Killing effects of 5-Aza-CdR combined with EBNA1-DC vaccine-induced lymphocytes on nasopharyngeal carcinoma C666-1 cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨EB病毒核抗原1（EBNA1） mRNA修饰的DC（EBNA1-DC）诱导的淋巴细胞联合甲基化抑制剂5-Aza-CdR对鼻咽癌C666-1细胞的杀伤作用。方法：以构建的EBNA1-pCDNA3.1质粒为模板，体外转录获得EBNA1 mRNA，通过脂质体转染至健康人外周血来源DC，构建EBNA1-DC疫苗。流式细胞术检测转染后DC表型及5-Aza-CdR处理后的C666-1细胞凋亡情况。实时无标记动态细胞分析技术检测EBNA1-DC疫苗诱导的淋巴细胞联合5-Aza-CdR的特异性抗肿瘤活性。结果：转染EBNA1 mRNA后EBNA1-DC表面EBNA1阳性率为（59.3±5.85）%，HLA-DR的表达与未转染DC相比显著升高[（84.9±5.5）% vs （68.0±5.8）%，P=0.026]，CD80的表达也显著升高[（88.2±3.9）% vs （61.1±4.4）%，P=0.015]。低剂量5-Aza-CdR处理后的C666-1细胞凋亡情况与未处理的细胞相比无显著差异。经低浓度5-Aza-CdR预处理的C666-1细胞中IRF7基因表达与未处理的细胞相比显著升高（P=0.000 1）。与空载的DC相比，EBNA1-DC诱导的淋巴细胞对EBV阳性表达的C666-1细胞具有更强的特异性杀伤活性（P=0.049）；经低浓度5-Aza-CdR预处理的C666-1细胞对EBNA1-DC诱导的特异性免疫杀伤更敏感（P=0.019）。结论：5-Aza-CdR与EBNA1-DC疫苗联合可显著增强对C666-1细胞的特异性免疫杀伤，本研究为开拓以mRNA为基础的DC疫苗及其在临床综合治疗中的应用转化提供前期研究基础。

Characteristics and functions of long-term passaged human umbilical cord mesenchymal stem cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨传至10代（P10）的人脐带来源间充质干细胞（P10-hUC-MSC）的生物学特性及功能。方法：人脐带来源于厦门弘爱医院（伦理批号：HAXM-MEC-20201012-037-01），分离、收集、培养hUC-MSC并传代培养，收集P1-、P10-hUC-MSC，FCM检测hUC-MSC表型，细胞衰老β-半乳糖苷酶染色法及FCM法检测终末期细胞衰老与凋亡情况，秋水仙碱处理检测细胞染色体稳定性，体外成脂、成骨诱导实验检测其多向分化能力，以不同比例与外周血单个核细胞（PBMC）混合培养后FCM检测T细胞亚群及表型变化。结果：成功分离和培养的P10-hUC-MSC与P1-hUC-MSC的表型相似，表现为CD45、CD34、HLA-DR表达阴性而CD105、CD90阳性率≥95%。终末期的P1-hUC-MSC和P10-hUC-MSC均表现出β-半乳糖苷酶表达阳性和早期凋亡特征，细胞染色体核型一致且保持稳定，未发生转化现象。P1-、P10-hUC-MSC在体外都可被诱导分化成脂肪、成骨细胞。P10-hUC-MSC与PBMC以1∶1混合培养7 d后，可显著上调CD4+/CD8+ T细胞比值、CD4+ Treg细胞比例和PD-1表达（均P<0.01）。结论：长期传代的P10-hUC-MSC仍然保持其生物学特性和安全性，并具备多向分化能力及免疫调节能力，这为最大限度发挥hUC-MSC的临床放疗损伤修复与预防作用提供了前期实验依据和指导。

Effect of PD-1 knockout by CRISPR/Cas9 system on proliferation and IFN-γ secretion in human T lymphocytes / 中国肿瘤生物治疗杂志

@#Objective: : To explore the effect of PD-1 gene knockout by CRISPR/Cas9 system on the proliferation and IFN-γ secretion in human T cells. Methods: : The sequence of sgRNA targeting PD-1 was designed. The PD-1-sgRNA and Cas9 mRNA were synthesized by T7 RNApolymerase in vitro, and then the mixture of PD-1-sgRNAand Cas9 mRNAwas delivered into activated T cells by nucleofection. The efficiency of gene knockout was confirmed by sequencing. The phenotypes of T lymphocytes and the expression of PD-1 after gene knockout were analyzed by Flow cytometry. The proliferation of T lymphocytes was calculated by trypan blue counting. The level of IFN-γ secreted by T lymphocytes was detected by ELISA. Results： ：PD-1-sgRNA and Cas9 mRNA were successfully synthesized in vitro and delivered into T cells by nucleofection. Sequencing technology confirmed that the PD-1 gene sequence was edited and the editing efficiency was 58.3%. The expression of PD-1 on T lymphocyte surface was down-regulated successfully by CRISPR/Cas9 system [(9.6±1.85)% vs (16.2±2.05)%, P<0.05]. The knockout of PD-1 gene did not affect the proliferation and phenotype of T lymphocytes(P<0.05); However, compared with the control group, the level of IFN-γ secreted by T lymphocytes in the PD-1sgRNA group was significantly increased (P<0.01). Conclusion: : CRISPR/Cas9 system can successfully ablate PD-1 gene in human T lymphocytes, which could block the negative regulation of PD-1／PD-L1 and further promote the IFN-γ secretion in T cells.