| Objective: Ovarian cancer is one of the common gynecological malignancies with its highest mortality rate. Co-treatment of surgery, radiotherapy and chemotherapy has limited effects on the prognose of patients with ovarian cancer. The recurrence and metastasis of the malignant tumors are related with the low immune function of the body, so the immune gene therapy is more and more concerned at present. CD40 ligand is one of the immunological regulatory molecules, and the interaction of CD40 and CD40 ligand (CD40L) can induce the proliferation and the differentiation of T-cell, which can further activate the specific cytotoxic effects on tumor cells and induce the production of the cytokines. Our purpose is to check the in vivo antitumor effects of the forced expression of CD40L cDNA in murine ovarian cancer OVHM cells (CD40L- OVHM cells) by injecting them into the spleens of mice, and provide some experimental bases for the future clinical use of CD40L gene.Methods: 1 The growth rates of CD40L-OVHM cells, OVHM cells and DNA-pMKITneo-OVHM cells were detected by MTT method, and the characteristics of these three kinds of cells were observed by the inverted microscope.2 The expression of CD40 ligand on CD40L-OVHM cells and OVHM cells was analyzed by flow cytometry.3 HE staining was used as a pathological means to testify the established liver metastases by injecting OVHM cells into the spleens of mice.4 In vivo study: all the mice were divided into four groups: CD40L-OVHM cells group, DNA-pMKITneo-OVHM cells group, OVHM cells group and PBS control group. On the 10th day after inoculating, the mice from different groups were sacrificed and the expressions of CD11c on splenic cells were detected by flow cytometry, and so was the specific cytotoxicity of splenic cells by MTT method. On the 14th day after inoculating, the cytokines of IFN-γ, TNF-α, IL-4, IL-10 and IL-12 in the serum of mice from different groups were measured by enzyme-linked immunosorbent assay techniques. Moreover, On the 14th day after inoculating, the weights of mice livers and spleens were detected, and the liver metastases in mice were observed, the survival of mice in different groups were also observed.Results:1 There were no difference of CD40L-OVHM cells, OVHM cells and DNA-pMKITneo-OVHM cells, and so the proliferation rates of this three kinds of cells in vitro.2 CD40L could be detected on CD40L-OVHM cells, but not on OVHM cells.3 On the 14th day after inoculating OVHM cells into the spleens of mice, the primary tumors were found in spleens and a large number of metastatic nodules were also presented in their livers.The tumors in mice livers were confirmed to originate from the same tumors in mice spleens by pathology. Thus the mice liver-metastases models were established.4 On the 10th day after inoculating, the CD11c positive cells in splenic cells of CD40L-OVHM cells group, DNA-pMKITneo-OVHM cells group and OVHM cells group were significantly higher than that of PBS group (P<0.05). Moreover, the CD11c positive cells of CD40L-OVHM cells group (20.30±1.23%) was significantly higher than those of DNA-pMKITneo-OVHM cells group and OVHM cells group (10.69±0.89% and 10.56±0.85%) (P<0.05).5 The specific killing activities in different groups were shown as the following: the cytotoxicity rates of OVHM cells group, CD40L-OVHM cells group and DNA-pMKITneo- OVHM cells group were higher than that of PBS group (P<0.05). The cytotoxicity rate of CD40L-OVHM cells group was much higher than that of DNA-pMKITneo-OVHM cells group and OVHM cells group (P<0.05). There were no significant difference of the cytotoxicity rates between DNA-pMKITneo-OVHM cells group and OVHM cells group (P>0.05).6 The expression levels of IFN-γ, TNF-αand IL-12 in the CD40L-OVHM cells group were increased more than those in DNA-pMKITneo-OVHM cells group and OVHM cells group (P<0.05). However, the expression levels of IL-4 and IL-10 in the CD40L-OVHM cells group were increased much less than those in DNA-pMKITneo-OVHM cells group and OVHM cells group (P<0.05). There were no significant difference of the cytokines expression levels between DNA-pMKITneo-OVHM cells group and OVHM cells group (P>0.05).7 On the 14th day after inoculating, all the mice in DNA-pMKITneo-OVHM cells group and OVHM cells group respectively were found to have liver metastases and the primary tumors in spleens. In these two groups the average weights of livers were 3.07±0.19g and 3.10±0.21g respectively, the average weights of spleens were 1.25±0.14g and 1.23±0.16g respectively. There were no significant difference of the average weights of livers and spleens between DNA-pMKITneo-OVHM cells group and OVHM cells group (P>0.05). Liver metastases were found in 3 mice in CD40L-OVHM cells group (10 mice in each group). The number of nodules of liver metastases in CD40L-OVHM cells group were significantly less than that in DNA-pMKITneo-OVHM cells group and OVHM cells group, but the primary tumors in spleens were found in all the mice in CD40L-OVHM cells group. In CD40L-OVHM cells group the average weight of livers was 1.45±0.14g and the average weight of spleens was 0.58±0.10g. The average weights of livers and spleens of mice in CD40L-OVHM cells group were significantly lighter than that of DNA-pMKITneo-OVHM cells group and OVHM cells group (P<0.05). The average weights of livers and spleens of mice in PBS group were significantly lighter than that of OVHM cells group, CD40L-OVHM cells group and DNA-pMKITneo-OVHM cells group (P<0.05).8 No mice in PBS group died in the experimental stage, moreover, the survival time of mice in PBS group was also longer than that of other groups. All mice in DNA-pMKITneo- OVHM cells group and OVHM cells group died during the observing stage, and the survival times were 18±1 days and 17±1 days respectively. Only six mice in CD40L-OVHM cells group died, the other 4 mice survived till the end of the observing stage, the survival time of mice in CD40L-OVHM cells group (58±3days) was significantly longer than that of OVHM cells group and DNA-pMKITneo-OVHM cells group(P<0.05).Conclusions:1 We confirmed that the in vivo antitumor effects of the forced expression of CD40L in murine ovarian cancer cells (CD40L-OVHM) by injecting CD40L-OVHM cells into the spleens of mice.2 The proliferation and differentiation of DC cells in spleen and the induced specific cytotoxic effect of T cells in spleen could be enhanced by the expression of CD40L in murine ovarian cancer cells (CD40L-OVHM).3 The expression of CD40L in murine ovarian cancer cells (CD40L-OVHM) could regulate the immune function of peripheral blood cells and the immune balance of Th1 and Th2 cells by increasing the expression level of TNF-α, IL-12 and IFN-γand decreasing the expression level of IL-4 and IL-10, which further induce the protective immunity reaction and enhance the systemic antitumor effects in vivo.4 This study provided some basic grouds to study the application of CD40L gene vaccine, and for its future clinical use...
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