胎盘组织源单个核细胞免疫活性的研究

发布时间：2018-06-01 07:26

本文选题：胎盘 + 造血干细胞　；参考：《郑州大学》2007年硕士论文

【摘要】： 研究目的近年来，造血干细胞移植成为治疗恶性血液病、再生障碍性贫血、自身免疫性疾病，某些遗传性疾病及实体瘤的一种重要手段。据有关资料显示，仅我国就有400多万各类疾病患者等待造血干细胞移植，而造血干细胞的来源却很有限。骨髓、外周血和脐血是目前造血干细胞的主要来源，骨髓和外周血造血干细胞移植要求HLA配型完全一致，这即使是同胞间也只有25％的机率，在非血缘关系的人群中仅有1/400—1/10000的可能性，远远不能满足临床治疗的需要；脐血虽免疫原性较弱，移植后移植物抗宿主病(GVHD)的发生率低，但单份脐血中所含的造血干细胞数量有限，难以使一个成人获得造血干细胞重建，需要扩增后才能用于成人。所以寻找新的造血干细胞来源是个极待解决的问题。已有研究证实：胎盘中含有较多的造血干细胞，胎盘组织有支持造血干细胞生存的微环境。这表明通常丢弃的胎盘也有望成为HSC移植的资源。但胎盘组织中造血干细胞的含量有限，故胎盘造血干细胞进行体外扩增成为胎盘造血干细胞用于移植的一个前提，国内外已有相关报道。移植治疗后GVHD是影响移植治疗效果的重要原因之一，本研究比较了扩增后的胎盘及脐血单个核细胞和动员后的外周血的免疫活性，以了解胎盘作为干细胞源用于移植时GVHD的发生情况，为胎盘组织源造血干细胞用于移植提供理论依据。对象与方法 (一)对象 1．胎盘组织和与其相对应的脐血8份：在无菌条件下，分别采集郑州大学第一附属医院足月健康顺产分娩孕妇的胎盘及与其相对应的脐血。 2．外周血8份：在无菌条件下采集郑州大学第一附属医院和河南省肿瘤医院血液科干细胞移植健康供者的经G-CSF动员4-5后的外周血。 (二)方法 1．单个核细胞(MNC)制备：剪切胎盘组织呈1—2mm~3大小的组织块，用酶消化的方法经320目的细胞筛过滤制备胎盘组织源单个细胞悬液；用淋巴细胞分离液分别分离胎盘组织源、脐血源的MNC。 2．胎盘和脐血MNC的体外培养：将分离的胎盘MNC、脐血MNC用DMEM培养液悬浮，加入刺激因子，置37℃、体积浓度为5％CO_2、饱和湿度的培养箱中培养7天。 3．用淋巴细胞分离液将培养后的胎盘和脐血细胞再次分离MNC，同时分离动员后的外周血的MNC，，并为后续实验调好细胞浓度。 4．采用CCK—8检测混合淋巴细胞反应中胎盘、脐血和外周血三种来源细胞的增殖情况；采用流式细胞仪技术检测胎盘、脐血和外周血三种来源细胞中CD_3、CD_(19)、CD_(16)、CD_(56)、HLA-DR的表达。结果 1．单向混合淋巴细胞反应混合淋巴细胞反应中，扩增后的胎盘MNC组的细胞增殖显著低于其他两组(P0.001)，扩增后的脐血MNC组的细胞增殖显著高于扩增后的胎盘MNC，但却显著低于动员后的外周血(P0.001)。 2．表型分析扩增后的胎盘MNC中T淋巴细胞(CD_3~+细胞)、B淋巴细胞(CD_(19)~+细胞)比例少于扩增后的脐血MNC和动员后的外周血，与扩增后脐血MNC和动员后的外周血均有显著差异。扩增后的胎盘MNC中NK细胞(CD_(16)~+CD_(56)~+细胞)和HLA-DR+细胞比例最高，与扩增后脐血MNC和动员后的外周血有显著差异；脐血中NK细胞的含量最少。结论 1．单向混合淋巴细胞培养中，以扩增后的胎盘MNC组的细胞增殖最低，表明扩增后的胎盘MNC免疫活性最低。 2．扩增后的胎盘MNC中CD_3~+细胞，CD_(19)~+细胞的比例最低，表明扩增后的胎盘MNC免疫活性最低。 3．扩增后的胎盘MNC中成熟型NK细胞比例最高，可能有助于增加移植物抗白血病作用，但并不增加GVHD的发生率和严重程度。 4．扩增后的胎盘MNC可用于移植，若其用于移植，GVHD的发生率及严重 4．扩增后的胎盘MNC可用于移植，若其用于移植，GVHD的发生率及严重程度较扩增后的脐血MNC和动员后的外周血用于移植时低。
[Abstract]:In recent years, hematopoietic stem cell transplantation has become an important means for the treatment of malignant hematological diseases, aplastic anemia, autoimmune diseases, some hereditary diseases and solid tumors. According to the relevant data, only about 4000000 of the patients in our country are waiting for the transplantation of blood stem cells, while the source of hematopoietic stem cells is very good. Bone marrow, peripheral blood and umbilical blood are the main sources of hematopoietic stem cells at present. Bone marrow and peripheral blood stem cell transplantation require the same HLA matching, which is only 25% of the probability between the siblings, and the only possibility of 1/400 1/10000 in the unrelated population is far from meeting the needs of clinical treatment; umbilical blood is exempt. Pestilence is weak and the incidence of graft versus host disease (GVHD) after transplantation is low, but the number of hematopoietic stem cells in single cord blood is limited. It is difficult for one adult to reestablish hematopoietic stem cells and need to be amplified to be used in adults. Therefore, finding new hematopoietic stem cells is a problem to be solved. There are more hematopoietic stem cells in the disc and the placental tissue has a microenvironment to support the survival of the hematopoietic stem cells. This indicates that the usually discarded placenta is also expected to be a resource for HSC transplantation. However, the content of hematopoietic stem cells in placental tissue is limited, so the placental hematopoietic stem cells are amplified into a pre transplant of placental hematopoietic stem cells for transplantation. It has been reported at home and abroad. GVHD is one of the important factors affecting the effect of transplantation treatment after transplantation. This study compares the immune activity of the expanded placenta and cord blood mononuclear cells and mobilized peripheral blood in order to understand the occurrence of GVHD in the placenta as a stem cell source for transplantation and to stem the hematopoietic stem of placenta tissue. The use of cell for transplantation provides a theoretical basis.
