PSMA/4-1BBL共转染DC疫苗联合CIK细胞体外抑制前列腺癌的实验研究

In vitro induction of anti - tumor effect against prostate cancer by dendritic cells transfected with PSMA and 4 - 1BBL recombinant adenoviruses and CIK cells

目的:本研究通过复制缺陷性腺病毒介导的人前列腺特异性膜抗原基因(PSMA)和肿瘤坏死因子配体超家族成员(4-1BBL)体外共同转染树突状细胞(DC)后,与细胞因子诱导的杀伤细胞(CIK)共培养,观察其体外抑制前列腺癌的生物学效应。方法:将Ad-PSMA、Ad-4-1BBL、Ad-GFP按MOI=200∶1转染DC作为刺激细胞,分为共转染组、PSMA转染组、4-1BBL转染组、阴性对照组(Ad-GFP-DC组)以及未转染DC组,Western blot法检测各组DC细胞中PSMA和4-1BBL蛋白的表达;流式细胞仪分析CD80、CD83、CD86等表型变化;取患者外周静脉血单个核细胞中淋巴细胞用于CIK细胞的诱导培养,按DC∶CIK=1∶10比例将上述不同转染组DC加入CIK中,共同孵育96h(设普通DC刺激组和单独CIK细胞组为对照),收获的细胞作为效应细胞,流式细胞仪测定不同DC刺激组CIK细胞CD3、CD56表达率,ELISA法检测不同DC-CIK组培养上清IFN-γ、IL-10水平;PE-7AAD凋亡试剂盒检测不同DC-CIK组对人前列腺癌细胞株LNCap、Du145的细胞毒作用。结果:Western blot结果证实PSMA、4-1BBL可在DC上成功表达;各转染组DC表面CD80、CD83、CD86、HLA-DR共刺激分子表达上调,与未转染组相比较差异显著(P<0.05)。培养14d,不同DC疫苗刺激组CIK以及单独CIK细胞组中CD3+、CD56+、CD3+/CD56+T细胞比例均高于培养7d的CIK,差异显著(P<0.05),其中经不同DC疫苗刺激的CIK组上升较单独培养14d-CIK组更明显(P<0.05)。DC-CIK培养上清中PSMA/4-1BBL-DC-CIK组IFN-γ分泌量最高,达1176.10±14.37pg/5×106cells,IL-10分泌量最低,为75.14±2.01 pg/5×106cells,与其他各组比较,差异显著(P<0.05)。不同组别DCCIK作用于LNCap、Du145细胞24h后,荧光显微镜下观察细胞凋亡和坏死明显。同一组别DC疫苗刺激下的CIK细胞对LNCap的杀伤作用强于DU145细胞,差异显著(P<0.05)。而在针对同一种肿瘤细胞时,PSMA/4-1BBL-DC-CIK组的杀伤作用最强,LNCap细胞的凋亡率可达(24.56±1.68)%,Du145细胞凋亡率可达(12.67±0.94)%,与PSMA-DC-CIK组、4-1BBL-DC-CIK组相比,差异显著(P<0.05),而PSMA-DC-CIK和4-1BBL-DC-CIK组的前列腺癌细胞的凋亡率则无显著差异(P>0.05),但与GFP-DC-CIK、普通DC-CIK、和单独CIK组相比差异显著(P<0.05)。结论:本实验中Ad-PSMA/4-1BBL高效转染的DC具有成熟DC的典型特征,与CIK共培养后提高了CD3+/CD56+T细胞比例,IFN-γ分泌量显著增高,并增加了对PSMA阳性靶细胞的特异性杀伤,比普通DC-CIK及单独CIK具有更强的杀伤活性。两种肿瘤相关抗原转染DC与CIK共培养后可以更有效地提高CIK细胞的杀伤活性。

Objective：To use recombinant adenoviruses mediated prostate-specific membrane antigen （PSMA） and the T cell co-stimulatory molecule 4-1BBL into human dendritic cells （DCs） which co-cultured with cytokines induced killer cells（CIK） and then observe its biological effects against prostate cancer in vitro.Methods：We transfected DCs which from peripheral blood of healthy volunteers with recombinant adenoviruses at MOI.Allocating the dendritic cells to five groups：co-transfected group,PSMA-transfected group,4-1BBL-transfected group,negative control group （Ad-GFP-DC） and non-transfected DC group.The phenotype change of tranfected DC such as CD80,CD83 and CD86 was detected by FACS,and detected expression of PSMA and 4-1BBL protein by Western blot.To take the non-adherent cells from peripheral blood mononuclear cell to culture cytokines induced killer cells （CIK）.Then these tranfected DCs were mixed into the cytokines induced killer cells at the rate of 1∶ 10,after co-incubating with 96 hours（set normal DC-CIK and CIK group as control group）,the PSMA / 4-1BBL-DC-CIK,PSMA-DC-CIK,4-1BBL-DC-CIK,DC-CIK and CIK were taken as effector cells,the expression of CD3,CD56 on different group of effector cells were detected by FACS,the IFN-γ,IL-10 concentration which come from different groups of culture supernatant was measured by ELISA method.The antitumor effect（CTL effect） of different DC-CIK groups targeted at prostate cancer cells were analyzed by PE-7AAD apoptosis assay kit.Results：Western blot method confirmed that PSMA,4-1BBL protein can be successfully expressed on the DCs.The immunophenotype expressed on DCs,such as CD80,CD83,CD86,HLA-DR,were more significantly higher in transfected group than non-transfected DC group （P ＜ 0.05）.After 14 days,the CD3＋,CD56＋,CD3＋/CD56＋ T cell ratio of different DC-CIK group was higher than the normal 7d-CIK group,among them the CIK stimulated by different DC vaccine was higher than the normal 14d-CIK （P ＜ 0.05）.The secretion of IFN-γ in supernatant was greatly higher in PSMA/4-1BBL-DC-CIK group （1176.10 ± 14.37pg/5 × 106cells） than in single transfected DC-CIK group or normal DC-CIK group （P ＜ 0.05）,but IL-10 was lowest （75.14 ± 2.01 pg/5 × 106 cells）.Under fluorescent microscope LNCap and Du145 cells become circle and floating after induced by different DC-CIK group in 24 hours later,apoptosis and necrosis rate of LNCap cells was higher than Du145 cells after effected by the same DC-CIK group,the difference was significant （P ＜ 0.05）.The killing effect of PSMA/4-1BBL-DC-CIK group [apoptosis rate of LNCap cells：（24.56 ± 1.68） ％ ; apoptosis rate of Du145 cells：（12.67 ± 0.94） ％] was significantly higher than PSMA-DC-CIK group and 4-1BBL-DC-CIK group （P ＜ 0.05）.While the difference between PSMA-DC-CIK and 4-1BBL-DC-CIK group was not significant （P ＞ 0.05）,but the difference was significant compared with GFP-DC-CIK,normal DC-CIK and CIK group （P ＜ 0.05）.Conclusion：DCs transfected by Ad-PSMA/4-1BBL was also mature,after co-cultured with CIK cells,the ratio of CD3＋/CD56＋ T cell was improved and the secretion of IFN-γ was significantly increased,but also elicit potent anti-tumor immune responses specific for LNCap cells which highly express PSMA protein.In addition,the DCs which were infected with two kind of tumor-associated antigens would induce more effective CTL effect.