实验动物与比较医学 ›› 2024, Vol. 44 ›› Issue (2): 139-148.DOI: 10.12300/j.issn.1674-5817.2023.121
刘佳1(
), 叶岩荣2, 沈赟2, 唐启瑛3, 陈梅卿2, 易可慧4, 陈少壮2(
)()
收稿日期:2023-08-30
修回日期:2024-02-18
出版日期:2024-05-09
发布日期:2024-04-25
通讯作者:
陈少壮（1994—），男，本科，主管药师：药理学、药剂学、药品的合理应用及指导、药物临床应用与研究、神经科学、肿瘤学。E-mail：394290375@qq.com。ORCID：0009-0009-5801-1582作者简介:刘佳（1988—），女，硕士，主治医师，研究方向：中西医结合神经科学、中西医结合肿瘤学、中西医结合内科学、药物临床应用与研究。E-mail：z3335014322@sina.com
基金资助:
Jia LIU1(), Yanrong YE2, Yun SHEN2, Qiying TANG3, Meiqing CHEN2, Kehui YI4, Shaozhuang CHEN2()()
Received:2023-08-30
Revised:2024-02-18
Published:2024-04-25
Online:2024-05-09
Contact:
CHEN Shaozhuang (ORCID: 0009-0009-5801-1582), E-mail: 394290375@qq.com摘要：
目的 探究银杏内酯B调控缺血性脑卒中恢复期小鼠的脑内T细胞生物学特性及T细胞与胶质细胞间的作用机制。 方法 选取36只成年C57BL/6小鼠，随机分为假手术组（Sham组）、对照组（PBS组）和银杏内酯B组（GB组）。Sham组仅给予假手术处理；PBS组和GB组均采用线栓法制备大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）损伤即缺血性脑卒中模型，并于损伤后连续14 d分别鼻饲等体积的PBS和银杏内酯B溶液。采用转棒实验及神经功能评分法评估3组小鼠的神经功能变化；并于实验第15天取PBS组和GB组小鼠脑损伤区及周边皮层、胼胝体及纹状体的新鲜组织，采用单细胞测序方法评估该区域中T细胞及其亚群的生物学特性，并进一步探索T细胞、小胶质细胞和少突胶质细胞之间的相互作用及机制。 结果 与Sham组相比，PBS组和GB组小鼠的神经功能评分均显著上升（P＜0.001），掉落前运动时程均显著降低（P＜0.001）；与PBS组相比，GB组在缺血性脑损伤后5、10、15 d的神经功能评分有下降趋势，掉落前运动时程有上升趋势，尤其15 d时的掉落前运动时程显著上升（P＜0.05）。与PBS组相比，GB组小鼠在脑损伤后15 d时脑内T细胞增殖活性显著升高（P＜0.05），增殖性T细胞数量及脂质代谢水平均显著上调（P＜0.05），所有T细胞的细胞外基质重塑显著增多（P＜0.05）；同时，GB组小鼠脑内T细胞与小胶质细胞、少突胶质细胞之间的相互作用以及小胶质细胞自身、小胶质细胞与少突胶质细胞之间的相互作用均显著增强，主要表现为CD74与巨噬细胞迁移抑制因子（macrophage migration inhibitory factor，MIF）、集落刺激因子1受体（colony stimulating factor 1 receptor，CSF1R）与集落刺激因子1（colony stimulating factor 1，CSF1）的相互作用增强（P＜0.05），但T细胞的炎性水平与PBS组相比均无显著差异。 结论 采用线栓法MCAO损伤手术可成功构建小鼠缺血性脑卒中疾病模型。银杏内酯B可能通过调控小鼠脑内T细胞生物学特性及其与胶质细胞间的互作关系，促进小鼠脑损伤后的神经功能恢复。
中图分类号:
刘佳, 叶岩荣, 沈赟, 唐启瑛, 陈梅卿, 易可慧, 陈少壮. 银杏内酯B通过调控脑内T细胞特性及与胶质细胞间相互作用促进缺血性脑卒中小鼠的神经功能恢复[J]. 实验动物与比较医学, 2024, 44(2): 139-148.
Jia LIU, Yanrong YE, Yun SHEN, Qiying TANG, Meiqing CHEN, Kehui YI, Shaozhuang CHEN. Ginkgolide B Promotes Neural Function Recovery of Ischemic Stroke Mice by Regulating Characteristics of Brain T Cells and Their Interactions with Glial Cells[J]. Laboratory Animal and Comparative Medicine, 2024, 44(2): 139-148.
