白藜芦醇在烧伤血清诱导的肺微血管内皮细胞凋亡中的作用研究  

The role and mechanism research of resveratrol in the pulmonary microvascular endothelial cells apoptosis induced by burn serum

作　　者：肖厚安 周小茜[1] 张丽[1] 安鸿肇 刘晨[1] 王煜[1] 寇谦 邓旭东[1] 贾赤宇 

机构地区：[1]西安交通大学医学院附属西安市第九医院烧伤整形科,710054 [2]解放军第三○九医院烧伤整形科,北京100091

出　　处：《中华损伤与修复杂志（电子版）》2015年第4期9-13,共5页Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)

基　　金：国家自然科学基金面上项目(81372051);总参军事医学和老年病科研基金项目(ZCWS14B06)

摘　　要：目的探讨白藜芦醇在烧伤血清诱导的肺微血管内皮细胞(PMVEC)凋亡中的作用及可能的分子机制。方法 (1)取5只SD大鼠背部于95℃热水浴18 s制成30%总体表面积Ⅲ度烫伤模型,伤后24 h制备烧伤大鼠血清;另取5只SD大鼠不做处理,制备正常大鼠血清。(2)体外组织块法培养大鼠PMVEC,免疫组织化学法鉴定细胞。取第3代对数生长期的细胞分别接种6孔板和12孔板,各板均分为3组(每组设3个复孔):对照组、烧伤血清组、烧伤血清+白藜芦醇组。白藜芦醇在烧伤血清刺激前2 h加入(浓度20μmol/L)。(3)采用铺明胶的Transwell培养细胞后,酶联免疫吸咐法检测单层细胞通透性;采用吖啶橙-溴化乙啶法检测PMVEC的凋亡;RT-PCR检测炎症因子肿瘤坏死因子-α(TNF-α)以及白细胞介素-1β(IL-1β)的mRNA水平;Western blot法检测细胞沉默信息调节因子1(SIRT1)以及半胱氨酸天冬氨酸蛋白酶3(Caspase3)的表达。结果 (1)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(97±3)%,鉴定为PMVEC。(2)培养24 h后,对照组,烧伤血清组,烧伤血清+白藜芦醇组中细胞通透性分别为(87±16)、(220±11)、(131±11)ng/m L(F=207.40,P<0.05),细胞凋亡率分别为(5.2±2.3)%、(22.3±3.7)%、(11.7±2.3)%(F=69.07,P<0.05)。与对照组比较,烧伤血清组单分子层通透性、细胞凋亡率明显增加(t值分别为18.76、10.75,P值均小于0.05);烧伤血清+白藜芦醇组内皮细胞单分子层通透性、细胞凋亡率较烧伤血清组明显减少(t值分别为6.21、5.47,P值均小于0.05)。(3)培养24 h后,对照组,烧伤血清组,烧伤血清+白藜芦醇组TNF-α以及IL-1β的mRNA水平分别为1.0±0.1、3.8±1.3、1.6±0.7(F=22.47,P<0.05);1.0±0.1、3.3±1.5、1.7±0.4(F=13.01,P<0.05);SIRT1以及Cleaved Caspase3的蛋白表达量分别为1.0±0.3、2.8±0.5、3.7±0.6(F=69.99,P<0.05);1.0±0.2、3.3±0.5、1.9±0.6(F=28.78,P<0.05);与对照组比Objective To explore the resveratrol in burn serum induced pulmonary microvascular endothelial cells( PMVEC) apoptosis inhibition and the possible molecular mechanisms. Methods Take only 5 SD rats back in the 95 ℃ hot water bath 18 s made 30% TBSA Ⅲ degree scald model,and 24 h after preparation burns in the rat serum; take five male SD rats don' t do processing,preparation of normal rat serum; PMVEC cultivation in vitro in rats,burn serum stimulus model is set up,according to different stimuli were divided into control group( normal serum stimulus),burn serum group( burn serum stimulus),resveratrol( burn serum + resveratrol),resveratrol group 20 μmol / L) 2 h before burn serum stimulus to join. The spread of gelatin transwell culture cell,enzyme-linked immunoassay detection in single cell permeability. Using AO / EB method to detect apoptosis of PMVEC; RT-PCR detection of inflammatory factors of TNF alpha and beta IL-1 mRNA level; Western blot method to detect the expression of cell SIRT1 and Caspase3. Results( 1) The original generation of cultured cells grew more slowly,the paving stone sample growth; After the wedding is evenly distributed cell growth. Cell Ⅷ positive expression rate of clotting factors( 97 ± 3) %,identified as PMVEC.( 2) The training after 24 h,the control group,the burn serum group,burn serum + cell permeability respectively in resveratrol group 87 ± 11,16,220 ± 11 ng / m L( F = 207. 4,P < 0. 05),the rate of cell apoptosis,respectively( 5. 2 ± 2. 3) %,( 22. 3 ± 3. 7) %,( 11. 7 ± 2. 3) %( F = 69. 07,P < 0. 05). Compared with control group,burn serum monolayer permeability and cell apoptosis rate increased significantly( t value were 18. 76,10. 75,P values all below 0. 05); Burn serum +endothelial monolayer permeability of resveratrol group,cell apoptosis rate than burn serum group decreased significantly( t value were 6. 21,5. 47,P value is less than 0. 05).( 3) Training after 24 h,the control group,the burn serum group,burn serum TNF alpha + resveratrol group and the mRNA level

关 键 词：烧伤 内皮细胞 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]