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.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2021 Apr;29(2):567-573. doi: 10.19746/j.cnki.issn.1009-2137.2021.02.041.
To investigate the damaging of human umbilical vein endothelial cells (HUVEC) induced by antiplatelet integrin β3 antibodies in vitro.
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 integrin β3 antibodies from the serum of the patients. After HUVEC were treated by ITP patient serum (PS) containing anti-integrin β3 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.
Among 36 cases of serum from the chronic ITP patients, 5 patients' serum containing anti-integrin β3 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 down-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）.
Anti-integrin β3 serum can cause the damage and apoptosis of HUVEC through Akt signaling pathway，the apoptotic effects of anti-integrin β3 antibodies to HUVEC was effectively reversed by SC79.
体外研究抗血小板整合素β3抗体对人脐静脉内皮细胞（HUVEC）的损伤作用。
收集36例慢性免疫性血小板减少症（ITP）患者的血清，采用流式细胞术和血小板抗原单克隆抗体特异性固定分析法（MAIPA）从患者血清中收集抗血小板整合素β3抗体。用含抗整合素β3抗体的ITP患者血清（PS）处理HUVEC后，采用乳酸脱氢酶（LDH）法检测细胞损伤，流式细胞术检测细胞凋亡，逆转录定量实时PCR（RT-qPCR）检测凋亡相关基因Bax的表达，蛋白质免疫印迹法检测凋亡相关信号通路蛋白Akt及相关蛋白Bax的表达。用PS联合Akt激活剂SC79处理HUVEC，采用LDH法检测细胞损伤，流式细胞术检测细胞凋亡，RT-qPCR检测凋亡相关基因Bax的表达。
36例慢性ITP患者血清中，收集到5例含抗整合素β3抗体的血清。PS处理HUVEC后，LDH活性显著升高（P<0.05），细胞凋亡率升高（P<0.05），Bax基因和蛋白表达上调（P<0.05），pAkt蛋白表达下调（P<0.05）。与单独用PS培养的HUVEC相比，PS联合SC79处理的HUVEC的LDH活性显著降低（P<0.05），细胞凋亡率降低（P<0.05），Bax基因表达显著降低（P<0.05）。
抗整合素β3血清可通过Akt信号通路导致HUVEC损伤和凋亡，SC79可有效逆转抗整合素β3抗体对HUVEC的凋亡作用。
