中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2010年
3期
175-179
,共5页
张万福%胡大海%徐成峰%吕根法%董茂龙%赵周婷%朱雄翔
張萬福%鬍大海%徐成峰%呂根法%董茂龍%趙週婷%硃雄翔
장만복%호대해%서성봉%려근법%동무룡%조주정%주웅상
烧伤%胰岛素%内皮细胞%NF-κB
燒傷%胰島素%內皮細胞%NF-κB
소상%이도소%내피세포%NF-κB
Bums%Insulin%Endothelial cells%NF-kappa B
目的 了解胰岛素对烧伤血清诱导的血管内皮细胞NF-κB核移位的抑制作用及其相关机制.方法 体外培养人脐静脉内皮细胞(HUVEC).按照随机数字表法将细胞分为5组:空白对照组,不加任何刺激因素常规培养;正常血清对照组和烧伤血清刺激组,分别用含体积分数20%健康人血清、体积分数20%烧伤患者血清的培养液培养;烧伤血清+胰岛素处理组,在烧伤血清刺激组培养液成分的基础上添加胰岛素(终浓度1×10-7mol/L)进行培养;抑制剂预处理组,预先加入蛋白激酶B(PKB或Akt)特异性抑制剂LY294002(50μmol/L)孵育细胞,30 min后改用培养液(成分同烧伤血清+胰岛素处理组)培养.6 h后,采用扫描电镜观察各组HUVEC损伤情况;用流式细胞仪检测细胞凋亡率;蛋白质印迹法检测细胞胞质磷酸化κB-α抑制蛋白(p-IκB-α)和磷酸化Akt(p-Akt)水平,以及胞核NF-κB-p65的蛋白表达变化.结果 (1)扫描电镜观察:与空白对照组HUVEC比较,烧伤血清刺激组、抑制剂预处理组细胞收缩明显,细胞间呈锯齿状连接或连接消失,胞核结构不规整.正常血清对照组及烧伤血清+胰岛素处理组细胞结构有轻微改变,但细胞延展性及胞核结构明显好于烧伤血清刺激组.(2)细胞凋亡率:空白对照组为(15.7±2.2)%.烧伤血清刺激组为(28.5±2.3)%,烧伤血清+胰岛素处理组为(22.3±1.8)%,抑制剂预处理组为(29.7±2.4)%,均明显高于空白对照组(F=14.288,P<0.05或P<0.01);正常血清对照组细胞凋亡率为(17.0±2.5)%,与空白对照组比较差异无统计学意义(F=14.288,P>0.05).与烧伤血清刺激组相比,烧伤血清+胰岛素处理组细胞凋亡率明显降低(F=14.288,P<0.05).(3)蛋白表达水平:与空白对照组相比,烧伤血清刺激组和抑制剂预处理组细胞胞质p-IκB-α与胞核NF-κB-p65的蛋白表达水平明显升高,p-Akt表达水平明显下降;正常血清对照组和烧伤血清+胰岛素处理组3种蛋白水平均与空白对照组接近.结论 胰岛素通过调节磷脂酰肌醇3激酶/Akt信号通路抑制IκB-α磷酸化,继而限制NF-κB核移位,最终发挥改善内皮细胞功能的作用.
目的 瞭解胰島素對燒傷血清誘導的血管內皮細胞NF-κB覈移位的抑製作用及其相關機製.方法 體外培養人臍靜脈內皮細胞(HUVEC).按照隨機數字錶法將細胞分為5組:空白對照組,不加任何刺激因素常規培養;正常血清對照組和燒傷血清刺激組,分彆用含體積分數20%健康人血清、體積分數20%燒傷患者血清的培養液培養;燒傷血清+胰島素處理組,在燒傷血清刺激組培養液成分的基礎上添加胰島素(終濃度1×10-7mol/L)進行培養;抑製劑預處理組,預先加入蛋白激酶B(PKB或Akt)特異性抑製劑LY294002(50μmol/L)孵育細胞,30 min後改用培養液(成分同燒傷血清+胰島素處理組)培養.6 h後,採用掃描電鏡觀察各組HUVEC損傷情況;用流式細胞儀檢測細胞凋亡率;蛋白質印跡法檢測細胞胞質燐痠化κB-α抑製蛋白(p-IκB-α)和燐痠化Akt(p-Akt)水平,以及胞覈NF-κB-p65的蛋白錶達變化.結果 (1)掃描電鏡觀察:與空白對照組HUVEC比較,燒傷血清刺激組、抑製劑預處理組細胞收縮明顯,細胞間呈鋸齒狀連接或連接消失,胞覈結構不規整.正常血清對照組及燒傷血清+胰島素處理組細胞結構有輕微改變,但細胞延展性及胞覈結構明顯好于燒傷血清刺激組.(2)細胞凋亡率:空白對照組為(15.7±2.2)%.燒傷血清刺激組為(28.5±2.3)%,燒傷血清+胰島素處理組為(22.3±1.8)%,抑製劑預處理組為(29.7±2.4)%,均明顯高于空白對照組(F=14.288,P<0.05或P<0.01);正常血清對照組細胞凋亡率為(17.0±2.5)%,與空白對照組比較差異無統計學意義(F=14.288,P>0.05).與燒傷血清刺激組相比,燒傷血清+胰島素處理組細胞凋亡率明顯降低(F=14.288,P<0.05).(3)蛋白錶達水平:與空白對照組相比,燒傷血清刺激組和抑製劑預處理組細胞胞質p-IκB-α與胞覈NF-κB-p65的蛋白錶達水平明顯升高,p-Akt錶達水平明顯下降;正常血清對照組和燒傷血清+胰島素處理組3種蛋白水平均與空白對照組接近.結論 胰島素通過調節燐脂酰肌醇3激酶/Akt信號通路抑製IκB-α燐痠化,繼而限製NF-κB覈移位,最終髮揮改善內皮細胞功能的作用.
