人臍血基質(zhì)細胞對小鼠單倍體相合外周血干細胞移植GVHD調(diào)節(jié)作用
發(fā)布時間：2019-05-21 11:42
【摘要】：研究背景： 異基因造血干細胞移植（Allo-HSCT）是惡性及非惡性造血功能紊亂、某些免疫缺陷性疾病、自身免疫性、遺傳代謝性疾病等的有效治療手段[1]。過去的20年里，Allo-HSCT的臨床應(yīng)用發(fā)生了巨大變化，由原先的比較單一的人白細胞抗原（humanleucocyte antigen，HLA）相合的同胞間的清髓移植轉(zhuǎn)向一個更為復(fù)雜的領(lǐng)域，尤其在HLA半相合移植方面取得了重要進展，這一全新的移植方式有效解決了既往供者來源緊缺、配型困難問題，使得90%以上的患者可以在父母、子女、兄弟姐妹等親緣關(guān)系中找到合適供者[2]。 近來，針對HLA半相合造血干細胞移植方式提出了很多新的策略[3]。經(jīng)處理或者不經(jīng)處理的骨髓（bone marrow，BM）聯(lián)合或者不聯(lián)合外周血干細胞（peripheral bloodstem cell，PBSCs），成為臨床上最常見的干細胞來源[3][4]。然而，，骨髓采集物中含有大量紅細胞，當供受者血型不合時，進行移植之前需要去除骨髓中的紅細胞或者血漿。此外，骨髓采集為有創(chuàng)操作，采集過程中需要實施麻醉，增加了供者的痛苦和風險。PBSCs采集方便，有效避免了上述骨髓移植帶來的問題。外周血來源的移植物不僅具有豐富的CD34+細胞可大大提高植入率，且采集物比較干凈，受紅細胞影響小。然而，外周血中含有豐富的成熟T細胞，約為骨髓的5-10倍，大大增加了移植后GVHD的發(fā)生[5]。 我們實驗室前期工作中發(fā)現(xiàn)一種來源于人臍血的基質(zhì)細胞，稱為人臍血源基質(zhì)細胞（hUCBDSCs），hUCBDSCs可以在多種因子特定組合下實現(xiàn)體外的增殖擴增[6]。同時，hUCBDSCs表面高表達HLA-I分子，HLA-II類分子及其他共刺激分子表達較少，具有較低的免疫原性[7]。體外實驗發(fā)現(xiàn)其具有抑制異種T細胞增殖的功能，動物實驗證實hUCBDSCs具有減低小鼠MHC半相合骨髓造血干細胞移植后GVHD的能力[8][9]。本研究旨在探討hUCBDSCs對于小鼠MHC半相合PBSCT后GVHD的調(diào)節(jié)作用。 一．研究內(nèi)容及方法 1.參照科室常規(guī)方法，使用6%明膠聯(lián)合1.077g/LPercoll分離hUCBDSCs，特定的細胞因子組合下進行細胞體外培養(yǎng)擴增[8]； 2.雌性C57BL/6×BALB/c F1受鼠接受7.0Gy60Coγ射線照射后，經(jīng)尾靜脈輸注雄性供鼠C57BL/6來源的外周血單個核細胞（MNC）(1×106/只)、脾臟細胞(1×107/只)，構(gòu)建小鼠MHC半相合外周血干細胞移植后GVHD模型； 3.將體外培養(yǎng)擴增的hUCBDSCs（1×106/只）經(jīng)尾靜脈輸注到已構(gòu)建的MHC半相合外周血造血干細胞移植GVHD模型上，從小鼠生存率、臨床表現(xiàn)、靶器官（肝臟、小腸、皮膚）病理解剖評定各組小鼠GVHD發(fā)生情況； 4.流式細胞儀檢測移植后不同時間點（1周、2周、3周、4周）各組小鼠脾臟NK細胞比例，以及移植后3周小鼠脾臟Th1、Th2細胞比例，初步探索hUCBDSCs調(diào)節(jié)MHC半相合外周血造血干細胞移植后GVHD的作用機制。 二．主要研究結(jié)果 1.按照科室既往的方法，成功實現(xiàn)hUCBDSCs的體外培養(yǎng)擴增。 2.通過小鼠生存率、臨床表現(xiàn)、靶器官病理表現(xiàn)及嵌合率的檢測證明MHC半相合外周血造血干細胞移植GVHD模型構(gòu)建成功。 3. hUCBDSCs可顯著延長移植后實驗組小鼠生存時間，降低死亡率（P0.05）；hUCBDSCs可顯著降低移植后多個時相點實驗組小鼠臨床表現(xiàn)及靶器官組織病理GVHD評分（P0.05），提示聯(lián)合hUCBDSCs輸注可有效減輕小鼠MHC半相合PBSCT后GVHD反應(yīng). 4. hUCBDSCs可顯著增加移植后實驗組小鼠脾臟NK細胞、Th2細胞比例（P0.05），減低移植后Th1細胞比例（P0.05）。 三．主要結(jié)論 1. hUCBDSC可顯著減輕小鼠MHC半相合PBSCT后GVHD； 2. hUCBDSCs可能通過增加移植后小鼠脾臟NK細胞、Th2細胞比例同時減低Th1細胞比例來減輕小鼠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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R457.7
本文編號：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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R457.7
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本文編號：2482076
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