with the group transfected with pTRE-parathyroid hormone-related protein （1-36） and pTRE-parathyroid hormone-related protein （38-94）, the group transfected with pTRE- parathyroid hormone-related protein （107-139） expressed more proliferating cell nuclear antigen. For the group transfected with pTRE-parathyroid hormone-related protein （107-139）, the expressions of collagen type Ⅱ and Sox9 were significantly increased, while the expression of Indian hedgehog protein did not change, and the expressions of Ptc, collagen type X and bone morphogenetic protein 6 were decreased. The expression of the genes showed a dose-dependent manner. As parts of parathyroid hormone-related protein, parathyroid hormone-related protein （107-139） may promote the proliferation and inhibit the differentiation of precartilagenous stem cells by regulating the expression of Sox-9, Ptc and bone morphogenetic protein 6. However, parathyroid hormone-related protein （1-36） and （38-94） were useless in proliferation of precancerous stem cells. pTet-on system can regulate the expression of alien genes effectively.
出处
《中国组织工程研究》
CAS
CSCD
2013年第10期1814-1820,共7页
Chinese Journal of Tissue Engineering Research
基金
中央高校基本科研业务费专项资金资助项目(4101052)~~
关键词
干细胞
干细胞培养与分化
甲状旁腺相关蛋白
印第安刺猬蛋白
骨骺干细胞
增殖
分化
增殖细胞核抗原
其他基金
干细胞图片文章
stem cells
stem cell culture and differentiation
parathyroid hormone-related protein
Indian hedgehog protein
precartilaginous stem cells
proliferation
differentiation
proliferating cell nuclear antigen
other grants-supported paper
stem cell photographs-containing paper
