中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2014年
4期
320-324
,共5页
烧伤%急性肺损伤%细胞凋亡%微血管内皮细胞%活性氧
燒傷%急性肺損傷%細胞凋亡%微血管內皮細胞%活性氧
소상%급성폐손상%세포조망%미혈관내피세포%활성양
Burns%Acute lung injury%Apoptosis%Microvascular endothelial cells%Reactive oxygen species
目的 观察大鼠严重烧伤后及肺微血管内皮细胞(PMVEC)经烧伤血清刺激后活性氧水平,探讨活性氧与PMVEC凋亡的关系. 方法 (1)取24只SD大鼠按随机数字表法(分组方法下同)分为假伤组3只、烧伤组21只,烧伤组大鼠背部造成30％ TBSAⅢ度烫伤,假伤组大鼠致假伤.伤后6、12、24、36、48、60、72 h按随机数字表法分别取3只烧伤组大鼠,腹主动脉取血,ELISA法检测血清中活性氧含量;假伤组大鼠行相同检测.(2)取5只SD大鼠按前述方法造成烫伤,伤后24 h制备烧伤大鼠血清;另取5只SD大鼠不作处理,制备健康大鼠血清.(3)切取20只SD大鼠幼鼠肺外边缘组织,组织块法培养细胞,免疫组织化学法鉴定细胞.取第3代对数生长期PMVEC分别接种于6孔板和12孔板,均分为4组(每组设3个复孔):正常血清组、烧伤血清组,无血清内皮细胞专用培养液中分别加入体积分数15％健康大鼠血清、体积分数15％烧伤大鼠血清培养;正常血清+锰四(4-苯甲酸)卟啉(MnTBAP)组、烧伤血清+MnTBAP组,在前2组的基础上再分别加入100 μmol/L MnTBAP培养.培养24 h后,流式细胞仪检测6孔板中细胞内活性氧含量,吖啶橙-溴化乙啶染色观察12孔板中细胞凋亡情况并计算凋亡率.对数据行单因素方差分析、LSD-t检验. 结果 (1)烧伤组大鼠伤后24、36、48、60、72 h血清中活性氧含量分别为(187 ±21)、(235 ±22)、(231 ±25)、(291 ±20)、(315±23) nmol/mL,显著高于假伤组的(141±19) nmol/mL(t值分别为7.86、9.57、13.87、14.98、18.40,P值均小于0.01).(2)原代培养细胞生长较慢,呈铺路石样生长;传代后细胞呈均匀分布生长.细胞凝血因子Ⅷ阳性表达率为(96±5)％,鉴定为PMVEC.(3)培养24 h后,正常血清组、烧伤血清组、正常血清+ MnTBAP组、烧伤血清+MnTBAP组PMVEC中活性氧含量分别为798±40、1 294±84、763±59、926 ±42(F=93.01,P＜0.01),细胞凋亡率分别为(6.2±1.3)％、(57.3±6.7)％、(3.7±0.8)％、(28.7±5.7)％(F=224.50,P＜0.01).与正常血清组比较,烧伤血清组PMVEC中活性氧含量、细胞凋亡率明显增加(t值分别为10.40、49.06,P值均小于0.01);烧伤血清+MnTBAP组PMVEC中活性氧含量、细胞凋亡率较烧伤血清组明显减少(t值分别为7.48、23.94,P值均小于0.01). 结论 大鼠严重烧伤后血清中活性氧上升,烧伤大鼠血清刺激PMVEC可以导致细胞内活性氧以及细胞凋亡增加,应用MnTBAP清除活性氧可减少烧伤大鼠血清诱导的细胞凋亡.