목적 료해이도소대소상혈청유도적혈관내피세포NF-κB핵이위적억제작용급기상관궤제.방법 체외배양인제정맥내피세포(HUVEC).안조수궤수자표법장세포분위5조:공백대조조,불가임하자격인소상규배양;정상혈청대조조화소상혈청자격조,분별용함체적분수20%건강인혈청、체적분수20%소상환자혈청적배양액배양;소상혈청+이도소처리조,재소상혈청자격조배양액성분적기출상첨가이도소(종농도1×10-7mol/L)진행배양;억제제예처리조,예선가입단백격매B(PKB혹Akt)특이성억제제LY294002(50μmol/L)부육세포,30 min후개용배양액(성분동소상혈청+이도소처리조)배양.6 h후,채용소묘전경관찰각조HUVEC손상정황;용류식세포의검측세포조망솔;단백질인적법검측세포포질린산화κB-α억제단백(p-IκB-α)화린산화Akt(p-Akt)수평,이급포핵NF-κB-p65적단백표체변화.결과 (1)소묘전경관찰:여공백대조조HUVEC비교,소상혈청자격조、억제제예처리조세포수축명현,세포간정거치상련접혹련접소실,포핵결구불규정.정상혈청대조조급소상혈청+이도소처리조세포결구유경미개변,단세포연전성급포핵결구명현호우소상혈청자격조.(2)세포조망솔:공백대조조위(15.7±2.2)%.소상혈청자격조위(28.5±2.3)%,소상혈청+이도소처리조위(22.3±1.8)%,억제제예처리조위(29.7±2.4)%,균명현고우공백대조조(F=14.288,P<0.05혹P<0.01);정상혈청대조조세포조망솔위(17.0±2.5)%,여공백대조조비교차이무통계학의의(F=14.288,P>0.05).여소상혈청자격조상비,소상혈청+이도소처리조세포조망솔명현강저(F=14.288,P<0.05).(3)단백표체수평:여공백대조조상비,소상혈청자격조화억제제예처리조세포포질p-IκB-α여포핵NF-κB-p65적단백표체수평명현승고,p-Akt표체수평명현하강;정상혈청대조조화소상혈청+이도소처리조3충단백수평균여공백대조조접근.결론 이도소통과조절린지선기순3격매/Akt신호통로억제IκB-α린산화,계이한제NF-κB핵이위,최종발휘개선내피세포공능적작용.
Objective To study the inhibitory effects of insulin on nuclear factor-kappa B (NF-κB) nuclear translocation of vascular endothelial cells induced by burn serum and its correlative mechanism. Methods Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and divided into 5 groups: blank control group (BC, ordinary culture without any stimulation) , normal serum control group (NS, cultured with nutrient solution containing 20% healthy human serum) , burn serum stimulation group (BS, cultured with nutrient solution containing 20% burn human serum) , burn serum + insulin treatment group (BI, cultured with nutrient solution containing 20% burn human serum and 1 × 10-7 mol/L insulin) ,inhibitor pretreatment group [IP, pretreated with 50 μmol/L protein kinase B (Akt) specific inhibitor LY-294002, then cultured with the same medium as used in BI group 30 minutes later] according to the random number table. Six hours later, the injury and apoptosis of HUVECs was respectively observed by the scanning electron microscope and determined by the flow cytometry. Meanwhile, the phosphorylation of inhibitor kappa B-α (p-IκB-α) and Akt (p-Akt) in cytoplasm, and the content of NF-κB-p65 in nucleus were determined with Western blot. Results (1) Compared with those in BC group, HUVECs in BS group shrank obviously with irregular nuclear structure, and intercellular links jagged or vanished. Slight change was observed in HUVECs structure in NS and BI groups, with the cell ductility and nuclear structure much better than those in BS group. (2) The apoptosis rates of HUVECs in BS group [(28. 5 ± 2. 3) %] , BI group [(22. 3 ±1.8)%], and IP group [(29. 7 ± 2. 4) %] were all obviously higher than that in BC group [(15.7 ±2.2)% , F =14.288, P <0.05otP <0.01]. There was no significant statistical difference between NS group [(17. 0 ± 2. 5) %] and BC group in apoptosis rate (F = 14. 288 , P > 0. 05). The apoptosis rate of HUVECs in BI group was obviously lower than that in BS group (F = 14. 288 , P <0.05). (3)Compared with those in BC group, the protein expressions of p-IκB-α in cytoplasm and NF-κB-p65 in nucleus were up-regulated, and the protein expression of p-Akt in cytoplasm was down-regulated in BS and IP groups. The expression levels of the three proteins in NS and BI groups were close to those in BC group. Conclusions Insulin could inhibit the IκB phosphorylation, and then restrict NF-κB nuclear translocation and improve the vascular endothelial cells function accordingly through regulating phosphatidylinositol 3 ki-nase/Akt pathway.
