| Objective: 1 Fundamental research: To study whether the dendritic cell vaccine from fusion of one strain of bladder carcinoma cells with dendritic cells can induce specific antitumor CTL reaction to another bladder carcinoma cell line in vitro, whether the vaccine can inhibit the growth of another strain of implanted bladder carcinoma cells in mice,so to understand the effect and mechanism of cross immunity from the DC vaccine2. Clinic research: To explore the value of combined determination of urinary tumor markers, which reflect the different stages of tumor genesis and progress , to detect bladder transitional cell carcinoma(BTCC), . Materials and Methods1.Fundamental research: The passage cultures of bladder carcinoma cell strain 5637 and T-24 was done;According to Inaba's methods and the principle of adherence, pDC was obtained from bone marrow of BALB/c mice and was given a primary culture stimulated by the mice rGM-CSF and IL-4 in the medium with 10% FCS-RPMI1640; The phenotype analysis of DC was assessed by immunofluorescence, flow cytometry and scanning electron microscope technology respectively; DC vaccine which presenting the whole 5637 cellular antigen was obtained by fusion of DCs and 5637 cells in vitroby PEG; Using nylon wool collumn assay to acquire T effector cells from the spleen of BALB/c mice and then the specific CTL activity to 5637 cell strain or the specific cross CTL activity to T-24 cell strain,which induced by mixed culture of DC/5637 hybridoma cells and T cells, was tested by MTT reduction assay: the T effector cells obtained from incubation of T cells with DC/5637, DC, 5637 cells respectively and 5637 cells were used as target cells , so three experimental groups were group DC/5637-5637, group DC and group 5637. Then substituting 5637 cells with T-24 cells as target cells, the fourth experimental group was group DC/5637-T-24. Using 10%FCS RPMI-1640 medium as blank, cultured 5637 without effector cells suspention as control group, T effector cells suspension without target cells as regulation group, each group'OD was determined by Benchmark plate reader at 570nm of wavelength. The blank's OD was regarded as 0 and the percentage of specific CTL activity was determined by the following equation:percentage of CTL activity ={1-[(OD of experimental group - OD of regulation group)/OD of control group]}×100%.In the study of cross immune activity of DC vaccine to tumor animal models in BALB/c-nu mice, the experimental animals were divided into five groups at random:(1)blank control group:10 mice, female and male all in half, were vaccinated with RPMI-1640+PBS. (2)DC group: 10 female mice were inoculated with cultured DCs. (3) dead 5637 cells group: 10 mice, female and male all in half,were inoculated with apoptotic 5637 cells and supernate. (4)5637 experimental group:15 mice with 10 female and 5 male were vaccinated with DC/5637 hybridoma cells.Above mentioned four groups' animals were challenged by 5637 cells and established implanted tumor animal model. (5) T-24 experimental group: matched with 5637 experimental group , 15 mice with 10 female and 5 male were vaccinated with DC/5637 hybridoma cells too ,but challenged by T-24 cells and set up T-24 transplantation tumor. One week after first subcutaneous vaccination, allmice were vaccinated again and another seven days later the mice were challenged by subcutaneous injection of IX 10V0. 2ml 5637 cells (group one to group four) or by 1 X lO'/O. 2ml T-24 cells(group five). The number of mice which acquired implanted tumor in each group were counted and the size of implanted tumor were recorded. Another 49 days later following tumor cells' challenge, the mortality of each group were calculated and the mice with tumor were sacrificed and the weight of tumor were weighed. The soft ware of SPSS for windows was used for statistics analysis The statistical significance of differences in the percentage of bearing cancer mice or the mortality in each group was determined using the exact probabilities in 2X2 table, two-sided P\0.05 was considered significant. The size and weight of implanted tumor of each group was recorded as mean+SD, Student' s paired t test or One Way ANOVA was used for hypothesis testing between different groups, level of a test was 0.05 (a=0. 05) , two-sided F(0. 