| Ovarian cancer is one of the common female genital cancers. In the ovarian cancers, epithelial ovarian cancer(EOC) accounts for about 90%, and its mortality rate ranks the first in all kinds of genital tumors. Patients with early ovarian cancer often lack specific symptoms, which is usually diagnosed at an advanced stage. Only 5-20% of the patients with advanced-stage ovarian cancer could survive 5 years after initial diagnosis. But the survival rate of patients diagnosed in early stage could reach up to 90%. Most of the patients, after cytoreductive surgery combined with platinum-based chemotherapy, could be alleviated in a period of time, but finally goes to be resistant to chemotherapy and die. Therefore, new early diagnostic screening indicator finding and more effective treatments development to improve the survival rate of patients with ovarian cancer are urgently needed.Mesothelin(MSLN), with molecular weight of 40 KDa, is a cell surface glycoproteins, usually expressed in a body cavity surface of mesothelial cells and overexpressed in some malignant tumors such as mesothelioma, pancreatic cancer, ovarian cancer, however it is not expressed in other normal tissues. Recent studies have confirmed MSLN plays an important role in survival, invasion of tumor cells and tumor progress. MSLN has high affinity with CA125 and the interaction between the two molecules involved in cell adhesion, and peritoneal tumor metastasis. Soluble MSLN-related protein(soluble mesothelin related protein, SMRP), with molecular weight of 42-45 KDa is an isoform of MSLN protein coded by MSLN gene, which is stopped early in the translation process and shredded off from the cell surface. SMRP with small molecular weight, not only can be detected in the serum, but also can be detected in ascites and urine. Therefore, SMRP may become an early diagnostic screening indicator for ovarian cancer, and its tissue-specific expression manner makes it an ideal target for ovarian cancer immunotherapy.Cancer vaccines are an important part of the immunotherapy, which induces specific immune response by the tumor cells or tumor antigens to kill tumor cells. Dendritic cell(DC) vaccine is a tumor vaccine. DCs are the most powerful in vivo antigen-presenting cells, capable of taking a tumor antigen, induce generation of cytotoxic T lymphocytes(CTLs), whereby a strong specific anti-tumor cell-mediated immunity. In recent years, research on ovarian cancer DC vaccine has made some achievements.In this present study MSLN levels of ovarian cancer, benign epithelial ovarian tumor, normal human serum and urine were detected and compared with the clinical evaluation of serum CA125; meanwhile the clinical value of combined serum CA125 with urine and serum MSLN for epithelial ovarian cancer diagnosis were evaluated, respectively.Then the full-length c DNA of MSLN was cloned using PCR technique. After the sequencing confirmation by sequencing, the endotoxin MSLN plasmid was extracted, and then lentiviral vectors containing MSLN c DNA was prepared and identified. Finally, the DCs were infected by lentiviral with the full-length c DNA of human MSLN to make a new DC epithelial ovarian cancer vaccine. The strong expression of MSLN was verified through western Blot technique which, as MSLN delivery cells was employed to induce T lymphocytes(CTLs) with a MSLN-specific cytotoxicity, then the killing effect of CTLs specific to epithelial ovarian cancer cells was identified through a series of methods including LDH release assay, flow cytometry, IFN-γ detection etc. The objective of this study is expected to propose new ideas for early diagnosis and gene therapy of ovarian cancer. Part 1 the diagnosis value of serum and urine mesothelin inepithelialovarian cancerObjectives: To investigate the levels of SMRP expression in serumand urine in patients with epithelial ovarian cancer or benign epithelial ovarian tumors and healthy females and compare with the diagnostic value of serum CA125; to make an objective evaluation by combining the levels of SMRP in serum and urine with serum CA125 as the clinical value of detecting epithelial ovarian cancer.Methods: ELISA was used to detect the SMRP levels in serum and urine in 46 cases of epithelial ovarian cancer patients, 36 cases of benign epithelial ovarian cancer patients and 35 healthy females. Meanwhile the serum CA125 level was detected by using chemiluminescence. The differences along with these three detection methods for SMRP level were analysed in different pathological types, in different clinical stage and in different histological grade of epithelial ovarian cancer patients. And the differences of these three methods in different histological types of benign epithelial ovarian tumors were also analysed. The effectiveness of these three detection methods was evaluated by plotting the ROC curve. The diagnostic values of SMRP combined with CA125 in serum and in urine were also evaluated, respectively.Results:1 