| Summary: | 碩士 === 長庚大學 === 醫學生物技術研究所 === 97 === In order to invade the tissues where metastases can develop, tumor cells must penetrate basement membranes and the extracellular matrix (ECM). The proteolytic enzymes, such as matrix metalloproteinases (MMPs) have been implicated in invasive processes of metastasis. Particularly, the expression and activity of MMP-2 and MMP-9 contribute many forms of human malignancy. Usually, invasion of tumor cells into Matrigel has been used to evaluate the invasive ability of tumors. However, the success of performing such classical invasion assay depends upon the sample preparation and the procedure is time-consuming. Zymography is used to identify proteolytic activity in enzymes separated in polyacrylamide gels. It has been used extensively in the qualitative evaluation of proteases present in tumors. Here we have adopted the zymography technology and combined with two-dimensional electrophoresis and MALDI-TOF analysis, and tried to develop a method not only to quantify the expression of but also to identify the proteolytic enzymes in tumors. It showed that the activity of reMMP-2 could be identified as low as 10pg by zymography, while Western blot only detected 10ng of reMMP-2. Additionally, the utilization of commercial ‘Clean-up’ reagents to concentrate reMMP2 appeared to be better than that of TCA precipitation. In addition, the culture supernatant of B16-F0 as well as B16-F10 cells rather than their cell lysate contained more protease activity. The activity of the proteolytic enzymes might not directly associate with the growth of tumor cells. The supplement of FBS indeed increased the activities of soluble enzymes in culture supernatant. Although the mass spectrographic analysis is ongoing to identify which gelatinases are involved in the current zymographic analysis of B16-F0 and B16-F10. These results provide some basis to further establish a detecting and identifying platform for active proteolytic enzymes in tumor metastasis.
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