超活化血小板裂解液对类风湿关节炎患者成纤维细胞样滑膜细胞影响的实验研究  

Eexperimental study of the influence of super-activated platelet lysate on fibroblast-like synovial cells in patients with rheumatoid arthritis

作　　者：赵启瑞 那馨宇 王文波[1] Qirui Zhao;Xinyu Na;Wenbo Wang(Department of Orthopedic Joint and Sports Medicine Ward,the First Affiliated Hospital of Harbin Medical University,Harbin 150001,China)

机构地区：[1]哈尔滨医科大学附属第一医院关节外科与运动医学科,哈尔滨150001

出　　处：《中华解剖与临床杂志》2021年第6期696-702,共7页Chinese Journal of Anatomy and Clinics

摘　　要：目的探讨超活化血小板裂解液(sPL)对类风湿关节炎患者成纤维细胞样滑膜细胞(RA-FLS)的影响。方法选取哈尔滨医科大学附属第一医院2018年1月—2019年3月6例类风湿关节炎患者作为研究对象,其中男3例、女3例,年龄26~49岁、中位年龄33岁,受累关节功能分级均为Ⅱ级。收集患者静脉血标本,先进行两次离心分离获得富血小板血浆(PRP);再加入0.08 mmol/L CaCl2,37℃孵育激活血小板;然后经过冷冻、融化、离心、过滤除菌、去除纤维蛋白原,获得sPL。培养RA-FLS,分为对照组和2.5%、5%、10%sPL组,分别与含有0、2.5%、5%、10%sPL的培养液培养48 h。采用酶联免疫吸附试验(ELISA)检测各组细胞中炎症因子白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α、IL-1β的浓度;细胞计数试剂盒(CCK)-8法测定各组细胞增殖活性;流式细胞术测定各组细胞的凋亡率;体外小管生成实验检测各组细胞血管生成能力;Transwell实验检测各组细胞迁移能力和侵袭能力;Western blot法测定各组细胞增殖细胞核抗原(PCNA)、细胞周期蛋白D1(CyclinD1)、B细胞淋巴瘤/白血病-2蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)、基质金属蛋白酶(MMP)2、MMP-9、血管内皮生长因子(VEGF)和VEGF受体-2(VEGFR2)的表达情况。结果(1)对照组、2.5%sPL组、5%sPL组、10%sPL组细胞培养基中IL-6浓度分别为(80.18±11.67)、(59.94±9.50)、(46.60±8.04)、(60.67±9.24)pg/mL,TNF-α浓度分别为(70.75±9.14)、(54.56±7.81)、(43.27±6.30)、(53.99±8.60)pg/mL,IL-1β浓度分别为(64.18±9.90)、(46.97±8.79)、(36.28±7.44)、(47.66±8.15)pg/mL,组间比较差异均有统计学意义(F=12.186、11.934、10.709,P值均<0.01);2.5%sPL组、5%sPL组、10%sPL组细胞中IL-6、TNF-α、IL-1β的表达水平均明显低于对照组,差异均有统计学意义(P值均<0.05);5%sPL组炎症因子表达水平低于2.5%sPL组和10%sPL组,差异均有统计学意义(P值均<0.05)。(2)对照组、2.5%sPL组、5%sPL组、10%sPL组RA-FLObjective This study aimed to observe the effect of super activated platelet lysate(sPL)on rheumatoid arthritis fibroblast-like synoviocyte(RA-FLS).Methods Six patients with rheumatoid arthritis from January 2018 to March 2019 in the First Affiliated Hospital of Harbin Medical University were selected as the research objects.The patients comprised three males and three females,aged from 26 years old to 49 years old,with a median age of 33 years old.The affected classification of joint function was gradeⅡ.The patients'venous blood samples were collected and centrifuged twice to separate platelet-rich plasma.The platelets were activated with 0.08 mmol/L CaCl2 and then subjected to two cycles of freezing and thawing.Finally,fibrinogen was removed through temperature-controlled coagulation.The cultured RA-FLS were divided into the control group,2.5%sPL group,5%sPL group,and 10%sPL group,which were incubated with 0,2.5%,5%,and 10%sPL for 48 h,respectively.Enzyme-linked immunosorbent assay was used to detect the concentration of inflammatory factors interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and interleukin-1β(IL-1β)in cell culture medium.Cell counting kit-8 was used to measure the proliferation activity of RA-FLS.Flow cytometry was used to measure the cell apoptosis rate.The angiogenic ability was tested by in vitro tubule formation experiment.The cell scratch test was used to detect the migration ability of RA-FLS.The Transwell experiment was used to detect the invasion ability of RA-FLS.Western blot was used to determine the expression of proliferating cell nuclear antigen(PCNA),Cyclin D1,B-cell lymphoma/leukemia gene-2(Bcl-2),Bcl-2-associated X protein(Bax),matrix metalloprotein(MMP)-2,MMP-9,vascular endothelial growth factor(VEGF),and vascular endothelial growth factor receptor 2(VEGFR2).Results(1)The IL-6 concentrations in the cell culture medium of the control group,2.5%sPL group,5%sPL group,and 10%sPL group were(80.18±11.67)pg/mL,(59.94±9.50)pg/mL,(46.60±8.04)pg/mL,(60.67±9.24)pg/mL,respective

关 键 词：关节炎 类风湿 超活化血小板裂解液 细胞生物学行为 血管生成 炎症因子 

分 类 号：R593.22[医药卫生—内科学]