骨髓间充质干细胞在致敏与非致敏BALB/c小鼠造血干细胞移植中的应用

doi:10.3969/j.issn.2095-4344.2015.32.024

摘要/Abstract

摘要：

背景：造血干细胞移植对多种疾病具有治疗作用，但其取材不便，且细胞数量受年龄限制等原因，故而应用具有一定局限性。
目的：探索骨髓间充质干细胞在致敏与非致敏BALB/c小鼠造血干细胞移植中的应用价值。
方法：将BALB/c小鼠骨髓细胞在体外进行分离，采用贴壁培养的方法获得间充质干细胞，使用流式细胞仪对细胞表面的分子标记进行检测。应用异基因脾细胞输注方法建立致敏动物模型，用绿色荧光染料标记骨髓间充质干细胞，分别移植到致敏和非致敏的受体小鼠体内，并在移植后的不同时间点对间充质干细胞的归巢情况进行检测。对致敏BALB/c小鼠进行照射预处理，联合应用异基因骨髓细胞与同基因间充质干细胞移植，观察BALB/c小鼠的生存情况。
结果与结论：移植48 h后，间充质干细胞在致敏受体和非致敏受体小鼠分别归巢于脾脏和骨髓。在造血干细胞的移植实验中，致敏BALB/c小鼠接受异基因骨髓细胞与同基因骨髓间充质干细胞联合移植，结果显示致敏BALB/c小鼠全部在移植后12-15 d死亡，生存的中位时间是14 d，而仅接受异基因骨髓细胞移植的致敏BALB/c小鼠的中位生存时间为13 d。说明细胞移植后在致敏受体内间充质干细胞主要归巢为脾脏和骨髓，联合应用间充质干细胞移植对异基因造血干/祖细胞植入致敏受体体内并没有起到有效的促进作用。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 干细胞, 移植, 骨髓, 间充质干细胞, 造血干细胞, 移植, 致敏受体, 流式细胞仪, 致敏动物模型

Abstract:

BACKGROUND: Hematopoietic stem cell transplantation has therapeutic effects on many diseases, but its application has some limitations, such as cell harvesting and age-limited number of cells.
OBJECTIVE: To investigate the application value of bone marrow mesenchymal stem cells in hematopoietic stem cell transplantation for sensitized and non-sensitized BALB/c mice.
METHODS: Bone marrow cells derived from BALB/c mice were isolated and cultured in vitro to harvest mesenchymal stem cells using adherent method. The cell surface markers were detected by flow cytometry. A murine model of sensitization was established by transfusion of allogeneic spleen cells. Mesenchymal stem cells labeled with green fluorescent dye were transplanted into non-sensitized and sensitized recipient mice, and the homing of mesenchymal stem cells in vivo was monitored at different time points post transplantation. Additionally, under irradiation pretreatment, sensitized BALB/c mice under irradiation were subjected to combined transplantation of allogeneic bone marrow cells and syngeneic mesenchymal stem cells. Survival rate of BALB/c mice was monitored daily.
RESULTS AND CONCLUSION: At 48 hours after transplantation, mesenchymal stem cells in sensitized and non-sensitized recipients were homing to the spleen and bone marrow, respectively. In the experiment of hematopoietic stem cell transplantation, the sensitized recipients died at 12-15 days after combined
transplantation, with a median of 14 days; however, the sensitized recipients only undergoing allogeneic bone marrow cell transplantation had a survival median of 13 days. These findings indicate that the transplanted mesenchymal stem cells in sensitized recipients are mainly homing to the spleen and bone marrow, but the combination transplantation cannot enhance the transplantation of allogeneic hematopoietic stem/progenitor cells in sensitized recipients.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Immunization, Hematopoietic Stem Cell Transplantation, Spleen, Cell Transplantation

中图分类号:

R394.2

郭小伟，关晨霞. 骨髓间充质干细胞在致敏与非致敏BALB/c小鼠造血干细胞移植中的应用[J]. 中国组织工程研究, 2015, 19(32): 5215-5219.

Guo Xiao-wei, Guan Chen-xia. Application of bone marrow mesenchymal stem cells in hematopoietic stem cell transplantation for sensitized and non-sensitized BALB/c mice[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(32): 5215-5219.

