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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 effect of conditioned medium (CM) of lung adenocarcinoma A549 cells on osteoclast differentiation and proliferation of human peripheral blood CD14 + monocytes in vitro and to explore the role of Notch pathway related factors. Methods: CD14 + monocytes from peripheral blood of healthy adults were cultured with α-MEM cells containing macrophage colony stimulating factor (M-CSF) as control group. M-CSF and human lung adenocarcinoma A549 cells CM Peripheral blood CD14 + monocytes were cultured as A549 CM group; M-CSF, A549 cells CM and gamma-secretase inhibitor 3,5-difluorophenylacetyl-L-alanyl-S-benzene Peripheral blood CD14 + monocytes were cultured in N- (N- (N- (3,5-dil uorophenacetyl) -L-alanyl] -S-phenylglycine t-butyl ester, DAPT group. After 6 days of culture, each group was added with RANKL (receptor activator for nuclear factor-κB ligand) for further culture. Real-time fluorescent quantitative PCR was used to detect the expression of tartrate-resistant acid phosphatase (TRAP), cathepsin K (CK), calcitonin receptor (CTR) and Notch The mRNA expression of HES-1 and HEY-1 was detected by ELISA. The formation of osteoclasts was detected by TRAP staining. The osteolytic function of osteoclasts was observed by scanning electron microscopy. The expression of NICD in Notch active fragments was detected by immunofluorescence. Detection of CD14 + monocyte proliferation. Results: The expression of TRAP, CK and CTR in A549 CM group and the fluorescence intensity of TRAP + multinucleated cells, osteolytic area and NICD of Notch active fragment in A549 CM group were significantly higher than those in control group (P <0.05), while those in DAPT group were significantly higher than those in A549 CM group (P <0.05). The mRNA expression of HES-1 and HEY-1 in A549CM group was significantly higher than that in DAPT group and control group (P <0.05), but there was no significant difference between the two groups Group cell proliferation rate was higher than the control group, while DAPT group cell proliferation rate was the highest. Conclusion: A549 CM may promote the differentiation of CD14 + monocytes into osteoclasts through activation of Notch pathway. Meanwhile, activation of Notch pathway may inhibit the proliferation of CD14 + monocytes.