中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2013年
2期
158-161
,共4页
孙立%陈旭林%郭峰%王飞%刘晟%梁勋%王仁素%王永杰%孙业祥
孫立%陳旭林%郭峰%王飛%劉晟%樑勛%王仁素%王永傑%孫業祥
손립%진욱림%곽봉%왕비%류성%량훈%왕인소%왕영걸%손업상
烧伤%枯否细胞%高迁移率族蛋白质类%晚期糖基化终末产物受体%促炎性细胞因子
燒傷%枯否細胞%高遷移率族蛋白質類%晚期糖基化終末產物受體%促炎性細胞因子
소상%고부세포%고천이솔족단백질류%만기당기화종말산물수체%촉염성세포인자
Burns%Kupffer cells%High mobility group proteins%Receptor for advanced glycation end products%Pro-inflammatory cytokine
目的 探讨高迁移率族蛋白B1(HMGB1)对严重烧伤大鼠枯否细胞(KC)分泌促炎性细胞因子的影响及晚期糖基化终末产物受体(RAGE)在该过程中的作用. 方法 将32只SD大鼠背部浸于98℃水中12 s,制成30％ TBSAⅢ度烫伤(以下称烧伤).伤后24 h,处死大鼠分离肝脏KC(32个样本),以每孔1×106个接种至24孔板中.(1)取部分细胞按随机数字表法分为2组,每组样本数为8.对照组,加入1 mL PBS液培养；HMGB1组,加入1 mL浓度为100 ng/mL HMGB1刺激.培养48 h后,采用蛋白质印迹法检测细胞表面RAGE的表达(结果以灰度值比值表示).(2)取部分细胞采用随机数字表法分为4组,每组样本数为8.对照组,加入1 mL PBS液培养；HMGB1组,加入1 mL浓度为100 ng/mL HMGB1刺激；HMGB1+抗RAGE抗体组,经1 mL浓度为20 μg/mL抗大鼠RAGE单克隆抗体孵育2h,加入1 mL浓度为100 ng/mL HMGB1刺激；HMGB1+重组大鼠RAGE/Fc嵌合体(rrRAGE/Fc)组,将0.5 mL浓度为100 ng/mL HMGB1与0.5 mL浓度为5μg/mL rrRAGE/Fc孵育2h后,作用于细胞.培养48 h后,采用ELISA法检测细胞培养上清液中TNF-α和IL-1β的含量,RNA印迹法检测细胞内TNF-α和IL-1β mRNA的表达水平(结果以灰度值比值表示).对数据进行单因素方差分析、t检验及LSD检验. 结果 (1)培养48 h后,HMGB1组细胞RAGE表达水平(1.036 ±0.101)明显高于对照组(0.191 ±0.024,t=-23.158,P=0.000).(2) HMGB1组、HMGB1+抗RAGE抗体组以及HMGB1+ rrRAGE/Fc组细胞培养上清液中TNF-α含量分别为(10.59±1.39)、(9.91±1.68)、(11.51±2.27) ng/mL,IL-1β含量分别为(2.49±0.33)、(2.08±0.32)、(2.42±0.42) ng/mL；细胞内TNF-α mRNA水平分别为0.311 ±0.009、0.301±0.047、0.326±0.016,IL-1βmRNA水平分别为0.237±0.021、0.244±0.041、0.245±0.013,3组间比较差异均无统计学意义(P值均大于0.05),均显著高于对照组[细胞培养上清液中TNF-α和IL-1β含量分别为(2.69±0.14)、(0.43±0.05) ng/mL,细胞内TNF-α和IL-1β mRNA水平分别为0.140±0.022、0.077±0.005,P值均小于0.01]. 结论 HMGB1可引起严重烧伤大鼠KC分泌促炎性细胞因子TNF-α和IL-1β,但RAGE在这一过程中未起主导作用.