图/表（结果） 2

2.1 间充质干细胞的表型使用流式细胞仪检测第4代骨髓间充质干细胞(图1)，发现骨髓间充质干细胞阳性表达Scal⁺、CD29⁺、CD105⁺和CD44⁺，但是不表达造血细胞表面标记CD11b^-、CD34^-。

2.2 间充质干细胞的归巢情况流式细胞仪结果发现，移植后6 h，间充质干细胞主要归巢于BALB/c小鼠的脾脏和外周血。移植后24 h，间充质干细胞主要归巢于BALB/c小鼠脾脏和骨髓，移植后48 h，致敏组骨髓间充质干细胞主要归巢于BALB/c小鼠的脾脏，非致敏组骨髓间充质干细胞主要归巢于BALB/c小鼠的骨髓。致敏组小鼠脾脏间充质干细胞数量在移植后6，24 h时比非致敏组多。说明骨髓间充质干细胞在非致敏与致敏受体归巢情况存在差异，见图2。 2.3 间充质干细胞在致敏受体造血干细胞移植的作用单纯进行照射但是没有移植的小组的BALB/c小鼠在进行照射后10-16 h全部死亡，说明该组的处理方案中BALB/c小鼠的致死因是8 Gy ⁶⁰Co照射；对非致敏受体进行8 Gy ⁶⁰Co照射预处理，并移植1×10⁷异基因供体骨髓细胞后28 d时，小鼠依然存活，说明1×10⁷异基因供体骨髓细胞足量；对致敏组BALB/c小鼠进行8 Gy ⁶⁰Co照射预处理，并移植1×10⁷异基因供体骨髓细胞后12-15 d，小鼠全部死亡，生存中位数是时间13 d。另对致敏小鼠进行8 Gy 60 Co照射预处理，并联合移植1×10⁷异基因骨髓细胞和1×10⁶同基因骨髓间充质干细胞后12-16 d，小鼠全部死亡，生存中位数时间为14 d。2组致敏BALB/c小鼠的生存时间差异无显著性意义(P > 0.05)，说明联合应用间充质干细胞移植对异基因造血干/祖细胞植入致敏受体体内并没有起到有效的促进作用。

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引言

材料方法

讨论

根据相关的临床调查发现，致敏是导致实体器官移植失败的重要因素，致敏受者体内的抗体对肾移植物的存活具有重要的影响^[12-19]。目前，全球面临的一个亟待解决的问题是怎样促进造血干/祖细胞在致敏受者体内植入。国外有研究人员对这一问题进行了探讨，他们对动物进行了实现，实验显示导致造血干细胞移植出现排斥的主要原因是由抗体介导的体液免疫^[20-24]。间充质干细胞是一群非造血细胞，具有促进造血和调节免疫的作用及多向分化潜能^[25]。研究显示，间充质干细胞在细胞和体液免疫中能发挥一定的效果，它能抑制免疫反应的发生，以促使受者的致敏程度降低。研究结果还证实了间充质干细胞能有效抑制致敏受者的移植排斥作用。由此可见，间充质干细胞对治疗敏受者移植排斥具有一定的效果^[12^，^26-29]。本实验体外分离了BALB/c小鼠的骨髓细胞，并且成功培育出了间充质干细胞，证实了骨髓间充质干细胞发展到第4代所具备的免疫抑制功能十分强大^[30-36]。作者通过对BABL/c小鼠输注异基因脾细胞的方法建立了致敏模型，对同基因骨髓间充质干细胞使用荧光染料羧基荧光素二醋酸盐琥珀酰亚胺酯进行染色标记，选取正常BALB/c小鼠为非致敏受体，检测了间充质干细胞移植后在BALB/c小鼠体内的归巢情况^[37-43]。结果发现，移植后第48小时，在致敏组中，骨髓间充质干细胞主要归巢于BALB/c小鼠的脾脏，而在非致敏组中，骨髓间充质干细胞主要归巢于BALB/c小鼠的骨髓，说明骨髓间充质干细胞在非致敏与致敏受体体内归巢情况有明显差异，间充质干细胞的归巢情况受到移植受者体内的微环境影响^[44-45]。临床上的大量实验研究显示，异基因造血干/祖细胞在移植受者体内植入情况受到间充质干细胞的影响，间充质干细胞对其具有促进作用，这与间充质干细胞促进造血生成等具有一定的关系^[46]。本实验对间充质干细胞在致敏与非致敏BALB/c小鼠造血干细胞移植中的应用价值进行了研究，结果发现联合应用间充质干细胞和异基因骨髓细胞进行移植后，经照射预处理后的致敏BALB/c小鼠依然全部死亡了，说明联合应用间充质干细胞移植对异基因造血干/祖细胞植入致敏受者体内并没有起到有效的促进作用^[47]。造成间充质干细胞对致敏受体造血干细胞移植作用的阴性结果的可能原因很多，如：移植受者的致敏程度、间充质干细胞输注时间和剂量等^[48]。目前，临床上研制出了多种治疗高敏患者的方法，如：免疫抑制剂、免疫吸附和单克隆抗体等。已经有研究人员通过实验证实了将以上方法联合应用在降低受者的致敏程度、促进移植物的植入和降低排斥的发生率等方面具有一定的效果^[49]。有研究显示使用CTLA4免疫球蛋白和抗CD154单克隆抗体对信号途径具有协同和刺激作用，能明显抑制体液免疫和细胞免疫。能否联合应用间充质干细胞输注和免疫抑制剂等途径治疗致敏受者需要进行深入的研究^[50]。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程