小鼠间充质干细胞抑制急性移植物抗宿主病的机制

发布时间：2018-01-04 15:35

本文关键词：小鼠间充质干细胞抑制急性移植物抗宿主病的机制　出处：《中国人民解放军军事医学科学院》2008年博士论文　论文类型：学位论文

【摘要】： 间充质干细胞(MSC)是最先从骨髓中分离出来的一群具有自我更新和多向分化潜能的成体干细胞。近年来许多学者在骨髓以外的组织器官中也分离出MSC,包括肌肉、脂肪、骨实质、胰腺、胸腺、肺脏、脐带和胎盘等几乎机体所有的组织和器官。MSC在特定的诱导条件下能够向骨、软骨、脂肪、肌肉等多种间充质系统细胞分化;体外和体内实验均证明了MSC及其分化细胞可给予造血细胞结构和功能上的支持,具有造血支持作用。MSC具有的这些功能使之成为了组织工程和骨髓移植细胞治疗的研究热点。造血干细胞移植是一些难治性血液病(白血病、再障、免疫缺损病、极重度放射病等)最有效且唯一的解决途径。急性移植物抗宿主病(aGvHD)是异体造血干细胞移植(allo-HSCT)常见的并发症和死亡原因。近年来,MSC强大的免疫调节功能逐渐受到人们的重视,这促使了MSC进入临床应用于aGvHD的治疗。目前,疗效良好,在美国已进入Ⅲ期临床。但由于小鼠MSC分离和培养困难的瓶颈,限制了MSC用于常用的小鼠模型进行体内免疫调节功能机制的研究。本研究的主要目的是探讨小鼠骨实质MSC的生物学特性及其抑制aGvHD的机制。具体研究内容包括建立一种新的小鼠MSC分离培养的方法—胶原酶消化后的骨实质分离小鼠MSC,并对细胞的一般形态、免疫表型、分化能力、支持造血和免疫调节功能进行鉴定;建立小鼠aGvHD模型,给予模型小鼠尾静脉共输注MSC,观察输注后小鼠平均生存时间,病理变化确定MSC抑制aGvHD的效果,并在此基础上对MSC抑制aGvHD的体内机制作进一步探讨。本实验中,采用了骨片消化法分离培养小鼠MSC。培养中,纤维样的细胞从骨片周围爬出,渐渐长满瓶皿;这些细胞有较均一的表型,是一群非造血(CD45~-),非巨噬(CD11b~-),非内皮(CD31~-),仅表达一些细胞粘附分子(CD29~+、CD44~+、CD105~+)的干细胞(Sca-1~+)群体,经传代后能够保持较稳定的细胞表型;具有较稳定的克隆形成能力;经诱导能够向脂肪、成骨、软骨细胞分化。进一步对这种方法培养的细胞进行功能检测,结果表明:它们体外具有与骨髓基质细胞相似的造血支持功能和抑制ConA或异种细胞刺激的淋巴细胞增殖的能力。MSC输注可延长异种皮肤移植的平均生存时间,说明此种小鼠骨实质来源的MSC具有体内免疫调节能力。 aGvHD发生的病理生理过程可划分为三个阶段。阶段Ⅰ:预处理作用,在移植之前的放化疗作用造成机体上皮和内皮的损伤,使抗原呈递细胞(APC)活化成熟;阶段Ⅱ:供者T细胞接受APC呈递的抗原,活化,增殖;阶段Ⅲ:炎性因子和细胞杀伤作用损伤靶器官。机体的损伤可进一步活化APC,使疾病进入一个渐进的恶性循环,所以,aGvHD一旦发生,就很难控制。应用异种脾细胞尾静脉输注建立小鼠aGvHD模型,发现MSC共输注延长了aGvHD小鼠的平均生存时间,并呈剂量依赖关系;改善了aGvHD小鼠靶器官的病理改变。进一步的对脾细胞表型及功能的检测表明:MSC输注降低了aGvHD小鼠脾脏CD11b~+细胞MHCⅡ和早期活化分子CD69的表达;降低了CD3~+细胞早期活化分子CD69的表达和脾细胞的增殖;降低了脾细胞的杀伤功能。MSC提高了脾脏CD4~+细胞的比例,晚期降低了CD8~+细胞的比例,从而明显提高了CD4~+/CD8~+的比值;CD4~+CD25~+调节性T细胞(Tregs)活化后表达FoxP3,临床观察,aGvHD患者单独Tregs数量与疾病严重程度无相关性,原因在于aGvHD患者存在着Tregs的数量严重减少和T淋巴细胞的大量增殖。Tregs的靶细胞是CD8~+T淋巴细胞,患者的外周血和靶器官FoxP3~+/CD8~+的比值与疾病严重程度有密切的相关性。我们的实验结果与此相互验证:MSC仅略微提高了Tregs的比例,但Tregs/CD8~+的比值却被显著提高。 aGvHD的发生发展有三个必要条件,是在1966年界定的Billingham三原则:1.宿主必须处于免疫抑制或缺陷状态;2.移植物中含有免疫功能的细胞;3.宿主和供者之间存在着不相容性抗原。在1989年提出,并在1993年确定并加入的第四条原则:效应细胞必须能迁移到aGvHD靶器官,称为Billingham的修订原则,此修订原则强调了效应细胞迁移到aGvHD靶器官的重要性。令我们感兴趣的是:MSC输注增加了aGvHD小鼠二级淋巴器官(SLO)的T淋巴细胞的数量。表达CD62L和CCR7分子是T淋巴细胞进入SLO的先决条件。体外的混合淋巴细胞反应中,MSC的存在,使T细胞(包括Tregs)保持了幼稚细胞的表型(CD62L~+/CCR7~+),并且保持了这些细胞的由二级淋巴组织趋化因子(SLC)引导的细胞迁移。树突状细胞(DC)是体内免疫反应的启动者,表达CCR7对DC的成熟并进一步向SLO迁移至关重要。在体外DC诱导过程中,MSC的存在降低了DC的CD80、CD86、CD40、CDIa~b的表达并明显降低了CCR7的表达,并且这种DC向SLC的迁移作用也相应的减弱。以上过程主要是通过MSC分泌的可溶性分子来实现的。T细胞活化和DC的迁移功能的发挥总是与细胞骨架重排相伴行的。MSC共培养减少了T细胞极化和伪足的形成,减少了DC的极化和树突的形成,降低其相关信号通路蛋白Racl和Cdc42的活化可能使其原因。相一致地,MSC输注使aGvHD小鼠SLO中T细胞保持了幼稚(CD62L~+/CCR7~+)的表型,并且降低了SLO中DC的比例和CCR7的表达。用转入eGFP的MSC输注来示踪输注后MSC的体内分布,发现MSC主要分布于外周组织和器官,而极少进入SLO,MSC极少表达CD62L和CCR7可能是这一现象的原因。综上所述,此种简单易行方法所培养的小鼠骨实质MSC,符合MSC的特征,具有MSC功能,可进一步用于小鼠生理和病理模型的研究;MSC从aGvHD发-生的三个阶段均能抑制其发展,改变T淋巴细胞亚群的比例可能是MSC体内发挥免疫调节的机制之一;通过改变T和DC的迁移功能对MSC抑制aGvHD起到了重要作用。本研究的创新点:1.应用一种新方法培养小鼠MSC,突破了小鼠MSC培养困难的瓶颈;2.将MSC应用于小鼠aGvHD模型,进行MSC体内免疫调节功能的机制探讨;3.发现MSC在体外,体内均能改变T细胞和DC的迁移功能,此改变是MSC抑制aGvHD的机制之一。此研究对MSC移植发挥体内免疫调节的机制提供了新线索,为MSC更好地应用于临床治疗奠定基础。
