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

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-FLS增殖率分别为87.33%±10.98%、76.17%±8.18%、60.83%±7.99%、75.83%±8.45%,细胞凋亡率分别为6.51%±1.16%、16.23%±2.75%、29.69%±3.80%、16.37%±2.29%,小管形成数分别为(41.00±7.55)、(26.67±4.16)、(11.67±3.51)、(26.00±6.56)个,迁移细胞数目分别为(443.00±54.06)、(282.33±31.66)、(154.64±23.18)、(292.00±35.03)个,侵袭细胞数目分别为(243.30±27.39)、(67.00±12.53)、(22.33±8.74)、(79.33±14.98)个,组间比较差异均有统计学意义(F=8.795、38.095、34.278、29.352、94.772, P值均<0.05);2.5%sPL组、5%sPL组、10% sPL组RA-FLS细胞增殖率、血管管腔形成数量、迁移细胞数目和侵袭细胞数目均低于对照组,细胞凋亡率均高于对照组,差异均有统计学意义(P值均＜0.05);5% sPL组中细胞增殖率、小管形成数、迁移细胞数目和侵袭细胞数目均明显低于2.5% sPL组和10% sPL组,细胞凋亡率则高于2.5% sPL组和10% sPL组,差异均有统计学意义(P值均＜0.05)。(3)对照组、2.5%sPL组、5%sPL组、10%sPL组RA-FLS PCNA、CyclinD1、Bax、Bcl-2、MMP-2、MMP-9、VEGF和VEGFR2蛋白相对表达量比较,差异均有统计学意义(P值均＜0.01);2.5%sPL组、5%sPL组、10% sPL组细胞中PCNA、CyclinD1、Bcl-2、MMP-2、MMP-9、VEGF和VEGFR2蛋白相对表达量均低于对照组,Bax蛋白相对表达量均高于对照组,差异均有统计学意义(P值均＜0.05);5% sPL组PCNA、CyclinD1、Bcl-2、MMP-2、MMP-9、VEGF和VEGFR2蛋白相对表达量均低于2.5% sPL组和10% sPL组,而Bax蛋白相对表达量则高于2.5% sPL组和10% sPL组,差异均有统计学意义(P值均＜0.05)。结论 sPL对RA-FLS的增殖、迁移、侵袭、血管生成以及炎症因子的产生具有抑制作用,对RA-FLS凋亡具有促进作用,且SPL对RA-FLS的生长抑制效果与SPL浓度有关。

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

Objective 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 grade Ⅱ. 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, respectively; their TNF-α concentrations were (70.75±9.14) pg/mL, (54.56±7.81) pg/mL, (43.27±6.30)pg/mL, and (53.99±8.60)pg/mL, respectively; and their IL-1β concentrations were (64.18±9.90) pg/mL, (46.97±8.79) pg/mL, (36.28±7.44) pg/mL, and (47.66±8.15) pg/mL, respectively. The differences were statistically significant (F=12.186, 11.934, 10.709; all P values＜0.01). A comparison of the two groups showed that the concentrations of inflammatory factors IL-6, TNF-α, and IL-1β in the 5% sPL group were lower than those in the other groups (all P values＜0.05). (2) The proliferation rates of RA-FLS in the control group, 2.5% sPL group, 5% sPL group, and 10% sPL group were 87.33%±10.98%, 76.17%±8.18%, 60.83%±7.99%, and 75.83%±8.45%, respectively; the apoptosis rates were 6.51%±1.16%, 16.23%±2.75%, 29.69%±3.80%, and 16.37%±2.29%, respectively; the amounts of tubule formation were 41.00±7.55, 26.67±4.16, 11.67±3.51, and 26.00±6.56, respectively; the numbers of migrating cells were 443.00±54.06, 282.33±31.66, 154.64±23.18, and 292.00±35.03, respectively; and the numbers of invasive cells were 243.30±27.39, 67.00±12.53, 22.33±8.74, and 79.33±14.98, respectively. The differences were statistically significant (F=8.795, 38.095, 34.278, 29.352, 94.772; all P values＜0.05). A comparison between the two groups revealed that the cell proliferation rate, the number of migrating cells, and the number of invasive cells in the 5% sPL group were lower than those of the other groups (all P values＜0.05), while the apoptosis rate was higher than those of the other groups (all P values＜0.05). The angiogenic ability of RA-FLS decreased after treatment with SPL, and the 5% SPL group was better than the 2.5% and 10% SPL groups. (3) The expression levels of PCNA, Cyclin D1, Bax, Bcl-2, MMP-2, MMP-9, VEGF, and VEGFR2 of RA-FLS in the control group, 2.5% sPL group, 5% sPL group, and 10% sPL group were significantly different (all P values＜0.05). A comparison between the two groups demonstrated that the expression levels of PCNA, Cyclin D1, Bcl-2, MMP-2, MMP-9, VEGF, and VEGFR2 in the 5% sPL group were lower than those in the other groups (all P values＜0.05), while the expression of Bax was higher than that in the other groups (all P values＜0.05).Conclusions sPL can inhibit the proliferation, migration, invasion, angiogenesis, and production of inflammatory factors of RA-FLS and promote the apoptosis of RA-FLS. Moreover, the inhibitory effect of sPL on the growth of RA-FLS is related to the concentration of SPL.