| Background.Ovarian cancer is one of the most common gynecologic malignancy. Most of the patients are diagnosed at an advanced stage and the mortality rate ranks first in gynecological malignancies. The absences of obvious symptoms and signs, specific and effective early screening methods are considered to be the major reasons for it.Cytoreductive surgery, postoperative platinum and paclitaxel based chemotherapy is the conventional first-line treatment. However, the patients’ clinical outcome following the treatments is not ideal. The 5 year recurrence rate and overall survival have not been significantly improved. According to the clinical research, drug resistance and the postoperative residual lesions are the major obstacles account for it. With the multidisciplinary development, tumor immunotherapy has gradually become the focus of clinical medicine. Among them, adoptive cellular immunotherapy is considered to be an ideal strategy to advanced ovarian cancer patients, who come up with low immunity after chemotherapy. It can specifically eliminate minimal residual disease, prevent tumor recurrence and reverse drug-resistance to chemotherapy, which result in the improvement of the clinical outcome in patients with ovarian carcinoma. Therefore, in preliminary studies, our team established a specific cytotoxic T lymphocytes(CTL) large-scale culture system with HER2 / neu antigen peptide, combining Cp G ODN(CPG Oligodeoxynucleotide) and ginsenoside Rg1 as immune adjuvants in vitro.Experiment in vitro confirmed the high antigen specificity and inhibiting effect. The specific CTL can destroy the normal cell cycle of ovarian cancer cell SKOV-3 and cause it to be stuck in the S stage. At the same time, it can also regulate the expression of apoptosis gene, induced apoptosis of SKOV-3 cells. In addition, specific CTL can also inhibit the expression of the related proteins of PI3K/Akt pathway, which can play a role in resisting ovarian cancer and reversing the drug-resistance of SKOV-3.Objective.To verify the inhibitory effect of specific CTL on the migration and invasion of ovarian cancer cell line SKOV-3 in vitro, the killing and inhibiting effect on ovarian cancer cells in tumor bearing nude mice and the influence on the apoptosis and chemotherapy resistance signaling pathway in vivo.Methods.Part 1. The experiment was divided into three groups, specific CTL group, PBMC group and blank control group. In specific CTL group, we used the specific CTL which was come from PBMCs that induced by HER2 / neu antigen peptide, Cp G ODN and ginsenoside Rg1 obtaining from healthy volunteers peripheral blood. In PBMC group, we used the unstimulated healthy volunteers’ peripheral blood mononuclear lymphocytes. The same amount of normal saline was used in the blank control group as reference. In the scratch test, specific CTL and SKOV-3, PBMC and SKOV-3 were co culture respectively. Determine the extent of 24 h and 48 h wound healing to detect the inhibitory effect of the specific CTL on the migration of SKOV-3 cells. Transwell assay was applied to study the inhibitory effect of the specific CTL on the invasion ability of SKOV-3 cells. After the co cultured of specificity of CTL and SKOV-3, PBMC and SKOV-3 cells for 24 hours, the number of SKOV-3 cells through the polycarbonate membrane were detected. The less amounts of SKOV-3 cells through the polycarbonate membrane, the stronger inhibition effect is.Part 2. The subcutaneous tumor model of ovarian carcinoma in nude mice was established. They were randomly divided into three groups(specific CTL group,PBMC group, blank control group) and the corresponding treatments were given by local injection. After 7 days of treatment, the nude mice were sacrificed on the eighth day. Measure the size and weight of the subcutaneous tumors, calculate tumor inhibition rate and the T/C value. Determine the effectiveness of treatment according to the results. At the same time, the peritoneal ovarian cancer nude mice model were established. They were randomly divided into three groups(specific CTL group,PBMC group, blank control group) and the corresponding treatments were given by intraperitoneal injection. After 7 days of treatment, the nude mice were sacrificed on the eighth day. The tumor lesions in the abdominal cavity were collected. Record the weight of the tumor and calculate tumor inhibition rate.Part 3. The subcutaneous tumor tissues and peritoneal tumor tissues were collected from the above groups. Immunohistochemical staining were used to observed the expression of PI3 K, Akt, Bcl-2 and Caspase-3 for semi quantitative detection.According to the level of the corresponding protein expression, the effect of specific CTL on SKOV-3 apoptosis and drug resistance was identified.Results.Part 1. The scratch test showed that the migration distance of SKOV-3 cells in specific CTL group was significantly less than that of PBMC group and blank control group at 24 hours and 48 hours. Transwell experiment results show that the number of the SKOV-3 cells in specific CTL group through the polycarbonate membrane at 24 hours was significantly lower than that in peripheral blood mononuclear cells(PBMC) group(P < 0.05) and blank control group(P < 0.05),the difference between the groups was statistically significant.Part 2. The subcutaneous tumor size of specific CTL group was obviously smaller than that of PBMC group(P < 0.05) and blank control group(P < 0.05), the difference between the groups was statistically significant. According to the results,the weight of the subcutaneous tumor in specific CTL group was less than that in group PBMC(P < 0.05) and blank control group(P < 0.05). Compared with the control group, the tumor inhibition rate was IR=54.23%. The T/C value is 29.80%,less than 40%. The treatment was effective. In the peritoneal ovarian cancer nude mice model, the weight of specific CTL group was less than that of PBMC group(P< 0.05) and blank control group(P < 0.05). Compared with the control group, the tumor inhibition rate was IR=70.61%.Part 3. The expression of PI3 K protein and Akt protein in ovarian cancer tissues of specific CTL group were lower than those in PBMC group(P < 0.05) and blank control group(P < 0.05). There was significant difference between the two groups.The specific CTL group, PBMC group and blank control group were all weakly positive in Bcl-2 protein, and there was no statistical difference between each two groups. The expression of Caspase-3 protein in ovarian cancer tissues was significantly higher than that in PBMC group(P < 0.05) and blank control group(P< 0.05), and the difference between the two groups was statistically significant.Conclusion.The specific cytotoxic T lymphocytes, which come from the large-scale culture system induced by HER2 / neu antigen peptide, Cp G ODN(CPG Oligodeoxynucleotide) and ginsenoside Rg1 in vitro, can inhibit the migration and invasion of ovarian cancer cells in vitro, and it also has a certain inhibitory effect on the ovarian cancer cells in nude mice ovarian cancer model. The specific CTL may promote tumor apoptosis and reverse drug resistance by affecting the activation of PI3K/Akt pathway and Caspase-3 protein in ovarian cancer cells in nude mice. But the influence on the expression of apoptosis related protein Bcl-2 is little. |
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