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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 antitumor effect of DC vaccine silenced by SOCS1 gene on mice bearing melanoma and the anti-tumor effect of IL-10 on the anti-tumor effect of DC vaccine in tumor microenvironment. Methods DCs were isolated from mouse femurs and GM-CSF and IL-4 cells were induced to differentiate into DCs. Lentivirus was transfected into Len-SOCS1-shRNA lentivirus with Silencing SOCS1 gene. DC vaccine containing SOCS1 silencing was loaded with TRP2 antigen peptide. Induction of maturation, flow cytometry analysis of DC cell surface MHC Ⅱ and CD86 expression, real-time PCR analysis of DC cells SOCS1, IL-10 and IL-12p40 expression. The tumor-bearing mice model was established by B16 cells or B16 (B16-IL-10 - / -) which reduced the expression of IL-10 gene, and the DC vaccine was injected intratumorally (3 × 106 cells / Survival. Tumor infiltrating lymphocytes were separated by discontinuous gradient centrifugation. The distribution of CD8 + T cells was observed by flow cytometry. The cytotoxic activity of CTLs was analyzed by using cytotoxic method. Results After Len-SOCS1-shRNA lentivirus infection, the expression of SOCS1 was down-regulated by 80%. The expression of MHCⅡ and CD86 in DCs with SOCS1 down-regulation was increased, but there was no significant difference compared with that in control group. Down-regulation of SOCS1 expression was decreased IL-10 expression of DC cells, increasing the expression of IL-12p40. Silencing SOCS1 DC vaccine had no significant effect on the survival rate of B16 tumor-bearing mice, but significantly increased the survival rate of B16-IL-10 - / - tumor-bearing mice (P <0.05). DC vaccine that down-regulated SOCS1 expression can not only increase the number of tumor infiltrating CD8 + T lymphocytes in B16-IL-10 - / - bearing mice, but also promote the secretion of IFN-γ and CTL activity of CD8 + T cells. Conclusion Silencing SOCS1 can enhance the activity of DC vaccine. However, IL-10 in tumor microenvironment remains a factor that limits the effective anti-tumor effect of this DC vaccine.