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投稿时间：2019-05-27 网络发布日期：2020-01-20
投稿时间：2019-05-27 网络发布日期：2020-01-20
中文摘要: 目的 在动物整体水平上分析甲状旁腺激素(PTH)对成骨的调节作用,探讨Glu/Asp丰富羧基端域Cbp/p300相互作用反式激活因子1(CITED1)在骨代谢中的调节作用。
方法 以间断小剂量PTH皮下注射的野生型C57BL小鼠为实验组,同体积生理盐水皮下注射的野生型C57BL小鼠为对照组。处死后取股骨检测小鼠的骨代谢表型,如皮质骨、松质骨的厚度和骨量;采用酶联免疫吸附法(ELISA)测量小鼠血清中Ⅰ型原胶原肽(P[KG-*2]Ⅰ[KG-*4]NP)、骨钙素(OC)、骨碱性磷酸酶(BALP)、抗酒石酸酸性磷酸酶(TRAP)的浓度。
取1日龄野生型C57BL小鼠头盖骨制备颅骨成骨细胞，分别经含PTH（PTH处理组）、含PTH和蛋白激酶A(PKA)信号通路特异性抑制剂H 89 2HCl (共处理组)及不含此两制剂（未处理组）的培养基培养，
Real-time PCR 和Western blot检测PTH处理组、共处理组、对照组颅骨成骨细胞中骨代谢相关分子的表达。
结果 显微CT定量测量结果,实验组小鼠反映皮质骨及松质骨的骨量和骨厚度指标均较对照组明显增高(P<0.05,P<0.01)。ELISA检测结果示,实验组小鼠外周血清中P[KG-*2]Ⅰ[KG-*4]NP、OC、BALP水平较对照组显著增加,TRAP浓度较对照组显著降低(P<0.01,P<0.05)。Real-time PCR结果显示,共处理组颅骨成骨细胞OC、碱性磷酸酶(ALP)的mRNA表达较PTH处理组显著下降,TRAP的mRNA表达较PTH处理组增加（P均<0.01）。Western blot细胞定位结果发现,不用PTH刺激的未处理组,CITED1蛋白均定位于细胞浆中;PTH处理组,CITED1明显转向细胞核;加H 89 2HCl的共处理组CITED1向胞核转移被抑制。
结论 PTH可能通过cAMP/蛋白激酶A(PKA)信号通路促进共转录因子CITED1出入细胞核,特异性地调节OC及ALP基因的表达,参与骨代谢的调节。
Abstract:Objective To analyze the regulatory effect of parathyroid hormone (PTH) on osteogenesis and the effect of Glu/Asp-rich carboxy terminal domains Cbp/p300 interacting transactivator 1(CITED1) in bone metabolism.
Methods Wild-type C57BL mice subcutaneously injected with low-dose PTH were designed as experimental group,and wild type C57BL mice injected subcutaneously with the same volume of normal saline were served as control group.After being killed, the femur was taken to detect the bone metabolic phenotype,such as the thickness and bone mass of cortical bone and cancellous bone.
The concentrations of procollagen Ⅰ peptide (P[KG-*2]Ⅰ[KG-*4]NP),osteocalcin (OC),bone alkaline phosphatase (BALP) and tartrate resistant acid phosphatase (TRAP) were measured by ELISA.One-day-old wild-type C57BL mice cranium was used to prepare osteoblasts, which were cultured with PTH (PTH treatment group), PTH and protein kinase A (PKA) signal pathway specific inhibitor H 89 2HCl(co-treatment group)and culture medium without the two preparations (untreated group),respectively. Real-time PCR and Western blot were used to detect the expression of bone metabolism-related molecules in mice cranial osteoblasts of the three groups.
Results The bone mass and thickness of cortical and cancellous bone in experimental group were significantly higher than those in control group (P<0.05,P<0.01).ELISA results showed that the peripheral serum levels of PⅠNP,OC and BALP in experimental group were significantly higher than those in control group,and TRAP level was significantly lower than that in control group (P<0.01,P<0.05).Real-time PCR results showed that the mRNA expressions of OC and ALP in co-treatment group were significantly lower than those in PTH treated group,and the mRNA expression of TRAP was significantly higher than that in PTH treated group(all P<0.01).Western blot results untreated that in untreated group CITED1 protein was located in the cytoplasm of cells,while in PTH treatrment group CITED1 protein was obviously located in the nucleus of cells,and CITED1 nuclear transfer was inhibited in co-treatment group.
Conclusion PTH may promote the co-transcription factor CITED1 in and out of the nucleus through the cAMP/protein kinase A (PKA) signaling pathway, specifically regulate the expression of OC and ALP genes and participate in the regulation of bone metabolism.
keywords: Parathyroid hormone Osteoporosis Glu/Asp-rich carboxy terminal domains Cbp/p300 interaction transactivator 1 Protein kinase A inhibitor Bone metabolism
文章编号： 中图分类号： 文献标志码：A
基金项目:国家自然科学基金(81802237);深圳科创委基金(JCYJ20160425104432398)
| Author Name | Affiliation |
| QIU Yi-yan* ,ZHENG Yu-chen,GUO Wei-zhuang,ZHOU Wen-yu,YAN Bin,YANG Xin-jian | * Department of Orthopaedics,Shenzhen Second People′s Hospital,Shenzhen,Guangdong 518000,China |
引用文本：
邱奕雁,郑羽晨,郭伟壮,等.cAMP/PKA/CITED1信号通路介导的甲状旁腺激素在骨代谢中的作用机制[J].中国临床研究,2020,33(1):10-14.
邱奕雁,郑羽晨,郭伟壮,等.cAMP/PKA/CITED1信号通路介导的甲状旁腺激素在骨代谢中的作用机制[J].中国临床研究,2020,33(1):10-14.