CAJ | 학술논문

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(MTT)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,PBS 100μl,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 100μl+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.
목적 관찰인도호저단백(Ihh)화갑상방선격소상관태(PTHrP)신호통로대대서전연골간세포(PSCs)분화화증식적조공작용.방법 응용면역자주사선기술획득대서PSCs.새서람(MTT)비색법사선Ihh신호조단제배왕파명(Cyclopamine,Cyclo)적최저유효농도,PTHrP진핵질립(pEGPF-N1-PTHrP)전염PSCs.세포분4조:대조조(Control,PBS 100μl,n=3)、Ihh신호조단조(Ihh-,20nmol/L Cyclo 100μl,n=3)、PTHrP신호증강조(PTHrP+,1.0μg pEGPF-N1-PTHrP,n=3)화쌍중간예조(Ihh-/PTHrP+,20nmol/L Cyclo 100μl+1.0μg pEGPF-N1-PTHrP,n=3).간예2주후응용역전록-취합매련반응(RT-PCR)검측Ihh、PTHrP、Ⅱ형효원(ColⅡ)적mRNA표체,BrdU참입법측세포증식,면역조직화학검측ColⅡ표체,아신람염색검측산성당알다당함량.결과 성공획득PSCs,사선획득Cyclo최저유효공작농도위20nmol/L;1.0μg PTHrP진핵질립전염효솔>60%,전염후고표체해기인;Ihh-조ColⅡ기인화단백표체명현하강,PTHrP+조화Ihh-/PTHrP+조ColⅡ기인화단백표체은정,산성당알다당분비다;BrdU실험현시Ihh-조화Ihh-/PTHrP+조증식솔분별위(23.26±3.96)%화(21.65±5.12)%,상대Control조(35.42±3.79)%차이유통계학의의(P<0.01),이량조간차이무통계학의의(P>0.05).결론 Ihh-PTHrP신호통로삼여대PSCs분화화증식적조공,Ihh신호가독립조공PSCs적증식,단대기분화적조공의뢰PTHrP신호적작용.
Objective To observe the regulatorty effects of Indian hedgehog-parathyroid hormone-related 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 ColⅡ. BrdU incorporation method was applied to examine the DNA synthesis. Immunocytochemistry was used to assay ColⅡ protein expression level, and sulfate glycosaminoglycan was measured by Alcian blue dying. Results 20 nmol/L Cyclo could efficiently block Ihh pathway. pEGPF-N1-PTHrP tansfection efficiency was over 60%. ColⅡ 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.