纤黏连蛋白重组多肽CH50抑制黑色素瘤MMP-2和MMP-9表达、激活的研究

Recombinant Polypeptide CH50 of Fibronectin Inhibits the Expression and Activation of Matrix Metalloproteinase 2 and 9 in Melanoma

目的研究纤黏连蛋白重组多肽CH50对黑色素瘤B16细胞侵袭能力的影响,探讨CH50多肽抑制肿瘤生长、侵袭的机制。方法体外培养小鼠黑色素瘤B16细胞、小鼠腿部皮下注射B16细胞建立肿瘤动物模型。采用明胶电泳法检测B16细胞和黑色素瘤组织中基质金属蛋白酶MMP-2和MMP-9的表达和激活;以CH50多肽体外处理B16细胞或体内表达CH50,观察CH50下调MMPs表达以及对肿瘤细胞侵袭能力的抑制作用。结果B16细胞在体外培养条件下主要表达MMP-2,而在肿瘤微环境中则同时表达MMP-2和MMP-9。肿瘤组织中MMPs的表达明显高于体外培养B16细胞。CH50多肽对体外培养B16细胞的MMPs表达和激活无明显抑制作用,但处理后的B16细胞进入体内后表达MMPs的能力受到明显抑制。体内转染表达的CH50多肽亦可明显抑制肿瘤表达MMPs、并抑制肿瘤侵袭能力。结论纤黏连蛋白重组多肽CH50可以抑制肿瘤微环境中基质金属蛋白酶MMP-2和MMP-9的表达和激活,从而抑制黑色素瘤生长及侵袭能力。

Objective To explore the effect of a recombinant polypeptide of fibronectin, CH50, on the invasive ability of melanoma B16 cells, and examine the mechanism by which CH50 inhibits the growth and invasion of tumor. Methods B16 ceils was cultured in vitro and the mouse model of melanoma was established by s. c. injection of 2 ×10^5 of B16 cells into C57BL/6 mouse. Then the expression and activity of MMP2 and MMP9 in the culture supernatant and melanoma tissue were detected by gelatin zymography. To observe the effect of CH50 on MMPs, CH50 polypeptide was added to the cultured B16 cells and CH50-expressing vector plasmid or mock plasmid was transfected to tumor site. The cultured ceils and tumor tissues were analyzed by gel zymography. Results The B16 cells mainly expressed MMP2 in vitro, while both MMP2 and MMP9 were abundantly expressed in melanoma tissue. The MMP expression in tumor tissue was significantly higher than that in cultured B16 cells. After treatment with CH50, both production and activation of MMP2 and MMIx） were decreased in melanoma tissue, but no obvious change was observed in cultured B16 cells. After the treatment, the expression of BI6 cells were significantly inhibited in vivo. The CH50 could also obviously inhibit the MMP expression of tumor and reduce the invasiveness of tumors. Conclusion CH50 can effectively suppress both the expression and activation of MMP2 and MMP9 in tumor microenvironment, therefore it may act as a potent inhibitor for the prevention of the invasion and metastasis of melanoma.