NK细胞的程序性扩增及功能鉴定

发布时间：2018-03-15 07:39

本文选题：NK细胞　切入点：程序性分化　出处：《中国科学技术大学》2017年博士论文　论文类型：学位论文

【摘要】：自然杀伤细胞(NK细胞)是固有免疫系统中的一种重要细胞,在抗病毒、抗肿瘤过程中发挥着重要作用。在造血干细胞移植过程中,NK细胞是最早出现的免疫细胞,发挥着重要的抗白血病细胞效应,而且不会引发机体移植物抗宿主病。因此,NK细胞在白血病过继转输治疗方面具有重要的应用前景。体外扩增大量功能性NK细胞并获得有效的治疗效果是目前NK细胞临床应用研究的热点。就这些问题,我们进行了相关的实验,得到以下结果:1、通过采用脐血CD34+干细胞,应用不同的细胞因子组合使其最终分化为NK细胞。经过五周的培养,NK细胞的比例可达到90%以上,扩增约7000倍。通过对不同阶段培养细胞差异表达的基因进行聚类及相关性分析,我们发现前三周细胞和后两周细胞之间的基因表达有明显不同,根据这一现象,我们将NK细胞的体外分化过程分别命名为程序一和程序二。2、对已报道的转录因子进行检测,发现程序二的细胞与原代NK细胞具有相似的转录因子表达谱,表明所培养的细胞可以作为研究NK细胞发育分化的体外模型。进而对两个分化阶段的细胞进行差异表达的转录因子分析,我们发现程序一的细胞主要聚集锌指结构域的转录因子,而程序二的细胞主要聚集基础结构域家族和螺旋-转向-螺旋结构域家族的转录因子。对相应的转录因子进行分析,表明程序一为NK细胞前体,程序二调控NK细胞的转化以及功能成熟。3、对不同阶段培养细胞的趋化因子受体及细胞因子受体表达谱进行分析,我们发现程序二,即分化的NK细胞表达相对高水平的NK细胞功能相关受体,表明此细胞具有发挥相应NK细胞活化及杀伤功能的潜能。4、随后我们对不同阶段细胞杀伤性分子的表达进行分析检测,同样发现分化的NK细胞表达更高水平的炎性细胞因子及杀伤相关的功能分子,并且该细胞具有较强的细胞杀伤活性。5、对所做的芯片进行深层次的挖掘,我们发现分化的NK细胞表达大量的SLAM家族的受体,包括CD244、CD48、CRACC和NTBA。具有较强杀伤功能的外周血NK及脐带血NK细胞高表达以上的SLAM家族受体。并且,培养的细胞表达有SAP和EAT-2,确保SLAM家族受体可以传递活化性信号。6、通过RT-PCR和流式细胞术进行检测,我们发现血液系统肿瘤细胞系相较于实体瘤细胞系,表达更高水平的SLAM家族对应配体,CD48、CD244、CRACC和NTBA。同样的,临床白血病病人的白血病细胞也有相应配体的表达,提示NK细胞可通过SLAM家族受配体的相互作用更有效杀伤白血病细胞。7、应用过表达或干扰SLAM家族配体的细胞进行细胞结合和杀伤实验,我们发现SLAM家族受配体的相互作用可以促进NK细胞与靶细胞的结合及促进其对靶细胞的杀伤。对临床白血病病人SLAM家族配体的表达水平与NK细胞的杀伤能力进行检测,发现白血病细胞上SLAM家族配体表达水平越高,NK细胞的杀伤能力越强。结合上述的实验结果,我们建立了一个体外大规模分化扩增NK细胞的体系,并且发现在NK细胞的发育分化过程中,无论在转录因子、细胞受体及功能方面都有明显的分区,存在一种程序性的分化模式。对分化的NK细胞进行检测,发现其表面表达较高水平的SLAM家族受体,与之相对应的白血病细胞上表达相应的配体,并且该受配体的存在可明显增强NK细胞与靶细胞的结合,及促进NK细胞对靶细胞的杀伤。上述结果提示NK细胞在白血病过继转输中有更好的治疗效果,并且白血病细胞表面SLAM家族配体的表达可作为NK细胞治疗有效性的潜在预测指标。
[Abstract]:Natural killer cells (NK cells) is a kind of important cells in the innate immune system plays an important role in antiviral and antitumor process. After hematopoietic stem cell transplantation in NK cells is the earliest immune cells play an important effect of anti leukemia cells, but not cause the body graft host disease. Therefore, it has important application prospect of NK cell transfusion therapy in the treatment of leukemia. A large number of adoptive in vitro functional NK cells and obtain effective treatment is currently a hot research and clinical application of NK cells. These problems, we have done experiments, we obtained the following results: 1, through the use of umbilical cord blood CD34+ stem cells, using different cytokine combinations which eventually differentiate into NK cells. After five weeks of incubation, the proportion of NK cells can reach more than 90%, was about 7000 times. Through the different stages of fine culture Clustering analysis and correlation of cell differentially expressed genes, we found the expression between three weeks before and two weeks after cell genes are significantly different, according to this phenomenon, we will in vitro differentiation of NK cells are named for the program and procedures to detect two.2 transcription factors have been reported, found that cell program two and primary NK cells with similar transcription factor expression profiles show that the cultured cells can be used as an in vitro model of NK cell development and differentiation. Then the two stages of differentiation cell factor analysis of differentially expressed transcripts, we found a main program of cell aggregation of zinc finger transcription factor