脐血输注修复放疗免疫抑制的实验动物研究

发布时间：2018-06-20 12:52

本文选题：人脐血干细胞 + 免疫损伤小鼠模型　；参考：《昆明医科大学》2017年硕士论文

【摘要】：[目的]临床放化疗所致的免疫抑制是患者不良预后的主要原因,寻找一种修复免疫抑制安全有效的方法能够提高患者的治疗效果和预后。本实验通过建立放疗免疫抑制小鼠模型,观察骨髓干细胞的输注对放疗免疫抑制小鼠的修复作用,再观察人脐血干细胞输注后对裸鼠的影响。为脐血用于临床治疗放化疗所致的造血细胞损伤和免疫抑制病人提供理论依据和实验基础。[方法]采用放疗照射的方法建立小鼠骨髓细胞损伤和免疫抑制模型,随后用血细胞仪检测外周血白细胞、流式细胞仪检测T淋巴细胞亚群、胸腺细胞凋亡率,HE染色观察小鼠骨髓病理切片的变化。将骨髓干细胞通过尾静脉输注至免疫抑制小鼠体内后,利用血细胞分析仪分析外周血血常规,流式细胞仪检测T淋巴细胞亚群(CD3+、CD4+、CD8+)、ELISA试剂盒检测血清免疫球蛋白IgM变化情况,并观察生存状态和存活率。人脐血干细胞输注后对裸鼠的影响:选择NOD/SCID小鼠,经4Gy剂量的x线照射后,随机分为实验组和对照组,分选脐血有核细胞,通过尾静脉输注至实验组裸小鼠,流式细胞仪检测人源的CD45、CD3和CD19分子表达,并观察小鼠存活情况。[结果]6.0Gy照射组小鼠30d存活率为100%; 8.0Gy照射组小鼠12天后死亡2只,30d存活率为10% ; 10.0Gy照射组小鼠30d存活率为0。因此,确定研究导致小鼠骨髓细胞和免疫功能损伤的放疗照射剂量为8.0Gy。小鼠骨髓干细胞输注至放疗后小鼠体内后,60d存活率为80%,并且移植后小鼠未观察到移植物抗宿主病(GVHD)等现象,而未经输注干细胞的小鼠60d存活率为10%;输注干细胞后小鼠外周血白细胞数、淋巴细胞比例及CD3+T细胞亚群较未经移植的小鼠高(P0.05);在输注干细胞后第15天,CD4+%、CD8+%和免疫球蛋白IgM最低,随后逐渐升高(P0.05);骨髓干细胞移植后能降低辐射后小鼠外周血单核细胞比例及CD4+/CD8+比值(P0.05)。人脐血干细胞输注入4Gy剂量的x线照射实验组NOD/SCID裸小鼠后,同对照组的裸鼠比较,实验组裸鼠30d存活率为70%,对照组裸鼠30d的存活率为10%。并且,通过流式细胞仪,我们在实验组的裸小鼠体内检测到了人源的CD45、CD3、CD 19分子表达。[结论]本实验成功建立放疗致小鼠骨髓细胞损伤和免疫抑制模型;输注同种系小鼠骨髓造血干细胞具有造血重建和免疫功能修复作用;异种(人)脐血干细胞能够显著延长放射后裸鼠的生存期,在裸鼠实验研究中提示异种(人)脐血干细胞有重建造血和免疫系统的征象。
[Abstract]:[objective] Immunosuppression caused by clinical radiotherapy and chemotherapy is the main cause of poor prognosis. Finding a safe and effective method to repair immunosuppression can improve the therapeutic effect and prognosis of patients. In this study, we established an immunosuppressive mouse model of radiotherapy to observe the repair effect of bone marrow stem cell infusion on radiotherapy immunosuppressive mice and the effect of human umbilical cord blood stem cell infusion on nude mice. To provide theoretical and experimental basis for the clinical treatment of hematopoietic cell injury and immunosuppressive patients caused by radiotherapy and chemotherapy. [methods] the model of bone marrow cell injury and immunosuppression in mice was established by radiotherapy. The peripheral blood leukocytes were detected by blood cell analyzer and T lymphocyte subsets were detected by flow cytometry. Thymocyte apoptosis rate and HE staining were used to observe the changes of bone marrow sections in mice. Bone marrow stem cells were infused into immunosuppressive mice via tail vein. The peripheral blood routine was analyzed by hematology analyzer, and the changes of serum immunoglobulin IgM were detected by flow cytometry (FCM) and Elisa kit for detecting T lymphocyte subsets (CD3, CD3, CD 4, CD 4, CD 8). The survival state and survival rate were observed. The effect of human umbilical cord blood stem cell infusion on nude mice: NOD / SCID mice were randomly divided into experimental group and control group after X-ray irradiation of 4 Gy dose. Flow cytometry was used to detect the expression of CD45, CD3 and CD19, and to observe the survival of mice. [results] the 30-day survival rate of mice in 6.0Gy irradiation group was 100, that in 8.0Gy irradiation group was 10% after 12 days, and that in 10.0Gy irradiation group was 0. Therefore, the radiation dose of radiation induced bone marrow cell and immune function injury in mice was determined to be 8.0 GY. The survival rate of mouse bone marrow stem cells was 80% in vivo after radiotherapy, and GV HDD was not observed in mice after transplantation, such as graft-versus-host disease (GV HDD). The survival rate of mice without infusion of stem cells in 60 days was 10 and the number of peripheral blood leukocytes in mice after infusion of stem cells was 10. The percentage of lymphocytes and CD3 T cell subsets were higher than that of untransplanted mice (P 0.05), and on the 15th day after infusion of stem cells, CD4% and IgM were the lowest. After transplantation of bone marrow stem cells, the percentage of peripheral blood monocytes and CD4 / CD8 ratio of mice were decreased gradually, and the ratio of CD _ 4 / CD _ 8 to CD _ 4 / CD _ 8 was decreased after transplantation of bone marrow stem cells. Human umbilical cord blood stem cells were injected with 4 Gy dose of x-ray to irradiated nod / SCID nude mice. Compared with the nude mice of the control group, the survival rate of the nude mice in the experimental group was 70 and that in the control group was 10. By flow cytometry, we detected the expression of human CD45, CD3, and CD19 in nude mice of experimental group. [conclusion] the model of bone marrow cell injury and immunosuppression induced by radiotherapy in mice was successfully established, and the allogeneic bone marrow hematopoietic stem cells were infused with hematopoietic reconstitution and immune function repair. Xenogeneic (human) cord blood stem cells could significantly prolong the survival time of nude mice after radiation. In the experimental study of nude mice, the xenogeneic (human) cord blood stem cells showed signs of reconstitution of hematopoiesis and immune system.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5

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