血液淤积红细胞裂解诱导椎板切除术后硬膜外瘢痕增生的机制研究  

Mechanism of epidural scar hyperplasia after laminectomy induced by blood siltation

作　　者：王浩然 刘军[1] 白云峰 孙锦鹏 华峰 李明哲 Wang Haoran;Liu Jun;Bai Yunfeng;Sun Jinpeng;Hua Feng;Li Mingzhe(Department of Orthopaedics,The Second Affiliated Hospital of Nanjing Medical University,Nanjing 210011,China)

机构地区：[1]南京医科大学第二附属医院骨科

出　　处：《中华骨科杂志》2023年第8期516-525,共10页Chinese Journal of Orthopaedics

基　　金：国家自然科学基金项目(81671563, 82172486);江苏省"六大人才高峰"项目(WSW-04);骨科相关疾病防治研究项目(江苏省华侨公益基金会)。

摘　　要：目的探讨小鼠椎板切除术后切口血液淤积红细胞破裂释放白细胞介素33(interleukin-33, IL-33)诱导硬膜外瘢痕组织增生的机制。方法取6~8周龄野生型小鼠12只, 制作T12~L2椎板切除模型, 随机分为假手术组、生理盐水干预手术组、单纯红细胞干预手术组、全血干预手术组。术后第30天取切口区域标本行HE染色和Masson染色, 观察是否出现血液淤积;并通过免疫印迹技术检测四组术后第30天的瘢痕组织中纤连蛋白水平, 采用免疫组化染色方法观察术后硬膜外瘢痕增生程度。取野生型小鼠以及IL-33敲除小鼠红细胞, 按生理盐水孵育或红细胞裂解液裂解处理方法分为野生型小鼠红细胞生理盐水对照组、IL-33敲除小鼠红细胞生理盐水对照组、野生型小鼠红细胞裂解组、IL-33敲除小鼠红细胞裂解组, 通过免疫印迹技术检测四组红细胞中的IL-33水平。抽取小鼠血液后进行梯度离心提取较为纯净的红细胞, 按红细胞小鼠来源类型及干预措施分为野生型小鼠红细胞空白对照组、野生型小鼠相对对照组(仅加二抗, 检验是否有非特异性结合)、野生型小鼠红细胞组、IL-33敲除小鼠红细胞组(检验一抗是否有抗原特异性), 行免疫荧光染色以及流式细胞仪检测IL-33水平。结果椎板切除术后HE染色和Masson染色示手术组小鼠切口区域局部有血液淤积, 全血或红细胞干预小鼠术后切口区域的硬膜外瘢痕增生程度更高, 尤其是全血干预组;野生型红细胞生理盐水对照组、IL-33敲除红细胞生理盐水对照组及IL-33敲除红细胞裂解组几乎检测不到IL-33的表达, 而野生型红细胞裂解组可检测到明显的IL-33表达;免疫荧光染色示野生型小鼠红细胞膜内及膜上均有IL-33表达, 而其他三组几乎检测不到IL-33或极少量的非特异性表达, 四组IL-33免疫荧光强度分别为0.62±0.41, 60.17±4.39, 16.78±7.43和0.61±0.03(F=281.90, P<0.001), �Objective To explore the mechanism of epidural scar tissue hyperplasia induced by erythrocyte rupture and release of interleukin-33(IL-33)after laminectomy in mice.Methods In the zoological experiment,the operation group(Laminectomy)and the sham operation group were set,and HE staining and Masson staining were performed to test for blood accumulation in the operation area after laminectomy in mice.Then 12 wild-type mice with 6-8 week old were selected and divided into 4 groups:the sham operation group,the operation group(normal saline control),the pure red blood cell intervention operation group,the whole blood intervention operation group.The normal saline(100 mg/kg)was injected into the postoperative area.The red blood cells or whole blood with the same volume were injected into the postoperative area in the pure red blood cell intervention group and the whole blood intervention group.The postoperative recovery of mice in each group was observed.The levels of fibronectin in the postoperative scar tissues of mice in four groups were detected by western blot technology,and the degree of postoperative epidural scar hyperplasia was directly observed by immunohistochemistry.In the cytological experiment,the wild-type mouse erythrocyte normal saline group,the control group of IL-33 knockout mouse erythrocyte normal saline,the wild-type mouse erythrocyte lysis group,and the IL-33 knockout mouse erythrocyte lysis group were set.The levels of IL-33 in the red blood cells of four groups were detected by western blot.Then,a blank wild-type mouse erythrocyte control group,a wild-type mouse relative to the control group(only secondary antibody added to test for non-specific binding),a wild-type mouse erythrocyte group and an IL-33 knockout mouse erythrocyte group(to test for antigen specificity of the primary antibody)were set.Immunofluorescence staining was performed on the erythrocytes of four groups and the level of IL-33 was detected by flow cytometry.Results HE staining and Masson staining after laminectomy showed that

关 键 词：椎板切除术 瘢痕 红细胞 白细胞介素33 

分 类 号：R687.3[医药卫生—骨科学]