南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (1): 23-33.doi: 10.12122/j.issn.1673-4254.2026.01.03
赵贝1(
), 吕政仪2(
), 季丁汝1, 田书心1, 吴雨欣1, 李星震1, 周杰1, 方剑乔1, 梁宜1(
)
收稿日期:2025-05-26
出版日期:2026-01-20
发布日期:2026-01-16
通讯作者:
梁宜
E-mail:984722585@qq.com;aprilv_kuco@icloud.com;liangyiwww@126.com
作者简介:赵 贝,硕士,中医师,E-mail: 984722585@qq.com基金资助:
Bei ZHAO1(
), Zhengyi LÜ2(
), Dingru JI1, Shuxin TIAN1, Yuxin WU1, Xingzhen LI1, Jie ZHOU1, Jianqiao FANG1, Yi LIANG1(
)
Received:2025-05-26
Online:2026-01-20
Published:2026-01-16
Contact:
Yi LIANG
E-mail:984722585@qq.com;aprilv_kuco@icloud.com;liangyiwww@126.com
Supported by:摘要:
目的 观察慢性术后疼痛(CPSP)小鼠疼痛行为学指标的时间动态特征,并从背根神经节(DRG)层面初步筛选介导CPSP的关键物质。 方法 采用足底切口术(INC)联合足背注射前列腺素E2(PGE2)建立慢性术后疼痛小鼠(I/P)模型。根据手术方式与给药不同,将小鼠随机分为假手术+生理盐水组(SI/NS)、假手术+PGE2组(SI/PGE2)和手术+PGE2组(I/P),观察实验小鼠造模后不同时间点机械缩足阈(PWTs)、热缩足潜伏期(PWLs)和冷缩足反应时间(WDs)变化。根据给药和取材时间不同,将小鼠分为SI/NS组、模型第1天组、模型第8天组,利用RNA-Seq技术建立CPSP早期(PGE2诱导后第1天)和维持期(PGE2诱导后第8天)小鼠背根神经节基因表达谱,通过样本相关性分析、差异表达基因分析、富集分析筛选重要介导物质,采用RT-qPCR和ELISA检测目标物质基因及蛋白表达情况。根据手术方式与给药不同,将小鼠随机分为SI/P+Veh组、I/P+Veh组、I/P+药物组,通过鞘内注射CX3CL1中和抗体(Ab)或CX3CR1选择性拮抗剂(JMS-17-2)等药理学实验,探索下游相关受体的作用。 结果 I/P小鼠足底切口疼痛在术后14 d内恢复,经PGE2二次诱发后PWTs持续降低至PGE2注射后第10天WDs持续延长至第12天;PWLs未发生持续性变化。生信分析显示,CPSP早期和维持期实验小鼠DRG差异表达基因为975、895个,共同参与介导的交集差异基因524个。富集分析表明“细胞膜”富集基因数目最多,涉102个基因;“质膜”富集显著性最大(P<0.001);“CX3C 趋化因子受体结合”等条目富集因子最大(Rich Factor=1)。与SI/NS组相比,CPSP早期趋化因子相关差异基因中表达差异最大为Cx3cl1(FC Cx3cl1 =2.08),其次为Cxcl14(FC Cxcl14 =1.99)。RT-qPCR和ELISA结果显示CPSP早期I/P小鼠患侧DRG中Cx3cl1、Cxcl14基因和相关蛋白表达较SI/NS组增加(P<0.05),维持期小鼠则较早期组降低(P<0.05)。PGE2诱导前鞘内注射CX3CL1中和抗体可阻止CPSP发生。单次鞘内注射CX3CR1拮抗剂可一定程度翻转CPSP维持期疼痛。 结论 CPSP小鼠机械痛觉异常、冷痛觉异常持续至PGE2注射后第10天,热痛觉敏化不显著。背根神经节趋化因子尤其是CX3CL1可能介导慢性术后疼痛发生。
赵贝, 吕政仪, 季丁汝, 田书心, 吴雨欣, 李星震, 周杰, 方剑乔, 梁宜. 慢性术后痛小鼠痛行为时间动态特征及背根神经节关键物质筛选[J]. 南方医科大学学报, 2026, 46(1): 23-33.
Bei ZHAO, Zhengyi LÜ, Dingru JI, Shuxin TIAN, Yuxin WU, Xingzhen LI, Jie ZHOU, Jianqiao FANG, Yi LIANG. Temporal changes of chronic postsurgical pain in mice: the regulatory role of CX3CL1 in the dorsal root ganglion[J]. Journal of Southern Medical University, 2026, 46(1): 23-33.