[Abstract]:Mesenchymal stem cells (MSC) were isolated from bone marrow of a group of self-renewal and multilineage differentiation potential of adult stem cells. In recent years, many scholars in the bone marrow tissues also isolated MSC, including muscle, fat, bone substance, pancreas, thymus, lung, umbilical cord and placenta. Almost all body tissues and organs of.MSC to bone, cartilage, fat in the induction of specific conditions, such as muscle mesenchyme cell differentiation; in vitro and in vivo experiments have demonstrated the MSC and differentiated cells can give hematopoietic cell structure and function support, support hematopoiesis.MSC has these features enable it has become a research hotspot in tissue engineering and cell therapy. Bone marrow transplantation of hematopoietic stem cell transplantation is some refractory hematological diseases (leukemia, aplastic anemia, immunodeficiency disease, severe radiation sickness etc.) the most effective and unique solution No way. Acute graft-versus-host disease (aGvHD) is allogeneic hematopoietic stem cell transplantation (allo-HSCT) common complications and causes of death. In recent years, immune MSC strong regulatory function has attracted more and more attention. This prompted the MSC treatment in clinical application in aGvHD. At present, the United States has good curative effect. In phase III clinical. But because of the mouse MSC isolate and culture difficult bottleneck, restrict the research on MSC in murine model of in vivo immune mechanism.
The main purpose of this study is to explore the biological characteristics mechanism of mouse bone MSC and inhibition of aGvHD. The research contents include bone substance isolated from mouse MSC after collagenase digestion method and establish a novel mouse MSC were isolated, and the general form of cell immunophenotype, differentiation ability, support hematopoiesis and immune identification regulating function; establish the mouse model of aGvHD, given the model of mouse tail vein infusion of MSC were observed after infusion, average survival time of mice, the pathological changes of MSC inhibit the effects of aGvHD, to further explore the in vivo of MSC inhibiting aGvHD production and on this basis.
In this experiment, using a bone chip digestion method of isolation and culture of mouse MSC. fibroblast like cells in culture, to climb out from the bone around, gradually covered the bottle dish; these cells have the phenotype of a uniform, is a group of non hematopoietic (CD45~-), non macrophage (CD11b~-), endothelium (CD31~-), only the expression of some cell adhesion molecules (CD29~+, CD44~+, CD105~+) stem cells (Sca-1~+) group, after passage can maintain a stable phenotype; with stable clone forming ability; Osteogenic induced by fat, cartilage cell differentiation. Further these cells function test results they show that in vitro with the average survival time is similar to bone marrow stromal cells support hematopoiesis function and inhibit ConA or xenogeneic cells stimulated lymphocytes proliferation ability of.MSC infusion can prolong the xenograft skin, the mouse bone from MSC It has the ability to regulate the immune system in the body.
