体外培养人脐血单个核细胞生物学活性的初步研究

发布时间：2019-07-04 17:20

【摘要】：背景: 临床上对于肿瘤等恶性疾病的治疗手段,已从传统的手术、放疗、化疗扩展到了生物免疫治疗。在这一领域中,细胞治疗占主导地位。脐血以其丰富的干细胞、较弱的免疫细胞抗原性、简单易行的采集和保存等多方面的优势,逐渐取代骨髓,而成为近年细胞治疗中细胞的主要来源。因此脐血细胞的生物学性能、免疫学特性、杀伤肿瘤细胞(特别是血液系统肿瘤)的机制,成为了研究的热点。目的: 研究人脐血林巴细胞体外经丝裂原刺激培养后的生物学、免疫学等方面的特性,并与健康人外周血淋巴细胞进行比较,为脐血淋巴细胞输注治疗在临床上的应用提供客观的指标和理论基础。材料和方法: Ficoll密度梯度离心法分离的人脐血及外周血单个核细胞,体外无血清体系培养5天,同时设加PHA(植物血凝素)和不加PHA两组。观察细胞克隆生长情况,并在培养前和培养后不同时间点收集细胞和培养上清。台盼兰染色法计算活细胞数;流式细胞术检测细胞的表型特征;ELIsA法检测培养上清中IFN-γ含量;Trizol提取细胞的RNA,逆转录成cDNA后,用FRQ-PCR法进行IFN-γ及β-actin基因扩增,相对定量地比较细胞内IFN-γ mRNA转录以情况。结果: 1.MNC体外生长情况:PHA(+)组UCBMNC在培养过程中细胞花团的体积、数量、密集程度不断增加;PBMNC细胞花团的生长情况远不如UCBMNC旺盛。PHA(-)组UCBMNC在培养过程中未见明显细胞花团,但细胞始终布满培养孔;PBMNC无细胞花团,培养后期细胞分布甚至变得稀疏。 2.MNC细胞数量的扩增:PHA(+)组UCBMNC细胞扩增的最大值为
文内图片：图IUCBPHAG)组，培养1天

图片说明：图IUCBPHAG)组，，培养1天
[Abstract]:Background: the clinical treatment of malignant diseases such as tumor has been extended from traditional surgery, radiotherapy and chemotherapy to bioimmunotherapy. Cell therapy dominates in this field. Cord blood has gradually replaced bone marrow because of its rich stem cells, weak immune cell antigenicity, simple collection and preservation, and has become the main source of cells in cell therapy in recent years. Therefore, the biological performance and immunological characteristics of cord blood cells, and the mechanism of killing tumor cells (especially blood system tumors) have become the focus of research. Objective: to study the biological and immunological characteristics of human umbilical cord blood Limba cells stimulated by mitogen in vitro and to compare them with those of healthy peripheral blood lymphocytes, so as to provide an objective index and theoretical basis for the clinical application of cord blood lymphocytes infusion therapy. Materials and methods: human umbilical cord blood and peripheral blood mononuclear cells isolated by Ficoll density gradient centrifugation were cultured in serum-free system for 5 days. PHA (plant hemagglutinin) and no PHA were added at the same time. The cell clone growth was observed, and the cell and culture supernatant were collected at different time points before and after culture. The number of living cells was calculated by trypan blue staining, the phenotypic characteristics of the cells were detected by flow cytometry, the content of IFN- 纬 in the culture supernatant was detected by ELIsA assay, and the IFN- 纬 and 尾-actin genes were amplified by FRQ-PCR method after RNA, reverse transcription into cDNA, and the transcription of IFN- 纬 mRNA in the cells was compared relatively quantitatively. Results: the volume, number and density of UCBMNC cells in: PHA () group increased continuously during the culture process of 1.MNC in vitro, and the growth of PBMNC cells was much less exuberant than that of UCBMNC. PHA (-) group UCBMNC did not see obvious cell flower clusters during the culture process, but the cells were always covered with culture pores, and the cell distribution even became sparse in the later stage of culture. The maximum value of UCBMNC cell amplification in: PHA () group was as follows: the number of UCBMNC cells was amplified by the number of UCBMNC cells.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

【参考文献】