重组FN多肽CH50抑制黑色素瘤B16细胞体内转移的研究

CH50,a Recombinant Polypeptide of Fibronectin,Inhibits the Metastasis of Melanoma B16 Cells In Vivo

目的研究重组纤黏连蛋白(FN)多肽CH50对小鼠黑色素瘤B16细胞体内转移的影响,以探讨CH50多肽抑制肿瘤转移的可能分子机制。方法体外培养黑色素瘤B16细胞,用荧光染料CFSE标记,接种脾脏后24h取脾、肝、肺做冰冻切片,观察肿瘤细胞在3种组织中的侵袭情况。从脾脏接种B16细胞,建立体内肿瘤转移动物模型,采用基于流体动力学的体内基因转染方法于小鼠体内表达CH50多肽,RT-PCR检测CH50 mRNA在肝组织的表达,Western印迹检测CH50多肽的表达。通过比较原位肿瘤结节及转移结节在数量、大小、分布上的差异及检测原位肿瘤组织中MMP-2、MMP-9表达差异,观察CH50多肽的治疗效果。结果注射24h后即可在脾脏形成荧光结节。pCH510质粒通过尾静脉注射后,可在肝组织中检测到CH50 mRNA及CH50多肽的表达。从脾脏接种B16细胞后第14天可在脾脏形成原发肿瘤,至第35天肝脏表面已形成转移瘤结节,成功建立了体内器官间(脾转肝)肿瘤转移动物模型。体内转染表达CH50多肽能抑制肿瘤生长、侵袭和转移,抑制原位肿瘤结节中MMP-2、MMP-9的表达。结论CH50多肽可以通过对MMP-2、MMP-9蛋白表达的抑制作用来抑制黑色素瘤B16细胞的成瘤能力和体内侵袭、转移能力。

Objective The aim of this study is to investigate the inhibitory effect of recombinant polypeptide of fibronectin, CH50, on the metastasis of melanoma B16 ceils. Methods The cultured B16 cells were labeled with fluorescent dye CFSE, and then inoculated into spleen of mice. Twenty hours later, the spleen, liver and lung were removed for observation of metastatic B16 cells. The CHS0-expressing vector was efficiently transfected into liver of tumor-bearing mice by using hydrodynamics-based gene delivery technique. The expression of transfected vector in the liver was determined by RT-PCR and Western-blot. And the curative effect of the expressed CH50 was evaluated in terms of the number, size, and distribution of the black tumor nodes and the expression of MMP- 2, MMP-9. Results Twenty hours after the injection of tumor cells, the obvious fluorescence was observed in spleen. After the injection of plasmid pCHS10, the expression of mRNA and CH50 polypeptide could be detected. The primary tumor was formed ld days after the injection of B16 cells into spleen, and the metastatic liver tumor nodes were formed 35 days later. Nevertheless, the expressed CH50 polypeptide produced an obvious inhibitory effect on tumor growth, invasion and metastasis. After administration of pCH510, the expressions of MMP-2, MMP-9 in tumor were obviously decreased as compared to controls. Conclusion CHS0 polypeptide can inhibit the formation ofB16 tumor nodes, bition of tumorous