抗血小板整合素β3抗体诱导内皮细胞凋亡机制的研究  

Apoptosis of Endothelial Cells Induced by Anti-Platelet Integrinβ3 Antibody

作　　者：王健玉 王明镜[2] 孙平[3] 孙妍 王学哲 胡晓梅[2] 全日城[2] LIANG Simon-Xun WANG Jian-Yu;WANG Ming-Jing;SUN Ping;SUN Yan;WANG Xue-Zhe;HU Xiao-Mei;QUAN Ri-Cheng;LIANG Simon-Xun(Department of Biochemistry and Molecular Biology,College of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,Liaoning province,China;Department of Hematology,Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China;Department of Hematology,Jining First People's Hospital,Jining 272000,Shandong Province,China;Department of Clinical Laboratory,The First Affiliated Hospital of Jinzhou Medical University,Jinzhou 121001,Liaoning province,China;Department of pathoanatomy,College of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,Liaoning province,China)

机构地区：[1]锦州医科大学基础医学院生物化学与分子生物学教研室,辽宁锦州121000 [2]中国中医科学院西苑医院血液科,北京100091 [3]济宁市第一人民医院血液科,山东济宁272000 [4]锦州医科大学第一附属医院临床检验科,辽宁锦州121001 [5]锦州医科大学基础医学院病理解剖教研室,辽宁锦州121000

出　　处：《中国实验血液学杂志》2021年第2期567-573,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金(NO.81370619);国家重点基础研究发展计划(973计划)项目(2015CB554403)。

摘　　要：目的:探讨抗血小板整合素β3抗体对人脐静脉血管内皮细胞(HUVEC)的损伤及其相关机制。方法:收集36例慢性免疫性血小板减少症患者的血清,通过流式细胞术和单克隆抗体特异性俘获血小板抗原技术(MAIPA)筛选出含抗整合素β3抗体的患者血清。用抗整合素β3血清处理HUVEC后,乳酸脱氢酶(LDH)活性测定法检测HUVEC损伤情况,流式细胞术检测HUVEC凋亡情况,逆转录实时荧光定量PCR(RT-q PCR)检测HUVEC凋亡相关基因Bax表达情况,Western blot检测细胞凋亡相关信号通路蛋白Akt和相关蛋白Bax的表达情况。用抗整合素β3血清结合Akt激活剂SC79处理HUVEC后,LDH活性测定法检测HUVEC损伤情况,流式细胞术检测HUVEC的凋亡情况,RT-q PCR检测HUVEC凋亡相关基因Bax的表达情况。结果:从36份患者血清中筛选出5份含抗整合素β3抗体血清,用这些血清处理HUVEC后,细胞外LDH活性增加(P<0.05),细胞凋亡比例增加(P<0.05),Bax蛋白和mRNA表达水平上调(P<0.05),p Akt蛋白表达下调(P<0.05)。抗整合素β3血清结合SC79处理HUVEC后,与单独使用抗整合素β3血清处理组相比,细胞外LDH活性显著降低(P<0.05),HUVEC凋亡比例显著下降(P<0.05),Bax基因表达下降(P<0.05)。结论:抗整合素β3抗体在体外能够引起HUVEC的损伤和凋亡,其作用机制可能与抑制Akt信号通路有关,且Akt激活剂SC79能够在体外抑制抗整合素β3抗体引起的HUVEC损伤和凋亡。Objective:To investigate the damaging of human umbilical vein endothelial cells(HUVEC)induced by antiplatelet integrinβ3 antibodies in vitro.Methods:The serum from 36 chronic ITP patients were collected,flow cytometry and monoclonal antibody specific immobilization of platelet antigen(MAIPA)assay were used to collect antiplatelet integrinβ3 antibodies from the serum of the patients.After HUVEC were treated by ITP patient serum(PS)containing anti-integrinβ3 antibodies,the cell damage was detected by Lactate dehydrogenase(LDH)assay,cell apoptosis was detected by flow cytometry,the expression of apoptosis-related gene Bax was detected by Reverse transcription-Quantitative real-time PCR(RT-qPCR),and expression of Apoptosis-related signaling pathway protein Akt and related protein Bax were detected by Western blot.HUVEC were treated by PS combined with Akt activator SC79,the cells damage were detected by LDH assay,apoptosis of the cells were detected by flow cytometry,the expression of apoptosis-related gene Bax was detected by RT-qPCR.Results:Among 36 cases of serum from the chronic ITP patients,5 patients’serum containing anti-integrinβ3 antibodies were collected.After HUVEC was treated by PS,the viability of LDH was significant increased(P<0.05),so as for the apoptosis of the cells(P<0.05),the expression of gene and protein of Bax was increased up-regulated(P<0.05),the protein expression of pAkt was dowm-regulated(P<0.05).Comparing with HUVEC cultured with PS alone,the viability of LDH of HUVEC treated by PS combined with SC79 was significantly reduced(P<0.05),so as for the apoptosis of the cells(P<0.05),and gene expression of Bax was significantly decreased(P<0.05).Conclusion:Anti-integrinβ3 serum can cause the damage and apoptosis of HUVEC through Akt signaling pathway,the apoptotic effects of anti-integrinβ3 antibodies to HUVEC was effectively reversed by SC79.

关 键 词：免疫性血小板减少症 抗整合素β3抗体 AKT 凋亡 内皮细胞 

分 类 号：R558.2[医药卫生—血液循环系统疾病] R329[医药卫生—内科学]