MMPs Expressions Of Lung Cancer Cell In Three-dimensional Cell Culture

Now the morbidity and mortality of the bronchogenic carcinoma is the highest in all kinds of malignant tumors, and its pathogenesis has been the warm spots for reseach recently. The matrix metalloproteinase (MMPs) are a family of zinc-dependent proteinases involved in the degradation of the extracellular matrix. The MMPs have been implicated in the processes of growth, invasion, and metastasis of tumor, and are frequently overexpressed in malignant tumors, and have been associated with an aggressive malignant phenotype and hazardous prognosis in patients with cancer. We study the MMPs expression of lung cancer cell in three-dimensional culture, to investigate the role and mechanism of MMPs in the processes of growth, invasion, and metastasis of lung cancer.We established the three-dimensional collagen gels culture of lung cancer cell. In this study, nonsmall-cell lung cancer cell strain, H460, were cultured. The typeⅠcollegen were extractrd from the tail of mice. The H460 cell was cultured at cell concentration of 4×10~5/mL in collagen solution including iso-osmia salt, DMEM and 0.75mg/mL collagen. We compared the three-dimensional culture with the plane culture in the biological feature of H460. The growth state of cell was observed and recorded by camera in 24h, 72h, 120h. H460 was cultured with the mononuclearcell isolated from the peripheral blood of healthy volunteers and the MRC-5, human embryonic lung fibroblast cell strain, respectively. The MMP-2, MMP-9 in cell culture supernatant were detected by gelatinase zymography and the activity of collagenic degradation were calculated by determining oxyproline in cell culture supernatant. In addition, the tumor necrosis factor-α(TNF-α) was added into the H460 cultured in three-dimensional collagen gels. The MMP-2, MMP-9 in cell culture supernatant was measured by methods mentioned above. Finally the MMP-2, MMP-9 expressed in peripheral blood serum of 36 non small-cell lung cancer (NSCLC) patients were detected by gelatinase zymography, compared with that of 10 healthy volunteers. We also determined the MMP-2, MMP-9 expressed in pleural fluid of 23 patients with malignant pleural fluid and 15 patients with nonmalignant pleural fluid. The strap gray scale of MMP-2 was measured throuth the Bandscan's software.The three-dimensional collagen gels culture of lung cancer cell was established and the H460, mononuclearcell and MRC-5 grew well. The expression of MMP-2, MMP-9 in three dimensional cultured was higher than in plane culture. When mononuclearcell was co-culture with the H460 cell, the expression of MMP-2, MMP-9 were higher, compared with H460 cultured lonely. When the mononuclearcell was cultured lonely, the expression of MMP-2, MMP-9 was not detected. The degradation of collagen in the co-culture group was maximum and the mononuclearcell group was minimum. And the difference was significant (P<0.05). When the MRC-5 cell was co-culture with the H460 cell, the expression of MMP-2, MMP-9 in culture supernatant was lower than in that of the MRC-5 cultured lonely,. The degradation of collagen in the group of MRC-5 cultured lonely was maximum and in group of the H460 cultured lonely was minimum, it was mediate in the co-culture group. The differences among the three groups was significant, too (P<0.05). The expression of MMP-2, MMP-9 in cell culture supernatant with TNF-αwas higher than in culture medium without TNF-α. The activity of collagen degradation in cell culture supernatant with TNF-αwas higher than that without TNF-α(P<0.05). The MMP-2 was expreesed in serum of NSCLC patients and healthy volunteers. The strap gray scale was no difference between the two groups (P>0.05). The rusult of electrophresis and scale gray displayed that the MMP-2, MMP-9 expressed in malignant pleural fluid specimens were maximum, that in tuberculous pleurisy were mediate, and that in transudate pleural fluid were minimum. The strap gray scale of MMP-2 was so. The difference was significant between malignant pleural fluid and transudate pleural fluid (P<0.05), but was no significant between malignant pleural fluid and tuberculous pleurisy (P>0.05).The three-dimensional collagen gels culture model of lung cancer cell is similar to internal environment, so it is good method to study expression of MMPs. When mononuclearcell and lung cancer cell are co-cultured, the expression of MMP-2, MMP-9 are up. When lung fibroblast and lung cancer cell are co-cultured, the expression of MMP-2, MMP-9 are increased, but the TIMPs maybe predominant. TNF-αcan stimulate cancer cell to express MMPs. The expression of MMP-2 in blood serum is no significant difference between NSCLC patients and healthy volunteers. The overexpression of MMP-2, MMP-9 in pleural fluid is hinted malignant disease.