沉默SOCS1联合rAAV/PSA基因修饰的树突状细胞疫苗治疗前列腺癌的实验研究  

Vaccination treatment of prostate cancer by dendritic cells with silenced suppressor of cytokine signaling 1 and infected with recombinant adeno-associated virus/prostate specific antigen

作　　者：宋浩杰[1] 宋琴 施为建[1] 吕成伟[2] 蒋凤莲[1] 王丽[1] 李全泳[1] SONG Haojie;SONG Qin;SHI Weijian(Department of Oncology,Boai Hospital of Zhongshan,Guangdong,Zhongshan 528403,China)

机构地区：[1]广东省中山市博爱医院肿瘤科,528403 [2]南方医科大学南方医院临床医学实验研究中心

出　　处：《河北医药》2023年第20期3056-3060,共5页Hebei Medical Journal

基　　金：国家自然科学基金项目(编号:81071847);广东省科技计划项目(编号:2008B030301156);广东省中山市医学科研项目(编号:2021A020054)。

摘　　要：目的研究沉默细胞因子信号抑制因子1(suppressor of cytokine signaling 1,SOCS1)基因联合rAAV/PSA基因修饰的树突状细胞(dendritic cell,DC)对前列腺癌LNCaP细胞的体外杀伤效应。方法应用重组腺相关病毒(rAAV)介导的RNA干扰技术沉默人DC的SOCS1基因的表达,行Western blot检测基因沉默效果。rAAV-shRNA-SOCS1和rAAV/PSA感染DC,将DC细胞分为Control-DC组、rAAV/PSA-DC组、SOCS1(-)+rAAV/PSA-DC组。采用系列细胞因子诱导DC成熟,诱导细胞毒性T淋巴细胞(CTL)。流式细胞术检测分析各组DC细胞表型及CTL细胞的免疫表型;ELISA法检测各组DC细胞培养上清中细胞因子白介素-10(IL-10)及IL-12 p70的分泌水平,各组CTL细胞释放γ-干扰素(IFN-γ)的水平;LDH法检测各组CTL细胞对前列腺癌LNCaP靶细胞的杀伤效率。结果rAAV-shRNA-SOCS1感染DC后,可有效下调DC的SOCS1表达水平。与对照组比较,沉默SOCS1联合rAAV/PSA基因修饰的DC细胞培养上清中细胞因子IL-12 p70的分泌水平显著增高,IL-10的分泌水平明显下降(P<0.05);该组DC表面分子CD80、CD83和CD86的表达明显上调(P<0.05)。该组DC诱导的CTL中CD8^(+)、CD8^(+)CD69^(+)T细胞的比例明显增高,而CD4^(+)T细胞、CD4^(+)CD25^(+)FoxP3^(+)Treg细胞的比例显著降低(P<0.05),IFN-γ的释放水平明显增高(P<0.05),对PSA阳性的前列腺癌靶细胞LNCaP具有更强的杀伤活性(P<0.05),且具有抗原特异性。结论沉默SOCS1联合rAAV/PSA基因修饰的DC疫苗可以产生高效而特异性的抗前列腺癌免疫效应。Objective To study the cell killing effect of dendritic cells(DCs)with silenced SOCS1(suppressor of cytokine signaling 1)and infected with recombinant adeno-associated virus(rAAV)/prostate specific antigen(PSA)on the in vitro prostate cancer cell line LNCaP.Methods SOCS1 in human DCs was silenced by rAAV-mediated RNA interference,and the silencing effect was detected by Western blot.DCs were infected with negative control,rAAV/PSA or rAAV-shRNA-SOCS1 plus rAAV/PSA lentivirus.A series of cytokines were used to induce DC maturation and cytotoxic T lymphocytes(CTLs).The immunophenotypes of DCs and CTLs in each group were analyzed by flow cytometry.Relative levels of cytokines interleukin-10(IL-10)and interleukin-12p70(IL-12p70)in the cell supernatant of DCs,and the level of Interferon-γ(IFN-γ)released by CTLs were detected by enzyme-linked immunosorbent assay(ELISA).The cytotoxicity of CTLs against LNCaP cells was tested by lactate dehydrogenase(LDH)assay.Results The rAAV-shRNA-SOCS1 infection significantly downregulated SOCS1 in human DCs.Compared with those of the control group,significantly higher level of IL-12p70 and lower IL-10,as well as the upregulated surface markers CD80,CD83 and CD86 were detected in DCs transfected with rAAV-shRNA-SOCS1 plus rAAV/PSA lentivirus(all P<0.05).After transfection of rAAV-shRNA-SOCS1 plus rAAV/PSA lentivirus in DCs,the proportion of CD8^(+)and CD8^(+)CD69^(+)T cells in CTLs was significantly elevated,and that of CD4^(+)T cells,CD4^(+)CD25^(+)FoxP3^(+)Tregs was significantly reduced(P<0.05).Moreover,the released level of IFN-γwas significantly enhanced,and the killing effect on PSA-positive LNCaP cells was more pronounced with the antigen specificity(all P<0.05).Conclusion DC vaccine with silenced SOCS1 and rAAV/PSA infection can induce effective and antigen-specific antitumor immunity effect against prostate cancer cells in vitro.

关 键 词：细胞因子信号抑制因子1 基因沉默 腺相关病毒 前列腺癌特异性抗原 前列腺癌 疫苗 

分 类 号：R737.25[医药卫生—肿瘤]