目的 探討高遷移率族蛋白B1(HMGB1)對嚴重燒傷大鼠枯否細胞(KC)分泌促炎性細胞因子的影響及晚期糖基化終末產物受體(RAGE)在該過程中的作用. 方法 將32隻SD大鼠揹部浸于98℃水中12 s,製成30％ TBSAⅢ度燙傷(以下稱燒傷).傷後24 h,處死大鼠分離肝髒KC(32箇樣本),以每孔1×106箇接種至24孔闆中.(1)取部分細胞按隨機數字錶法分為2組,每組樣本數為8.對照組,加入1 mL PBS液培養；HMGB1組,加入1 mL濃度為100 ng/mL HMGB1刺激.培養48 h後,採用蛋白質印跡法檢測細胞錶麵RAGE的錶達(結果以灰度值比值錶示).(2)取部分細胞採用隨機數字錶法分為4組,每組樣本數為8.對照組,加入1 mL PBS液培養；HMGB1組,加入1 mL濃度為100 ng/mL HMGB1刺激；HMGB1+抗RAGE抗體組,經1 mL濃度為20 μg/mL抗大鼠RAGE單剋隆抗體孵育2h,加入1 mL濃度為100 ng/mL HMGB1刺激；HMGB1+重組大鼠RAGE/Fc嵌閤體(rrRAGE/Fc)組,將0.5 mL濃度為100 ng/mL HMGB1與0.5 mL濃度為5μg/mL rrRAGE/Fc孵育2h後,作用于細胞.培養48 h後,採用ELISA法檢測細胞培養上清液中TNF-α和IL-1β的含量,RNA印跡法檢測細胞內TNF-α和IL-1β mRNA的錶達水平(結果以灰度值比值錶示).對數據進行單因素方差分析、t檢驗及LSD檢驗. 結果 (1)培養48 h後,HMGB1組細胞RAGE錶達水平(1.036 ±0.101)明顯高于對照組(0.191 ±0.024,t=-23.158,P=0.000).(2) HMGB1組、HMGB1+抗RAGE抗體組以及HMGB1+ rrRAGE/Fc組細胞培養上清液中TNF-α含量分彆為(10.59±1.39)、(9.91±1.68)、(11.51±2.27) ng/mL,IL-1β含量分彆為(2.49±0.33)、(2.08±0.32)、(2.42±0.42) ng/mL；細胞內TNF-α mRNA水平分彆為0.311 ±0.009、0.301±0.047、0.326±0.016,IL-1βmRNA水平分彆為0.237±0.021、0.244±0.041、0.245±0.013,3組間比較差異均無統計學意義(P值均大于0.05),均顯著高于對照組[細胞培養上清液中TNF-α和IL-1β含量分彆為(2.69±0.14)、(0.43±0.05) ng/mL,細胞內TNF-α和IL-1β mRNA水平分彆為0.140±0.022、0.077±0.005,P值均小于0.01]. 結論 HMGB1可引起嚴重燒傷大鼠KC分泌促炎性細胞因子TNF-α和IL-1β,但RAGE在這一過程中未起主導作用.
목적 탐토고천이솔족단백B1(HMGB1)대엄중소상대서고부세포(KC)분비촉염성세포인자적영향급만기당기화종말산물수체(RAGE)재해과정중적작용. 방법 장32지SD대서배부침우98℃수중12 s,제성30％ TBSAⅢ도탕상(이하칭소상).상후24 h,처사대서분리간장KC(32개양본),이매공1×106개접충지24공판중.(1)취부분세포안수궤수자표법분위2조,매조양본수위8.대조조,가입1 mL PBS액배양；HMGB1조,가입1 mL농도위100 ng/mL HMGB1자격.배양48 h후,채용단백질인적법검측세포표면RAGE적표체(결과이회도치비치표시).(2)취부분세포채용수궤수자표법분위4조,매조양본수위8.대조조,가입1 mL PBS액배양；HMGB1조,가입1 mL농도위100 ng/mL HMGB1자격；HMGB1+항RAGE항체조,경1 mL농도위20 μg/mL항대서RAGE단극륭항체부육2h,가입1 mL농도위100 ng/mL HMGB1자격；HMGB1+중조대서RAGE/Fc감합체(rrRAGE/Fc)조,장0.5 mL농도위100 ng/mL HMGB1여0.5 mL농도위5μg/mL rrRAGE/Fc부육2h후,작용우세포.배양48 h후,채용ELISA법검측세포배양상청액중TNF-α화IL-1β적함량,RNA인적법검측세포내TNF-α화IL-1β mRNA적표체수평(결과이회도치비치표시).대수거진행단인소방차분석、t검험급LSD검험. 결과 (1)배양48 h후,HMGB1조세포RAGE표체수평(1.036 ±0.101)명현고우대조조(0.191 ±0.024,t=-23.158,P=0.000).(2) HMGB1조、HMGB1+항RAGE항체조이급HMGB1+ rrRAGE/Fc조세포배양상청액중TNF-α함량분별위(10.59±1.39)、(9.91±1.68)、(11.51±2.27) ng/mL,IL-1β함량분별위(2.49±0.33)、(2.08±0.32)、(2.42±0.42) ng/mL；세포내TNF-α mRNA수평분별위0.311 ±0.009、0.301±0.047、0.326±0.016,IL-1βmRNA수평분별위0.237±0.021、0.244±0.041、0.245±0.013,3조간비교차이균무통계학의의(P치균대우0.05),균현저고우대조조[세포배양상청액중TNF-α화IL-1β함량분별위(2.69±0.14)、(0.43±0.05) ng/mL,세포내TNF-α화IL-1β mRNA수평분별위0.140±0.022、0.077±0.005,P치균소우0.01]. 결론 HMGB1가인기엄중소상대서KC분비촉염성세포인자TNF-α화IL-1β,단RAGE재저일과정중미기주도작용.