图1 大脑中动脉栓塞（MCAO）损伤后各组小鼠神经功能的变化注：A，记录小鼠掉落前在转棒上表现出的最高的运动评分，评分越高，表示神经功能越差；可见术后5、10、15 d时GB组的神经功能评分较PBS组有下降趋势，但差异无统计学意义（P > 0.05）。B，记录小鼠在转棒上掉落前的运动时程，时间越长，表示神经功能恢复得越好；可见术后5、10、15 d时GB组的掉落前运动时程较PBS组有上升趋势，且在15 d时差异有统计学意义（*P＜0.05）。Sham组（n=6），仅假手术处理；PBS组（n=15），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲PBS；GB组（n=15），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲银杏内酯B溶液。
Figure 1 Changes of neurological function of different groups of mice after middle cerebral artery occlusion （MCAO）Note： A, The highest neurological scores of each mouse before falling from the rotarod were recorded. A higher score indicates poorer neurological function. It was observed that the neurological scores of the GB group showed a decreasing trend compared to the PBS group on days 5, 10, and 15 after MCAO, but there was no statistically significant difference (P>0.05). B, The duration of motor activity on the rotarod before falling was recorded for mice. Longer durations indicated better recovery of neurological function. It was evident that on days 5, 10, and 15 after MCAO, the pre-fall motor durations of the GB group showed an increasing trend compared to the PBS group, and there was a statistically significant difference at day 15 (*P<0.05). Sham group (n=6): Received sham surgeries only. PBS group (n=15): Prepared with the filament method to create a model of MCAO, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=15): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.
图2 U score评估大脑中动脉栓塞（MCAO）造模后各组小鼠脑内T细胞的增殖情况注：A，GB组和PBS组小鼠受损脑组织中总的T细胞U评分比较显示，GB组的T细胞增殖活性较PBS组显著升高（*P＜0.05）；B，半监督UMAP降维可视化数据显示出所有T细胞，从单细胞维度观察细胞增殖情况；C，比较两组小鼠脑组织中反映CD8+ T细胞、γδT（GDT）细胞、辅助性T细胞、初始T细胞、自然杀伤（NK）细胞、NKT细胞、增殖性T细胞增殖情况的U评分，GB组较PBS组的增殖性T细胞显著增加（P＜0.05）。PBS组（n=6），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲PBS；GB组（n=6），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲银杏内酯B溶液。
Figure 2 U score assessment of T cells proliferation in the brains of mice after middle cerebral artery occlusion（MCAO）Note： A, Comparison of overall T-cell U scores in the damaged brain tissues of the GB and PBS group revealed a significant increase in T-cell proliferative activity in the GB group compared to the PBS group (*P<0.05). B， Semi-supervised uniform manifold approximation and projection（UMAP） dimensionality reduction visualization displayed all T-cells, enabling observation of cellular proliferation at the single-cell level. C, Comparison of the proliferation of CD8+ T cells, γδT （GDT) cells, helper T cells, naive T cells, natural killer（NK） cells, NKT cells, and proliferative T cells in the brain tissues of both groups based on U scores revealed a significant increase in the proliferative T cells in the GB group compared to the PBS group (P < 0.05). PBS group (n=6): Prepared with the filament method to create a model of MCAO, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=6): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.
图3 U score评估大脑中动脉栓塞（MCAO）造模后各组小鼠脑内T细胞的细胞外基质重塑情况注：A，GB组和PBS组小鼠受损脑组织中反映T细胞细胞外基质重塑情况的U评分比较，GB组的T细胞的细胞外基质重塑较PBS组显著增多（*P＜0.05）；B，半监督UMAP降维可视化数据显示出所有T细胞，从单细胞维度观察细胞的细胞外基质重塑情况；C，比较两组小鼠脑组织中反映CD8+ T细胞、γδT（GDT）细胞、辅助性T细胞、初始T细胞、自然杀伤（NK）细胞、NKT细胞、增殖性T细胞的细胞外基质重塑情况的U评分，GB组所有细胞外基质重塑与PBS组相比均显著增加（P＜0.05）。PBS组（n=6），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲PBS；GB组（n=6），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲银杏内酯B溶液。
Figure 3 U score assessment for extracellular matrix remodeling of T cells after middle cerebral artery occlusion （MCAO）Note： A, Comparison of U scores for extracellular matrix remodeling of T cells in the damanged brain tissues of the GB and PBS groups showed significantly increased remodeling in the GB group compared to the PBS group (*P <0.05). B, Semi-supervised UMAP dimensionality reduction visualization revealed all T cells, enabling observation of extracellular matrix remodeling at the single-cell level. C, Comparison of U scores for extracellular matrix remodeling among CD8+ T cells, γδT （GDT) cells, helper T cells, naive T cells, natural killer （NK） cells, NKT cells, and proliferative T cells in the brain tissues of both mouse groups showed a significant increase in extracellular matrix remodeling across all cell types in the GB group compared to the PBS group (P < 0.05). PBS group (n=6): Prepared with the filament method to create a model of MCAO, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=6): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.
