中国组织工程研究

中国组织工程研究 ›› 2024, Vol. 28 ›› Issue (7): 1007-1014.doi: 10.12307/2024.101

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

脊髓损伤模型小胶质细胞的高效移植替换策略

曾凡卓1，李雨欣2，孙嘉晨3，谷欣阳2，文  山1，田  鹤4，梅晰凡1   

  1. 1锦州医科大学附属第三医院骨科，辽宁省锦州市   121000；2复旦大学脑科学转化研究院，医学神经生物学国家重点实验室，上海市   200032；3锦州医科大学附属第一医院，辽宁省锦州市   121000；4辽宁省医学检测与药物研发协同创新中心，辽宁省锦州市   121000
  • 收稿日期:2022-12-15 接受日期:2023-02-18 出版日期:2024-03-08 发布日期:2023-07-15
  • 通讯作者: 梅晰凡，博士，教授，锦州医科大学附属第三医院骨科，辽宁省锦州市 121000
  • 作者简介:曾凡卓，男，1997年生，湖北省襄阳市人，汉族，锦州医科大学骨外科学在读硕士，主要从事脊髓损伤及小胶质细胞研究。
  • 基金资助:
    国家自然科学基金面上项目(82072165，82272256)，项目负责人：梅晰凡

Efficient strategies for microglia replacement in spinal cord injury models

Zeng Fanzhuo1, Li Yuxin2, Sun Jiachen3, Gu Xinyang2, Wen Shan1, Tian He4, Mei Xifan1   

  1. 1Department of Orthopedics, Third Affiliated Hospital of Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China; 2State Key Laboratory of Medical Neurobiology, Institute of Brain Science Transformation, Fudan University, Shanghai 200032, China; 3First Affiliated Hospital of Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China; 4Liaoning Medical Testing and Drug R&D Collaborative Innovation Center, Jinzhou 121000, Liaoning Province, China
  • Received:2022-12-15 Accepted:2023-02-18 Online:2024-03-08 Published:2023-07-15
  • Contact: Mei Xifan, MD, Professor, Department of Orthopedics, Third Affiliated Hospital of Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China
  • About author:Zeng Fanzhuo, Master candidate, Department of Orthopedics, Third Affiliated Hospital of Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 82072165, 82272256 (to MXF)

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摘要：


文题释义：

Mr BMT-X Ray：通过X射线辐射清髓实现的骨髓移植替代小胶质细胞。传统的骨髓移植方式为致死性辐射后直接移植骨髓细胞，导致移植率较低，仅为2%，而复旦大学彭勃教授等人开发的新型移植方式Mr BMT可实现高达92%的移植替换比率，但这种替换方式是全中枢的，对于脊髓损伤来说，这种方式可能会导致同时替换非治疗区域如脑内的细胞。为此，该实验改良了彭勃教授的移植方式，具体来说就是在移植时使用5 mm厚的铅板遮住小鼠的头部，实验证明可以实现仅替换小鼠的脊髓小胶质细胞，且替换效率也高达84.8%，将这种方式命名为Mr BMT-X Ray。
Mr BMT-Busulfan：通过白消安化疗清髓实现的骨髓移植替换小胶质细胞。传统的骨髓移植方式除辐射清髓移植外还有使用化疗药清髓移植，而复旦大学彭勃教授等人开发的移植方式Mr BMT虽然可以实现高效率替换，但致死性的辐射可能会损伤中枢导致炎症等情况。为此，该实验改良了彭勃教授的移植方式，使用化疗药白消安替代X射线辐射清髓，实验证明同样可以实现小鼠中枢神经系统的高效替换，将这种方式命名为Mr BMT-Busulfan。


背景：由于脊髓损伤的发生率逐年升高，且脊髓损伤后神经再生困难，因此如何促进脊髓损伤的恢复和提高脊髓损伤后干细胞及其他治疗细胞的移植效率，一直是临床及科研的研究热点和重点。

目的：建立脊髓损伤模型小鼠脊髓小胶质细胞高效率移植替换的方式。
方法：选取8-10周龄CX3CR1 creER-/+::LSL-BDNF-/+-tdTomato小鼠，CX3CR1 +/GFP小鼠，β-actin GFP小鼠及C57BL/6J野生型小鼠。按照实验要求随机分为6组：①假手术组：只掀除小鼠椎板而不做损伤，使用8只基因型为C57BL/6J野生型小鼠；②脊髓打击损伤组：使用8只基因型为C57BL/6J野生型小鼠；③脊髓钳夹损伤组，使用8只基因型为C57BL/6J野生型小鼠；④连体共生脊髓打击损伤组：通过手术将血液发绿色荧光的β-actin GFP小鼠与C57BL/6J野生型小鼠分别缝合在一起，使用8只β-actin GFP小鼠和8只C57BL/6J野生型小鼠；⑤Mr BMT-X Ray组(PLX5622清除脊髓小胶质细胞加X射线辐射清髓移植组)：将4只CX3CR1 creER-/+::LSL-BDNF-/+-tdTomato小鼠骨髓细胞移植给8只C57BL/6J野生型小鼠并进行脊髓打击损伤造模；⑥Mr BMT-Busulfan组(PLX5622清除脊髓小胶质细胞加Busulfan清髓移植组)：将4只CX3CR1 +/GFP小鼠的骨髓细胞移植给8只C57BL/6J野生型小鼠，只观察细胞移植替换比例，不进行脊髓损伤造模处理。假手术组、脊髓打击损伤组、脊髓钳夹损伤组小鼠在脊髓损伤后第14天灌流取材，连体共生脊髓打击损伤组小鼠在脊髓损伤后第7天灌流取材，Mr BMT-X Ray组小鼠在脊髓损伤后第28天灌流取材，Mr BMT-Busulfan组在移植后第28天灌流取材，取材部位为以小鼠脊髓T10节段为中心共1.2 cm长的脊髓；为观察Mr BMT-X Ray组和Mr BMT-Busulfan组是否会发生脑部的移植替换，故需额外留存小鼠的脑组织。通过使用转基因小鼠、连体共生及免疫荧光检测损伤区域移植和替换的细胞数目与比例。

