人脐血基质细胞对小鼠单倍体相合外周血干细胞移植GVHD调节作用
发布时间：2019-05-21 11:42
【摘要】：研究背景： 异基因造血干细胞移植（Allo-HSCT）是恶性及非恶性造血功能紊乱、某些免疫缺陷性疾病、自身免疫性、遗传代谢性疾病等的有效治疗手段[1]。过去的20年里，Allo-HSCT的临床应用发生了巨大变化，由原先的比较单一的人白细胞抗原（humanleucocyte antigen，HLA）相合的同胞间的清髓移植转向一个更为复杂的领域，尤其在HLA半相合移植方面取得了重要进展，这一全新的移植方式有效解决了既往供者来源紧缺、配型困难问题，使得90%以上的患者可以在父母、子女、兄弟姐妹等亲缘关系中找到合适供者[2]。 近来，针对HLA半相合造血干细胞移植方式提出了很多新的策略[3]。经处理或者不经处理的骨髓（bone marrow，BM）联合或者不联合外周血干细胞（peripheral bloodstem cell，PBSCs），成为临床上最常见的干细胞来源[3][4]。然而，，骨髓采集物中含有大量红细胞，当供受者血型不合时，进行移植之前需要去除骨髓中的红细胞或者血浆。此外，骨髓采集为有创操作，采集过程中需要实施麻醉，增加了供者的痛苦和风险。PBSCs采集方便，有效避免了上述骨髓移植带来的问题。外周血来源的移植物不仅具有丰富的CD34+细胞可大大提高植入率，且采集物比较干净，受红细胞影响小。然而，外周血中含有丰富的成熟T细胞，约为骨髓的5-10倍，大大增加了移植后GVHD的发生[5]。 我们实验室前期工作中发现一种来源于人脐血的基质细胞，称为人脐血源基质细胞（hUCBDSCs），hUCBDSCs可以在多种因子特定组合下实现体外的增殖扩增[6]。同时，hUCBDSCs表面高表达HLA-I分子，HLA-II类分子及其他共刺激分子表达较少，具有较低的免疫原性[7]。体外实验发现其具有抑制异种T细胞增殖的功能，动物实验证实hUCBDSCs具有减低小鼠MHC半相合骨髓造血干细胞移植后GVHD的能力[8][9]。本研究旨在探讨hUCBDSCs对于小鼠MHC半相合PBSCT后GVHD的调节作用。 一．研究内容及方法 1.参照科室常规方法，使用6%明胶联合1.077g/LPercoll分离hUCBDSCs，特定的细胞因子组合下进行细胞体外培养扩增[8]； 2.雌性C57BL/6×BALB/c F1受鼠接受7.0Gy60Coγ射线照射后，经尾静脉输注雄性供鼠C57BL/6来源的外周血单个核细胞（MNC）(1×106/只)、脾脏细胞(1×107/只)，构建小鼠MHC半相合外周血干细胞移植后GVHD模型； 3.将体外培养扩增的hUCBDSCs（1×106/只）经尾静脉输注到已构建的MHC半相合外周血造血干细胞移植GVHD模型上，从小鼠生存率、临床表现、靶器官（肝脏、小肠、皮肤）病理解剖评定各组小鼠GVHD发生情况； 4.流式细胞仪检测移植后不同时间点（1周、2周、3周、4周）各组小鼠脾脏NK细胞比例，以及移植后3周小鼠脾脏Th1、Th2细胞比例，初步探索hUCBDSCs调节MHC半相合外周血造血干细胞移植后GVHD的作用机制。 二．主要研究结果 1.按照科室既往的方法，成功实现hUCBDSCs的体外培养扩增。 2.通过小鼠生存率、临床表现、靶器官病理表现及嵌合率的检测证明MHC半相合外周血造血干细胞移植GVHD模型构建成功。 3. hUCBDSCs可显著延长移植后实验组小鼠生存时间，降低死亡率（P0.05）；hUCBDSCs可显著降低移植后多个时相点实验组小鼠临床表现及靶器官组织病理GVHD评分（P0.05），提示联合hUCBDSCs输注可有效减轻小鼠MHC半相合PBSCT后GVHD反应. 4. hUCBDSCs可显著增加移植后实验组小鼠脾脏NK细胞、Th2细胞比例（P0.05），减低移植后Th1细胞比例（P0.05）。 三．主要结论 1. hUCBDSC可显著减轻小鼠MHC半相合PBSCT后GVHD； 2. hUCBDSCs可能通过增加移植后小鼠脾脏NK细胞、Th2细胞比例同时减低Th1细胞比例来减轻小鼠MHC半相合PBSCT后GVHD。
[Abstract]:Study Background: Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is an effective means of treatment for malignant and non-malignant hematopoietic function disorders, certain immune-deficient diseases, autoimmune diseases, and genetic metabolic diseases[1] ]. In the past 20 years, the clinical application of the Allo-HSCT has changed greatly, and the clear marrow transplantation between the sibling of the single human leucocyte antigen (HLA) and the single human leukocyte antigen (HLA) is shifted to a more complex field, especially in the case of HLA-half-consistent transplantation. The new transplanting method effectively solves the problem of the prior donor's shortage and the difficulty of matching, so that more than 90% of the patients can find the right donor in the related relationship of parents, children, siblings and the like[2]. ]. Recently, many new strategies have been put forward for HLA-half-consistent hematopoietic stem cell transplantation. [3]. The peripheral blood stem cells (PBSCs), which are treated or not treated, are the most common source of stem cells in clinical practice[3]. [4]. However, a large amount of red blood cells are contained in the bone marrow collection, and red blood cells or red blood cells in the bone marrow need to be removed prior to transplantation, In addition, the bone marrow is collected as an invasive operation, and the anesthesia is required in the acquisition process, and the pain of the donor is increased. And the bone marrow transplantation is effectively avoided. The peripheral blood-derived graft not only has abundant CD34 + cells, can greatly improve the implantation rate, However, the peripheral blood contains abundant mature T cells, which is about 5-10 times of the bone marrow, which greatly increases the occurrence of GVHD after transplantation. [5]. In our lab, a matrix cell derived from human umbilical cord blood, called human umbilical cord blood-derived stromal cells (hUCBDSCs), can be used for the proliferation of human umbilical cord blood in a variety of factors. The expression of HLA-I, HLA-II and other co-stimulatory molecules on the surface of the hUCBDSCs was low, and the expression of HLA-II and other co-stimulatory molecules was low. [7]. In vitro, it was found that it has the function of inhibiting the proliferation of different T cells, and the animal experiments confirm that hUCBDSCs have the ability to reduce GVHD after the transplantation of the murine MHC semi-consistent bone marrow hematopoietic stem cells[7]. 