| Gene | Sequence (5′-3′) |
|---|---|
| Cx3cl1 forward | CAGAGGAGCAGGCAGGACAG |
| Cx3cl1 reverse | CTTCAGAGCAGGAGAGACCCATC |
| Cxcl14 forward | TCCGGTCAGCATGAGGCTCC |
| Cxcl14 reverse | CACCCTATTCTTCGTAGACC |
| Ccr5 forward | GTTGTTTTGGAGAACGCCCC |
| Ccr5 reverse | CAACACTGCTCCGAAACTGC |
| Il7 forward | CTAGCAACTGGCAAGGAGGAC |
| Il7 reverse | CCTGTAAGTGGAAGCATGGC |
| β-actin forward | ACTGGAACGGTGAAGGTGAC |
| β-actin reverse | AGAGAAGTGGGGTGGCTTTT |
表1 RT-qPCR引物序列
Tab.1 Primer sequences for RT-qPCR
| Gene | Sequence (5′-3′) |
|---|---|
| Cx3cl1 forward | CAGAGGAGCAGGCAGGACAG |
| Cx3cl1 reverse | CTTCAGAGCAGGAGAGACCCATC |
| Cxcl14 forward | TCCGGTCAGCATGAGGCTCC |
| Cxcl14 reverse | CACCCTATTCTTCGTAGACC |
| Ccr5 forward | GTTGTTTTGGAGAACGCCCC |
| Ccr5 reverse | CAACACTGCTCCGAAACTGC |
| Il7 forward | CTAGCAACTGGCAAGGAGGAC |
| Il7 reverse | CCTGTAAGTGGAAGCATGGC |
| β-actin forward | ACTGGAACGGTGAAGGTGAC |
| β-actin reverse | AGAGAAGTGGGGTGGCTTTT |
图1 CPSP模型的建立和小鼠疼痛行为学指标的变化
Fig.1 Temporal changes of pain-related behaviors in the mouse models of chronic postsurgical pain (CPSP). A: Schematic diagram of plantar incision for inducing CPSP in mice. B: Changes in mechanical paw withdrawal thresholds (PWTs) after plantar incision and after PGE2 injection in each group. C: Changes in thermal paw withdrawal latencies (PWLs) after plantar incision and after PGE2 injection in each group. D: Changes in cold withdrawal durations (WDs) of the mice after plantar incision and after PGE2 injection. INC: Incision. *P<0.05, **P<0.01 I/P vs SI/NS; #P<0.05, ##P<0.01 I/P vs SI/P; &&P<0.01 SI/P vs SI/NS.
图2 不同时间点I/P小鼠DRGs基因表达情况
Fig.2 Gene expression profile in dorsal root ganglion (DRGs) of the mice with CPSP at different time points. A: Heat map of correlation coefficient of each sample (Numbers in the box are Pearson correlation coefficients between the corresponding two samples). B: Heat map and hierarchical clustering of differential gene expression in each sample (P<0.05 and |log2FC|>0.3 were the threshold criteria for differentially expressed genes). C: Venn diagram of DEGs between groups.
图3 I/P小鼠DRGs交集差异基因GO和KEGG富集分析
Fig.3 GO and KEGG enrichment analysis of differentially expressed genes at the intersection DEGs in CPSP mice. A: GO enrichment bar chart of the intersection DEGs between Day1vs. SI/NS and Day8 vs. Day1 (Ranked by the number of significantly enriched genes). B: GO enrichment scatter plot of intersection DEGs. C: KEGG enrichment bar chart of intersection DEGs. D: KEGG enrichment scatter plot of intersection DEGs.
图4 不同时间点CPSP小鼠DRGs趋化因子相关基因表达情况
Fig.4 Expression of chemokine-related genes in DRGs of mice with CPSP at different time points. A: Volcano plot of differentially expressed genes in day 1 and SI/NS groups (Chemokine-related genes are marked). B: Volcano plot of differentially expressed genes in day 8 and day 1 groups. C: Table of differential genes associated with chemokines between the groups. All chemokine-related genes are extracted using GO terms.
图5 各组小鼠CPSP不同时期靶物质基因表达情况
Fig.5 Gene and protein expression of Cx3c11 and Cxc114 in different stages of CPSP. A: Cx3cl1 mRNA expression levels in ipsilateral DRGs of the mice at each time point. B: Cxcl14 mRNA expression levels in ipsilateral DRGs of the mice at each time point. C: CX3CL1 protein expression in ipsilateral DRGs of the mice at each time point. D: CXCL14 protein expression in ipsilateral DRGs of the mice at each time point. *P<0.05, **P<0.01.
图6 各组实验小鼠鞘内注射CX3CL1中和抗体的镇痛效应
Fig.6 Intrathecal injection of CX3CL1 neutralizing antibody (5 µg/10 µL) 10 min prior to PGE2 injection significantly modulates mechanical PWTs in INC/PGE2 mice. **P<0.01 I/P+Veh vs SI/P+Veh group; #P<0.05, ##P<0.01 I/P+Ab vs I/P+Veh group; &P<0.05, &&P<0.01 I/P+Ab vs SI/P+Veh group. Ab: CX3CL1-neutralizing antibody; Veh: Vehicle, goat IgG control (1 µL/10 µL).
图7 选择性拮抗CX3CR1对实验小鼠痛行为的影响
Fig.7 Effects of intrathecal injection of JMS17-2 (75 µg/10 µL), a selective CX3CR1 antagonist, on Day 7 post-PGE2 injection, on PWTs in CPSP mice. *P<0.05, **P<0.01 I/P+Veh vs SI/P+Veh; #P<0.05, ##P<0.01 I/P+JMS vs SI/P+Veh; &&P<0.01 I/P+JMS vs I/P+Veh. I/P: INC/PGE2; SI/P: Sham INC/PGE2; JMS: CX3CR1-selective antagonist JMS17-2; Veh: Corn oil vehicle.
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