摘要
目的探讨自然杀伤(natural killer,NK)细胞激活对蛛网膜下腔出血大鼠神经损伤的影响及其与Wnt/β-catenin通路的关系。方法46只大鼠随机分为假手术组15只,抗体对照组15只,NK细胞激活组16只,其中抗体对照组和NK细胞激活组采用改良血管内穿刺法建立蛛网膜下腔出血大鼠模型;假手术组术中仅穿刺,不刺破血管。建模成功后即刻,NK细胞激活组给予0.5 mg/25 g程序性死亡配体1单抗腹腔注射,抗体对照组腹腔注射等体积IgG2a抗体,假手术组腹腔注射等体积生理盐水。建模成功后24 h,采用改良Carcia JH评分表评估3组大鼠神经损伤情况,采用干湿法测定大鼠脑组织湿重/干重值,采用流式细胞术检测脑组织NK细胞浸润情况,采用ELISA法检测脑组织白细胞介素(interleukin,IL)-6、IL-1β、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平,采用HE染色行脑组织病理检查,采用Western blot法检测脑组织Wnt1、Wnt3a、Wnt10、β-catenin、细胞周期蛋白D1(cyclin D1)蛋白相对表达量。结果抗体对照组和NK细胞激活组大鼠改良Carcia JH评分[(8.12±0.43)、(12.15±0.82)分]和脑组织NK细胞百分比[(14.14±1.32)%、(28.24±1.45)%]低于假手术组[(17.55±0.44)分、(35.25±1.82)%](P<0.05),抗体对照组低于NK细胞激活组(P<0.05);NK细胞激活组和抗体对照组大鼠脑组织湿重/干重值(4.58±0.55、5.41±0.54)、IL-6[(32.54±4.54)、(56.85±6.25)ng/L]、TNF-α[(28.64±3.25)、(46.35±4.05)ng/L]、IL-1β[(35.62±2.51)、(50.04±2.44)ng/L]水平高于假手术组[3.15±0.42、(9.25±3.15)ng/L、(10.02±2.15)ng/L、(17.62±1.52)ng/L](P<0.05),抗体对照组高于NK细胞激活组(P<0.05)。假手术组脑皮层和神经元正常;抗体对照组皮层组织疏松,神经元肿胀、收缩变性,部分伴核变形、固缩;NK细胞激活组皮层组织结构及神经元异常改变较抗体对照组减轻。抗体对照组和NK细胞激活组大鼠脑组织Wnt1、Wnt3a、Wnt10、β-catenin、cyclin D1蛋白相对表达量低于假手术组(P<0.05),抗体对照组低于NK细胞激活组(P<0.05)。结论NK细胞激活可减轻蛛网膜下腔出血大鼠神经损伤,作用机制可能与激活Wnt/β-catenin通路有关。
Objective To investigate the influence of natural killer(NK)cells activation on nerve injury in rats with subarachnoid hemorrhage and its relationship with Wnt/β-catenin pathway.Methods Forty-six rats were randomly divided into sham-operation group(n=15),antibody control group(n=15)and NK cells activation group(n=16).In antibody control group and NK cells activation group,modified intravascular puncture method was used to establish subarachnoid hemorrhage rat models,while in sham-operation group,only puncture was performed with no rupture of blood vessel.Immediately after modeling,NK cells activation group received intraperitoneal injection of 0.5 mg/25 g programmed death-ligand 1 monoclonal antibody,antibody control group received intraperitoneal injection of equal volume of IgG2 a antibody,and sham-operation group received intraperitoneal injection of equal volume of normal saline.In 24 h after modeling,nerve injury was assessed by modified Carcia JH scale in three groups,the wet/dry weight of brain tissue was detected by dry wet method,the infiltration of NK cells in brain tissue was detected by flow cytometry,and the levels of interleukin(IL)-6,IL-1βand tumor necrosis factor-α(TNF-α)in brain tissue were detected by ELISA.HE staining was used to perform histophathological examination.The relative expressions of Wnt1,Wnt3 a,Wnt10,β-catenin and cyclin D1 were detected by Western bolt method.Results The modified Carcia JH scale scores and NK cells percentages were lower in antibody control group(8.12±0.43,(14.14±1.32)%)and NK cells activation group(12.15±0.82,(28.24±1.45)%)than those in sham-operation group(17.55±0.44,(35.25±1.82)%)(P<0.05),and lower in antibody control group than those in NK cells activation group(P<0.05).The wet/dry weight value,IL-6,TNF-αand IL-1βwere higher in NK cells activation group(4.58±0.55,(32.54±4.54)ng/L,(28.64±3.25)ng/L,(35.62±2.51)ng/L)and antibody control group(5.41±0.54,(56.85±6.25)ng/L,(46.35±4.05)ng/L,(50.04±2.44)ng/L)than those in sham-operation group(3.15±0.42,(9.25±3.15)ng/L,(10.02±2.15)ng/L,(17.62±1.52)ng/L)(P<0.05),and higher in antibody control group than those in sham-operation group(P<0.05).The cortex and neurons were normal in sham-operation group.In antibody control group,the cortical tissue was loose,neurons swelled and contracted,and some of them were accompanied with nuclear deformation and pyknosis.In NK cells activation group,the cortical tissue structure and neurons were improved compared with antibody control group.The relative expressions of Wnt1,Wnt3 a,Wnt10,β-catenin and cyclin D1 were lower in antibody control group and NK cells activation group than those in sham-operation group(P<0.05),and lower in antibody control group than those in NK cells activation group(P<0.05).Conclusion Activation of NK cells can alleviate the nerve injury in subarachnoid hemorrhage rats,and the mechanism may be correlated with the activation of Wnt/β-catenin pathway.
作者
王芳
王喜玲
陈文丽
WANG Fang;WANG Xi-ling;CHEN Wen-li(Department of Neurology,Zhengzhou People’s Hospital,Zhengzhou,Henan 450000,China;Department of Neurology,the Second Provincial Hospital of Henan Provincial People’s Hospital,Zhengzhou,Henan 450000,China;Department of Neurology,Zhengzhou Hospital of Traditional Chinese Medicine,Zhengzhou,Henan 450000,China)
出处
《中华实用诊断与治疗杂志》
2021年第8期801-805,共5页
Journal of Chinese Practical Diagnosis and Therapy
