Study On The Ctla-4 Monoclonal Antibody On The Nude Mice That Were Bearing With T Lymphadenoma Jurkat Cells

Objective: Clinical trials have already proved, CD4+CD25+ regulatory T cells (CD4+CD25+regulatory T cell, Tr) is an inhibitory regulator of the immune cells, can inhibit the body's own tumor cells recognize tumor effector cell development and activation, in themediated immune tolerance and tumor immunity and tumor escape may play an important role in untreated non-Hodgkin's lymphoma (non-Hodgkin lymphoma, NHL) in patients with peripheral blood CD4+CD25+ Tr cell ratio was significantly higher in normal control group CD4+CD25+ Tr cell ratio compared with untreated patients with CD4+CD25+ Tr cell ratio decreased significantly after chemotherapy were complete remission (CR) and partial remission (PR) CD4+CD25+ Tr cell ratio and pre-treatment were significantly different. Peripheral blood of patients newly diagnosed NHL and the CD4+CD25+ Tr cell ratio and patient age, sex, PS status, clinical stage, histological type did not find significant correlation between.Through the results of the above can be considered, CD4+CD25+ Tr cell ratio is a better reflection of cellular immune function in reference to one of the indicators. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is the activation of T cell surface expression of a transmembrane glycoprotein molecules, and B7 and CD28 are both members of the immunoglobulin superfamily. Modern immunology study found that complete the initial T-cell activation requires a dual signal stimulation requires both T cell antigen receptor (TCR) provided by antigen-specific signal that T cells, TCR and the APC processing of antigen peptides-MHC complexes with (first signal), but also must have non-specific co-stimulatory antigens signal (second signal), if the lack of a second auxiliary signal, will allow T cells in clonal anergy (anergy) status. Although the CTLA-4 in activated T cell membrane expression levels of CD28 is only 1/30 ~ 1/50, but its affinity with the B7 is CD28 and B7 for more than 20 times, is an important T cell activation and regulation of molecules. CTLA-4 monoclonal antibody can block CTLA-4 and T-cell binding sites, thereby blocking T-cell activation, proliferation, in recent years, CTLA-4 monoclonal antibody as an effective treatment has demonstrated its broadapplication prospects. CTLA-4 monoclonal antibody in a variety of autoimmune diseases, transplant anti-rejection, gene therapy on a wide range of clinical research, but in malignant tumors, particularly hematologic malignancies is also rare in the application of research.This test by CTLA-4 monoclonal antibody in nude mice bearing lymphoma blocked CD4+ CD25+ Tr cell activation, proliferation, thus breaking the body's tolerance of tumor cells, inhibit tumor cell growth. At the same time explore the CTLA-4 monoclonal antibody in lymphoma cell proliferation, apoptosis and its possible mechanism for lymphoma targeted therapy to provide new ideas.Methods:1 Cell culture of JurkatJurkat was cultured in RPMI1640 medium, supplemented with 10% heated-inactivated fetal bovine serum, at 37℃in a humidified of 5% CO2 in-air atmosphere. The cells were passaged every two days.2 Construct human Jurkat cell xenografts in nude mice.The node mice of 5-week-old were radiated at 4Gy, after 3 days, Jurkat cells were injected in the BALB/c nude mice's right department of shoulder(3×107 once) to establish nude mouse xenograft model. When the tumor was grown enough, the node mice were killed and the tumor was removed and cut it up into little pieces for 2-3 mm under sterile condition, and hypo-injected them on the back of 10 5-week-oid node mice. 10 days later, the tumor was grown well, and divided into 2 groups randomly, experimental group and control group, each group were 5 node mice. When the volume of the tumor was grown at 100 mm3, and we started to treatment.3 Peritoneal injectionsThe mice were injected with CTLA-4 monoclonal antibody for 2 days.4 To measure the volume of the transplanted tumor after treatment. To measure the volume of the transplanted tumor, twice a week, and measured it for 4 weeks.5 To killed the node miceTo killed the node mice after treatment for 4 weeks, and removed the transplanted tumor divided into 5 parts, colleted peripheral blood for 1 ml and anticoagulant with EDTA-K2.6 Morphology of Jurkat cells in transplanted tumors were observed under optical microscope after HE stainingThe tumor