regulatory T cells in local immune microenvironment of renal cell carcinoma negative regulation and its mechanism

Title: regulatory T cells in local immune microenvironment of renal cell carcinoma negative regulation and its mechanismAuthor: LI Jin-fengDegree-granting units: Fudan UniversityKeywords: regulatory: 5418, eukaryotic expression plasmids: 3892, renal cell carcinoma: 3315, regulation: 3141, Mechanism: 3043, renal cell carcinoma: 2807 transfection: 2451, cancer occurred: 2280, negative : 1773, high expression: 1769Summary:The first part of the kidney with local tumor Treg tumor prognosis and tumor COX-2 Expression inPurpose of this section from the cellular level to understand the local immune microenvironment in the kidney T lymphocytes subtypes, the number of Magnetic lifter clinical indicators of renal carcinoma and prognosis, and tumors with COX-2 expression correlation analysis, of local tumor regulatory T cells increased the possible mechanism.Methods This study collected in January 1999 - December 2005 in Zhongshan Hospital, Fudan University underwent radical nephrectomy or partial nephrectomy for renal cell carcinoma patient data, including randomly selected complete follow-up data of 125 cases of renal cell paraffin , by means of high-throughput tissue microarray and immunohistochemical staining of tumor with adjacent micro-environment CD4 ~ + T cells, CD8 ~ + T cells, FOXP3 ~ + regulatory T cells, GranzymeB ~ +-activated the number of cytotoxic T cells, COX-2 expression and tumor of the clinical parameters and prognosis, and to explore the COX-2 expression and regulatory T cells in correlation.Results (1) infiltration of different immune cells in patients with different forms of kidney cancer cells to spread or aggregate form, with adjacent lymphocytic infiltration is more apparent. (2) Univariate survival analysis showed that age, gender and kidney cancer prognosis, tumor and adjacent CD4 ~ + T cells, CD8 ~ + T cells, Granzyme B ~ + activated cytotoxic T http://www.999magnet.com/products/131-magnetic-lifter cells and prognosis in renal cell carcinoma nothing to do; while the adjacent regulatory T cells increased with the overall survival and disease progression-free survival was significantly negatively correlated (P <0.001, P <0.001), tumors with high expression of COX-2 but also with overall survival and disease progression-free survival rate was significantly negatively correlated (P = 0.021, P = 0.027). However, regulatory T cells in the tumor, adjacent kidney COX-2 expression and prognosis. Tumor TNM stage, tumor nuclear grade and tumor size were associated with renal prognosis. (3) multivariate Cox survival analysis showed that adjacent regulatory T cells increased, increased tumor TNM stage (Ⅲ, Ⅳ period), increased tumor nuclear grade (Grade 3 and 4) and tumor diameter increased (≥ 7cm) tips kidney cancer prognosis. Correlation analysis showed that the adjacent number of regulatory T cells and tumor TNM stage and tumor diameter was positively correlated (P = 0.001, P = 0.002), tumor Granzyme B ~ + T cell number and tumor nuclear grade was positively correlated (P = 0.024), while the level of COX-2 expression and tumor TNM stage and tumor nuclear grade was positively correlated (P = 0.018, P = 0.013); adjacent regulatory T cells and tumor COX-2 expression was positively correlated (P <0.001 .)Conclusion The results show that kidney cancer and adjacent regulatory T cells increased with increasing tumor TNM stage (Ⅲ, Ⅳ period), increased tumor nuclear grade (Grade 3 and 4) and tumor diameter increased (≥ 7cm) can be used as kidney cancer, an independent indicator of poor prognosis. Adjacent regulatory T cells and tumor COX-2 expression was positively correlated, based on the source of PGE_2 COX-2 can induce non-regulatory T cells to regulatory T cell transformation, the kidney may be induced by the same mechanism to generate regulatory T cells , thus contributing to tumor immune escape.The second part of the kidney source and mechanism of local Treg2.1 pcDNA3.1-hCOX-2 eukaryotic expression plasmidTo