8][9]. The purpose of this study is to explore the GVHD of hUCBDSCs in mice with MHC half-consistent PBSCT tone one's action . Study content and method 1. Reference to the general method of the department, use 6% gelatin in combination with 1.077 g/ L Percoll to separate hUCBDSCs, specific cytokine combination to carry out the cell in vitro culture amplification[8];2. female C57BL/6/ BALB/ c F1 mice receiving 7.0 G After the irradiation of y60Co X-ray, the peripheral blood mononuclear cells (MNC) (1-106/ only) and spleen cells (1-107/ only) of the C57BL/6-derived male mice were infused by the tail vein, and the mouse MHC-half-consistent peripheral blood was constructed. GVHD model after cell transplantation;3. The cultured hUCBDSCs (1-106/ only) in vitro were transfused to the constructed MHC semi-consistent peripheral blood stem cell transplantation GVHD model, and the pathological anatomy of the target organ (liver, small intestine, skin) was evaluated from the survival rate, clinical manifestation, target organ (liver, small intestine, skin) of the mouse. The percentage of NK cells in the spleen and the ratio of Th1 and Th2 cells in the spleen of the mice at different time points (1 week,2 weeks,3 weeks and 4 weeks) were detected by flow cytometry. dry and fine GV after cell transplantation The mechanism of action of HD. II. Main study results 1. According to the prior methods of the department, In vitro culture and amplification of hUCBDSCs was successfully achieved.2. The MHC half-phase was demonstrated by the test of the survival rate, the clinical manifestation, the pathological expression of the target organ and the fitting rate. 3.hUCBDSCs could significantly prolong the survival time of the experimental group and lower the death rate (P0.05). The expression and the pathological GVHD score of the target organs (P0.05), suggesting that the combined hUCBDSCs infusion may 4. hUCBDSCs could significantly increase the NK cell and Th2 cell ratio in the spleen of the experimental group after transplantation (P 0. 05), The ratio of Th1 cells after transplantation was decreased (P0.05). UCBDSC can significantly reduce the GVHD after the mouse MHC half-consistent PBSCT;2. hUCBDSCs may be the same as that of the NK cells and the Th2 cells in the spleen of the mouse after the transplantation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R457.7
本文编号：2482076
[Abstract]:Study Background: Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is an effective means of treatment for malignant and non-malignant hematopoietic function disorders, certain immune-deficient diseases, autoimmune diseases, and genetic metabolic diseases[1] ]. In the past 20 years, the clinical application of the Allo-HSCT has changed greatly, and the clear marrow transplantation between the sibling of the single human leucocyte antigen (HLA) and the single human leukocyte antigen (HLA) is shifted to a more complex field, especially in the case of HLA-half-consistent transplantation. The new transplanting method effectively solves the problem of the prior donor's shortage and the difficulty of matching, so that more than 90% of the patients can find the right donor in the related relationship of parents, children, siblings and the like[2]. ]. Recently, many new strategies have been put forward for HLA-half-consistent hematopoietic stem cell transplantation. [3]. The peripheral blood stem cells (PBSCs), which are treated or not treated, are the most common source