中华骨科杂志
中華骨科雜誌
중화골과잡지
CHINESE JOURNAL OF ORTHOPAEDICS
2014年
1期
70-77
,共8页
甲状旁腺素%破骨细胞%基因表达%凝集素类
甲狀徬腺素%破骨細胞%基因錶達%凝集素類
갑상방선소%파골세포%기인표체%응집소류
Parathyroid hormone%Osteoclasts%Gene expression%Agglutinins
目的 探讨甲状旁腺素(parathyroid hormone,PTH)(1-34)对UMR106成骨细胞表达破骨细胞抑制性凝集素(osteoclast inhibitory lectin,OCIL)基因mRNA的调节及其信号转导机制.方法 培养大鼠UMR106成骨细胞,采用PTH(1-34)及蛋白激酶A(PKA)、蛋白激酶C(PKC)、钙离子、MAPK通路的激动剂或阻断剂干预细胞不同时间后,收集细胞提取总RNA,实时荧光定量PCR检测OCIL mRNA的表达水平.结果 PTH(1-34)以剂量和时间依赖方式促进OCIL mRNA表达.10 nmol/L PTH(1-34)作用6h后促进OCIL mRNA表达,24 h上调效应最显著,约为对照组[未加入PTH(1-34)]的2.8倍.PTH(1-34)对OCIL mRNA表达促进作用的起始时间及高峰时间均晚于其对RANKL的诱导和对OPG的抑制.蛋白激酶A通路激动剂福司可林(FSK)、双丁酰基环腺苷磷酸(db-cAMP)及钙离子载体A23187均不同程度促进OCIL mRNA表达,最大上调效应分别约为对照组的4.2、4.5和5.1倍.蛋白激酶C激动剂佛波酯(PMA)作用早期(2～6 h)抑制OCILmRNA表达,作用6h后最大抑制率达50％;PMA作用后期(24 h)对OCIL mRNA的抑制作用逆转为促进效应.PKA阻断剂KT5720、钙调蛋白(CaMK)阻断剂W-7、钙调蛋白激酶Ⅱ阻断剂KN-62及丝裂原激活的蛋白激酶(MAPK)阻断剂PD98059均下调PTH(1-34)诱导的OCIL mRNA表达,最大抑制率分别为56％、61％ 、63％和50％.各信号通路间存在交互作用.MAPK通路阻断剂PD98059减少PKA激动剂FSK或db-cAMP诱导的OCIL mRNA表达,抑制率分别为98％和63％;但不影响A23187诱导的OCIL mRNA水平增高.结论 PKA通路、钙/钙调蛋白通路和MAPK通路可介导PTH(1-34)诱导的OCIL mRNA表达.
目的 探討甲狀徬腺素(parathyroid hormone,PTH)(1-34)對UMR106成骨細胞錶達破骨細胞抑製性凝集素(osteoclast inhibitory lectin,OCIL)基因mRNA的調節及其信號轉導機製.方法 培養大鼠UMR106成骨細胞,採用PTH(1-34)及蛋白激酶A(PKA)、蛋白激酶C(PKC)、鈣離子、MAPK通路的激動劑或阻斷劑榦預細胞不同時間後,收集細胞提取總RNA,實時熒光定量PCR檢測OCIL mRNA的錶達水平.結果 PTH(1-34)以劑量和時間依賴方式促進OCIL mRNA錶達.10 nmol/L PTH(1-34)作用6h後促進OCIL mRNA錶達,24 h上調效應最顯著,約為對照組[未加入PTH(1-34)]的2.8倍.PTH(1-34)對OCIL mRNA錶達促進作用的起始時間及高峰時間均晚于其對RANKL的誘導和對OPG的抑製.蛋白激酶A通路激動劑福司可林(FSK)、雙丁酰基環腺苷燐痠(db-cAMP)及鈣離子載體A23187均不同程度促進OCIL mRNA錶達,最大上調效應分彆約為對照組的4.2、4.5和5.1倍.蛋白激酶C激動劑彿波酯(PMA)作用早期(2～6 h)抑製OCILmRNA錶達,作用6h後最大抑製率達50％;PMA作用後期(24 h)對OCIL mRNA的抑製作用逆轉為促進效應.PKA阻斷劑KT5720、鈣調蛋白(CaMK)阻斷劑W-7、鈣調蛋白激酶Ⅱ阻斷劑KN-62及絲裂原激活的蛋白激酶(MAPK)阻斷劑PD98059均下調PTH(1-34)誘導的OCIL mRNA錶達,最大抑製率分彆為56％、61％ 、63％和50％.各信號通路間存在交互作用.MAPK通路阻斷劑PD98059減少PKA激動劑FSK或db-cAMP誘導的OCIL mRNA錶達,抑製率分彆為98％和63％;但不影響A23187誘導的OCIL mRNA水平增高.結論 PKA通路、鈣/鈣調蛋白通路和MAPK通路可介導PTH(1-34)誘導的OCIL mRNA錶達.
