肺癌细胞条件培养液活化Notch通路促进破骨细胞分化  

Conditioned medium of lung cancer cells stimulates the osteoclast differentiation by activating Notch pathway

作　　者：王汝杰[1] 沈伟伟[1] 黄晨[2] 刘复州[1] 胡旭[1] 张超[1] 初同伟[1] 周跃[1] 

机构地区：[1]第三军医大学第二附属医院骨科,重庆400037 [2]成都军区成都总医院骨科,四川成都610083

出　　处：《肿瘤》2014年第3期216-222,共7页Tumor

基　　金：国家自然科学基金资助面上项目(编号:81271979)

摘　　要：目的 :体外检测肺腺癌A549细胞条件培养液(conditioned medium,CM)对人外周血CD14+单核细胞破骨分化和增殖的影响,探讨Notch通路相关因子在其中所起的作用。方法 :用含巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)的α-MEM细胞培养液培养健康成人外周血CD14+单核细胞,作为对照组;用M-CSF和人肺腺癌A549细胞CM培养人外周血CD14+单核细胞,作为A549 CM组;用M-CSF、A549细胞CM和γ-分泌酶抑制剂3,5-二氟苯乙酰-L-丙氨酰-S-苯基甘氨酸t-丁酯{N-[N-(3,5-dil uorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester,DAPT}培养人外周血CD14+单核细胞,作为DAPT组。各组均在培养6 d后加入核因子κB受体活化因子配体(receptor activator for nuclear factor-κB ligand,RANKL)继续培养。实时荧光定量-PCR法检测破骨细胞标志基因抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)、组织蛋白酶K(cathepsin K,CK)、降钙素受体(calcitonin receptor,CTR)及Notch靶基因HES-1、HEY-1的mRNA表达;TRAP染色法检测破骨细胞形成;扫描电子显微镜下观察破骨细胞溶骨功能;免疫荧光法检测Notch活性片段NICD的表达情况;CCK-8法检测CD14+单核细胞增殖情况。结果 :A549 CM组TRAP、CK和CTR表达水平以及TRAP+多核细胞数、溶骨面积和细胞核内Notch活性片段NICD的荧光强度均较对照组明显上调(P<0.05),而DAPT组较A549 CM组明显下调,但仍高于对照组(P<0.05);A549CM组HES-1和HEY-1 mRNA的表达水平明显高于DAPT组和对照组(P<0.05),后2者无明显差异;A549CM组细胞增殖率高于对照组,而DAPT组的细胞增殖率最高。结论 :肺腺癌A549 CM可能通过活化Notch通路,促进CD14+单核细胞向破骨细胞分化;同时Notch通路活化可抑制CD14+单核细胞增殖。Objective： To investigate the effects of conditioned medium （CM） of lung cancer cell line A549 on the proliferation and oseoclast differentiation of human peripheral blood CD14-positive monocytes, and explore the role of Notch signaling in this process. Methods： CD14-positive monocytes obtained from human peripheral blood were incubated with ^-MEM containing macrophage colony- stimulating factor （M-CSF） as the control group. The conditioned medium （CM） of lung cancer cell line A549 was used to incubate CD14-positive monocytes with M-CSF, named as A549 CM group. The Notch pathway blocking agent N-[N-（3,5-difluorophenacetyl）-L-alanyl]-S-phenylglycine t-butyl ester （DAPT） was used to treat monocytes with A549 CM and M-CSF, named as DAPT group. Receptor activator for nuclear factor-KB ligand （RANKL） was supplied to all groups after 6 d. The expression levels of osteoclast marker genes tartrate-resistant acid phosphatase （TRAP）, cathepsin K （C/O, calcitonin receptor （CTR） and Notch key target genes hairy and enhancer of split-1 （HES-1） and HES-related with YRPF motif-1 （HEY-1） were measured by real-time fluorescence quantitative PCR. The TRAP staining was used to detect the formation of osteoclast, and the bone resorption was evaluated by scanning electron microscopy. The fluorescent expression of active fragment N^cD was determined by immunofluorescence. The proliferation of CD14-positive monocytes was detected by cell counting kit-8 （CCK-8） method. Results： The mRNA expression levels of TRAP, CK and CTR, the number oI TRAP＋ multinuclear cells and the area of bone resorption, as well as the nuclear fluorescence intensity of N^cDin A549 CM group were upregulated as compared with the control group （P 〈 0.05）, and these^indexes in DAPT group were down-regulated as compared with A549 CM group, but still higher than those in the control group （P 〈 0.05）. The mRNA expression levels of HES-1 and HEY-1 in A549 CM group were higher than those in DAPT group and

关 键 词：g肺肿瘤 破骨细胞 受体 NOTCH 细胞分化 细胞增殖 

分 类 号：R734.2[医药卫生—肿瘤] R730.6[医药卫生—临床医学]