重组纤维连接蛋白多肽CH50对黑色素瘤B16细胞侵袭能力的抑制作用及机制

Inhibitory Effect of Recombinant Polypeptide of Fibronectin CH50 on Invasion Ability of Melanoma B16 Cells and the Mechanism

目的研究重组纤维连接蛋白多肽CH50对黑色素瘤B16细胞侵袭能力抑制作用及其机制。方法将小鼠黑色素瘤B16细胞用CFSE标记,经尾静脉注射接种小鼠,分别在6 h和24 h取肺组织作冰冻切片,观察肿瘤细胞在肺部聚集和对肺组织侵袭情况;接种15 d后解剖小鼠取肺,观察B16细胞在肺表面形成转移结节数量差异;观察CH50多肽对B16细胞结合纤维连接蛋白和纤维蛋白原的影响;RT-PCR法检测B16细胞中转移相关基因的表达水平;zymography法检测MMP-9水平。结果CH50多肽短时间作用于B16细胞,注射细胞6 h后肺组织荧光结节数显著减少;而作用较长时间,注射细胞24 h后肺组织荧光结节数减少更为明显。CH50多肽处理过的B16细胞在肺部形成转移结节明显减少。CH50多肽能够显著抑制B16细胞结合纤维连接蛋白及结合后的细胞铺展能力及B16细胞结合纤维蛋白原的能力,能够显著下调B16细胞cdc2、αv、β3、MMP-9等基因的表达与MMP-9分泌。结论CH50多肽能够抑制黑色素瘤B16细胞的侵袭能力,抑制瘤细胞与黏附分子结合,抑制转移相关基因的表达,从而使肿瘤细胞的生物学特征发生改变。

Objective To investigate the inhibitory effect of recombinant polypeptide of fibronectin CH50 on melanoma B16 cells and the mechanism. Methods Melanoma B16 cells were labeled with CFSE, and injected into mouse through tail vein. The lungs were removed 6 h and 24 h later and used for frozen sectioning. The accumulation of B16 cells in lung and the invasion of them into lung tissue were observed. The difference of metastatic loci formed by B16 cells in lung was observed 15 days after tumor inoculation. The effect of CH50 on the binding of B16 cells to fibronectin and fibrinogen was detected. The expression levels of metastasis-related genes in B16 cells were detected by RT-PCR. Zymography was used to analyze the activity of MMP-9. Results The interaction of CH50 with B16 cells within a short period of time could inhibit the accumulation of B16 cells in lung, and result in the significant decrease of fluorescent loci in lung 6 h after the injection of tumor cells. The action of CH50 on B16 cells for a longer time could inhibit the invasion ability of B16 cells into tissue, resulting in more significant decrease of fluorescent loci in lung 24 h after the injection of tumor cells. The lung metastasis of B16 cells was significantly suppressed after treatment with CH50. Polypeptide CH50 could inhibit the binding of B16 cells to fibronectin and fibrinogen, and the spreading of cells after the binding. The expression of genes such as cdc2, αv, β3 and MMP-9 by B16 cells was down-regulated and the activity of MMP-9 was significantly decreased by CH50. Conclusion Polypeptide CH50 can inhibit the invasion ability of melanoma B16 cells. The main mechanisms involve the inhibition of the binding of tumor cells to adhesive molecules and down-regulation of the expression of genes related to metastasis, which change the biological characteristics of tumor cells.