cAMP-PKA-CREB信号通路在骨形态发生蛋白9诱导小鼠间充质干细胞成骨分化中的作用  

Role of cAMP-PKA-CREB signal pathway in regulation of osteogenic differentiation of murine mesenchymal stem cells by bone morphogenetic protein 9

作　　者：宋涛[1,2] 刘跃亮[1,2] 赵艳芳[1,2] 王文娟[1,2] 徐静[1,2] 罗进勇[1,2] 

机构地区：[1]重庆医科大学检验医学院 [2]重庆医科大学检验医学教育部重点实验室重庆市重点实验室,重庆400016

出　　处：《中国生物制品学杂志》2014年第2期189-193,196,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(31071304;30800658)

摘　　要：目的探讨cAMP-PKA-CREB信号通路在骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导小鼠间充质干细胞(mesenchymal stem cells,MSCs)C3H10T1/2成骨分化过程中的作用及其机制。方法将C3H10T1/2细胞分别加入不同浓度的cAMP-PKA-CREB信号通路抑制剂H89(1、2.5、5和10μmol/L),检测其对碱性磷酸酶(alkaline phosphatase,ALP)活性的影响;通过ALP定量和钙盐沉积试验分别检测H89对BMP9诱导C3H10T1/2细胞早期和晚期成骨分化的影响;经Western blot法检测H89对C3H10T1/2细胞中磷酸化CREB、骨钙素(Osteocalcin,OCN)和成骨关键转录因子Runx2表达水平的影响;通过Wentern blot及荧光素酶活性的检测,观察H89对经典信号通路BMPs-smad1/5/8的影响。结果随着H89浓度的增加,对BMP9诱导的C3H10T1/2细胞ALP的抑制作用明显增强(P<0.05),且呈剂量依赖性;ALP定量和钙盐沉积试验结果表明,H89可明显抑制BMP9诱导的C3H10T1/2细胞早期及晚期成骨分化;H89可显著抑制BMP9诱导的C3H10T1/2细胞中磷酸化CREB、OCN及Runx2蛋白的表达(P<0.05),与AdBMP9组比较,H89对经典BMPs-smad1/5/8信号通路无明显影响(P>0.05)。结论阻断cAMP-PKA-CREB信号通路可抑制BMP9诱导的MSCs C3H10T1/2的成骨分化,为BMP9的临床应用奠定了理论基础。Objective To investigate the role of cAMP-PKA-CREB signaling pathway in regulation of osteogenic differen- tiation of murine bone marrow mesenchymal stem cells（MSCs）C3H10T1 / 2 by bone morphogenetic protein 9（BMP9）as well as the relevant mechanism. Methods C3H10T1 / 2 cells were divided into six groups. The cells in blank control group were treated,while those in GFP,GFP ＋ DMSO,BMP9 ＋ DMSO,BMP9 ＋ H89（10 μmol / L）and BMP9 groups were infected with 30%AdGFP,30% AdGFP ＋ DMSO,BMP9,30% BMP9 ＋ DMSO,30% AdBMP9 ＋ 10 μmol / L H89 and 30% adBMP9 respectively,then determined for alkaline phosphatase（ALP）activity and subjected to calcium salt sedimentation test,based on which the effects of H89 on early and late osteogenic differentiations of C3H10T1 / 2 cells induced by BMP9 were observed. Meanwhile,C3H10T1 / 2 cells were divided into blank control,GFP,BMP9 and BMP9 ＋ H89（10 μmol / L） groups,and observed for effect of H89 on expression levels of phosphorylated CREB,osteocalcin（OCN）and osteogenic key transcription factor Runx2 by Western blot,and for that on classical signaling pathway BMPs-smad1 / 5 / 8 by Western blot and test for luciferase report gene. Results H80 showed dose-dependent inhibitory effect on BMP9-induced ALP activity in C3H10T1 / 2 cells（P〈 0. 05）. The ALP activity in BMP9 ＋ H89（10 μmol / L）group was significantly lower than that in blank control group（P〈 0. 05）. The calcium salt sedimentation level in BMP9 ＋ H89（10 μmol / L）group was significantly lower than those in BMP9 ＋ DMSO and BMP9 groups. H89 inhibited the expressions of phosporylatd CREB,OCN and Runx2,and the relative expression levels of various proteins in BMP9 group showed significant difference with those in BMP ＋ H89（10 μmol / L）group（P〈 0. 05）. H89 showed no significant effect on classical signaling pathway BMPs-smad1 / 5 / 8. The relative expression levels of phosphorylated smad1 / 5 / 8 in BMP9 and BMP9 ＋ H89 groups showed no significant difference（P〈 0

关 键 词：骨形态发生蛋白质 信号通路 间充质干细胞 分化 

分 类 号：Q254[生物学—细胞生物学]