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为了探讨IL-6(interleukin-6,IL-6)/STAT3(signal transducer and activator of transcription3,STAT3)信号通路对骨肉瘤细胞增殖和迁移的作用,该研究采用ELISA检测人脐带间充质干细胞(human umbilical cord-derived mesenchymal stem cells,h UC-MSCs)和Saos-2细胞白介素-6的表达量。Western blot检测Saos-2细胞STAT3、p-STAT3蛋白质的表达水平。RT-PCR检测PCNA、Cyclin D1、Survivin、STAT3基因的转录水平。细胞计数法和CCK-8法检测Saos-2细胞的增殖能力。划痕实验和Transwell实验检测Saos-2细胞的迁移能力。结果显示,h UC-MSCs IL-6表达量明显高于Saos-2细胞;h UC-MSCs的条件培养基(conditioned medium,CM)和IL-6均能激活STAT3,IL-6中和抗体则明显削弱了h UC-MSCs-CM的激活作用;用IL-6中和抗体或AG490抑制STAT3的活性后,Saos-2细胞的增殖相关基因PCNA、Cyclin D1、Survivin的m RNA表达水平明显下调,其增殖和迁移能力明显下降。以上表明,脐带间充质干细胞能分泌IL-6激活,STAT3信号通路促进骨肉瘤细胞的体外增殖和迁移。
To investigate the role of the IL-6/STAT3 signal transducer and activator of transcription 3 (STAT3) signaling pathway in the proliferation and migration of osteosarcoma cells, this study used ELISA to detect human umbilical cord mesenchymal stem cells ( Human umbilical cord-derived mesenchymal stem cells, hUC-MSCs) and Saos-2 cells, interleukin-6 expression levels. The expression of STAT3 and p-STAT3 protein in Saos-2 cells was detected by Western blot. RT-PCR was used to detect the transcription of PCNA, Cyclin D1, Survivin and STAT3 genes. Cytokine counting and CCK-8 assay were used to determine the proliferation ability of Saos-2 cells. Scratch test and Transwell assay were used to detect the migration ability of Saos-2 cells. The results showed that the expression of IL-6 in hUC-MSCs was significantly higher than that in Saos-2 cells; the conditioned medium (CM) and IL-6 in hUC-MSCs could activate STAT3, IL-6 neutralizing antibody. The activation of hUC-MSCs-CM was significantly attenuated; after inhibition of STAT3 activity with IL-6 neutralizing antibody or AG490, the mRNA expression levels of proliferation-associated genes PCNA, Cyclin D1, and Survivin in Saos-2 cells were significantly downregulated. Its ability to multiply and migrate significantly decreased. The above shows that umbilical cord mesenchymal stem cells can secrete IL-6 activation, and STAT3 signaling pathway promotes the proliferation and migration of osteosarcoma cells in vitro.