树突状细胞感染PSMA/4-1BBL基因重组腺病毒后的免疫功能变化

Changes in immune function of dendritic cells infected by recombinant adenovirus containing PSMA and 4-1BBL genes

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 changes in immune function of dendritic cells（DCs） infected by recombinant adenovirus containing human prostate specific membrane antigen（PSMA） and 4-1BB ligand （4-1BBL） genes.Methods： The empty Ad-GFP infected immature DCs at different multiplicities of infection（MOI）,and the optimal MOI was determined.DCs from peripheral blood of healthy volunteers were infected with recombinant adenoviruses at the optimal MOI.The DCs were allocated into five groups： co-infected group（Ad-PSMA/4-1BBL-DC）,PSMA-infected group（Ad-PSMA-DC）,4-1BBL-infected group（Ad-4-1BBL-DC）,negative control group（Ad-GFP-DC） and non-infected group.Morphological changes of DCs were observed under a fluorescence microscope;and the phenotype changes of CD80,CD83,CD86 of tranfected DCs were detected by FACS,and the expressions of PSMA and 4-1BBL proteins was detected by Western blotting.IL-12 concentrations of DC supernatant in different groups were measured by ELISA.Allogenic T cell proliferations stimulated with DCs in different groups and the cytotoxic effect on prostate cancer cell lines LNCap,Du145 and 22RV were analyzed by CCK-8 assay.Results： The optimal MOI was 200.The immunophenotype expressed in the co-infected DC group,such as CD80,CD83,CD86,HLA-DR,was significantly higher than that expressed in the other DC groups（[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]%;P0.05）.The secretion of IL-12 in supernatant was greatly higher in the co-infected group than that in single-infected group or non-infected DC group（[134.29±2.22] vs [79.51±1.60],[70.33±1.13],[69.67±1.43],[28.88±2.97] pg;P0.05）.At the same rate of DCs to T,the co-infected group had a more significant increase in allogenic T cell proliferation than other single-infected group or non-infected group（P0.05）.The cytotoxic rate of PSMA/4-1BBL-DC-CTL against PSMA positive target LNCap cells was significantly stronger than that against PSMA negative target cells such as DU145 and 22RV cells（P0.05）,and also higher than that of PSMA-DC-CTL and 4-1BBL-DC-CTL（P0.05）.Conclusion： Ad-PSMA/4-1BBL-infected DCs not only own high secretion of IL-12,but also stimulate and enhance the cytotoxic effect of tumor specific CTLs against PSMA positive prostate cancer cells which highly express PSMA protein.In addition,the DCs infected with Ad-PSMA and Ad-4-1BBL induce more effective tumor specific CTLs than single-infected DCs.