CAJ | 학술논문

目的：研究重度烧伤大鼠血清刺激后巨噬细胞 Notch1蛋白表达变化，及其细胞分泌因子白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的水平变化。方法选取成年雄性 SD 大鼠24只，随机分为假伤组、烧伤24 h 组和烧伤7 d 组，每组8只。烧伤组 SD 大鼠造成约30％总体表面积（TBSA）Ⅲ度烧伤，分别于伤后24 h、7 d 收集血清；假伤组大鼠用37℃水浴，水浴后24 h 取其血清作为对照。用上述各组血清制成20％培养液，刺激小鼠源巨噬细胞系 RAW264．7，分别于加入血清刺激后即刻和刺激4、8、12、24、48 h 后收取细胞及其上清液。另外用含20％假伤血清＋脂多糖（100 ng／mL）培养液刺激巨噬细胞，于相同时间点收样。Western Blot 检测各时间点巨噬细胞中Notch1蛋白表达变化，酶联免疫吸咐试验检测刺激24 h 后培养上清液中 IL-6及 TNF-α的含量变化。结果（1）假伤组血清刺激后即刻和刺激4、8、12、24、48 h 后，Notch1蛋白表达无明显变化；（2）烧伤24 h 组血清刺激后，Notch1蛋白表达明显升高，并随时间延长而达高峰；（3）烧伤7 d 组血清刺激后， Notch1蛋白表达无明显变化，与假伤组血清刺激结果类似；（4）假伤组血清＋脂多糖混合刺激后， Notch1蛋白表达明显增加；（5）烧伤24 h 组血清及假伤组血清＋脂多糖，上清液中 IL-6和 TNF-α含量均明显高于假伤组血清及烧伤7 d 组血清。结论烧伤血清刺激下的巨噬细胞 Notch1信号被激活，IL-6及 TNF-α分泌能力增强，且这种激活可被脂多糖刺激所模拟。
목적：연구중도소상대서혈청자격후거서세포 Notch1단백표체변화，급기세포분비인자백세포개소-6（IL-6）화종류배사인자-α（TNF-α）적수평변화。방법선취성년웅성 SD 대서24지，수궤분위가상조、소상24 h 조화소상7 d 조，매조8지。소상조 SD 대서조성약30％총체표면적（TBSA）Ⅲ도소상，분별우상후24 h、7 d 수집혈청；가상조대서용37℃수욕，수욕후24 h 취기혈청작위대조。용상술각조혈청제성20％배양액，자격소서원거서세포계 RAW264．7，분별우가입혈청자격후즉각화자격4、8、12、24、48 h 후수취세포급기상청액。령외용함20％가상혈청＋지다당（100 ng／mL）배양액자격거서세포，우상동시간점수양。Western Blot 검측각시간점거서세포중Notch1단백표체변화，매련면역흡부시험검측자격24 h 후배양상청액중 IL-6급 TNF-α적함량변화。결과（1）가상조혈청자격후즉각화자격4、8、12、24、48 h 후，Notch1단백표체무명현변화；（2）소상24 h 조혈청자격후，Notch1단백표체명현승고，병수시간연장이체고봉；（3）소상7 d 조혈청자격후， Notch1단백표체무명현변화，여가상조혈청자격결과유사；（4）가상조혈청＋지다당혼합자격후， Notch1단백표체명현증가；（5）소상24 h 조혈청급가상조혈청＋지다당，상청액중 IL-6화 TNF-α함량균명현고우가상조혈청급소상7 d 조혈청。결론소상혈청자격하적거서세포 Notch1신호피격활，IL-6급 TNF-α분비능력증강，차저충격활가피지다당자격소모의。
Objective To investigate the dynamic expression of Notch1 signaling and the secretion of interleukin-6 (IL-6)and tumor necrosis factor alpha(TNF-α)from macrophage RAW264.7 cells induced by severe burn rat serum.Methods Twenty-four male Sprague-Dawley rats were divided into sham burn group and burn at 24 h and 7 d groups randomly,each group had eight rats.The rats of burn groups were made the third degree burns about 30% total body surface area.The serum of burn rats were collected at 24 h and 7 d after burn.The sham burn group was employed 37℃ water-bath and the serum was treated as a control.The mixed culture medium contained 20% burned serum,RAW264.7 cells were cultivated with each group of burned serum for immediate moment,4,8,1 2,24,48 h.In addition,1 00 ng/mL lipopolysaccharide +20% sham burn serum were applied to culture medium,which were employed to cultivate RAW 264.7.The Notch1 signaling of macrophages were detected after cultivation and the secretion of IL-6 and TNF-αin culture supernatants of each group was measured by enzyme linked immunosorbent assay.Results (1 )The expression of Notch1 protein had no obvious change at immediate moment,4,8, 1 2,24,48 h after stimulation of sham burn serum.(2)In burn at 24 h group,the expression of Notch 1 protein was significantly increased and as time progresses the signaling peaked.(3)In burn at 7 d group, the expression of Notch 1 protein had no obvious change,which was flat as the sham burn serum group.(4) In the mixed culture medium of lipopolysaccharide +sham burn serum group,the level of Notch1 signaling was significantly increased after stimulation.(5)The contents of IL-6 and TNF-αin burn at 24 h group and in lipopolysaccharide +sham burn serum group were significantly higher than those of the sham burn serum group and the burn at 7 d group.Conclusion The Notch1 signaling and the secretion of IL-6 and TNF-αof macrophage could be stimulated by burn serum and this kind of high level signaling could be imitated by lipopolysaccharide.