摘要
目的探讨哺乳动物雷帕霉素靶蛋白(mammaliantargetofrapamycin,mTOR)相关调节蛋白(regulatoryassociatedproteinofmTOR,Raptor)对小鼠髓系细胞破骨分化与骨吸收的影响。方法本研究为随机对照试验,于2020年1月至2021年6月,选取健康B6/C57小鼠10只,随机分为敲除组与对照组,每组5只。其中敲除组通过基因敲除构建髓系细胞特异性敲除Raptor小鼠模型。两组小鼠正常饲养后处死通过试剂盒分离髓系细胞,并通过RANKL诱导形成破骨细胞。通过免疫印迹试验(Westernblot)检测破骨分化相关基因转录因子(spi-1 proto-oncogene,PU.1)、组织蛋白酶K(cathepsinK,CTSK)以及Fos蛋白(c-Fos)的蛋白水平;通过反转录-聚合酶链反应(RT-PCR)法检测破骨分化相关基因PU.1、CTSK、c-Fos的mRNA水平;通过酶联免疫吸附试验(ELISA)法检测血清中骨吸收指标I型胶原C端肽(C-telopeptideoftypeI,CTXI)、I型胶原N端肽(N-telopeptideoftypeI,NTX1)及尿羟脯氨酸(U-Hydroxyproline,U-HYP)评估骨吸收情况。结果Westernblot结果显示,敲除Raptor后小鼠髓系细胞中破骨分化相关基因PU.1、CTSK、c-Fos的蛋白表达量显著降低,RT-PCR结果显示细胞中PU.1、CTSK、c-FosmRNA表达水平的变化与Westernblot结果趋势一致;ELISA结果显示,相比对照组,Raptor敲除小鼠血清CTXI[(185.53±36.69)pg/ml比(124.58±25.77)pg/ml,t=3.040,P=0.016]、NTX1[(2.89±0.37)μg/L比(2.21±0.42)μg/L,t=2.717,P=0.026]及U-HYP[(20.51±2.85)μg/L比(14.49±1.84)μg/L,t=3.968,P=0.004]表达量显著降低。结论敲除Raptor能够抑制小鼠髓系细胞破骨分化与骨吸收,证实TOR相关调节蛋白Raptor在破骨细胞分化与骨吸收中发挥关键作用。
Objective To investigate the effects of mammalian target of rapamycin mTOR related regulatory protein Raptor on osteoclast differentiation and bone resorption in mouse myeloid cells.Methods This study was a randomized controlled trial.From January 2020 to June 2021,ten healthy B6/C57 mice were selected and randomly divided into a knockout group and a control group,with 5 mice in each group.In the knockout group,the myeloid cell-specific knockout Raptor mouse models were constructed by gene knockout.After normal feeding,the mice in both groups were sacrificed and their myeloid cells were isolated by the kit,and the osteoclasts were induced by RANKL.The protein levels of osteoclast differentiation related genes spi-1 proto-oncogene(PU.1),cathepsin K(CTSK),and c-Fos were detected by Western blot.The MRNA levels of osteoclast differentiation related genes PU.1,CTSK,and C-Fos were detected by reverse transcription-polymerase chain reaction(RT-PCR).The C-telopeptide of type I(CTXI),N-telopeptide of type I(NTX1),and U-Hydroxyproline(U-HYP)in serum were detected by enzyme-linked immunosorbent assay(ELISA)to evaluate the bone resorption.Results The Western blot results showed that the protein expression levels of osteoclast differentiation related genes PU.1,CTSK,and C-FOS in the mice's myeloid cells were significantly decreased after Raptor knockout.The RT-PCR results showed that the expression levels of PU.1,CTSK,and C-FOs mRNA in the cells were consistent with the trend of Western Blot results.The ELISA results showed that the expressions of CTXI[(185.53±36.69)pg/ml vs.(124.58±25.77)pg/ml,t=3.040,P=0.016],NTX1[(2.89±0.37)μg/L vs.(2.21±0.42)μg/L,t=2.717,P=0.026],and U-HYP[(20.51±2.85)μg/L vs.(14.49±1.84)μg/L,t=3.968,P=0.004]in the serum of the Raptor knockout mice were significantly decreased.Conclusion Raptor knockout can inhibit osteoclast differentiation and bone resorption of myeloid cells in mice,suggesting that Raptor plays a key role in osteoclast differentiation and bone resorption.
作者
赖玲林
李素娟
胡楚璇
陈燕
陈力
何娟
卢慧勤
Lai Linglin;Li Sujuan;Hu Chuxuan;Chen Yan;Chen Li;He Juan;Lu Huiqin(Department of Clinical Research,Guangdong Second People's Hospital,Guangzhou 510317,China;Department of Pharmacy,Guangdong Second People's Hospital,Guangzhou 510317,China)
出处
《国际医药卫生导报》
2021年第23期3614-3617,共4页
International Medicine and Health Guidance News
基金
广东省医学科学技术研究基金项目(B2019081)。
关键词
RAPTOR
髓系细胞
破骨分化
骨吸收
Raptor
Myeloid cells
Osteoclast differentiation
Bone resorption