construct pcDNA3.1-hCOX-2 eukaryotic expression plasmid.Methods pSG5-COX-2 as a template, PCR to obtain full-length human COX-2cDNA, EcoRI, BamHI double digestion was cloned into eukaryotic expression vector pcDNA3.1, the restriction enzyme digestion and DNA sequence analysis Analysis of recombinant plasmid.Results The recombinant eukaryotic expression plasmid pcDNA3.1-hCOX-2 by restriction enzyme digestion, electrophoresis shows about hCOX-2 1800bp fragment and the 5.4kb fragment of the pcDNA3.1 vector, sequencing confirmed that the fragment 1800bp Gene-bank registered with the COX-2 in the same sequence.Conclusion We successfully constructed pcDNA3.1-hCOX-2 eukaryotic expression plasmid can be used for subsequent transfection.2.2 pcDNA3.1-hCOX-2 in renal cancer cell lines transfected with COX-2 expression and the resulting supernatant PGE_2 enhancementObjective To determine the common renal cancer cell lines COX-2 expression, the selection of high COX-2 expression in renal cancer cell lines transfected into the follow-up experiments; the pcDNA3.1-hCOX-2 eukaryotic expression plasmid liposome lipofectamine 2000 transfection COX -2 High expression of renal cancer cell lines, understanding of COX-2 mRNA and protein effects, especially for tumor supernatant PGE_2 secretion; transfected with different doses were added to NS-398, to understand their supernatants PGE_2 secretion.Method (1) the introduction of two common kidney cancer cell line :786-0 and OS-RC-2, were cultured in vitro. Semi-quantitative RT-PCR of renal cancer cell lines was determined two kinds of COX-2 mRNA expression levels; Western-blot method of determination of two kinds of kidney cancer cell lines the expression of COX-2 protein levels. (2) pcDNA3.1-hCOX-2 eukaryotic expression plasmid transfected by liposome lipofectamine2000 high COX-2 expression in renal cancer cell line OS-RC-2; 72 hours after transfection, semi-quantitative RT-PCR determination renal carcinoma cell line OS-RC-2 in COX-2 mRNA expression levels, Western-blot of COX-2 protein levels, to determine the transfection efficiency. (3) ELISA method for detection of renal cancer cell lines transfected with OS-RC-2 secretion of PGE_2 transfection groups were added to 12.5μM, 25μM, 50μM, 100μM NS-398, ELISA method to detect the content of the supernatant PGE_2 change.Results (1) the two renal cancer cell lines COX-2mRNA and protein expression were different. Semi-quantitative RT-PCR determination of the results: in two renal cancer cell lines COX-2/beta-actin compare ,786-0 and OS-RC-2 ratios were 0.2756 ± 0.01318,0.4667 ± 0.02159, the former for the latter expression of 59.1%, resulting in statistically significant difference (P <0.05). Western-blot determination results: in two renal cancer cell lines COX-2/GAPDH compare ,786-0 and OS-RC-2 ratios were 0.2333 ± 0.02183,0.4686 ± 0.04294, the former for the latter expression of the 50 %, resulting in statistically significant difference (P <0.05). Subsequent transfection experiments selected renal cancer cell line OS-RC-2. (2) renal carcinoma cell line OS-RC-2 transfected with pcDNA3.1-hCOX-2 eukaryotic expression plasmid COX-2mRNA and protein expression were significantly increased. Semi-quantitative RT-PCR results: 72 hours after transfection of COX-2 mRNA in the transfected group, idling and control groups COX-2/beta-actin ratios were 0.7922 ± 0.07139,0.4325 ± 0.02045,0.4739 ± 0.02377, twenty-two comparison showed that transfection group was significantly higher than the idling and control groups, statistically significant difference (P <0.05); the latter two no more statistically significant difference (P> 0.05). Western-blot determination results: 72 hours after transfection of COX-2 protein in transfected group, idling and control groups COX-2/GAPDH ratios were 0.8593 ± 0.1332,0.4321 ± 0.03362,0.4533 ± 0.05883, pairwise comparison showed that transfection group was significantly higher than the idle group and control group, statistically significant difference (P <0.05); after statistically no significant difference between the two (P> 