Experimental Study Of SKOV3 Cells With UC-MSCS Transduced With An Recombinant Adenoviral Vector Expressing Interleukin12 In Vitro

Objective: This experiment, which uses UC-MSCs as target carrier for gene therapy, is aimed at constructing UC-MSCs Transduced with an Recombinant Adenoviral Vector Expressing Interleukin 12 and producing mIL-12 with biologic activity in vitro by cultivating SKOV3 and UC-MSCs in vitro. It attempts to observe the influence of AdIL-12-MSCs on the growing appearance, in vitro generation, cell cycle as well as apoptosis of the SKOV3 cells, and approach the effect of UC-MSCs on SKOV3 as target carrier for gene therapy so as to provide a new target for the gene therapy of ovarian cancer.Methods:1 Cultivate ovarian cancer cell line SKOV3 and UC-MSCs as well as 293 cell in vitro in order to observe the change of cell appearance and cell multiplication through microscope upside down day by day.2 Enlarge the recombinant adenovirus over and over again to the required amount, apply UC-MSCs Transduced with an Recombinant Adenoviral Vector Expressing Interleukin12 as transfection group. In the meantime, set up a Ad-MSCs group and UC-MSCs group transfested by an alone viral vector.3 Take advantage of Western Blotting to test out the expression of protein in IL-12 existing in the AdIL-12-MSCs,Ad-MSCs as well as UC-MSCs group.4 Make use of RT-PCR to test out the expression of protein in IL-12 existing in the group AdIL-12-MSCs,Ad-MSCs as well as UC-MSCs .5 Inoculate SKOV3 onto the clear supernatant liquid of AdIL-12-MSCs which was transfected 24 hours later to act as transfection group, meantime, Inoculate SKOV3 onto the clear supernatant liquid of UC-MSCs which was transfected 24 hours later to act as control group.6 Observe and compare with a inverted microscope the growing appearance of SKOV3 cells 24~72 hours later both in the transfection group and control group so as to identify the influence of AdIL-12-MSCs on the growing appearance of SKOV3 cells.7 Use MTT to test out the effect of AdIL-12-MSCs on the multiplication in vitro of the SKOV3 cells.8 With the help of flow cytometer, inspect the effect of AdIL-12-MSCs on the mitotic cycle and cell apoptosis of the SKOV3 cells.9 Adopt t-test in the SPSS 13. 0 to inspect the significance of the difference between the detection groups. The data will be displayed in the form of x±s with P<0.05 as the standard of the differences'significance.Results:1 The results of testing IL-12 protein in the the tranfected AdIL-12-MSCs using Western Blotting are as follows: there are clear straps in the cells in AdIL-12-MSCs group, while there is no expression of IL-12 protein in the Ad-MSCs and UC-MSCs group, which indicates that IL-12 gene has expressed successfully in the protein level in AdIL-12-MSCs.2 The results of RT-PCR are that AdIL-12-MSCs show masccline straps at 389bp, which fits the length of IL-12 Amplification fragment and suggests that IL-12 gene has expressed successfully in the mRNA level in AdIL-12-MSCs while there is no expression of IL-12 mRNA in the Ad-MSCs and UC-MSCs group.3 After testing the growing situation of SKOV3 cell with inverted microscope 24~72 hours after it has been cultivated, we can see that in the transfection group, the growth of SKOV3 cell was obviously restrained, the adherence rate decreased, cell density lowered, and cell apophysis lessened. The cell appearance of SKOV3 has no obvious change in the control group.4 Use MTT to test the cell multiplication for 24 to 72 hours of SKOV3 in both transfection and control group to get the following result: with time going by, the restraining ability on the SKOV3 cell in the transfection group was obviously raised, with(47.56±2.31)% 72 hours later, which has great statistical significance(P<0.05).5 Inspecting the effect of AdIL-12-MSCs on the mitotic cycle of the SKOV3 cells with the help of flow cytometer can lead to the listed results: compared with the control group, the SKOV3 cell in the transfection group in the S+G2 period has a lower rate, while the cell rate in the G0/G1 period was lowered. So we can conclude that AdIL-12-MSCs can restrain the increase of the SKOV3 cell.6 Inspecting the effect of AdIL-12-MSCs on the cell apoptosis of the SKOV3 cells with the help of flow cytometer can lead to the listed results: the apoptosis rate of the SKOV3 cell in the transfection group was(9.72±1.38)%, which was much higher that (2.69±0.45)% in the control group. So that it has great statistical significance(P<0.05).Conclusion: This experiment, which uses UC-MSCs as target carrier for gene therapy, constructed successfully the AdIL- 12-MSCs cell system and observed that the Ectogenesis IL-12 can restrain obviously the multiplication of the SKOV3 cell and led to its apoptosis. This cell system can be further applied to the research on the gene treatment for tumor and provide a new target for the gene treatment and cytoimmunity treatment for the ovaries epidermis tumor.