肿瘤微环境中IL-10限制SOCS1基因沉默的树突状细胞疫苗的抗肿瘤作用  

Inhibiting effect of IL-10 in tumor microenvironment on anti-tumor activity of SOCS1-silenced DC vaccine

作　　者：王智华[1] 尤红煜[2] 宋淑霞[2] 

机构地区：[1]河北省人民医院老年病重点实验室,河北石家庄050012 [2]河北医科大学免疫教研室,河北省实验动物重点实验室,河北石家庄050017

出　　处：《细胞与分子免疫学杂志》2013年第4期379-383,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81071710)

摘　　要：目的观察SOCS1基因沉默的DC疫苗对荷黑素瘤小鼠的抗肿瘤作用及肿瘤微环境中IL-10对该DC疫苗抗瘤作用的影响。方法从小鼠股骨分离骨髓细胞,GM-CSF和IL-4联合诱导DCs分化,然后感染携带沉默SOCS1基因的Len-SOCS1-shRNA慢病毒;用TRP2抗原肽负载沉默SOCS1的DC细胞制备DC疫苗,LPS诱导成熟,流式细胞仪分析DC细胞表面MHCⅡ和CD86的表达,real-time PCR分析该DC细胞的SOCS1、IL-10和IL-12p40表达。用B16细胞或降低IL-10基因表达的B16(B16-IL-10-/-)制备荷瘤小鼠模型,瘤内注射DC疫苗(3×106/只),观察肿瘤生长和荷瘤小鼠生存期。采用不连续梯度离心法分离肿瘤浸润淋巴细胞,流式细胞仪观察CD8+T细胞的分布;并采用微量细胞毒的方法分析CTL活性。结果 Len-SOCS1-shRNA慢病毒感染DC后,可使SOCS1表达下调80%;下调SOCS1表达的DC细胞MHCⅡ和CD86表达有增加趋势,但与对照组DC相比无明显差异;下调SOCS1表达可降低DC细胞的IL-10表达,提高IL-12p40的表达。沉默SOCS1的DC疫苗对B16荷瘤小鼠的生存率没有明显影响,但可显著提高B16-IL-10-/-荷瘤小鼠的生存率(P<0.05)。下调SOCS1表达的DC疫苗不仅可提高B16-IL-10-/-荷瘤小鼠肿瘤浸润CD8+T淋巴细胞数,还可促进CD8+T细胞IFN-γ的分泌及CTL活性。结论沉默SOCS1可提高DC疫苗的活性,但肿瘤微环境的IL-10依然是限制该DC疫苗有效发挥抗瘤作用的因素。Objective To observe the anti-tumor effect of suppressors of cytokine signaling 1 （SOCS1）-silenced dentritic cell （DC） vaccines in melanoma-bearing mice, and the influence of IL-10 in the tumor microenvironment on DC vaccine action. Methods To obtain SOCSt-silenced DCs, DCs derived from mouse bone marrow cells ex vivo were induced to differentiation in the presence of granulocyte-macrophage colony-stimulating factor （GM-CSF） and IL-4, and then transduced with Len-SOCSt-shRNA or control Len-GFP lentiviruses. The SOCSt-silenced DCs were loaded by TRP2 peptide to prepare the DC vaccine, which was induced to mature by LPS. The DCs were analyzed by flow cytometry （FCM） for surface expressions of MHC I1 and CD86 and by real-time PCR for the expressions of SOCS1, IL-t0 as well as IL-12p40. B16 or IL-10- silenced B16 （IL-10-/-） cells were inoculated into C57BL/6 mice. Five days later, the mice were randomly divided into 3 groups （PBS-DC, Len-DC and SOCSt-shRNA-DC groups） and injected with 1 × 106/100 μL per mouse of the transduced DCs or PBS-DCs. We observed the tumor growth and the survival of the tumor-bearing mice. Tumor-infiltrating leukocytes （TIL） were isolated from tumor tissues using the discontinuous gradient centrifugation and the distribution of CD8 ＋T was analyzed by FCM; IFN-γ secretion and CTL activity were detected by the ELISpot and the standard microcytotoxicity assay, respectively. Results SOCSt expression in DCs was down-regulated by 80% after Len-SOCSI-shRNA lentivirus infection. In the DCs with down-regulated SOCS1 expression, the expressions of MHC I1 and CD86 increased a little, which did not differ significantly from the control DCs, and IL-10 level dropped and IL-12p40 went up significantly compared with the control DCs. There was no any effect of SOCS]-silenced DCs on the survival of melanoma-bearing mice, however, the survival of B16-1L-10-/--bearing mice was promoted（ P 〈 0.05）. The further investigation showed that SOCSI-shRNA DCs raised the number of CD

关 键 词：细胞因子信号抑制因子1 基因沉默 树突状细胞 IL-10 

分 类 号：R392.12[医药卫生—免疫学] R739.5[医药卫生—基础医学]