0.05). (3) ELISA detected in transfected tumor cells secreted significantly higher than the supernatant PGE_2 idle and control groups, respectively, 6.330 ± 1.181,0.2817 ± 0.03181,0.3030 ± 0.03246, pairwise comparison showed that transfection group was significantly higher than idling and control groups, statistically significant difference (P <0.05); after statistically no significant difference between the two (P> 0.05). Application of transfected NS-39812.5μM, 25μM, 50μM, 100μM after, PGE_2 contents were 2.906 ± 0.5892,0.6484 ± 0.09880,0.4189 ± 0.06513,0.2221 ± 0.04094 ng / ml, pairwise comparison showed that transfection group was significantly higher than transfected +12.5 μMNS-398 group, transfected +25 μMNS-398 group, transfected +50 μMNS-398 group, transfected +100 μMNS-398 group, statistically significant difference (P <0.05).Conclusion (1) renal cancer cell lines 786-0 and OS-RC-2 are COX-2 expression, but the former, whether COX-2 expression at the mRNA level or protein levels were lower than the latter, subsequent transfection experiments OS-choice RC-2 as target cells. (2) constructed pcDNA3.1-hCOX-2 eukaryotic expression plasmid by liposome lipofectamine 2000 transfection renal carcinoma cell line OS-RC-2, OS-RC-2 increase in COX-2 expression levels. (3) pcDNA3.1-hCOX-2 eukaryotic expression plasmid enhances renal carcinoma cell line transfected with OS-RC-2 supernatant PGE_2 secretion, and the application of NS-398 in a dose-dependent reduction in OS-transfected supernatant PGE_2 RC-2 secretion.2.3 pcDNA3.1-hCOX-2 kidney cell lines transfected with supernatant-induced CD4 ~ + FOXP3 ~-T cells into TregObjective renal carcinoma cell line transfected with OS-RC-2 supernatants whether cells induce CD4 ~ + FOXP3T to CD4 ~ + FOXP3 ~ + regulatory T cell transformation.Method of density gradient centrifugation to obtain peripheral blood mononuclear cells, immunomagnetic cell sorting to obtain CD4 ~ + CD25 ~ - effector T cells and CD4 ~ + CD25 ~ + regulatory T cells, RT-PCR and FACS detection of cell sorting Purity. anti-CD3/CD28 antibodies and inactivated APC cell activation conditions, the separation of CD4 ~ + CD25 ~-T cells were positive with PGE_2 control group, transfection group, +100 μM transfected with control antibody, transfection +100 μMNS- 398, the negative control group of supernatant were incubated 96 hours, FACS detection of cells in each group in the CD4 ~ + FOXP3 ~ + regulatory T cells.Results (1) Immunomagnetic cell sorting to obtain stable CD4 ~ + CD25 ~ - effector T cells and CD4 ~ + CD25 ~ + regulatory T cells, both purity greater than 90%, which is gene-specific expression of FOXP3 and protein. (2) supernatant of transfected cells can be significantly induced CD4 ~ + FOXP3 ~-T cells to CD4 ~ + FOXP3 ~ + regulatory T cell transformation, 96 hours after the CD4 ~ + FOXP3 ~ + regulatory T cells accounts for CD4 ~ + T cell ratio was 30.00 ± 2.618%, with the transfection supernatant +100 μMNS-398 group were incubated CD4 ~ + FOXP3 ~ + regulatory T cells to produce significantly lower 96 hours after the CD4 ~ + FOXP3 ~ + regulatory T cells accounted for CD4 ~ + T cell ratio of only 7.990 ± 1.227%.Conclusion (1) immunomagnetic beads cell sorting to obtain stable CD4 ~ + CD25 ~ - effector T cells and CD4 ~ + CD25 ~ + regulatory T cells, because CD4 ~ + CD25 ~ + regulatory T cell-specific gene expression of FOXP3 and protein, some follow-up to CD4 ~ + FOXP3 ~ + regulatory T cells represent a CD4 ~ + CD25 ~ + regulatory T cells, CD4 ~ + CD25 ~ - effector T cells in FOXP3 expression was negative, follow-up part of the CD4 ~ + FOXP3 ~ -T cells represent CD4 ~ + CD25 ~ - effector T cells. (2) supernatant of transfected renal carcinoma cell line induced CD4 ~ + FOXP3 ~-T cells to CD4 ~ + FOXP3 ~ + regulatory T cell transformation, and NS-398 can be reduced transfected cell supernatant-induced renal regulatory T cells to produce, suggesting that COX-2 inhibitors can reduce the generation of regulatory T cells, induced anti-tumor immune response to local adjuvant treatment of kidney cancer.Degree Year: 2009