Notch信号通路调控骨肉瘤细胞增殖的机制研究

Study on the mechanism of Notch signaling pathway regulating the proliferation of osteosarcoma cells

目的观察Notch通路阻断剂DAPT和Notch通路激动剂Jagged1对人骨肉瘤143B细胞和MG63细胞体外增殖和体内成瘤的影响,探讨Notch信号通路在骨肉瘤细胞增殖中的调控作用。方法体外培养人骨肉瘤143B细胞和MG63细胞,采用CCK-8法、集落形成试验以及BrdU染色法检测DAPT和Jagged1对两种细胞增殖的影响;在体内利用裸鼠成瘤试验,观察DAPT对于骨肉瘤细胞体内成瘤能力的调控作用,并对肿瘤组织进行切片染色,观察肿瘤组织恶性程度的变化情况。结果CCK-8法检测结果表明DAPT显著抑制143B细胞和MG63细胞的增殖,且和浓度呈正相关,但10μM的DAPT与20μM的DAPT对143B细胞的增殖抑制能力无明显差异。肿瘤集落形成试验结果表明DAPT能够显著抑制肿瘤集落的形成。BrdU染色结果显示DAPT组的BrdU阳性细胞明显少于对照组和Jagged1组,而Jagged1组的阳性细胞数明显多于对照组。体内裸鼠成瘤实验结果显示,与对照组相比,DAPT组能够明显抑制肿瘤大小,Ki67阳性率降低。HE染色结果显示,DAPT组肿瘤组织中的细胞核数量和血管数量明显减少,且组织内部更加疏松。结论Notch信号通路参与调控人骨肉瘤143细胞和MG63细胞的体外增殖与体内成瘤行为,抑制Notch通路的表达可能成为骨肉瘤治疗的重要靶点。

Objective To observe the effects of Notch pathway blocker DAPT and Notch pathway agonist Jagged1 on the proliferation of human osteosarcoma cell line 143 B and MG63 in vitro and tumor formation in vivo,and to explore the regulatory role of Notch signaling pathway in the proliferation of osteosarcoma cells.Methods Human osteosarcoma cell line 143 B and MG63 were cultured in vitro.CCK-8 assay,Colony-formation units(CFU)assay and BrdU labeling assay were used to detect the effects of DAPT and Jagged1 on the proliferation of these two kinds of cells.In vivo,nude mice tumor xenografts assay was used to observe the regulation of DAPT on the tumorigenesis ability of osteosarcoma cells and the tumor tissue sections were stained to observe the changes in the malignancy of tumor tissues.Results CCK-8 assay results showed that DAPT significantly inhibited the proliferation of 143 B and MG63 cells,and was positively correlated with the concentration,but there was no significant difference in the inhibitory ability of 10μM DAPT and20μM DAPT on the proliferation of 143 B cells.Colony-formation units(CFU)assay showed that DAPT can significantly inhibit the formation of tumor colonies.BrdU labeling assay showed that the number of BrdU positive cells in DAPT group was significantly less than that in control group and Jagged1 group.The results of nude mice tumor xenografts assay showed that compared with the control group,the DAPT group could significantly inhibit the tumor size and the positive rate of Ki67 was reduced.HE staining results showed that the number of nuclei and blood vessels in tumor tissue of DAPT group was significantly reduced,and the tumor tissue became looser.Conclusion Notch signaling pathway is involved in regulating the proliferation and tumorigenesis of human osteosarcoma 143 cells and MG63 cells.Inhibiting the expression of Notch pathway may become an important target for the treatment of osteosarcoma.