The pathophysiological process of the occurrence of aGvHD can be divided into three stages. The first phase: effect of pretreatment, the epithelial and endothelial damage caused by chemotherapy in action before transplantation, the antigen presenting cells (APC) activation; stage II: donor T cells underwent APC antigen presentation, activation, proliferation; phase III the inflammatory factors and cell killing effect of damage to the target organ. The damage of organism can further activate APC, bringing the disease into a progressive vicious spiral, so once aGvHD occurs, it is difficult to control. The application of intravenous injection of xenogeneic spleen cells in mice aGvHD model showed that MSC co infusion prolonged the average survival time of aGvHD mice, in a dose-dependent manner; improve the pathological changes of target organs of aGvHD mice. The spleen cells showed that the phenotype and function of further MSC infusion reduced aGvHD mouse spleen CD11b~+ cells and early activation of MHC II The expression of CD69 molecules; reduce early CD3~+ cell proliferation and activation of CD69 molecules and expression of spleen cells; reduce the spleen cell killing function of.MSC increased the proportion of CD4~+ cells in spleen, late reduced the proportion of CD8~+ cells, resulting in a significant increase of the ratio of CD4~+/CD8~+; CD4~+CD25~+ regulatory T cells (Tregs) expression and clinical observation FoxP3, after activation, no correlation with aGvHD Tregs alone the number and severity of disease in patients with aGvHD due to the number of Tregs CD8~+ is severely reduced T lymphocyte target cell proliferation of.Tregs and T lymphocytes, there was a close correlation between the ratio of patients with disorders of the peripheral blood and the severity of target organ of FoxP3~+/CD8~+. We the experimental results and the mutual authentication: MSC only slightly increased the proportion of Tregs, but the ratio of Tregs/CD8~+ was significantly increased.
There are three necessary conditions for the occurrence and development of aGvHD in 1966, is defined by the Billingham three principles: 1. host must be in immunodeficiency state; immune function contains 2. cells in the graft; 3. between host and donor incompatible with antigen. In 1989, fourth principles and determined to join in 1993: effector cells must be able to migrate to the target organ of aGvHD, known as the revised Billingham principles, this revision principle emphasizes the importance of the aGvHD effect cells migrate to target organs. It is interesting for us: MSC infusion increased aGvHD mice two secondary lymphoid organs (SLO) the number of T lymphocytes and CD62L. The expression of CCR7 is a prerequisite for T cell into SLO. The mixed lymphocyte reaction in vitro in the presence of MSC, T cells (including Tregs) maintain phenotype of immature cells (CD62L~+/CCR7~+), and keep these The cell is composed of two lymphoid tissue chemokine (SLC) cell migration guide. Dendritic cells (DC) is an immune response in the body to start, CCR7 on the maturation of DC expression and further to SLO migration is essential. In vitro induced by DC in the process of MSC was decreased in the presence of DC CD80, CD86, CD40. The expression of CDIa~b and decreased the expression of CCR7, DC and the migration effect of SLC also decreased. The above process is the main migration function of soluble molecules through the secretion of MSC to achieve the.T cell activation and DC play is always accompanied with the cytoskeletal rearrangement of.MSC co culture to reduce the formation of T cell polarization and actin, reducing the formation of polarization and dendritic DC, reduce the activation of the signal pathway related proteins Racl and Cdc42 may be the reason. Consistently, MSC infusion of T cells in aGvHD mice SLO keep the naive (CD62L~+/CCR7~+) Phenotypes, and reduced the proportion of DC and CCR7 expression in SLO. The MSC distribution in eGFP was used to trace the distribution of MSC after infusion. It was found that MSC was mainly distributed in peripheral tissues and organs, but rarely entered SLO. MSC rarely expressed CD62L and CCR7, which may be the reason for this phenomenon.
In conclusion, this simple method of cultured mouse bone MSC, with the characteristics of MSC, with MSC function, can be used for further research of the physiology and pathology of mice; MSC from aGvHD - three stage students can restrain the development and changes of T lymphocyte subsets ratio may be one mechanism of in vivo MSC immune regulation; through the migration function changes of T and DC on the inhibition of MSC aGvHD played an important role.
The innovation of this research: 1. the application of a new method of culturing mouse MSC, break through the bottleneck of the difficulty in the culture of mouse MSC; 2. the application of MSC in a rat model of aGvHD, discusses the mechanism of MSC in immune function; 3. MSC in vitro, and in vivo changes of T cells and DC transfer function, this change is one of the mechanisms of MSC inhibition of aGvHD.
This study provides new clues for the mechanism of MSC transplantation to play the immune regulation in the body and lays the foundation for the better application of MSC to clinical treatment.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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