敲降CD36抑制白血病细胞培养上清液介导的血小板活化

Knockdown of CD36 inhibits leukemia cell culture supernatant-mediated platelet activation

目的:评估干扰CD36表达白血病细胞培养上清液对血小板活化的影响及其机制。方法:利用L1210小鼠白血病细胞上清液培养血小板4、6、12、24 h,以普通培养基培养血小板作为对照,通过流式细胞术检测血小板活化标志物P-选择素(CD62P)的表达,WB法检测CD36表达,确定上清液活化血小板最佳时间。构建CD36干扰载体转染至活化的血小板中,实验分为对照组、模型组、CD36干扰空载体组(si-CD36 NC)、CD36干扰组(si-CD36)、抑制剂组(i CRT3)、抑制剂+CD36干扰组(iCRT3+si-CD36),CCK-8法检测血小板活力,流式细胞术检测血小板中CD62P表达,WB法检测血小板中PECAM-1、CD36、β-catenin蛋白表达。结果:L1210小鼠白血病细胞上清液活化血小板最佳时间为12 h。与对照组相比,模型组血小板活力、CD62P表达、PECAM-1、CD36、β-catenin蛋白表达均显著上升(均P<0.01)。与模型组相比,si-CD36和iCR73组血小板活力、CD62P表达、PECAM-1、CD36、β-catenin蛋白表达均显著下降(均P<0.01)。与iCRT3组相比,iCRT3+si-CD36组变化更为显著。结论:CD36干扰抑制β-catenin蛋白表达,协同Wnt/β-catenin通路抑制剂,进而抑制小鼠白血病细胞上清液介导的血小板活化。

Objective:To evaluate the effect and mechanism of CD36 interference on platelet activation mediated by leukemia cell culture supernatant.Methods:Platelets were cultured with supernatant from L1210 murine leukemia cells for 4,6,12,and 24 hours,with platelets cultured in regular medium as the control.To determine the optimal time for supernatant-mediated platelet activation,flow cytometry was used to detect the expression of the platelet activation marker P-selectin(CD62P),and Western blot(WB)was used to detect CD36 expression.A CD36 interference vector was constructed and transfected into activated platelets.The cells were divided into the following groups:control group,model group,CD36 interference empty vector group(si-CD36 NC),CD36 interference group(si-CD36),inhibitor group(iCRT3),and inhibitor+CD36 interference group(iCRT3+si-CD36).CCK-8 assay was used to detect platelet viability,flow cytometry was used to detect CD62P expression in platelets,and WB was used to detect the expression of PECAM-1,CD36,andβ-catenin proteins in platelets.Results:The optimal time for platelet activation mediated by L1210 murine leukemia cell supernatant was 12 hours.Compared with the control group,the platelet viability,CD62P expression,and protein expression of PECAM-1,CD36,andβ-catenin were significantly increased in the model group(all P<0.01).Compared with the model group,platelet viability,CD62P expression,and protein expression of PECAM-1,CD36,andβ-catenin were significantly decreased in the si-CD36 and iCRT3 groups(all P<0.01).The changes were more pronounced in the iCRT3+si-CD36 group compared to the iCRT3 group.Conclusion:CD36 interference inhibitsβ-catenin protein expression and,in combination with a Wnt/β-catenin pathway inhibitor,further inhibits murine leukemia cell supernatant-mediated platelet activation.