The SMRP levels in serum and urine of epithelial ovarian cancer patients were significantly higher than that of benign epithelial ovarian cancer patients and healthy(P <0.05).2 Serum SMRP levels in serous carcinoma were significantly higher than that in mucinous carcinoma and endometrioid carcinoma(P <0.05), while the difference of serum SMRP levels between mucinous carcinoma and endometrioid carcinoma was not significant(P> 0.05). Furthermore there were no differences of urine SMRP levels when comparing along with these three types patients(P> 0.05).3 SMRP levels in serum and urine of patients with advanced cancer was significantly higher than those in patients with early(P <0.05).4 Serum and urine SMRP levels in group G3 were significantly higher in group G1 and G2(P <0.05), while there was no significant difference between group G1 and G2(P> 0.05).5 The ROC curves of SMRP in serum and urine and serum CA125 were built. The area under ROC(AUC) curve of serum SMRP was 0.822, standard error 0.42, 95% confidence interval(0.740-0.905); the AUC of urine SMRP was 0.787, standard error 0.46, 95% confidence interval(0.697-0.877); the AUC of serum CA125 was 0.814, standard error 0.49, 95% confidence interval(0.719-0.910).6 The sensitivity of serum CA125 was the highest(76.1%), but the specificity was the lowest(85.5%). The sensitivity of serum SMRP(71.7%) was slightly lower than serum CA125, but the specificity of serum SMRP(89.8%) was increasing when comparing over its. The specificity of urine SMRP is the highest(93.4%) with the lowest sensitivity(67.4%).7 The Kappa consistency test was conducted for these three indicators. The Kappa value of serum SMRP was 0.617(P=0.000), urinary SMRP 0.628(P= 0.000), CA125 0.590(P = 0.000).8 The test sensitivity of CA125 serum SMRP in parallel with the joint highest(93.2%), urinary SMRP and CA125 series combined detection highest specificity(99%). Combined to make three slightly increased sensitivity, but it does not improve specificity.Conclusions:1 SMRP in epithelial ovarian cancer serum was significantly higher than in benign epithelial tumor group and the healthy control group; serum SMRP in serous carcinoma patients was significantly high comparing with that in mucinous carcinoma patients and endometrioid carcinoma patients; serum SMRP in advanced cancer patients was significantly higher than in patients with early stage cancer; the worse histological grade in patients, the higher the serum SMRP.2 Urine SMRP levels in epithelial ovarian cancer patients were significantly higher than that of benign epithelial tumor group and the healthy control group; urinary SMRP levels in serous carcinoma patients were higher than those from mucinous carcinoma and endometrioid carcinoma, however the difference was not significant; urine SMRP levels in advanced cancer patients was significantly higher than in patients with early stage cancer; the higher the SMRP levels in urine, the worse the histological grade.3 Among the three indicators, serum CA125 is the most sensitive but least specific. The sensitivity of serum SMRP is slightly lower than CA125 but its specificity improved. Specificity of urine SMRP is the highest, but the sensitivity is the lowest. For diagnosis consistency, urine SMRP is better than serum SMRP and serum CA125, while serum SMRP is slightly better than CA125, which confirmed the application value of serum and urine SMRP in for the diagnosis of epithelial ovarian cancer.4 The joint detection of combined in parallel of serum SMRP and CA125 has the highest sensitivity, meanwhile the most specificity was obtained from the joint detection of combined in series of urine SMRP and CA125. Both Serum and urine SMRP tests could improve early diagnosis of ovarian cancer by using CA125 only. Part 2 Cloning of mesothelin c DNA and Construction of lentiviral vectorsObjectives: Construction of lentiviral vectors with mesothelin full-length c DNA.Methods: The cloned MSLN c DNA by using PCR, after sequencing confirmation, was transformed into lentiviral expression vector to make endotoxin lentivirus vectors with MSLN c DNA which was then titered.Results:1 Approximately 2000 bp PCR product, after two rounds of PCR and sequencing check, was confirmed to be a full-length c DNA sequence coding MSLN which has a coding region of 1893 bp encoding a 630 amino acid protein.2 PCR product of MSLN coding c DNA ends coding region and each containing Age I restriction sites Xho I and after double digestion of the plasmid vector p BOB ligation reaction to obtain a lentivirus for transfection of p BOB-MSLN plasmid vector, and using a micro spectroscopic photometer K5600 detection plasmid purity.3 The lentiviral vector with full-length MSLN c DNA was successfully constructed. The titers of virus particles measured using quantitative PCR method were 6.56 × 106 ± 2.1 × 105.Conclusions1 The PCR was conducted to amplify the c DNA of MSLN when c DNA library containing encoding