目的 觀察大鼠嚴重燒傷後及肺微血管內皮細胞(PMVEC)經燒傷血清刺激後活性氧水平,探討活性氧與PMVEC凋亡的關繫. 方法 (1)取24隻SD大鼠按隨機數字錶法(分組方法下同)分為假傷組3隻、燒傷組21隻,燒傷組大鼠揹部造成30％ TBSAⅢ度燙傷,假傷組大鼠緻假傷.傷後6、12、24、36、48、60、72 h按隨機數字錶法分彆取3隻燒傷組大鼠,腹主動脈取血,ELISA法檢測血清中活性氧含量;假傷組大鼠行相同檢測.(2)取5隻SD大鼠按前述方法造成燙傷,傷後24 h製備燒傷大鼠血清;另取5隻SD大鼠不作處理,製備健康大鼠血清.(3)切取20隻SD大鼠幼鼠肺外邊緣組織,組織塊法培養細胞,免疫組織化學法鑒定細胞.取第3代對數生長期PMVEC分彆接種于6孔闆和12孔闆,均分為4組(每組設3箇複孔):正常血清組、燒傷血清組,無血清內皮細胞專用培養液中分彆加入體積分數15％健康大鼠血清、體積分數15％燒傷大鼠血清培養;正常血清+錳四(4-苯甲痠)卟啉(MnTBAP)組、燒傷血清+MnTBAP組,在前2組的基礎上再分彆加入100 μmol/L MnTBAP培養.培養24 h後,流式細胞儀檢測6孔闆中細胞內活性氧含量,吖啶橙-溴化乙啶染色觀察12孔闆中細胞凋亡情況併計算凋亡率.對數據行單因素方差分析、LSD-t檢驗. 結果 (1)燒傷組大鼠傷後24、36、48、60、72 h血清中活性氧含量分彆為(187 ±21)、(235 ±22)、(231 ±25)、(291 ±20)、(315±23) nmol/mL,顯著高于假傷組的(141±19) nmol/mL(t值分彆為7.86、9.57、13.87、14.98、18.40,P值均小于0.01).(2)原代培養細胞生長較慢,呈鋪路石樣生長;傳代後細胞呈均勻分佈生長.細胞凝血因子Ⅷ暘性錶達率為(96±5)％,鑒定為PMVEC.(3)培養24 h後,正常血清組、燒傷血清組、正常血清+ MnTBAP組、燒傷血清+MnTBAP組PMVEC中活性氧含量分彆為798±40、1 294±84、763±59、926 ±42(F=93.01,P＜0.01),細胞凋亡率分彆為(6.2±1.3)％、(57.3±6.7)％、(3.7±0.8)％、(28.7±5.7)％(F=224.50,P＜0.01).與正常血清組比較,燒傷血清組PMVEC中活性氧含量、細胞凋亡率明顯增加(t值分彆為10.40、49.06,P值均小于0.01);燒傷血清+MnTBAP組PMVEC中活性氧含量、細胞凋亡率較燒傷血清組明顯減少(t值分彆為7.48、23.94,P值均小于0.01). 結論 大鼠嚴重燒傷後血清中活性氧上升,燒傷大鼠血清刺激PMVEC可以導緻細胞內活性氧以及細胞凋亡增加,應用MnTBAP清除活性氧可減少燒傷大鼠血清誘導的細胞凋亡.
목적 관찰대서엄중소상후급폐미혈관내피세포(PMVEC)경소상혈청자격후활성양수평,탐토활성양여PMVEC조망적관계. 방법 (1)취24지SD대서안수궤수자표법(분조방법하동)분위가상조3지、소상조21지,소상조대서배부조성30％ TBSAⅢ도탕상,가상조대서치가상.상후6、12、24、36、48、60、72 h안수궤수자표법분별취3지소상조대서,복주동맥취혈,ELISA법검측혈청중활성양함량;가상조대서행상동검측.(2)취5지SD대서안전술방법조성탕상,상후24 h제비소상대서혈청;령취5지SD대서불작처리,제비건강대서혈청.(3)절취20지SD대서유서폐외변연조직,조직괴법배양세포,면역조직화학법감정세포.취제3대대수생장기PMVEC분별접충우6공판화12공판,균분위4조(매조설3개복공):정상혈청조、소상혈청조,무혈청내피세포전용배양액중분별가입체적분수15％건강대서혈청、체적분수15％소상대서혈청배양;정상혈청+맹사(4-분갑산)계람(MnTBAP)조、소상혈청+MnTBAP조,재전2조적기출상재분별가입100 μmol/L MnTBAP배양.배양24 h후,류식세포의검측6공판중세포내활성양함량,아정등-추화을정염색관찰12공판중세포조망정황병계산조망솔.대수거행단인소방차분석、LSD-t검험. 결과 (1)소상조대서상후24、36、48、60、72 h혈청중활성양함량분별위(187 ±21)、(235 ±22)、(231 ±25)、(291 ±20)、(315±23) nmol/mL,현저고우가상조적(141±19) nmol/mL(t치분별위7.86、9.57、13.87、14.98、18.40,P치균소우0.01).(2)원대배양세포생장교만,정포로석양생장;전대후세포정균균분포생장.세포응혈인자Ⅷ양성표체솔위(96±5)％,감정위PMVEC.(3)배양24 h후,정상혈청조、소상혈청조、정상혈청+ MnTBAP조、소상혈청+MnTBAP조PMVEC중활성양함량분별위798±40、1 294±84、763±59、926 ±42(F=93.01,P＜0.01),세포조망솔분별위(6.2±1.3)％、(57.3±6.7)％、(3.7±0.8)％、(28.7±5.7)％(F=224.50,P＜0.01).여정상혈청조비교,소상혈청조PMVEC중활성양함량、세포조망솔명현증가(t치분별위10.40、49.06,P치균소우0.01);소상혈청+MnTBAP조PMVEC중활성양함량、세포조망솔교소상혈청조명현감소(t치분별위7.48、23.94,P치균소우0.01). 결론 대서엄중소상후혈청중활성양상승,소상대서혈청자격PMVEC가이도치세포내활성양이급세포조망증가,응용MnTBAP청제활성양가감소소상대서혈청유도적세포조망.
