中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2009年
1期
36-41
,共6页
宋达疆%黄晓元%任利成%杨兴华%肖目张%王双
宋達疆%黃曉元%任利成%楊興華%肖目張%王雙
송체강%황효원%임리성%양흥화%초목장%왕쌍
RNA干扰%拟杆菌%脆弱%脓毒症%髓系细胞触发受体1%慢病毒载体
RNA榦擾%擬桿菌%脆弱%膿毒癥%髓繫細胞觸髮受體1%慢病毒載體
RNA간우%의간균%취약%농독증%수계세포촉발수체1%만병독재체
RNA interference%Bacteroides fragilis%Sepsis%Triggering receptor expressed on myeloid cells 1%Lentiviral vector
目的 了解髓系细胞触发受体1(TREM-1)基因重组慢病毒短发夹RNA(vshRNA)载体对脆弱拟杆菌脓毒症小鼠促炎因子表达及小鼠存活率的影响.方法 (1)构建TREM-1 vshRNA载体.经实验确定脓毒症小鼠模型制作条件:腹腔注射2.5×10<9CFU/mL灭活脆弱拟杆菌0.5 mL,刺激12 h.(2)115只小鼠按随机数字表法分为健康对照组(3只)和脓毒症模型组、绿色荧光蛋白(GFP)干扰组、TREM-1干扰组、TREM-1半量干扰组.后4组每组28只,先分别经尾静脉注射等渗盐水、GFP vshRNA、TREM-1 vshRNA 2×108TU、TREM-1 vshRNA 1×108TU;1 h后,将该4组小鼠制成脓毒症模型,健康对照组注射等体积等渗盐水.12 h后处死5组小鼠(每组3只),取血检测血浆TNF-α、IL-1β和IL-6含量;取肝组织标本检测TREM-1 mRNA及其蛋白的表达水平.后4组小鼠每组所余25只用于观察腹腔注射后72 h的存活率.(3)另建立脓毒症小鼠模型125只,其中100只分别于腹腔注射后1、2、4、6 h尾静脉注射TREM-1 vshRNA 2×108TU(每时相点25只),余下25只尾静脉注射等渗盐水为对照组.计算尾静脉注射后72 h小鼠存活率.结果 (1)与脓毒症模型组比较.TREM-1干扰组、TREM-1半量干扰组小鼠血浆TNF-α、IL-1β、IL-6含量均下降(P<0.05),以TREM-1干扰组下降最明显.GFP干扰组中各促炎因子含量与脓毒症模型组接近(P>0.05).(2)与GFP干扰组比较,TREM-1干扰组及其半量干扰组小鼠肝组织TREM-1 mRNA表达均明显下调(P<0.01),以TREM-1干扰组尤为明显(P<0.05).各组小鼠TREM-1蛋白表达情况与此一致.(3)脓毒症模型组、GFP干扰组小鼠腹腔注射后72 h存活率均为16%.TREM-1干扰组及其半量干扰组分别为76%和44%(P<0.05或P<0.01).(4)另建立的125只脓毒症小鼠模型中,对照组及腹腔注射后1、2、4、6 h组其尾静脉注射后72 h存活率分别为16%、72%、56%、40%、16%,其中1、2、4 h组明显高于对照组(P<0.05或P<0.01).结论 运用TREM-1 vshRNA干扰技术,可有效降低脆弱拟杆菌脓毒症小鼠肝组织TREM-1表达水平,抑制炎性反应,提高小鼠存活率.
目的 瞭解髓繫細胞觸髮受體1(TREM-1)基因重組慢病毒短髮夾RNA(vshRNA)載體對脆弱擬桿菌膿毒癥小鼠促炎因子錶達及小鼠存活率的影響.方法 (1)構建TREM-1 vshRNA載體.經實驗確定膿毒癥小鼠模型製作條件:腹腔註射2.5×10<9CFU/mL滅活脆弱擬桿菌0.5 mL,刺激12 h.(2)115隻小鼠按隨機數字錶法分為健康對照組(3隻)和膿毒癥模型組、綠色熒光蛋白(GFP)榦擾組、TREM-1榦擾組、TREM-1半量榦擾組.後4組每組28隻,先分彆經尾靜脈註射等滲鹽水、GFP vshRNA、TREM-1 vshRNA 2×108TU、TREM-1 vshRNA 1×108TU;1 h後,將該4組小鼠製成膿毒癥模型,健康對照組註射等體積等滲鹽水.12 h後處死5組小鼠(每組3隻),取血檢測血漿TNF-α、IL-1β和IL-6含量;取肝組織標本檢測TREM-1 mRNA及其蛋白的錶達水平.後4組小鼠每組所餘25隻用于觀察腹腔註射後72 h的存活率.(3)另建立膿毒癥小鼠模型125隻,其中100隻分彆于腹腔註射後1、2、4、6 h尾靜脈註射TREM-1 vshRNA 2×108TU(每時相點25隻),餘下25隻尾靜脈註射等滲鹽水為對照組.計算尾靜脈註射後72 h小鼠存活率.結果 (1)與膿毒癥模型組比較.TREM-1榦擾組、TREM-1半量榦擾組小鼠血漿TNF-α、IL-1β、IL-6含量均下降(P<0.05),以TREM-1榦擾組下降最明顯.GFP榦擾組中各促炎因子含量與膿毒癥模型組接近(P>0.05).(2)與GFP榦擾組比較,TREM-1榦擾組及其半量榦擾組小鼠肝組織TREM-1 mRNA錶達均明顯下調(P<0.01),以TREM-1榦擾組尤為明顯(P<0.05).各組小鼠TREM-1蛋白錶達情況與此一緻.(3)膿毒癥模型組、GFP榦擾組小鼠腹腔註射後72 h存活率均為16%.TREM-1榦擾組及其半量榦擾組分彆為76%和44%(P<0.05或P<0.01).(4)另建立的125隻膿毒癥小鼠模型中,對照組及腹腔註射後1、2、4、6 h組其尾靜脈註射後72 h存活率分彆為16%、72%、56%、40%、16%,其中1、2、4 h組明顯高于對照組(P<0.05或P<0.01).結論 運用TREM-1 vshRNA榦擾技術,可有效降低脆弱擬桿菌膿毒癥小鼠肝組織TREM-1錶達水平,抑製炎性反應,提高小鼠存活率.