tissues were treatment by fixation, dehydration, paraffin imbedding, and cutting sheet, HE staining, mounting, and then them were observed under optical microscope.7 Infiltration of CD4+ and CD8+ T cells in tumor tissues before and after CTLA-4 monoclonal antibody treatment from the mice were detected by immunohistochemistryThe tumor tissues were treatment by fixation, paraffin imbedding, and cutting sheet, SP staining. The assessment of positive was reference to the introduction of the reagent, the mon-antibody was replaced by PBS for the negative, as the Buffy intra-cellular was positive. Every section observed 5 Hp vf, each Hp vf was counted 200 cells, as the percentage of the positive was results.8 The expression of CD4+CD25+Tr in the peripheral blood before and after CTLA-4 monoclonal antibody treatment from the mice were detected with flow cytometryAnticoagulant blood 0.1ml, add CD4 and CD25 antibody, each for 20μl, incubation at room temperature for 30 min, and then add RBC lysate, the RBC was treatment by fixative and rupture of membrane reagent, the cell was detected by Expo 32 ADC.9 Expression ofβ-catenin, survivin, c-myc and cyclin D1 mRNA in Jurkat cells were detected by semi-quantitative RT-PCRExtract the total RNA of tumor tissue, identification of RNA purity, testing the integrity of RNA, primer design, all the RT-PCR primers were synthesized by Shanghai Bio-Engineering Company, total cellular RNA pre-heating, add reverse transcription-polymerase chain reaction (RT-PCR) (two-step) reverse transcription reaction to obtain cDNA. Further PCR amplification, PCR products were obtained, plus sample plus sample holes in the agarose gel, the electrophoresis 30 min, gel imaging system analysis and photographed images using Gel-Pro Analyzer 3.1 analysis software analysis of band optical Brightness, semi-quantitative analysis of their experiments, calculate the relative coefficient.10 Flow cytometry was used to measure cell apoptosis rate and cell cycles of Jurkat cellsPreparation of the nude mouse tumor tissue as a single cell suspension by flow cytometry on the cell cycle changes and apoptosis.11 To analysis statistic date use statistical software SPSS13.0Result:1 CTLA-4 monoclonal antibody could significantly inhibite the proliferation of Jurkat cells in vivo (P<0.01), the growth inhibition was in a time -dependent manner in one concentration.2 There were infiltration cells in the control group, the cell was moderately, the nuclear was round or in-regular round, karyokinesis was easy to see, but the experimental group were different from the control group by HE staining observed under optical microscope.3 CTLA-4 monoclonal antibody could reduced the ratio of the CD4+CD8+ cells in the tumor tissue. The infiltration of CD4 was (57.5±1.3%), and CD8+ was (31.7±1.1%)in the experimental group; the The infiltration of CD4 was (25.3±0.9%), and CD8+ was (18.4±0.7%)in the control group, there was statistically significant differences compared to the control group(P<0.05).4 CTLA-4 monoclonal antibody could reduced the ratio of the CD4+CD25+ Tr cells in the peripheral blood, the ratio of control group was (6.26±3.95)%, but the experimental group was(1.96±0.92)%, there was statistically significant differences compared to the control group(P<0.05).5 The results analized by semi-quantitative RT-PCR showed thatβ-catenin mRNA didn't have significant changes after Jurkat cells were treated with CTLA-4 monoclonal antibody for 4 weeks, there was no statistically significant differences compared to the control group (P>0.05). Butβ-catenin/TCF downstream target genes survivin, c-myc and cyclin D1 were down regulated (P<0.01).6 The results of FCM showed that CTLA-4 monoclonal antibody could arrest cell cycle of Jurkat cells at G0/G1 phase and induce cell apoptosis.Conclusion:1 CTLA-4 monoclonal antibody could significantly inhibite the proliferation of Jurkat cells in vivo, the growth inhibition was in a time -dependent manner in one concentration.2 CTLA-4 monoclonal antibody could reduce the infiltration of tumor cell in vivo.3 CTLA-4 monoclonal antibody could reduce the infiltration of CD4+CD8+ cells in the tumor tissue.4 CTLA-4 monoclonal antibody could reduce the ratio of CD4+CD25+ Tr cells in the peripheral blood.5 CTLA-4 monoclonal antibody could downregulate the expression of survivin, c-myc and cyclin D1 mRNA.6 CTLA-4 monoclonal antibody could arrest the Jurkat cells at G0/G1 phase, and increased the apoptosis of it.