Objective To investigate the effect of high mobility group box protein 1 (HMGB1) on the production of pro-inflammatory cytokines by Kupffer cell (KC) of rats with severe burn and the role of receptor for advanced glycation end products (RAGE) in the process.Methods Model of 30％ TBSA full-thickness burn was reproduced in 32 SD rats through immersing the back in 98 ℃ water for 12 s.KC (32 samples) was isolated from rat liver 24 h after injury and inoculated in 24-well plate in the concentration of 1 × 106 cell per well.(1) Cells were divided into control group (cultured with 1 mL PBS) and HMGB1 group (stimulated with 100 ng/mL HMGB1 in the volume of 1 mL) according to the random number table,with 8 samples in each group.At post culture hour (PCH) 48,the expression of RAGE (denoted as grey value ratio) was detected with Western blotting.(2) Another portion of cells were divided into control group (cultured with 1 mL PBS),HMGB1 group (treated with 100 ng/mL HMGB1 in the volume of 1 mL),HMGB1 + anti-RAGE antibody group (treated with 100 ng/mL HMGB1 in the volume of 1 mL after being pre-incubated with 20 μg/mL anti-RAGE monoclonal antibody in the volume of 1 mL for 2 hours),HMGB1 + recombinant rat RAGE/Fc chimera (rrRAGE/Fc) group (treated with the mixture of 100 ng/mL HMGB1 in the volume of 0.5 mL and 5 μg/mL rrRAGE/Fc in the volume of0.5 mL which were pre-incubated for 2 hours) according to the random number table,with 8 samples in each group.At PCH 48,the protein levels of TNF-α and IL-1β in supernatant were determined with enzyme-linked immunosorbent assay,while the mRNA expression of TNF-α and IL-1β (denoted as grey value ratio) were determined with Northern blotting.Data were processed with one-way analysis of variance,t test,and LSD test.Results (1)The expression of RAGE in HMGB1 group (1.036 ± 0.101) was significantly higher than that of control group at PCH 48 (0.191 ±0.024,t =-23.158,P =0.000).(2) In HMGB1 group,HMGB1 +anti-RAGE antibody group,and HMGB1 + rrRAGE/Fc group,the contents of TNF-αt in supernatant were respectively (10.59 ± 1.39),(9.91 ± 1.68),(11.51 ± 2.27) ng/mL; the contents of IL-1β in supernatant were respectively (2.49 ± 0.33),(2.08 ± 0.32),(2.42 ± 0.42) ng/mL; the mRNA levels of TNF-α in cells were respectively 0.311 ± 0.009,0.301 ± 0.047,0.326 ± 0.016 ; the mRNA levels of IL-1β in cells were respectively 0.237 ± 0.021,0.244 ± 0.041,0.245 ± 0.013.There were no statistically significant differences in the above indexes among these three groups (with P values all above 0.05).Their levels were all significantly higher than those of control group [with contents of TNF-α and IL-1β in supernatant respectively (2.69 ±0.14),(0.43 ±0.05) ng/mL,and mRNA levels of TNF-oα and IL-1β in cells respectively 0.140 ± 0.022,0.077 ± 0.005,P values all below 0.01].Conclusions HMGB1 can induce the production of pro-inflammatory cytokines TNF-α and IL-1β from the KC in rats with severe burn.However,RAGE does not play a predominant role in this process.