목적 탐토갑상방선소(parathyroid hormone,PTH)(1-34)대UMR106성골세포표체파골세포억제성응집소(osteoclast inhibitory lectin,OCIL)기인mRNA적조절급기신호전도궤제.방법 배양대서UMR106성골세포,채용PTH(1-34)급단백격매A(PKA)、단백격매C(PKC)、개리자、MAPK통로적격동제혹조단제간예세포불동시간후,수집세포제취총RNA,실시형광정량PCR검측OCIL mRNA적표체수평.결과 PTH(1-34)이제량화시간의뢰방식촉진OCIL mRNA표체.10 nmol/L PTH(1-34)작용6h후촉진OCIL mRNA표체,24 h상조효응최현저,약위대조조[미가입PTH(1-34)]적2.8배.PTH(1-34)대OCIL mRNA표체촉진작용적기시시간급고봉시간균만우기대RANKL적유도화대OPG적억제.단백격매A통로격동제복사가림(FSK)、쌍정선기배선감린산(db-cAMP)급개리자재체A23187균불동정도촉진OCIL mRNA표체,최대상조효응분별약위대조조적4.2、4.5화5.1배.단백격매C격동제불파지(PMA)작용조기(2～6 h)억제OCILmRNA표체,작용6h후최대억제솔체50％;PMA작용후기(24 h)대OCIL mRNA적억제작용역전위촉진효응.PKA조단제KT5720、개조단백(CaMK)조단제W-7、개조단백격매Ⅱ조단제KN-62급사렬원격활적단백격매(MAPK)조단제PD98059균하조PTH(1-34)유도적OCIL mRNA표체,최대억제솔분별위56％、61％ 、63％화50％.각신호통로간존재교호작용.MAPK통로조단제PD98059감소PKA격동제FSK혹db-cAMP유도적OCIL mRNA표체,억제솔분별위98％화63％;단불영향A23187유도적OCIL mRNA수평증고.결론 PKA통로、개/개조단백통로화MAPK통로가개도PTH(1-34)유도적OCIL mRNA표체.
Objective To investigate the regulation of parathyroid hormone(1-34) on mRNA expression of osteoclast inhibitory lectin (OCIL) gene in UMR106 osteoblastic-like cells and involved signaling pathway.Methods Rat UMR106 osteoblastic-like cells were cultured and treated with various concentration of PTH(1-34) and specific agonists or inhibitors of PKA,PKC,Ca2+/calmodulin-dependent protein kinase (CaMK) and mitogen-activated protein kinase (MAPK) signal pathways for indicated time intervals.Then the cells were gathered at indicated time points and total RNA were extracted.OCIL mRNA expression was analyzed using real-time PCR technique.Results PTH(1-34) stimulated OCIL mRNA expression in a time-and dose-dependentmanner.A dose of 10 nmol/L PTH(1-34) started to induce OCIL mRNA from 6 h,with a highest increase of about 2.8-fold vs.control group (without PTH treatment) at 24 h.The up-regulation of OCIL mRNA began and reached maximum later than RANKL induction and OPG suppression effected by PTH(1-34).Protein Kinase A (PKA) signaling activators forskolin(FSK) and dibutyryl cAMP (db-cAMP),as well as calcium ionophore A23187 all up-regulated OCIL mRNA with the maximal induction of about 4.2-fold,4.5-fold and 5.1-fold.Protein Kinase C (PKC) activator phorbol-12-myristate-13-acetate(PMA) reduced OCIL mRNA expression at the early stage(2-6 h),with the highest down-regulation of 50％ at 6 h.However,the inhibitory effect on OCIL mRNA turned into slightly stimulatory effect later (24 h).PKA inhibitor KT5720,calmodulin antagonist W-7,CaMK Ⅱ inhibitor KN-62 and mitogen-activated protein kinase (MAPK) inhibitor PD98059 all blocked PTH(1-34)-induced OCIL mRNA expression by the maximal reduction of 56％,61％,63％ and 50％ respectively.There also exist cross-talks between different signal pathways.MAPK inhibitor PD98059 blocked the expression of OCIL mRNA which was stimulated by PKA activators FSK or db-cAMP,with the reduction of 98％ and 63％ respectively,while the OCIL mRNA expression stimulated by A23187 remained unaffected.Conclusion PTH(1-34) increased OCIL mRNA expression in vitro through cAMP/PKA,Ca2+/CaMK and MAPK signaling pathways.
