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目的:观察复制缺陷型腺病毒介导的人前列腺特异性膜抗原(prostate specific membrane antigen,PSMA)基因和肿瘤坏死因子配体超家族成员4-1BB配体(4-1BB ligand,4-1BBL)基因体外共同感染的树突状细胞(dendritic cell,DC)的免疫学功能变化。方法:Ad-GFP空载体感染未成熟DC,确定最适MOI。将感染的健康志愿者外周血来源DC分为Ad-PSMA/4-1BBL-DC组(共感染组)、Ad-PSMA-DC组、Ad-4-1BBL-DC组、Ad-GFP-DC组以及未感染DC组,荧光倒置显微镜观察DC的形态学变化,流式细胞仪分析CD80、CD83、CD86等表型变化,Western blotting检测DC上PSMA和4-1BBL蛋白的表达,ELISA法检测各组DC上清的IL-12水平,CCK-8法检测各组DC刺激自体T淋巴细胞增殖能力及对人前列腺癌细胞株LNCap、Du145和22RV的细胞毒作用。结果:确定最佳MOI为200。共感染组DC表面CD80、CD83、CD86、HLA-DR共刺激分子均上调,明显高于未感染DC组[(38.72±0.99)%、(44.65±0.77)%、(63.60±0.75)%、(62.25±0.58)%vs(28.27±1.04)%、(28.08±1.16)%、(41.05±1.33)%、(46.87±1.12)%;P<0.05]。共感染组IL-12分泌水平明显高于单个基因重组腺病毒感染组及未感染组[(134.29±2.22)vs(79.51±1.60)、(70.33±1.13)、(69.67±1.43)、(28.88±2.97)pg;P<0.05]。DC∶T同一比例下,共感染组刺激自体T细胞增殖能力明显高于单感染组及未感染组(P<0.05)。PSMA/4-1BBL-DC-CTL对PSMA阳性的LNCap细胞的杀伤率明显高于对另两种PSMA阴性细胞DU145、22RV的杀伤率(P<0.05),也明显高于PSMA-DC-CTL组、4-1BBL-DC-CTL组的杀伤率(P<0.05)。结论:Ad-PSMA/4-1BBL感染后,DC不但高分泌IL-12,还能刺激和增强肿瘤特异性CTL对PSMA阳性前列腺癌细胞的杀伤作用;Ad-PSMA和Ad-4-1BBL共感染DC较单一感染DC能更有效诱导肿瘤特异性CTL。
OBJECTIVE: To observe the effect of replication-deficient adenovirus-mediated prostate specific membrane antigen (PSMA) gene and 4-1BB ligand (4-1BB ligand) on tumor necrosis factor ligand superfamily Changes of immunological function of dendritic cells (DCs) co-infected with genes in vitro. Methods: Ad-GFP empty vector was infected with immature DC to determine the optimal MOI. The peripheral blood DC from infected healthy volunteers were divided into Ad-PSMA / 4-1BBL-DC group (co-infected group), Ad-PSMA-DC group, Ad-4-1BBL-DC group and Ad-GFP-DC group The morphological changes of DCs were observed under fluorescent inverted microscope. The phenotypic changes of CD80, CD83 and CD86 were analyzed by flow cytometry. The expressions of PSMA and 4-1BBL protein were detected by Western blotting. DC supernatant IL-12 levels, CCK-8 method to detect DC stimulation of autologous T lymphocyte proliferation and the human prostate cancer cell lines LNCap, Du145 and 22RV cytotoxicity. Results: Determine the best MOI of 200. The CD80, CD83, CD86 and HLA-DR co-stimulatory molecules in the co-infected group were significantly higher than those in the non-infected DC group (38.72 ± 0.99%, 44.65 ± 0.77%, 63.60 ± 0.75%, 62.25 ± 0.58)% vs (28.27 ± 1.04)%, (28.08 ± 1.16)%, (41.05 ± 1.33)%, (46.87 ± 1.12)% respectively; P <0.05]. The level of IL-12 secretion in co-infected group was significantly higher than that in single-gene-infected group and non-infected group [(134.29 ± 2.22) vs (79.51 ± 1.60), (70.33 ± 1.13), (69.67 ± 1.43), (28.88 ± 2.97) pg; P <0.05]. Under the same ratio of DC: T, the ability of stimulating autologous T cells proliferation in co-infected group was significantly higher than those in single infection group and non-infected group (P <0.05). The cytotoxicity of PSMA / 4-1BBL-DC-CTL on PSMA-positive LNCap cells was significantly higher than that of PSMA-DC-CTL on the other two PSMA-negative cells DU145 and 22RV (P <0.05) , 4-1BBL-DC-CTL group killing rate (P <0.05). CONCLUSION: After Ad-PSMA / 4-1BBL infection, DC not only secretes IL-12, but also stimulates and enhances cytotoxicity of tumor-specific CTL on PSMA-positive prostate cancer cells. Infection with Ad-PSMA and Ad-4-1BBL DC can induce tumor-specific CTLs more effectively than single-infected DCs.