Object and method
(I) objects
1. placental tissue and 8 umbilical cord blood corresponding to it: under aseptic conditions, the placenta and its corresponding umbilical cord blood were collected from the First Affiliated Hospital of Zhengzhou University.
2. peripheral blood 8: under sterile conditions, the First Affiliated Hospital of Zhengzhou University and Henan oncology doctor were collected.
Stem cells transplantation from healthy Department of Hematology donors were mobilized by G-CSF for 4-5 of peripheral blood.
(two) method
1. mononuclear cell (MNC) preparation: the cut placenta tissue is 1 - 2mm~3 size tissue block. The placental tissue source suspension is prepared by the enzyme digestion method through 320 cell sieves, and the placental tissue source and the MNC. of the umbilical blood source are separated by the lymphocyte separation solution.
2. in vitro culture of placenta and umbilical cord blood MNC: the isolated placenta MNC, umbilical cord blood MNC was suspended in DMEM medium, added to the stimulating factor, placed at 37 C, the volume concentration was 5%CO_2, and the saturated humidity incubator was cultured for 7 days.
3. the MNC was separated from the cultured placenta and cord blood cells by lymphocyte separation solution, and the MNC of the mobilized peripheral blood was separated, and the cell concentration was adjusted for the follow-up experiment.
4. CCK - 8 was used to detect the proliferation of three kinds of cells from the placenta, umbilical blood and peripheral blood in mixed lymphocyte reaction. The flow cytometry was used to detect CD_3, CD_ (19), CD_ (16), CD_ (56), and HLA-DR in the placenta, umbilical cord blood and peripheral blood.
Result
In 1. unidirectional mixed lymphocyte reaction mixed lymphocyte reaction, the proliferation of the expanded placental MNC group was significantly lower than that of the other two groups (P0.001). The proliferation of MNC group after amplification was significantly higher than that after the amplification of placental MNC, but it was significantly lower than that of the mobilized peripheral blood (P0.001).
2. phenotypic analysis of T lymphocyte (CD_3~+ cells) and B lymphocyte (CD_ (19) + cells) in the placental MNC after amplification, the proportion of MNC and mobilized peripheral blood after amplification is less than that of amplified umbilical blood and mobilized peripheral blood. There is a significant difference between the MNC of umbilical cord blood and the peripheral blood after the amplification. The ratio of NK cells (CD_ (16) ~+CD_ (56) ~ + cells) and HLA-DR+ cells in the amplified placental MNC The highest cases were significantly different from those of the expanded cord blood MNC and mobilized peripheral blood, and the NK cells in umbilical cord blood were the least.
conclusion
1. in unidirectional mixed lymphocyte culture, the proliferation of the placental MNC group was the lowest, indicating that the MNC activity of the expanded placenta was the lowest.
2. the percentage of CD_ (19) ~ + cells in CD_3~+ cells after MNC amplification was the lowest, indicating that the MNC activity of the expanded placenta was the lowest.
The proportion of mature NK cells in MNC after 3. amplification is the highest, which may help to increase the anti leukemic effect of the graft, but does not increase the incidence and severity of GVHD.
4. the expanded placenta MNC can be used for transplantation. If it is used for transplantation, the incidence and severity of GVHD are serious.
4. expanded placental MNC can be used for transplantation. If it is used for transplantation, the incidence of GVHD and the severity of MNC in umbilical blood after amplification and the mobilization of peripheral blood are low for transplantation.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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