靶向抑制Akt对成骨细胞分化PI3K/Akt信号通路调控的研究  

Study of targeted inhibition of Akt on PI3K/Akt signal pathway regulating osteoblast differentiation

作　　者：陈巧玲[1] 白亦光[2] 别俊[1] 杨蜜[1] 张加勇 尚东琴 Chen Qiaoling;Bai Yiguang;Bie Jun;Yang Mi;Zhang Jiayong;Xiao Dongqin(Department of Oncology,Nanchong Central Hospital,Second Clinical Institute of North Sichuan Medical College;Department of Orthopaedics,Nanchong Central Hospital,Second Clinical Institute of North Sichuan Medical College;Institute of Tissue Engineering and stem cells,Nanchong 637000,China)

机构地区：[1]川北医学院第二临床医学院,南充市中心医院肿瘤科,南充637000 [2]川北医学院第二临床医学院,南充市中心医院骨科,南充637000 [3]组织工程与干细胞研究所,南充637000

出　　处：《广西医科大学学报》2020年第12期2123-2128,共6页Journal of Guangxi Medical University

基　　金：国家自然科学基金资助项目(No.82002289);四川省南充市校科技战略合作专项课题资助项目(No.18SXHZ0539);四川省卫计委课题资助项目(No.16PJ202)。

摘　　要：目的:观察蛋白激酶B(Akt)特异性抑制剂MK2206对成骨细胞分化的影响,探讨PI3K/Akt信号通路影响成骨细胞分化的分子机制。方法:通过CCK-8检测MK2206对骨髓间充质干细胞(BMSC)增殖的最大安全浓度;将BMSC分为两组,其中对照组使用OBM诱导处理,干预组加入含有3μmol/L的MK2206的OBM进行诱导处理,检测细胞碱性磷酸酶(ALP)活性和矿化能力,实时荧光定量PCR(qPCR)检测相关基因的表达水平。将细胞分为3组,其中OBM组仅加入成骨细胞诱导培养基培养;OBM+MK2206组使用含有3μmol/L MK2206的OBM培养;MK2206组中使用含有3μmol/L MK2206的普通培养基处理,采用Western blotting检测相关蛋白的表达情况。结果:CCK-8结果提示,在3.2μmol/L及以下浓度MK2206对BMSC的增殖没有影响;随着浓度的升高,ALP阳性的细胞数量逐渐下降;干预组的ALP表达、细胞外基质矿化能力、ALP mRNA和骨钙素(OCN)mRNA表达较对照组均明显减少(均P<0.05);与OBM组比较,OBM+MK2206组和MK2206组细胞中RUNX2、Collagen I和p-Akt蛋白均出现明显的降低,且MK2206组下降更加明显(均P<0.05)。结论:靶向抑制Akt在体外细胞培养中可通过PI3K/Akt信号通路明显下调成骨细胞相关基因ALP和OCN的表达,并抑制成骨细胞的分化成熟。Objective:To investigate the effect of protein kinase B(Akt)specific inhibitor MK2206on osteoblast differentiation and explore the molecular mechanism of PI3K/Akt signaling pathway on osteoblast differentiation.Methods:The maximum safe concentration of MK2206 on the proliferation of bone marrow mesenchymal stem cells(BMSCs)was detected by CCK-8.The BMSCs were divided into 3 groups:theosteoblast induction medium(OBM)+MK2206 group(intervention group),in which the cells were cultured in OBM containing 3μmol/LMK2206;the OBM group(control group),in which the cells were treated with OBM alone;and the MK2206 group,in which the cells were cultured in a conventional medium containing 3μmol/LMK2206.Alkaline phosphatase(ALP)activity and mineralization ability were detected.The related mRNA and protein expressions were determined by qPCR and Western blotting,respectively.Results:CCK-8 suggested that MK2206 had no impact on BMSCs proliferation atthe concentration of 3.2μmol/L and below.As the concentration increased,the number of ALP positive cells gradually decreased.The ALP activity and extracellular matrix mineralization ability as well as the ALP and osteocalcin(OCN)mRNA levels in the intervention group were decreased significantly compared with the control group(P<0.05).Compared with OBM group,the RUNX2,Collagen I and p-Akt protein levels in OBM+MK2206 group and MK2206 group were decreased,and the decrease was more significant in MK2206 group(P<0.05).Conclusion:Targeted inhibition of Akt can significantly down-regulate the expression of osteoblast related genes ALP and OCN and inhibit the differentiation of osteoblasts in vitro through PI3K/Akt signaling pathway.

关 键 词：蛋白激酶B 骨破坏 成骨细胞 信号通路 

分 类 号：R363.1[医药卫生—病理学]