Ihh信号通过甲状旁腺激素相关肽依赖和非依赖方式参与调控前软骨干细胞的分化和增殖  

Indian hedgehog regulates precartilainous stem cells differentiation and proliferation in PTHrP-dependent or independent manner

作　　者：许凯[1] 陈安民[1] 成薇婷[2] 郭风劲[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030 [2]华中科技大学同济医学院附属中西医结合医院肿瘤科

出　　处：《中华实验外科杂志》2011年第7期1048-1051,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30571872）

摘　　要：目的观察印度豪猪蛋白（Ihh）和甲状旁腺激素相关肽（PTHrP）信号通路对大鼠前软骨干细胞（PSCs）分化和增殖的调控作用。方法应用免疫磁珠筛选技术获得大鼠PSCs。噻唑蓝（M3T）比色法筛选Ihh信号阻断剂环王巴明（Cyclopamine，Cyclo）的最低有效浓度，PTHrP真核质粒（pEGPF—N1-PTHrP）转染PSCs。细胞分4组：对照组（Control，PBS100μl，n=3）、Ihh信号阻断组（Ihh^-，20nmol／L Cyclo 100μl，n=3）、PTHrP信号增强组（PTHrP^＋，1．0μg pEGPF—N1-PTHrP，n=3）和双重干预组（Ihh^-／PTHrP^＋，20nmol／L Cyclo 100tμ＋1．0μg pEGPF—N1-PTHrP，n=3）。干预2周后应用逆转录-聚合酶链反应（RT—PCR）检测Ihh、PTHrP、Ⅱ型胶原（ColⅡ）的mRNA表达，BrdU掺入法测细胞增殖，免疫组织化学检测ColⅡ表达，阿辛蓝染色检测酸性糖胺多糖含量。结果成功获得PSCs，筛选获得Cyclo最低有效工作浓度为20nmol／L；1．0μg PTHrP真核质粒转染效率〉60％，转染后高表达该基因；Ihh^-组ColⅡ基因和蛋白表达明显下降，PTHrP^＋组和Ihh^-／PTHrP^＋组ColⅡ基因和蛋白表达稳定，酸性糖胺多糖分泌多；BrdU实验显示Ihh^-组和Ihh／PTHrP^＋组增殖率分别为（23．26±3．96）％和（21．65±5．12）％，相对Control组（35．42±3．79）％差异有统计学意义（P〈0．01），而两组间差异无统计学意义（P〉0．05）。结论Ihh-PTHrP信号通路参与对PSCs分化和增殖的调控，Ihh信号可独立调控PSCs的增殖，但对其分化的调控依赖PTHrP信号的作用。Objective To observe the regulatorty effects of Indian hedgehog-parathyroid hormonerelated protein （Ihh-PTHrP） pathway on differentiation and proliferation of precartilainous stem cells （PSCs） Methods PSCs were isolated form neonatal rats by immunomagnetic separation system. Methyl thiazol tetrazolium （MTT） assay was used to choose proper concentration of cyclopamine （Cyclo）, inhibitor of Ihh pathway. Plasmids pEGPF-N1-PTHrP were transfected into PSCs to up-regulate PTHrP signaling. Four experiment groups were established： control （PBS 100 μl,n = 3 ） , Ihh^- （20 nmol/L Cyclo 100 μl, n =3）, PTHrP^＋ （ 1.0 μg pEGPF-N1-PTHrP,n =3） and Ihh^-/PTHrP^＋ （20 nmol/L Cyclo 100 μl ＋ 1.0 μg pEGPF-N1-PTHrP, n = 3）. After 2 weeks, reverse transcription-polymerase chain reaction （RT-PCR） was used to detect the mRNA level of PTHrP and Col Ⅱ. BrdU incorporation method was applied to examine the DNA synthesis. Immunocytochemistry was used to assay Col Ⅱ protein expression level, and sulfate glyco-saminoglycan was measured by Alcian blue dying. Results 20 nmol/L Cyclo could efficiently block Ihh pathway, pEGPF-N1-PTHrP tansfection efficiency was over 60%. Col Ⅱ mRNA and protein expression levels in Ihh^- group were significantly lower than in control group, while those in PTHrP^＋ and Ihh^-/PTHrP^＋ groups were stable. The deposition of typical cartilage extracellular matrix was only detected in PTHrP^＋ group and Ihh^-/PTHrP^＋ group. The growth rate of PSCs in Ihh^- group and Ihh /PTHrP^＋ group was （23.26 ±3. 96）% and （21.65 ±5. 12）% respectively, which was significantly lower than in control group [ （35.42 ±3.79）% ,P 〈 0. 01 ], but there was no significant difference between Ihh^- and Ihh^-/PTHrP^＋ groups （P 〉 0. 05 ）. Conclusion Ihh pathway plays an important role in PSCs differentiation and proliferation. PTHrP, as a downstream element of Ihh signaling pathway, mainly regulates cells differentiation.

关 键 词：前软骨干细胞 信号通路 分化 增殖 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]