摘要
目的:探讨Jagged1通过活化Notch通路对RAW 264.7细胞向破骨分化及增殖的影响。方法:将RAW 264.7细胞分三组培养:对照组(细胞培养基+鼠RANKL 50 ng/L)、Jagged1组(细胞培养基+鼠RANKL+Jagged1重组蛋白)、γ-分泌酶抑制剂(DAPT)组(细胞培养基+鼠RANKL+Jagged1重组蛋白+DAPT),实时荧光定量PCR检测各组破骨细胞标志基因组织蛋白酶K(Cathepsin K,CK)、抗酒石酸性磷酸酶(Tartrate-resistant acid phosphatase,TRAP)、降钙素受体(Calcitionin receptor,CTR)及Notch靶基因HES-1、HEY-1 mRNA的表达,TRAP染色鉴定破骨细胞,扫描电镜检测破骨细胞溶骨功能。免疫荧光检测NICD的表达变化,CCK-8检测RAW 264.7细胞增殖。结果:Jagged1组TRAP、CK、CTR及HES-1、HEY-1 mRNA的表达、TRAP+细胞数较对照组及DAPT组明显升高(P<0.05),而DAPT组与对照组均无明显变化;细胞免疫荧光显示Jagged1组NICD除表达于细胞膜、细胞质外,细胞核也有较高表达,对照组及DAPT组细胞核中NICD无明显表达;细胞培养48 h,Jagged1组细增殖较对照组及DAPT组出现明显抑制。结论:Jagged1通过活化Notch通路促进RAW 264.7向破骨细胞分化、抑制其增殖。
Objective：To study the role of Jagged1 and Notch signaling pathway played in the differentiation and proliferation of RAW 264.7 cells.Methods： RAW 264.7 cells were divided into three groups to culture：The control group：RAW 264.7 cells were threated with culture and RANKL.The Jagged1 group：RAW 264.7 cells were threated with recombinant protein Jagged 1 besides the control group.The DAPT group：RAW 264.7 cells were threated with DAPT besides the Jagged 1 group.The mRNA expression of osteoclast markers（TRAP,CK,CTR） and Notch key target genes （HES-1 and HEY-1） were measured by real-time PCR.The formation of osteoclast , bone resorption , Notch expression and proliferation of RAW 264.7 cells were detected by TRAP staining , scanning electron microscope ,immunofluorescence and cell counting kit-8 （ CCK-8 ）.Results： TRAP, CK, CTR , HES-1 and HEY-1 mRNA expression were significantly higher than the control group and DAPT group in Jadded 1 group （ P＆lt;0.05 ）.TRAP＋cell count ,osteolytic area was significantly increased in Jagged 1 group compared with control and DAPT group , and no significant difference observed between the last two groups.Immunofluorescence results showed high expression of N ^ICD in cell membrane and cytoplasm in all groups and additionally expressed in nucleus in Jadded 1 group.Cell proliferation was inhibited in Jagged 1 group also （ P＆lt;0.05 ）.Conclusion：Jagged1 promotes RAW264.7 cells osteoclast differentiation and inhibits proliferation by activating Notch signaling pathway .
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2014年第7期865-869,共5页
Chinese Journal of Immunology
基金
国家自然科学基金面上项目(No.81271979)
作者简介
王汝杰（1981年-），男，主要从事骨肿瘤及转移性骨肿瘤相关基础研究，E-mail：wangrujiell@163．com。
通讯作者及指导教师：初同伟（1966年-），男，教授，博士生导师，主要从事脊柱肿瘤相关基础研究及治疗，E-mail：chtw@sina．com
