肿瘤坏死因子-α调控血管平滑肌细胞向巨噬细胞表型转化及其在静脉血栓管壁重塑中的作用及机制

Effect and mechanism of phenotypic transformation to macrophage phenotype of vascular smooth muscle cell regulated by tumor necrosis factor-αon the venous wall remodeling after thrombosis

目的探讨肿瘤坏死因子-α(TNF-α)调节血管平滑肌细胞(VSMC)向巨噬细胞表型转化及其在静脉血栓管壁重塑中的作用及相关机制。方法选取2020年6—12月上海交通大学医学院附属第九人民医院收治的16例患者为研究对象,以血栓累及下肢浅静脉中膜部分作为试验组,邻近未受血栓累及的正常浅静脉中膜部分作为对照组,将16例血栓性浅静脉炎患者中的6例进行基因芯片分析,剩余10例患者进行巨噬细胞表型标志物mRNA的检测。并于上海交通大学医学院实验动物中心获得清洁级雄性SD大鼠22只进行动物实验,下腔静脉血栓造模成功的SD大鼠随机分为血栓组(n=12)和实验组(n=10)。收集人血栓性静脉炎静脉管壁组织,通过基因芯片和逆转录聚合酶链反应(RT-PCR)检测人静脉管壁VSMC巨噬细胞样表型标志物mRNA的表达。TNF-α作用人脐静脉平滑肌细胞(HUVSMC)3~4 d后,使用RT-PCR检测HUVSMC收缩表型、合成表型及巨噬细胞样表型标志物mRNA的表达。构建SD大鼠下腔静脉血栓模型,TNF-α局部处理血栓段静脉管壁24~48 h后,使用RT-PCR检测各组静脉管壁VSMC收缩表型、合成表型及巨噬细胞样表型标志物mRNA的表达。结果试验组人静脉管壁中膜巨噬细胞表型标志物白细胞分化抗原68(CD68)mRNA表达高于对照组,差异有统计学意义(P<0.05)。TNF-α作用HUVSMC 3~4 d后,试验组收缩表型α2-肌动蛋白(ACTA2)、转凝蛋白(TAGLN)和合成表型标志物I型胶原蛋白α1链(COL1A1)mRNA均低于对照组,差异均有统计学意义(P<0.05),其中收缩表型标志物的mRNA变化更为明显;TNF-α作用HUVSMC后,试验组巨噬细胞表型相关标志物CD68、半乳糖凝集素3(LGALS3)和炎症因子标志物肿瘤坏死因子(TNF)mRNA均明显高于对照组,差异均有统计学意义(P<0.05)。TNF-α处理下腔静脉血栓形成静脉管壁24 h后,实验组大鼠静脉管壁VSMC表型标志物中Tagln mRNA表达明显低于血栓组,差异有统计学意义(P<0.01),而两组大鼠Acta2、Col1a1 mRNA表达比较,差异均无统计学意义(P>0.05)。TNF-α处理下腔静脉血栓形成静脉管壁24 h后,实验组大鼠巨噬细胞表型标志物Cd68、Lgals3、Tnf mRNA均明显高于血栓组,差异均有统计学意义(P<0.01);TNF-α处理下腔静脉血栓形成静脉管壁48 h后,实验组大鼠巨噬细胞表型标志物分子Cd68、Lgals3 mRNA表达均明显低于血栓组,差异均有统计学意义(P<0.01),而Tnf mRNA表达明显高于血栓组(P<0.01)。结论血栓形成后静脉管壁VSMC可在TNF-α刺激下向巨噬细胞样表型转化,且该巨噬细胞样VSMC拥有分泌TNF-α的能力,并可能成为管壁炎症反应的重要细胞来源,加重炎症反应,为血栓形成后综合征(PTS)的防治干预靶点提供新策略。

Objective To investigate the effect of tumor necrosis factor-α(TNF-α)on the regulation of vascular smooth muscle cell(VSMC)phenotypic switch and related mechanisms in vascular remodeling after venous thrombosis.Method A total of 16 patients treated in Shanghai Ninth People's Hospital,Shanghai Jiaotong University School of Medicine from June to December 2020 were selected as the research object.The middle membrane part of the superficial vein of the lower extremity involved in thrombosis was taken as the experimental group,and the middle membrane part of the normal superficial vein adjacent to the non involved thrombosis was taken as the control group.6 cases of the 16 patients with superficial thrombophlebitis were analyzed by microarray,and the phenotypic markers mRNA of macrophages were detected in the remaining 10 patients.22 clean male SD rats were obtained from the experimental animal center of Medical College of Shanghai Jiaotong University for animal experiments.The SD rats with successful inferior vena cava thrombosis were randomly divided into thrombosis group(n=12)and test group(n=10).Microarray experiments and reverse transcription-polymerase chain reaction(RT-PCR)were used to detect the mRNA expression of VSMC macrophage-like phenotype markers from human superficial thrombotic vein specimens.Human umbilical vein smooth muscle cells(HUVSMC)were treated with TNF-αfor 3 and 4 days,RT-PCR was used to detect the mRNA expression of contractile,synthetic and macrophage-like phenotypic markers.SD rat inferior vena cava thrombosis model was established,after 24 to 48 hours of TNF-α local treatment of the venous wall of the thrombotic segment,the expression of VSMC contraction phenotype,synthetic phenotype and macrophage like phenotype markers mRNA in the venous wall of each group were detected by RT-PCR.Result The macrophage-like marker cluster of differentiation 68(CD68)of mRNA was significantly up-regulated in human superficial thrombotic vein specimens,the difference was statistically significant(P<0.05).After TNF-α stimulation of HUVSMC for 3 to 4 days,the contractile phenotype of actin alpha 2,smooth muscle,aorta(ACTA2),transgeli(TAGLN)of mRNA of the experimental group and synthetic phenotypic marker collagen type Ⅰ alpha 1 chain(COL1A1)of mRNA were lower than those in the control group,the differences were statistically significant(P<0.05),and the change of systolic phenotypic marker mRNA was more obvious;after TNF-α stimulation of HUVSMC,CD68,galectin 3(LGALS3)and tumor necrosis factor(TNF)of mRNA of the phenotype related markers of macrophages in the experimental group were significantly higher than those in the control group,the differences were statistically significant(P<0.05).TNF-α treated the venous wall after inferior vena cava thrombosis for 24 h,the mRNA expression of Tagln in the phenotypic marker of VSMC in the test group was significantly lower than that in the mRNA thrombosis group,the difference was statistically significant(P<0.01),there was no significant difference in the expression of Acta2 and Col1a1 in the two groups(P>0.05).TNF-α treated the venous wall after inferior vena cava thrombosis for 24 h,the mRNA expression of Tnf,Cd68,Lgals3 of macrophage phenotype markers in test group were significantly higher than those in thrombus group,the differences were statistically significant(P<0.01);TNF-α treated the venous wall after inferior vena cava thrombosis for 48 h,the mRNA expression of Cd68 and Lgals3 of macrophage phenotype markers in test group were significantly lower than that of the thrombus group,the differences were statistically significant(P<0.01),while the mRNA expression of Tnf was significantly higher than that of the thrombus group(P<0.01).Conclusion VSMC of the venous wall after thrombosis can be transformed into macrophage-like phenotype under TNF-α stimulation,and the macrophage-like VSMC has the ability to secrete TNF-α,and may be an important cell source of inflammatory response of the venous wall,aggravating the inflammatory response,providing a new strategy for the prevention and treatment of post-thrombotic syndrome(PTS)intervention target.