[Characteristics change of the human directional highly lymphatic metastasis ovarian carcinoma cell and venous endothelial cell after establishment of their condition cultrue and co-culture cell system].

OBJECTIVE

To establish the condition cultrue cell system and co- culture cell system with SKOV3/PM4, HUVEC and to study the changes of their biological characteristics.

METHODS

The cells of SKOV3/PM4 and HUVEC were labeled with green and red fluorescent respectively. The cell supernatant of SKOV3/PM4 and HUVEC were collected respectively as the condition medium (e.g: the cell supernatant of HUVEC cells was used as SKOV3/PM4 condition medium)and to establish the condition cultrue cell system and the co- culture cell system of the two cell lines. In the condition cultrue cell system, The morphological changes of cells were observed by HE staining to calculate the mitotic index. The ultrastructural changes of the two cells were observed by transmission electron microscopy (TEM). The growth curve of the cells was determined by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry was used to analyzed the cell cycles. In the co-culture cell system, the interaction of the two cells were detected by laser scanning confocal microscope (LSCM). The expression of matrix metalloproteinase- 2 (MMP- 2) and matrix metalloproteinase- 9 (MMP- 9) were detected by gelatin zymography.

RESULTS

Compared with the single culture SKOV3/PM4, the cells which cultured in HUVEC condition medium showed the increase of pseudopodia and nuclear division, the mitotic index respectively were [(4.8 ± 0.8)%, (11.2 ± 0.3)%; P < 0.05]. The growth rate was significantly increased. In cell cycles, it showed the declined cell ratio of G0/G1 phase, respectively [(69.4 ± 3.6)%, (48.4 ± 4.6)%; P < 0.05] and the raised cell ratio of G2/M phase, respectively [(5.2 ± 1.6)%, (24.9 ± 2.2)%; P < 0.05]. Compared with the single culture HUVEC, the cells which cultured in SKOV3/PM4 condition medium showed the significant morphological change and vacuolization in the cytoplasm, Nuclear division was increased and the mitotic index respectively were [(2.7 ± 0.5)%, (5.7 ± 0.6)%; P < 0.05]. The growth rate was slightly declined. In cell cycles, it showed the raised cell ratio in G0/G1 phase, respectively [(51.4 ± 2.2)%, (79.0 ± 4.1)%; P < 0.05] and the declined cell ratio in G2/M phase, respectively [(19.1 ± 1.2)%, (3.3 ± 0.5)%; P < 0.05]. After co-culture for 48 hours, spontaneous fusion between SKOV3/PM4 and HUVEC cell was observed by the laser confocal microscope. Gelatin zymography assay showed that MMP-2 was not expressed in HUVEC cells, low-expressed in SKOV3/PM4 cells and high-expressed in the co-culture SKOV3/PM4+HUVEC cells. The expression of MMP-2 in co-culture SKOV3/PM4+HUVEC cells and SKOV3/PM4 cells respectively were 1 885 ± 84 and 1 209 ± 114 (P < 0.05). But there were no MMP-9 expression in HUVEC cells, SKOV3/PM4 cells, and the co- culture SKOV3/PM4+HUVEC.

CONCLUSION

The characteristics of SKOV3/PM4 and HUVEC show significant changes after condition culture and co-culture, it may involve in the microenvironment of the cells and the intercellular crosstalk pathway.