中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
4期
706-708
,共3页
郭兵%杨斌%吴雨哲%王坤%李民%孙平%柯文波%郑启昌%宋自芳
郭兵%楊斌%吳雨哲%王坤%李民%孫平%柯文波%鄭啟昌%宋自芳
곽병%양빈%오우철%왕곤%리민%손평%가문파%정계창%송자방
γ-干扰素%血管平滑肌细胞%Fas配体%Fas%细胞凋亡
γ-榦擾素%血管平滑肌細胞%Fas配體%Fas%細胞凋亡
γ-간우소%혈관평활기세포%Fas배체%Fas%세포조망
Interferon-γ%Vascular smooth muscle cells%Fas ligand%Fas%Cell apoptosis
目的 探讨γ-干扰素(IFN-γ)对Fas配体(FasL)诱导大鼠血管平滑肌细胞(VSMCs)凋亡的促进作用和机制.方法 体外培养大鼠VSMCs,给予IFN-γ、FasL单独处理或两者联合处理后,采用Western blot、流式细胞学分析、Hoechst 33342染色等技术,检测IFN-γ对VSMCs凋亡、Fas表达和分布的影响.观察衣霉素预处理是否影响IFN-γ联合FasL诱导的VSMCs凋亡,探讨Fas膜转位在IFN-γ促进FasL诱导VSMCs凋亡中的作用.结果 体外条件下,Hoechst 33342染色显示,IFN-γ和FasL单独处理的凋亡诱导作用均较弱[凋亡率为(7.4±1.9)％、(6.8±2.5)％],与前者比较,IFN-γ联合FasL可显著诱导VSMCs凋亡[凋亡率为(20.1±2.2)％,P＜0.01].Western blot结果显示,经IFN-γ处理36、72 h后VSMCs总的Fas蛋白表达水平,相对表达量分别为0.469±0.129、0.383±0.045,与磷酸盐缓冲液(PBS)对照组(0.402 ±0.031)比较差异无统计学意义(P＞0.05),但可以诱导细胞表面Fas增多(0.266±0.042比0.042±0.017,P＜0.01)而细胞内Fas减少(0.193±0.038比0.426±0.074,P＜0.01).膜转运阻断剂衣霉素预处理可有效抑制IFN-γ和FasL联合处理诱导的VSMCs凋亡,凋亡率[(6.1±0.8)％]较对照组[(23.0±1.0)％]显著降低(P＜0.01).流式细胞学分析结果亦证实Hoechst染色结果.结论 IFN-γ通过诱导Fas受体膜转位,显著增强VSMCs对FasL的敏感性,促进VSMCs凋亡.
目的 探討γ-榦擾素(IFN-γ)對Fas配體(FasL)誘導大鼠血管平滑肌細胞(VSMCs)凋亡的促進作用和機製.方法 體外培養大鼠VSMCs,給予IFN-γ、FasL單獨處理或兩者聯閤處理後,採用Western blot、流式細胞學分析、Hoechst 33342染色等技術,檢測IFN-γ對VSMCs凋亡、Fas錶達和分佈的影響.觀察衣黴素預處理是否影響IFN-γ聯閤FasL誘導的VSMCs凋亡,探討Fas膜轉位在IFN-γ促進FasL誘導VSMCs凋亡中的作用.結果 體外條件下,Hoechst 33342染色顯示,IFN-γ和FasL單獨處理的凋亡誘導作用均較弱[凋亡率為(7.4±1.9)％、(6.8±2.5)％],與前者比較,IFN-γ聯閤FasL可顯著誘導VSMCs凋亡[凋亡率為(20.1±2.2)％,P＜0.01].Western blot結果顯示,經IFN-γ處理36、72 h後VSMCs總的Fas蛋白錶達水平,相對錶達量分彆為0.469±0.129、0.383±0.045,與燐痠鹽緩遲液(PBS)對照組(0.402 ±0.031)比較差異無統計學意義(P＞0.05),但可以誘導細胞錶麵Fas增多(0.266±0.042比0.042±0.017,P＜0.01)而細胞內Fas減少(0.193±0.038比0.426±0.074,P＜0.01).膜轉運阻斷劑衣黴素預處理可有效抑製IFN-γ和FasL聯閤處理誘導的VSMCs凋亡,凋亡率[(6.1±0.8)％]較對照組[(23.0±1.0)％]顯著降低(P＜0.01).流式細胞學分析結果亦證實Hoechst染色結果.結論 IFN-γ通過誘導Fas受體膜轉位,顯著增彊VSMCs對FasL的敏感性,促進VSMCs凋亡.
