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目的建立弥漫大B细胞淋巴瘤(diffuse large B lymphoma,DLBCL)小鼠模型,在肿瘤发生发展阶段中给予Th17细胞过继免疫治疗,检测肿瘤组织中趋化因子CXCL6/CCL2的表达,了解Th17细胞对DLBCL肿瘤微环境中CXCL6/CCL2表达的影响,及其与DLBCL发生发展的相关性。方法人生发中心B细胞(germinal center B cell,GCB)样DLBCL细胞株SUDHL-4进行传代培养后接种10只SCID小鼠建立DLBCL小鼠模型,观察小鼠肿瘤发生发展过程并得出DLBCL小鼠肿瘤中位发病时间T1及小鼠中位生存时间T2。体外培养Th17细胞,在接种肿瘤细胞株时予30只DLBCL小鼠注射Th17细胞行过继免疫,并分别于T0(接种Th17细胞后)、T1和T2这3个时间点处死小鼠,获得A0、A1和A2组,每组10只小鼠。实时定量PCR法检测DLBCL小鼠肿瘤组织CXCL6/CCL2表达,与同一时间点15只小鼠的生理盐水对照组(B0、B1和B2组,每组5只)进行对比,并对比T0、T1和T2这3个时间点各组CXCL6/CCL2的表达。结果 A0组、A1组和A2组CXCL6mRNA表达量分别为0.115±0.021、0.657±0.142和0.935±0.322,差异有统计学意义,F=7.013,P=0.025。B0组、B1组和B2组CXCL6mRNA表达量分别为0.175±0.024、0.321±0.011和0.631±0.027,差异有统计学意义,F=6.007,P=0.008。A0组CXCL6mRNA表达量与B0组对比差异无统计学意义,t=2.03,P=1.25;A1组明显高于B1组,t=6.43,P<0.01;而A2组也明显高于B2组,t=6.35,P<0.01。A0组、A1组和A2组CCL2mRNA表达量分别为0.133±0.015、0.654±0.534和0.928±0.322,差异有统计学意义,F=7.021,P=0.023。B0组、B1组和B2组CCL2mRNA表达量分别为0.124±0.013、0.327±0.025和0.628±0.322,差异有统计学意义,F=6.005,P=0.007。A0组CCL2mRNA表达量与B0组对比差异无统计学意义,t=1.98,P=1.75;A1组明显高于B1组,t=6.13,P<0.01;A2组也明显高于B2组,t=7.59,P<0.01。结论随着DLBCL肿瘤进程,CXCL6和CCL2的表达上升,CXCL6和CCL2可能是影响DLBCL肿瘤发生发展的因子,而Th17过继免疫治疗可以上调DLBCL肿瘤组织CXCL6和CCL2的表达,可能会对DLBCL肿瘤的发生发展产生影响。
Objective To establish a murine model of diffuse large B lymphoma (DLBCL) and to provide adoptive immunotherapy with Th17 cells in the developmental stage of tumorigenesis to detect the expression of chemokine CXCL6 / CCL2 in tumor tissues and to understand the effect of Th17 cell pair CXCL6 / CCL2 expression in DLBCL tumor microenvironment, and its relationship with the occurrence and development of DLBCL. Methods The germinal center B cell (DLBCL) cell line SUDHL-4 was subcultured and then inoculated with 10 SCID mice to establish DLBCL mouse model. The development and progression of the mouse tumor were observed and the expression of DLBCL mouse Tumor median time to onset T1 and median survival time T2 in mice. Th17 cells were cultured in vitro. Th17 cells were injected intraperitoneally with Th17 cells injected with tumor cell lines, and mice were sacrificed at T0 (after Th17 cells inoculation) and T1 and T2 respectively to obtain A0, Groups A1 and A2, 10 mice per group. Real-time quantitative PCR was used to detect the expression of CXCL6 / CCL2 in tumor tissues of DLBCL mice and compared with saline control group (B0, B1 and B2, 5 mice in each group) of 15 mice at the same time point. T0, T1 and T2 the three time points in each group CXCL6 / CCL2 expression. Results The expressions of CXCL6 mRNA in A0, A1 and A2 groups were 0.115 ± 0.021,0.657 ± 0.142 and 0.935 ± 0.322, respectively, with a significant difference (F = 7.013, P = 0.025). The expressions of CXCL6 mRNA in B0, B1 and B2 groups were 0.175 ± 0.024,0.321 ± 0.011 and 0.631 ± 0.027, respectively, with a significant difference (F = 6.007, P = 0.008). The expression of CXCL6 mRNA in A0 group was not significantly different from that in B0 group, t = 2.03, P = 1.25; A1 group was significantly higher than B1 group, t = 6.43, P <0.01; = 6.35, P <0.01. The expression levels of CCL2 mRNA in A0, A1 and A2 groups were 0.133 ± 0.015, 0.654 ± 0.534 and 0.928 ± 0.322, respectively, with a significant difference (F = 7.021, P = 0.023). The expression levels of CCL2 mRNA in B0, B1 and B2 groups were 0.124 ± 0.013,0.327 ± 0.025 and 0.628 ± 0.322, respectively, with a significant difference (F = 6.005, P = 0.007). The expression of CCL2 mRNA in A0 group was not significantly different from that in B0 group (t = 1.98, P = 1.75); A1 group was significantly higher than B1 group, t = 6.13, P <0.01; 7.59, P <0.01. Conclusions With the progression of DLBCL, the expressions of CXCL6 and CCL2 are up-regulated. CXCL6 and CCL2 may be the factors affecting the development of DLBCL. Th17 adoptive immunotherapy may up-regulate the expressions of CXCL6 and CCL2 in DLBCL and may affect the occurrence of DLBCL Development has an impact.