of stem cells in clinical practice[3]. [4]. However, a large amount of red blood cells are contained in the bone marrow collection, and red blood cells or red blood cells in the bone marrow need to be removed prior to transplantation, In addition, the bone marrow is collected as an invasive operation, and the anesthesia is required in the acquisition process, and the pain of the donor is increased. And the bone marrow transplantation is effectively avoided. The peripheral blood-derived graft not only has abundant CD34 + cells, can greatly improve the implantation rate, However, the peripheral blood contains abundant mature T cells, which is about 5-10 times of the bone marrow, which greatly increases the occurrence of GVHD after transplantation. [5]. In our lab, a matrix cell derived from human umbilical cord blood, called human umbilical cord blood-derived stromal cells (hUCBDSCs), can be used for the proliferation of human umbilical cord blood in a variety of factors. The expression of HLA-I, HLA-II and other co-stimulatory molecules on the surface of the hUCBDSCs was low, and the expression of HLA-II and other co-stimulatory molecules was low. [7]. In vitro, it was found that it has the function of inhibiting the proliferation of different T cells, and the animal experiments confirm that hUCBDSCs have the ability to reduce GVHD after the transplantation of the murine MHC semi-consistent bone marrow hematopoietic stem cells[7]. 8][9]. The purpose of this study is to explore the GVHD of hUCBDSCs in mice with MHC half-consistent PBSCT tone one's action . Study content and method 1. Reference to the general method of the department, use 6% gelatin in combination with 1.077 g/ L Percoll to separate hUCBDSCs, specific cytokine combination to carry out the cell in vitro culture amplification[8];2. female C57BL/6/ BALB/ c F1 mice receiving 7.0 G After the irradiation of y60Co X-ray, the peripheral blood mononuclear cells (MNC) (1-106/ only) and spleen cells (1-107/ only) of the C57BL/6-derived male mice were infused by the tail vein, and the mouse MHC-half-consistent peripheral blood was constructed. GVHD model after cell transplantation;3. The cultured hUCBDSCs (1-106/ only) in vitro were transfused to the constructed MHC semi-consistent peripheral blood stem cell transplantation GVHD model, and the pathological anatomy of the target organ (liver, small intestine, skin) was evaluated from the survival rate, clinical manifestation, target organ (liver, small intestine, skin) of the mouse. The percentage of NK cells in the spleen and the ratio of Th1 and Th2 cells in the spleen of the mice at different time points (1 week,2 weeks,3 weeks and 4 weeks) were detected by flow cytometry. dry and fine GV after cell transplantation The mechanism of action of HD. II. Main study results 1. According to the prior methods of the department, In vitro culture and amplification of hUCBDSCs was successfully achieved.2. The MHC half-phase was demonstrated by the test of the survival rate, the clinical manifestation, the pathological expression of the target organ and the fitting rate. 3.hUCBDSCs could significantly prolong the survival time of the experimental group and lower the death rate (P0.05). The expression and the pathological GVHD score of the target organs (P0.05), suggesting that the combined hUCBDSCs infusion may 4. hUCBDSCs could significantly increase the NK cell and Th2 cell ratio in the spleen of the experimental group after transplantation (P 0. 05), The ratio of Th1 cells after transplantation was decreased (P0.05). UCBDSC can significantly reduce the GVHD after the mouse MHC half-consistent PBSCT;2. hUCBDSCs may be the same as that of the NK cells and the Th2 cells in the spleen of the mouse after the transplantation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R457.7
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