목적 탐토γ-간우소(IFN-γ)대Fas배체(FasL)유도대서혈관평활기세포(VSMCs)조망적촉진작용화궤제.방법 체외배양대서VSMCs,급여IFN-γ、FasL단독처리혹량자연합처리후,채용Western blot、류식세포학분석、Hoechst 33342염색등기술,검측IFN-γ대VSMCs조망、Fas표체화분포적영향.관찰의매소예처리시부영향IFN-γ연합FasL유도적VSMCs조망,탐토Fas막전위재IFN-γ촉진FasL유도VSMCs조망중적작용.결과 체외조건하,Hoechst 33342염색현시,IFN-γ화FasL단독처리적조망유도작용균교약[조망솔위(7.4±1.9)％、(6.8±2.5)％],여전자비교,IFN-γ연합FasL가현저유도VSMCs조망[조망솔위(20.1±2.2)％,P＜0.01].Western blot결과현시,경IFN-γ처리36、72 h후VSMCs총적Fas단백표체수평,상대표체량분별위0.469±0.129、0.383±0.045,여린산염완충액(PBS)대조조(0.402 ±0.031)비교차이무통계학의의(P＞0.05),단가이유도세포표면Fas증다(0.266±0.042비0.042±0.017,P＜0.01)이세포내Fas감소(0.193±0.038비0.426±0.074,P＜0.01).막전운조단제의매소예처리가유효억제IFN-γ화FasL연합처리유도적VSMCs조망,조망솔[(6.1±0.8)％]교대조조[(23.0±1.0)％]현저강저(P＜0.01).류식세포학분석결과역증실Hoechst염색결과.결론 IFN-γ통과유도Fas수체막전위,현저증강VSMCs대FasL적민감성,촉진VSMCs조망.
Objective To investigate the effect of interferon-γ (IFN-γ) on Fas ligand (FasL)-induced apoptosis of rat vascular smooth muscle cells (VSMCs) and explore the underlying mechanism.Methods VSMCs isolated from rat aorta were cultured in vitro and treated with IFN-γor FasL alone,or both in combination,with or without the pretreatment of Tunicamycin.Hoechst 33342 staining and flow cytometric analysis of Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) staining were employed to define the effect of IFN-γon FasL-induced VSMCs apoptosis.Protein levels of surface Fas,cytoplasmic Fas and total Fas were detected by Western blotting in order to observe the expression and distribution of Fas.Results As shown by both Hoechst 33342 staining,apoptosis of VSMCs was only slightly induced by treatment with IFN-γor FasL alone [apoptotic index of (7.4 ± 1.9) ％ vs.(6.8 ± 2.5) ％],but significantly induced by IFN-γand FasL in combination [(20.1 ±2.2)％,both P ＜0.01].Increased surface Fas (relative level of 0.266 ± 0.042 vs.0.042 ± 0.017,P ＜ 0.01),decreased cytoplasmic Fas (0.193 ± 0.038 vs.0.426 ± 0.074,P ＜ 0.01) but unchanged total Fas expression (0.469 ± 0.129/0.383 ± 0.045 for 36 h/72 h vs.0.402 ± 0.031 for 0 h,both P ＞ 0.05) were detected by Western blotting after IFN-γ treatment.And pretreatment with membrane trafficking inhibitor Tunicamycin significantly diminished FasL-induced VSMCs apoptosis promoted by IFN-γ.The apoptotic index [(6.1 ± O.8)％] were less than that of control [(23.0 ± 1.0)％,P ＜0.01].Flow cytometric analysis also confirmed the results of Hoechst 33342 staining.Conclusion Instead of inducing VSMCs apoptosis directly,IFN-γchanges the Fas distribution by promoting translocation of Fas receptor to the cell membrane,which significantly enhances the sensitivity of VSMCs to FasL-induced apoptosis.