domain the two main program, cell aggregation based domain family and helix turn helix domain transcription factor family of transcription factors. The corresponding analysis shows that a program for NK cell The precursor, NK cell transformation and function control program of two mature.3, in different stages of cultured cells of chemokine receptor and cytokine receptor expression spectrum analysis, we found that the two program, the differentiation of NK cells and NK cells express high levels of relative function related receptor, shows that the cell has played the corresponding activation of NK cells and the cytotoxicity of potential.4, then we in different stages of the molecular expression of cell killing was detected, also found that the differentiation of NK cells and the expression of inflammatory cytokines and higher levels of cytotoxicity of related molecules, and the cells have strong cytotoxic activity of.5, deep mining to do the chip, we found the expression of a large number of SLAM family receptors, differentiated NK cells including CD244, CD48, CRACC and NTBA. have strong killing function of NK in the peripheral blood and umbilical cord blood NK cells with high expression The receptors of the SLAM family. And, the cultured cells expressed SAP and EAT-2, to ensure that the SLAM family receptors can transfer activation signals of.6, were detected by RT-PCR and flow cytometry, we found that tumor cells in blood system compared to the solid tumor cell lines, expression of SLAM family corresponding ligands, higher levels of CD48 CD244, CRACC and NTBA. in the same patient, clinical leukemia leukemia cells have the expression of the corresponding ligand, suggesting that the interaction of NK cells through the SLAM receptor ligand family more effective anti leukemia cell.7, application of overexpression or interference of ligands of the SLAM family cell binding and cytotoxicity assay, we found that the interaction of SLAM family by the ligand can promote the combination of NK and target cells and promote it to kill target cells. The expression level of SLAM in patients with leukemia family ligand NK cells can Force testing, found in leukemia cells SLAM family ligand expression level is higher, NK cell killing ability. According to the experimental results above, we established an in vitro differentiation of NK cell mass amplification system, and found in the development and differentiation of NK cells, both in transcription factors, cell receptors and function there are obvious partition, there is a procedural differentiation model. On the differentiation of NK cells were detected and found higher levels of SLAM receptor expression on the surface of the family, and the expression of the corresponding ligand corresponding to the leukemia cells, and the presence of ligand can significantly enhance the binding of NK cells and target cells, and promote the cytotoxicity of NK cells against target cells. These results suggest that NK cells have a better therapeutic effect in the transmission of leukemia and adoptive, leukemia cell surface ligands of the SLAM family table as an Potential predictors for the effectiveness of NK cell therapy.

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392

【相似文献】