活性氧对烧伤大鼠血清诱导肺微血管内皮细胞凋亡的影响

Effects of reactive oxygen species on apoptosis of pulmonary microvascular endothelial cells induced by burn rat serum

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。

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.