脊髓损伤模型小胶质细胞的高效移植替换策略  

Efficient strategies for microglia replacement in spinal cord injury models

作　　者：曾凡卓 李雨欣 孙嘉晨 谷欣阳 文山 田鹤 梅晰凡 Zeng Fanzhuo;Li Yuxin;Sun Jiachen;Gu Xinyang;Wen Shan;Tian He;Mei Xifan(Department of Orthopedics,Third Affiliated Hospital of Jinzhou Medical University,Jinzhou 121000,Liaoning Province,China;State Key Laboratory of Medical Neurobiology,Institute of Brain Science Transformation,Fudan University,Shanghai 200032,China;First Affiliated Hospital of Jinzhou Medical University,Jinzhou 121000,Liaoning Province,China;Liaoning Medical Testing and Drug R&D Collaborative Innovation Center,Jinzhou 121000,Liaoning Province,China)

机构地区：[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

出　　处：《中国组织工程研究》2024年第7期1007-1014,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(82072165,82272256),项目负责人:梅晰凡。

摘　　要：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;(5)Mr 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 cre ER^(-/+)::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;(6)Mr 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�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 eightβ-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

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 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R651.2