ABSTRACT
@#[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。
ABSTRACT
@#[Abstract] Autophagy, as an important intracellular metabolic pathway, has been proved to be ubiquitous in many kinds of cells. Its functional impairment can easily cause lots of diseases, such as cancer, leukemia, liver disease, diabetes and heart disease. In particular, autophagy is important for the development, differentiation and regulation of immune function of T lymphocytes. Abnormal autophagy of T lymphocytes can cause immune dysfunction and lead to diseases, such as inflammation, infection and autoimmunity. In view of the important role of autophagy in regulating T lymphocyte function and disease, this article illustrates the research progress on autophagy regulating the homeostasis, survival, proliferation, senescence, metabolism, immune function of T lymphocytes and many diseases, including tumors, in recent years.
ABSTRACT
@#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.