목적 료해수계세포촉발수체1(TREM-1)기인중조만병독단발협RNA(vshRNA)재체대취약의간균농독증소서촉염인자표체급소서존활솔적영향.방법 (1)구건TREM-1 vshRNA재체.경실험학정농독증소서모형제작조건:복강주사2.5×10<9CFU/mL멸활취약의간균0.5 mL,자격12 h.(2)115지소서안수궤수자표법분위건강대조조(3지)화농독증모형조、록색형광단백(GFP)간우조、TREM-1간우조、TREM-1반량간우조.후4조매조28지,선분별경미정맥주사등삼염수、GFP vshRNA、TREM-1 vshRNA 2×108TU、TREM-1 vshRNA 1×108TU;1 h후,장해4조소서제성농독증모형,건강대조조주사등체적등삼염수.12 h후처사5조소서(매조3지),취혈검측혈장TNF-α、IL-1β화IL-6함량;취간조직표본검측TREM-1 mRNA급기단백적표체수평.후4조소서매조소여25지용우관찰복강주사후72 h적존활솔.(3)령건립농독증소서모형125지,기중100지분별우복강주사후1、2、4、6 h미정맥주사TREM-1 vshRNA 2×108TU(매시상점25지),여하25지미정맥주사등삼염수위대조조.계산미정맥주사후72 h소서존활솔.결과 (1)여농독증모형조비교.TREM-1간우조、TREM-1반량간우조소서혈장TNF-α、IL-1β、IL-6함량균하강(P<0.05),이TREM-1간우조하강최명현.GFP간우조중각촉염인자함량여농독증모형조접근(P>0.05).(2)여GFP간우조비교,TREM-1간우조급기반량간우조소서간조직TREM-1 mRNA표체균명현하조(P<0.01),이TREM-1간우조우위명현(P<0.05).각조소서TREM-1단백표체정황여차일치.(3)농독증모형조、GFP간우조소서복강주사후72 h존활솔균위16%.TREM-1간우조급기반량간우조분별위76%화44%(P<0.05혹P<0.01).(4)령건립적125지농독증소서모형중,대조조급복강주사후1、2、4、6 h조기미정맥주사후72 h존활솔분별위16%、72%、56%、40%、16%,기중1、2、4 h조명현고우대조조(P<0.05혹P<0.01).결론 운용TREM-1 vshRNA간우기술,가유효강저취약의간균농독증소서간조직TREM-1표체수평,억제염성반응,제고소서존활솔.
Objective To investigate the effect of triggering receptor expressed on myeloid cells 1 (TREM-1) vshRNA vector on expression of inflammatory cytokines and survival rate in septic mice infected by Bacteroides fragilis. Methods (1) TREM-1 vshRNA vector was constructed. Bacteroides fragilis (2.5×109 CFU/mL, 0.5 mL) was intraperitoneally injected in each mouse, and septic model was repro-duced after 12 hours. (2)One hundred and fifteen mice were divided into healthy control group (n=3, HC), sepsis group (n=28, S) , TREM-1 vshRNA group(n=28, T), TREM-1 vshRNA hd group(n=28, Th), GFP group (n =28,G) according to random number table. Mice in S, T, Th, G groups were firstly injected with isotonic saline, TREM-1 vshRNA 2×108 TU, TREM-1 vshRNA 1×10>TU, GFP siRNA through tail vein, and then sepsis was induced after 1 hour. Mice in HC group were injected with equal vol-ume of isotonic saline through tail vein. Three mice in each group were sacrificed after 12 hours for determi-nation of plasma level of TNF-α, IL-1β and IL-6, and level of TREM-1 mRNA and its protein in hepatic tis-sue. The survival rate of other mice in each group was monitored for 72 hours. (3)In 125 mice sepsis was reproduced,among them 100 mice were injected with TREM-1 vshRNA 2×108 TU after 1, 2, 4, 6 hours through tail vein(25 mice at each time point), other 25 mice were injected with equal volume of isotonic saline as control. The survival rate of mice in each group was recorded 72 hours after injection. Results (1)Com-pared with those in S group, the plasma level of TNF-α, IL-1β and IL-6 lowered in T and Th groups(P < 0. 05) ,especially in T group,while those in G group showed no obvious difference(P >0.05). (2)Compared with those in G group, the level of TREM-ImRNA and its protein in hepatic tissue in T and Th groups de-creased(P <0. 01), especially in T group. (3)The survival rate of mice in S and G group was 16% , which was obviously lower than that in T and Th groups (76%, 44%, respectively, P < 0.05 orP < 0.01). (4) The survival rate of mice at 1,2, 4, 6 hours after injection was 72% , 56%, 40% , 16%, respectively, while all that except at 6 hour after injection were higher significantly than that of control(P <0.05 or P <0.01). Conclusions The intervention with TREM-1 vshRNA can effectively decrease hepatic level of TREM-1 in septic mice induced by Bacteroides fragilis, inhibit inflammatory response, and improve the survival rate.
