The Effect Of Anti-Endometrial Carcinama By DC Vaccine In Vivo

Objective: At present, there are 3 main methods of tumor therapy: operation, chemotherapy, radiotherapy. However, all of these cannot remove the last malignant cell, biotherapy is the optimal method to achieve this goal. The research on the dendritic cell (DC) vaccine of endometrial cacinoma is rare in both domestic and abroad.In the recent years, the project to use DC to cure tumor as the biotherapy and genetherapy has already been licensed to process into the 3 stage clinic experiment, which is more specific and more powerful to kill the tumor than LAK cell and CIK cell therapy. As far as endometrial carcinoma is concerned, the research on DC is rare.In this research, at first, we sensitize DC originated form cord blood monocyte by Ishikawa freeze-thawing antigen. The objective of this research is to observe whether the CTL activated by DC pulsed with Ishikawa endometrial cacinoma cell lysates has the therapeutic and preventic effect on the established Ishikawa endometrial cacinoma-bearing nude mice, and approach the mechanism initially.Materials and methods: Firstly, DC isolated from cord blood mononuclear cells(MNCs) of healthy term birth women were cultured and proliferated in vitro by using recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) and interleukin-4(rhIL-4) and tumor necrosis factor-A (TNF-α), and were pulsed with Ishikawa endometrial cacinoma cell lysates. On the 7th day, we collected the matured DC, mixed culturing with the isolated T cells from cord blood at the ratio of 1:10. Observe the morphous of DC and T lymphocytes with inverted microscope. Secondly, choose 15 nude mice randomly, to establish the model of Ishikawa transplantation tumor s.c. in nude mice. Select 10 tumor-bearing nude mice which tumor volume are almost 100mm3, divided into 2 groups, one is injected CTL activated by DC s.c.; the other is CTL activated by freeze thawing antigen. Inoculate every 7 days, ending up the experiment on the 21st day. The tumor volumes are measured every 3 days and draw the tumor growth curve. Compare the rate of tumor growth supression between 2 groups.Thirdly, divide the other 15 nude mice into 3 group, one group preventively innoculate the CTL activated by DC and the other groups innoculate the CTL activated by antigen, PRMI-1640 contained 10% medical serum s.c. on the leftback. 3 days later, the Ishikawa cells in the logarithmic growth phase were innoculate s.c. on the right back. The tumor volume was measured every 3 days. Remember the time when the implantation tumorigenesis.Forthly, observe the morphous of Ishikawa transplantation tumor by microscope; furthermore we observe the apoptosis ultramicrostructure by TEM.Fifthly, the apoptosis rate of the Ishikawa transplantation tumor in different therapy groups were assessed by flow cytometry.Sixly, the expression of Fas membrane protein in different therapy groups were also assessed by flow cytometry(FCM).Sevenly, the expression of Survivin in different therapy groups were analyzed by the immunohistochemistry SP.Results: 1 After cultured MNCs isolated from cord blood by using rhGM-CSF and rhIL-4 and TNF-α, and were pulsed with Ishikawa endometrial cacinoma cell lysates during 7 days, the matured DC which has the typical morphous. The CTL colon activated by the sensitized DC was larger and proliferated more rapidly than the CTL only activated by antigen.2 The Ishikawa tumor growth curve: The CTL group activated by matured DC was more efficient on the supression of the tumor growth than the CTL group activated by antigen .3 Comparion the tumor volumns between the different CTL therapy groups: After vaccinated the CTL activated by matured DC by the CTL activated by matured DC, the tumor volume were significantly diminished than before(P<0.05); as same as the CTL group activated by antigen.What's more, after vaccinated , the tumor volumes of the CTL activated by matured DC were obviously smaller than the CTL group activated by antigen (P<0.05). The tumor volumes of the 2 therapy groups on the 20th day all have the significant differences comparing with the control group(P<0.01).4 Immune preventive groups: After vaccination of the CTL activated by matured DC, only one mouse were found the subcutaneous implantation tumorigenesis. The others remained tumor-free until the experiment ends up. The rate of tumorigenesis is 16. 6 %; during the same period, after vaccinated, the CTL group activated by freeze-thrawing antigen were found the subcutaneous implantation tumorigenesis 4 days later, the rate is 63 %. While the tumorigenesis rate of the control group is 100%. There is significantly different between the CTL group activated by matured DC and the other 2 groups (P<0.05).5 TEM: The early apoptotic morphology changes of Ishikawa cells treated by the CTL activated by DC for 20d could be observed by TEM: acute angle project of the nuclear membrane; marked condensation and margination of nuclear chromatins; as well as formation fo vacuoles in cell cytoplam, but in the other 2 group, we didn't find the apoptotic morphology.6 Assessed the apoptotic rate by FCM: The apoptotic rate in the CTL group activated by DC was significantly different compared with the control group (P<0.01). The apoptotic rate in the CTL group activated by antigen was higer than the control group (P<0.05). The apoptotic rate in the CTL group activated by DC was significantly higher than the other CTL treated group (P<0.01).7 The expression of Fas: The expression of Fas in the CTL group activated by DC was the highest compared with the the CTL group activated by antigen and the control group (P<0.01), While there was no statistics diffirence in the other 2 groups (P>0.05).8 The expression of Survivn in endometrial cacinoma: The color intensity in the control group is strong positive, as well as weakly positive in the CTL group activated by antigen and negative in the CTL group activated by DC.Conclusions: 1 The CTL colon activated by the sen- sitized DC was larger and proliferated more rapidly than the CTL only activated by antigen. It instructs that DC can efficiently present antigen and promate the proliferation of the specific CTL.2 The CTL activated by DC could efficiently supress the growth of the subcutaneous implantated tumor, which was related to the innoculation frequency.3 The CTL activated by DC coule induce the apoptosis of the tumor cell. It means that DC is most suitable for the transplanted tumor of smaller size.4 Apoptosis is observed in the group treated with the CTL activated by DC.5 The CTL activated by DC could up-regulate the expression of Fas, which maybe the mechanism of the apoptosis.6 The CTL activated by DC could induce the apoptosis of the Ishikawa cells by down regulating the expression of Survivin.7 DC maybe release the suppression of the FAS through the downgradulate the expression of Survivin.8 The CTL activated by DC loaded antigen plays an important role in prevent the happeniss of the transplated tumors.