CAJ | 학술논문

目的通过体外观测柯萨奇病毒B3(CVB3)感染后髓系触发受体-1(TREM-1)在巨噬细胞中的表达,进一步探讨病毒性心肌炎炎症损伤的作用机制.方法 CVB3感染巨噬细胞后,按感染时间进行分组,检测各组TREM-1 mRNA的转录情况(具体分为0h、1h、4h、8h和12 h组),免疫印迹法检测TREM-1及其下游分子DAP-12蛋白的表达(具体分为0h、16h、24h和48 h组),用LP-17阻断TREM-1作为干预组,与单纯CVB3感染组(24h)和不感染CVB3的对照组比较,酶联免疫吸附法检测巨噬细胞分泌蛋白肿瘤坏死因子α(TNF-α)的量,用CCK8和Annexin V-FITC观察与巨噬细胞上清共培养后的心肌细胞的活力和凋亡.结果 CVB3感染后4h组TREM-1 mRNA表达量高于0h组(但差异无统计学意义),8 h组和12 h组则显著高于0h组(P均＜0.01),16 h组和24h组TREM-1蛋白表达水平显著高于0h组(P均＜0.01).CVB3感染24h组和48 h组TREM-1的下游信号分子DAP-12的蛋白表达也显著高于0h组(P均＜0.01).LP-17干预组巨噬细胞分泌的TNF-α的量明显少于CVB3感染组(24 h)(P＜0.01).LP-17干预组心肌细胞的活力显著高于CVB3感染组(24 h)(P＜0.01),细胞凋亡少于CVB3感染组(24 h)(P ＜0.01).结论 CVB3通过诱导巨噬细胞中TREM-1的表达产生过度的炎症反应,进一步引发心肌细胞的间接损伤是急性病毒性心肌炎的发病机制之一.
목적 통과체외관측가살기병독B3(CVB3)감염후수계촉발수체-1(TREM-1)재거서세포중적표체,진일보탐토병독성심기염염증손상적작용궤제.방법 CVB3감염거서세포후,안감염시간진행분조,검측각조TREM-1 mRNA적전록정황(구체분위0h、1h、4h、8h화12 h조),면역인적법검측TREM-1급기하유분자DAP-12단백적표체(구체분위0h、16h、24h화48 h조),용LP-17조단TREM-1작위간예조,여단순CVB3감염조(24h)화불감염CVB3적대조조비교,매련면역흡부법검측거서세포분비단백종류배사인자α(TNF-α)적량,용CCK8화Annexin V-FITC관찰여거서세포상청공배양후적심기세포적활력화조망.결과 CVB3감염후4h조TREM-1 mRNA표체량고우0h조(단차이무통계학의의),8 h조화12 h조칙현저고우0h조(P균＜0.01),16 h조화24h조TREM-1단백표체수평현저고우0h조(P균＜0.01).CVB3감염24h조화48 h조TREM-1적하유신호분자DAP-12적단백표체야현저고우0h조(P균＜0.01).LP-17간예조거서세포분비적TNF-α적량명현소우CVB3감염조(24 h)(P＜0.01).LP-17간예조심기세포적활력현저고우CVB3감염조(24 h)(P＜0.01),세포조망소우CVB3감염조(24 h)(P ＜0.01).결론 CVB3통과유도거서세포중TREM-1적표체산생과도적염증반응,진일보인발심기세포적간접손상시급성병독성심기염적발병궤제지일.
Objective To determine the expression of TREM-1 (triggering receptor expressed on myeloid cells-1 ) in macrophages after coxsackievirus B3 ( CVB3 ) infection and the cardiomyocytes viability after culturing with supernatant of macrophages in the absence and presence of TREM-1 inhibitor LP-17 to explore if TREM-1 is involved in the pathogenesis of CVB3 infection induced inflammation and cardiomyocytes injury.Methods TREM-1 mRNA and TREM-1 and DAP-12 protein expression in macrophages were detected by Real-time PCR at 0,1,4,8 and 12 h and by Western blot at 0,16,24 and 48 h post CVB3 infection. TNF-α secretion of macrophages was measure by ELISA,vitality and the apoptosis degree of cardiomyocytes was assessed by CCK8 and Annexin V-FITC after the cardiomyocytes were cultured with the supernatant of macrophages in normal control group,CVB3 infection group and LP-17 pretreated CVB3 infection group.Results TREM-1 mRNA expression was significantly upregalated at 4,8,and 12 h (peaked at 8 h) and TREM-1 protein expression was significantly upregulated at 16 and 24 h and returned to baseline level at 48 h after CVB3 infection.The protein expression of DAP-12,a direct downstream signaling molecule of TREM-1,also significantly increased at 24 and 48 h post CVB3 infection ( P ＜ 0.01 ).Level of macrophages secreted TNF-α post CVB3 infection was significantly reduced in LP-17 pretreated cells ( P ＜ 0.01 ),LP-17 pretreatment also significantly improved viability and significantly reduced apoptosis of cardiomyocytes cultured with supernatant of CVB3 infected macrophages (P ＜ 0.01 ).Conclusion TREM-1 might be an important mediator post CVB3 infection and a major player on inducing excess macrophages-related inflammation and resulting in an indirect injury to cardiomyocytes.