结果与结论：与传统的经外周血移植方式即连体共生脊髓打击损伤组小鼠的细胞移植效率(9.8%)相比，新型移植方式Mr BMT-X Ray和Mr BMT-Busulfan可以大幅度提高脊髓小胶质细胞的移植替换比例，分别可以达到84.8%和95.6%，差异有显著性意义(P < 0.05)。结果表明，新型的细胞移植方式Mr BMT-X Ray和Mr BMT-Busulfan可以实现脊髓小胶质细胞的高效率替换，可以改善传统细胞移植方式存在的细胞移植效率低、存活数目少及分化不清楚的问题，并且Mr BMT-X Ray可以实现仅替换小鼠的脊髓小胶质细胞，而Mr BMT-Busulfan则可以避免X射线辐射移植方式可能造成的脑部炎症和损伤。

https://orcid.org/0000-0001-6595-1331 (曾凡卓) 

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: 脊髓损伤, 细胞移植替换, 小胶质细胞, 巨噬细胞, Mr BMT-X Ray, Mr BMT-Busulfan

Abstract: BACKGROUND: As the incidence of spinal cord injury increases with the years and axon regeneration after spinal cord injury was very difficult. How to promote the recovery from spinal cord injury and improve the transplantation efficiency of stem cells and other therapeutic cells after spinal cord injury has been the focus of clinical and scientific research.  
OBJECTIVE: To establish the efficient transplantation and replacement of mouse spinal cord microglia in the spinal cord injury model.
METHODS: CX3CR1 creER-/+::LSL-BDNF-/+-tdTomato mice, CX3CR1+/GFP mice, β-actin GFP mice and C57 BL/6J wild-type mice at 8-10 weeks of age were selected. According to the requirements of the experiment, they were randomly divided into six groups. (1) Sham operation group: eight C57 BL/6J wild-type mice were used when only the lamina was removed without injury. (2) Spinal cord contusion injury group: eight C57 BL/6J wild-type mice were used. (3) Spinal cord crush injury group: eight C57 BL/6J wild-type mice were used. (4) Conjoined symbiotic spinal cord strike injury group: β-actin GFP mice with green fluorescent blood were surgically stitched together with C57 BL/6J wild-type mice, using eight β-actin GFP mice and eight C57 BL/6J wild-type mice. (5) Mr BMT-X Ray group (using PLX5622 to eliminate the spinal microglia and bone marrow transplantation with X-ray radiation): Bone marrow cells from four CX3CR1 creER-/+::LSL-BDNF-/+-tdTomato mice were extracted and transplanted into eight C57 BL/6J wild-type mice for spinal cord injury modeling. (6) Mr BMT-Busulfan group (using PLX5622 to eliminate the spinal microglia and bone marrow transplantation with Busulfan): Bone marrow cells from four CX3CR1+/GFP mice were transplanted into eight C57 BL/6J wild-type mice. The percentage of cell transplantation replacement in this group was observed, and the spinal cord injury model was not established in this group. The sham operation group, spinal cord contusion injury group and spinal cord crush injury group were sampled by perfusion on day 14 after spinal cord injury. The conjoined symbiotic spinal cord strike injury group was sampled by perfusion on day 7 after spinal cord injury. Mr BMT-X Ray group was sampled by perfusion on day 28 after spinal cord injury. Mr BMT-Busulfan group was sampled by perfusion on day 28 after transplantation. The sampling site was a 1.2 cm long spinal cord with the T10 segment as the center. In the Mr BMT-X Ray group and Mr BMT-Busulfan group, additional mouse brain tissue was retained to see if it would lead to brain transplantation and replacement. The number and proportion of transplanted and replaced cells in the damaged area were measured using transgenic mice, symbiosis and immunofluorescence.  
RESULTS AND CONCLUSION: Compared with the traditional peripheral blood transplantation (9.8%) of mice in the conjoined symbiotic spinal cord strike injury group, the new transplantation methods, Mr BMT-X Ray and Mr BMT-Busulfan, could greatly improve the proportion of spinal microglia transplantation and replacement, which could reach 84.8% and 95.6%, respectively. The difference was significant (P < 0.05). The results showed that Mr BMT-X Ray and Mr BMT-Busulfan could achieve efficient replacement of spinal microglia cells, and could improve the problems of low cell transplantation efficiency, few survival numbers and unclear differentiation of the traditional cell transplantation methods. In addition, Mr BMT-X Ray can only replace the microglia in the spinal cord, while Mr BMT-Busulfan could avoid brain inflammation and injury caused by X-ray radiation transplantation.

Key words: spinal cord injury, cell transplantation and replacement, microglia, macrophage, Mr BMT-X Ray, Mr BMT-Busulfan

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