可调控型反义基质金属蛋白酶9表达抑制人黑色素瘤细胞侵袭转移表型的研究

Down-regulation of metastatic phenotype in human melanoma cells by controlled expression of anti-sense matrix metalloproteinase 9

目的 探讨基质金属蛋白酶 (MMP) 9与肿瘤转移的相关性。方法 利用基因重组技术构建反义MMP 9cDNA四环素可调控型表达载体 ,用脂质体法转染反义MMP 9至转移性人黑色素瘤细胞株WM4 5 1(高表达MMP 9)。检测转染后细胞MMP 9表达水平的改变以及体外生长、侵袭、裸鼠体内成瘤及自发转移能力的变化。结果 转染反义基因后 ,WM4 5 1细胞MMP 9的表达及活性明显下降 ,同时MMP 2的表达也受到一定抑制 ,细胞生长速度、体外侵袭能力及裸鼠体内成瘤性及自发转移能力均受到一定程度抑制 ;运用四环素可以抑制四环素负调控逆转录病毒载体上的外源基因的表达。结论 反义MMP 9基因下调MMP 9的表达 ,可使人黑色素细胞转移能力受到一定程度的抑制 ,说明MMP 9在人黑色素瘤细胞转移过程中起重要作用。同时 。

Objective To investigate the correlation between matrix metalloproteinase 9 (MMP 9) expression and tumor invasion and metastasis as well, and to explore the potential application of controlled expression of target gene in tumor gene therapy. Methods One self contained tetracycline regulated retroviral vector containing anti sense cDNA of MMP 9 was constructed and transfected into a metastatic human melanoma cell line WM451 which expressed a high level of MMP 9. Techniques such as growth rate measurment, MTT assay, 3H thymidine incorporation, colony forming ability in soft agar, invasion assay in Boyden chamber, as well as zymography and Western blot were applied to analyze the expression of MMPs and behaviors of tumor cells in vitro before and after gene transfection. Tumorigenecity and spontaneous metastasis were tested in nude mice. Results In the presence of exogenous tetracycline, the transfected antisense MMP 9 did not affect the endogenous level of MMP 9 in WM451 cells, and showed no significant changes in cell behaviors in comparison with that of the vector transfected control cells. Nevertheless, withdrawal of tetracycline from the medium caused a significant down regulation of expression and activity ofMMP 9. The capacity of cell growth in vitro, colony forming ability in soft agar, invasion through Matrigel all were inhibited remarkably when compared with the controls. Spontaneous metastasis in nude mice was significantly inhibited. Conclusions Transfection of anti sense MMP 9 can down regulate the invasion and metastasis of melanoma cells both in vitro and in vivo, further clarifying the important role of MMP 9 in tumor progression.