图4 U score评估大脑中动脉栓塞（MCAO）造模后的各组小鼠脑内T细胞的脂质代谢情况注：A，GB组和PBS组小鼠受损脑组织中反映T细胞脂质代谢情况的U评分比较，GB组的T细胞脂质代谢水平较PBS组显著升高（*P＜0.05）；B，半监督UMAP降维可视化数据显示出所有T细胞，从单细胞维度观察细胞脂质代谢情况；C，比较两组小鼠脑组织中反映CD8+ T细胞、γδT（GDT）细胞、辅助性T细胞、初始T细胞、自然杀伤（NK）细胞、NKT细胞、增殖性T细胞脂质代谢情况的U评分，GB组较PBS组的增殖性T细胞的脂质代谢水平显著增加（P＜0.05）。PBS组（n=6），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲PBS；GB组（n=6），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲银杏内酯B溶液。
Figure 4 U score assessment for lipid metabolism of T cells in the brains of mice after middle cerebral artery occlusion （MCAO）Note：A, Comparison of U scores for overall T cell lipid metabolism in the damaged brain tissues of the GB and PBS groups showed a significant increase in lipid metabolism levels in T cells of the GB group compared to the PBS group (*P <0.05). B, Semi-supervised UMAP dimensionality reduction visualization displayed all T cells, enabling observation of lipid metabolism at the single-cell level. C, Comparison of U scores for lipid metabolism among CD8+ T cells, γδT （GDT) cells, helper T cells, naive T cells, natural killer （NK） cells, NKT cells, and proliferative T cells in the brain tissues of both mouse groups revealed a significant increase in lipid metabolism levels in proliferative T cells of the GB group compared to the PBS group (P < 0.05). PBS group (n=6): Prepared with the filament method to create a model of MCAO, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=6): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.
图5 U score评估大脑中动脉栓塞（MCAO）造模后的GB组、PBS组T细胞的炎性水平注：A，GB组和PBS组小鼠受损脑组织中反映T细胞炎性水平的U评分比较，GB组T细胞的炎性水平与PBS组无显著差异（nsP > 0.05）；B，半监督UMAP降维可视化数据显示出所有T细胞，从单细胞维度观察细胞的炎性水平；C，比较两组小鼠脑组织中反映CD8+T细胞、γδT（GDT）细胞、辅助性T细胞、初始T细胞、自然杀伤（NK）细胞、NKT细胞、增殖型T细胞炎性水平的U评分，GB组与PBS组T细胞的炎性水平均无显著差异。PBS组（n=6），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲PBS；GB组（n=6），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲银杏内酯B溶液。
Figure 5 U score assessment for inflammatory levels of T cells in the brains of mice after middle cerebral artery occlusion （MCAO）Note： A, Comparison of U scores for the overall inflammatory levels of T cells in the damaged brain tissues of the GB and PBS groups showed no significant difference (nsP>0.05) in the inflammatory levels of T cells between the GB group and the PBS group. B, Semi-supervised UMAP dimensionality reduction visualization revealed all T cells, enabling observation of inflammatory levels from a single-cell perspective. C, Comparison of U scores for the inflammatory levels of CD8+ T cells, γδT （GDT) cells, helper T cells, naive T cells, natural killer （NK） cells, NKT cells, and proliferative T cells in the brain tissues of both mouse groups indicated no significant differences in the inflammatory levels of T cells between the GB and PBS groups. PBS group (n=6): Prepared with the filament method to create a model of MCAO, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=6): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.
图6 通过细胞因子（A）和生长因子（B）的含量来判断T细胞与小胶质细胞、少突胶质细胞之间的相互作用注：相比于PBS组，GB组小鼠脑内T细胞与小胶质细胞、少突胶质细胞之间的相互作用，小胶质细胞自身、小胶质细胞与少突胶质细胞之间的相互作用均显著增强，主要表现为CD74与巨噬细胞迁移抑制因子（MIF）、集落刺激因子1受体（CSF1R）与集落刺激因子1（CSF1）的相互作用增强（*P＜0.05）。点的大小表示相互作用的百分比，颜色表示对数差异表达水平，由DotPlot函数计算。PBS组（n=6），采用线栓法制备大脑中动脉栓塞（MCAO）模型，并于损伤后连续14 d鼻饲PBS；GB组（n=6），采用线栓法制备MCAO模型，并于损伤后连续14 d鼻饲银杏内酯B溶液。
Figure 6 Assessment for interactions among T cells， microglia， and oligodendrocytes based on the levels of cytokines （A） and growth factors （B）Note：In comparison with the PBS group, the interactions between T cells and microglia, as well as oligodendrocytes, in the brains of mice from the GB group were significantly enhanced, including interactions within microglia and between microglia and oligodendrocytes. This was primarily manifested by increased interactions between CD74 and macrophage migration inhibitory factor 1（MIF), as well as macrophage migration inhibitory factor 1（CSF1R) and macrophage migration inhibitory factor 1（CSF1) (*P<0.05). The size of the dots represents the percentage of interaction, and the color represents the logarithmic differential expression levels, which are calculated using the DotPlot function. PBS group (n=6): Prepared with the filament method to create a model of middle cerebral artery occlusion （MCAO）, followed by daily intranasal administration of PBS for 14 days post-injury. GB group (n=6): Also prepared using the filament method for MCAO, followed by daily intranasal administration of ginkgolide B solution for 14 days post-injury.
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