05 was considered significant.2. Clinic research: Voided urine samples of 45 patients with BTCC were collected and divided into different aliquots for each tumor marker analysis. CYFRA21-1 and VEGF were measured by enzyme linked immunosorbent assay respectively, telomerase was determined by telomeric repeat amplification protocol. According to results previous to this study, cutoffs were obtained by the 95% percentile in controls with no evidence of disease, which give a 95% specificity for all biomarkers. Cutoff for CYFRA21-1 was 3. 5ng/ml, for VEGF, 105ng/gCr. The sensitivity of combined determination of urinary CYFRA21-1, telomerase and VEGF was compared with that of individual tumor marker and urinary cytology respectively, Chi-square test was used for hypothesis testing. Results1. Fundamental research: Through flushing mice bone marrow, DC precursors were obtained.By gently swirling the plates after 2h cultureand throwing away supernate and by aspirating 75% of the medium every two days and adding back fresh medium, nonadherent granulocytes were removed. DCs first loosely attached to firmly adherent macrophages, and then cellular aggregates with veil-or sheet-like processes of DC were observed. Stimulated by rGM-CSF and IL-4, DCs matured rapidly and on the 5th-7th days many typical dendritic cells were seen floating in the culture medium while adherent macrophages expanded in the cultures still remained firmly adherent to the culture surface. About lX108-2X106DCs were harvested from one mouse bone marrow and the staining reaction of cell surface marker CD80, CD86 by immunofluorescence were significant positive. By analysis of FCM the percentage of positive CD80 DCs after one week culture were 75%-97% and positive CD86 DCs, 70%-99%. SEM of a bone marrow-derived DC at day 7 of culture showed many slender surface processes. About 5X104 fusion cells of DCs with 5637 were harvested from total of 1X106 cells of mixture of DCs and 5637 (DC : 5637,10 : 1) by PEG. The percentage of fusion was about 20%. When radio of effector cells to target cells was 20 '. 1, the percentage of CTL activity induced by different stimulating cells was 86. 5% in group DC/5637-5637, 7. 3% in group DC, 4. 5% in group 5637,80.2% in group DC/5637-T-24 respectively. There was no significant difference between group DC/5637-5637 and group DC/5637-T-24,but when group DC/5637-5637 or group DC/5637-T-24 was compared with group DC or group 5637 , the difference was significant respectively(/K0.01, respectively). When E : T ratio was 40 : 1,the percentage of CTL activity in each group rose to 93.4%, 10. 5%, 6. 7%, 89.1% respectively. Compared to the result when E : T ratio was 20 I 1 in the same group, there was no significant difference respectively (P>0.05, respectively).At the end of study about cross immunity of DC vaccine used in tumor animal model, the percentage of bearing cancer mice was 100% in group1(blank),90% in group 2(DC group), 90% in group 3(dead 5637 cells group), 46. 7% in group 4(5637 experimental group), 53. 3% in group 5(T-24 experimental group) respectively. There was significant difference compared group 4 or group 5 with group 1 respectively(fKO. 05, respectively), but group 4 wasn' t significantly different from group 5(P>0. 05). The mortality in group 1 to group 5 was 40%, 20%, 20%, 6. 7%, 6. 7% respectively. The mean area of implanted tumor in each group was 103. 20mm2 ± 8.95mm2,107. 22mm2 ± 14. 25mm2,97. 00mm2 ± 12. 63mm2, 34.14mm2 + 14. 35mm2, 31.14mm2±13. 50mm2 respectively and the mean tumor weight, 8. 26g ±0. 72g, 8. 58g±l. 14g, 7. 76g+1.01g, 2. 73g±l. 14g, 2. 49g±1.08g, respectively. The mean area or weight of implanted tumor in 5637 experimental group showed no significant difference compared to T-24 experimental group. But each experimental group' s result was compared with that of each control group respectively, there was sigcificant difference.2. Clinic research: Sensitivities for diagnosing BTCC in this study were 82.2% for urinary CYFRA21-1, 77. 8% for telomerase, 84. 4% for VEGF. compared to that of cytology(44. 4%), there was significant difference respectively(K0. 005); The sensitivity of combined determination was 95. 6%, there was significant difference compared to that of urinary telomerase(0.025 |
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