Objective To observe the level of intracellular reactive oxygen species (ROS) in rats with severe burn and pulmonary microvascular endothelial cells (PMVECs) treated with serum of rat with burn injury,and to investigate the relationship between ROS and apoptosis of PMVECs.Methods (1)Twenty-four SD rats were divided into sham injury group (n =3) and burn group (n =21) according to the random number table (the same grouping method below).Rats in burn group were inflicted with 30％ TBSA full-thickness scald on the back,and rats in sham injury group were sham injured.Blood samples were collected from abdominal aorta at post injury hour 6,12,24,36,48,60,72 respectively from 3 rats of burn group.The serum content of ROS was assayed by ELISA.The same determination was performed in rats of sham injury group.(2) Five rats were subjected to scald injury as above,and burn serum was prepared 24 hours after injury.Another 5 rats without receiving any treatment were used to prepare normal serum.(3) Marginal pulmonary tissue was harvested from 20 SD young rats.Cells were cultured with tissue block method and indentified with immunohistochemical staining.The third passage of PMVECs in logarithmic phase were inoculated in 6-well plates and 12-well plates.PMVECs in both plates were divided into 4 groups:normal serum group,burn serum group,normal serum + MnTBAP group,and burn serum + MnTBAP group,with 3 wells in each group.Cells in the former 2 groups were respectively cultured with special nutrient solution of endothelial cells without serum added with 15％ healthy rat serum or 15％ burn rat serum.Cells in the latter 2 groups were cultured with the same culture conditions as in the former two groups correspondingly with addition of 100 μmol/L MnTBAP in the nutrient solution.After being cultured for 24 h,the content of ROS in PMVECs in 6-well plates was detected with flow cytometry.The apoptosis of PMVECs in 12-well plates was observed with acridine orange-ethidium bromide staining,and the apoptosis rate was calculated.Data were processed with one-way analysis of variance and LSD-t test.Results (1)The serum contents of ROS in rats of burn group were respectively (187 ± 21),(235 ± 22),(231 ± 25),(291 ±20),(315 ±23) nmol/mLat post injury hour 24,36,48,60,72,which were significantly higher than that in sham injury group [(141 ± 19) nmol/mL,with t values respectively 7.86,9.57,13.87,14.98,18.40,P values below 0.01].(2) Primary cells grew slowly and showed a cobblestone appearance.After passages,cells grew with orderly distribution.The positive rate of coagulation factor Ⅷ of cells was (96 ± 5)％,and thus they were identified as PMVECs.(3) In normal serum group,burn serum group,normal serum + MnTBAP group,and burn serum + MnTBAP group,the contents of ROS in PMVECs were respectively 798 ±40,1 294 ±84,763 ±59,926 ±42 (F =93.01,P ＜0.01),and the apoptosis rates of PMVECs were respectively (6.2 ±1.3)％,(57.3 ±6.7)％,(3.7 ±0.8)％,(28.7 ±5.7)％ (F =224.50,P ＜0.01) after being cultured for 24 h.Compared with those of normal serum group,the content of ROS and apoptosis rate of PMVECs in burn serum group increased significantly (with t values respectively 10.40 and 49.06,P values below 0.01).The content of ROS and apoptosis rate of PMVECs in burn serum + MnTBAP group were significantly lower than those in burn serum group (with t values respectively 7.48 and 23.94,P values below 0.01).Conclusions Serum content of ROS was increased in severely burned rats.Burn rat serum stimulation on PMVECs can lead to the increase of the intracellular ROS and induce apoptosis.However application of MnTBAP can scavenge ROS and reduce the apoptosis induced by burn rat serum.