human MSLN was used as template. The full-length MSLN c DNA sequence obtained was confirmed by sequencing the products after two rounds of PCR.2 Lentiviral vector system used in this experiment is four plasmid system p BOB(p CSC-SP-PW), and the lentiviral particles were packaged with 293 T cells. The lentiviral vector has the obvious advantages including a wide host range, large capacity of transfering gene fragment and difficult to induce a host immune response. The most importance is that exogenous gene can be efficiently integrated into the host chromosome by this carrier, so as to achieve persistent expression. The virus titer was satisfied after successfully constructing the lentiviral vector with full-length MSLN c DNA. Part 3 Study on the cytotoxicity of loading full-length MSLN c DNA DCvaccine on ovarian cancer cell lines OVCAR3 and SKOV3Objectives: Using mature DCs present MSLN to lymphocytes by constructed DC vaccine loaded with MSLN full-length c DNA to induce against MSLN-specific cytotoxic T lymphocytes cells(CTLs). The cytotoxicity of CTLs produced against epithelial ovarian cancer cells would be verified.Methods:1 Ovarian cancer cell lines OVCAR3 and SKOV3 were cultured. The expression of MSLN in these cell lines was detected with Western Blot method.2 The generation and detection of mature DCs: the monocytes were isolated in vitro from the fresh peripheral blood of healthy volunteers. Then the cytokines of GM-CSF, IL-4 and TNF-α had been added in culture medium of the monocytes. The mature DCs were harvested after 9 days and detected by detecting the expression of DCs surface molecules CD80, CD83 and CD86 using flow cytometry.3 Transfection of DCs by lentivirus with MSLN: The mature DCs were infected with the built lentiviral vector in part two(uninfected DCs as a negative control). The expression of MSLN in DCs was detected by Western Blot method.4 Obtain of CTLs cells: infected DCs with MSLN lentivirus and non infected DCs were added to human fresh peripheral blood monocytes and incubated with recombinant human IL-2. The CTLs were collected after seven days.5 Determination of CTLs function using LDH release assay: CTLs induced by DC-p BOB-1MSLN was incubated with OVCAR3 or SKOV3 in different ratio of E:T. The cytotoxicity of CTLs for OVCAR3 or SKOV3 cells was assessed by released LDH from injured OVCAR3 or SKOV3 cells.6 Detection of mortality of target cells by flow cytometry: The mortality rate of ovarian cancer cells OVCAR3 or SKOV3 by flow cytometry was used to validate the cytotoxicity of CTLs against ovarian cancer cells.7 IFN-γ release quantity detected by using enzyme-linked immunosorb-ent assay: The ability to produce IFN-γ of DC- p BOB-1MSLN induced CTLs was conducted by using enzyme-linked immunosorbent assay employing non-transfected DC induced CTLs as control.Results:1 MSLN expressed in both OVCAR3 and SKOV3 cells, of which the expression in ovarian cancer cell line OVCAR3 was higher than in SKOV3.2 Typical morphologies of matured DCs could be viewed under micro-scope after 9 days culture. CD molecule antibody on the surface of matured DCs was detected using flow cytometry. The results showed that of the DCs cells, 83.07% expressed CD80, while 85.80% expressed CD86, 52.42% expressed CD83.3 The expression of MSLN in DCs was identified using Western Blot. The results showed that MSLN only expressed in viral transfected DCs.4 The toxicity MSLN-specific CTLs to the two types of ovarian cancer cells significantly increased with the increase of E: T ratio. Meanwhile, the cytotoxicity to OVCAR3 was significantly higher than to SKOV3 at the ratio of 50: 1 and 10: 1, which indicated that with MSLN modification, the DCs induced CTLs performed high efficient MSLN-specific cytotoxicity against OVCAR3.5 Ovarian cancer mortality detected by using flow cytometry: Effector CTLs have largely died after completing the destruction; the natural mortality rates of two target cells, OVCAR3 and SKOV3, were 23.31%and 6.8% under natural release conditions. MSLN induced CTLs had very significant killing effects to both target cells, the killing rate improved markedly with the effector-target ratio increasing, and the mortality rate of OVCAR3 was higher than SKOV3; non-MSLN-induced CTLs also had significant killing effect against OVCAR3 and SKOV3.6 Compared with the control group, DC-p BOB-1MSLN induced CTLs released more IFN-γ. Comparing the two groups, the difference was significant(P <0.01).Conclusions:1 Strong MSLN expression was detected in transfected DCs suggesting that MSLN containing DC vaccine would have high efficiency.2 MSLN transfected DCs stimulated MSLN-specific CTLs could trigger more powerful cytotoxicity.3 The killing rate against ovarian cancer cell lines of MSLN-specific CTLs was significantly higher than normal CTLs, which indicated that MSLN containing DC vaccine has good application value and prospects for epithelial ovarian cancer treatment. |
PDF Full Text Request