中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2013年
6期
322-326
,共5页
卢娜娜%刘琪%顾立刚%周旭澎%吴珺%邱泽计%张洪春%晁恩祥%张沂
盧娜娜%劉琪%顧立剛%週旭澎%吳珺%邱澤計%張洪春%晁恩祥%張沂
로나나%류기%고립강%주욱팽%오군%구택계%장홍춘%조은상%장기
流感病毒性肺炎%疏风宣肺方%解表清里方%自然杀伤细胞%基因芯片%小鼠
流感病毒性肺炎%疏風宣肺方%解錶清裏方%自然殺傷細胞%基因芯片%小鼠
류감병독성폐염%소풍선폐방%해표청리방%자연살상세포%기인심편%소서
Pneumonia infected with influenza virus%Shufeng Xuanfei formula%Jiebiao Qingli formula%Natural killer cell%Gene chip%Mouse
目的 观察疏风宣肺方和解表清里方对流感病毒性肺炎小鼠自然杀伤细胞(NK细胞)毒性相关信号转导通路差异基因表达的调控作用.方法 将90只ICR小鼠按随机数字表法分为对照(N)组、模型(M)组、奥司他韦(C)组以及中药疏风宣肺方高、中、低剂量(SH、SM、SL)组和解表清里方高、中、低剂量(JH、JM、JL)组,每组10只.采用流感病毒亚甲型鼠肺适应株FM1滴鼻感染方法制备流感病毒性肺炎模型；N组以0.05 ml等渗盐水滴鼻.于制模后2h,C组给予奥司他韦11.375 mg· kg-1·d-1灌胃；SH、SM、SL组给予疏风宣肺方(主要药物金银花、连翘、板蓝根等)3.76、1.88、0.94 g· kg-1·d-1灌胃；JH、JM、JL组给予解表清里方(主要药物麻黄、石膏、生甘草等)4.36、2.18、1.09 g·kg-1·d-1灌胃；N组及M组则灌胃等量生理盐水；各组均0.2 ml/d,连用4d.提取肺组织总RNA,采用基因芯片筛选出NK细胞毒性信号转导通路相关的差异表达基因,以I表示信号强度；并采用荧光定量聚合酶链反应(PCR)和蛋白质免疫印迹试验(Western blotting)对部分基因进行验证.结果 在NK细胞毒性相关信号转导通路中,M组较N组上调差异基因43个.用药治疗后,SM组下调差异表达基因36个,JM组下调差异表达基因29个,SL组下调差异表达基因22个,JH组下调差异表达基因21个,SH组下调差异表达基因20个,JL组下调差异表达基因10个；SH 、SL、JH、JM、JL组表达下调的基因均包括在SM组的表达谱中,表明中剂量疏风宣肺方调控NK细胞毒性作用最显著.荧光定量PCR和Western blotting测定结果显示,与M组比较,疏风宣肺方与解表清里方各剂量组对肿瘤坏死因子-α (TNF-α)的mRNA及蛋白表达均有显著抑制作用,疏风宣肺方和解表清里方均以中剂量组最低(TNF-α mRNA:1.07±0.19、1.19±0.14比3.20±0.56,均P＜0.01).结论 流感病毒感染后在宿主细胞内增殖即成为带有病毒抗原的靶细胞,并激活NK细胞毒性信号转导通路的相关基因表达增加,被NK细胞识别并杀伤；两种方药可下调NK细胞中表达上调的差异基因,下调机制与其能明确减少流感病毒性肺炎体内靶抗原、减弱NK细胞识别活化作用有关.
目的 觀察疏風宣肺方和解錶清裏方對流感病毒性肺炎小鼠自然殺傷細胞(NK細胞)毒性相關信號轉導通路差異基因錶達的調控作用.方法 將90隻ICR小鼠按隨機數字錶法分為對照(N)組、模型(M)組、奧司他韋(C)組以及中藥疏風宣肺方高、中、低劑量(SH、SM、SL)組和解錶清裏方高、中、低劑量(JH、JM、JL)組,每組10隻.採用流感病毒亞甲型鼠肺適應株FM1滴鼻感染方法製備流感病毒性肺炎模型；N組以0.05 ml等滲鹽水滴鼻.于製模後2h,C組給予奧司他韋11.375 mg· kg-1·d-1灌胃；SH、SM、SL組給予疏風宣肺方(主要藥物金銀花、連翹、闆藍根等)3.76、1.88、0.94 g· kg-1·d-1灌胃；JH、JM、JL組給予解錶清裏方(主要藥物痳黃、石膏、生甘草等)4.36、2.18、1.09 g·kg-1·d-1灌胃；N組及M組則灌胃等量生理鹽水；各組均0.2 ml/d,連用4d.提取肺組織總RNA,採用基因芯片篩選齣NK細胞毒性信號轉導通路相關的差異錶達基因,以I錶示信號彊度；併採用熒光定量聚閤酶鏈反應(PCR)和蛋白質免疫印跡試驗(Western blotting)對部分基因進行驗證.結果 在NK細胞毒性相關信號轉導通路中,M組較N組上調差異基因43箇.用藥治療後,SM組下調差異錶達基因36箇,JM組下調差異錶達基因29箇,SL組下調差異錶達基因22箇,JH組下調差異錶達基因21箇,SH組下調差異錶達基因20箇,JL組下調差異錶達基因10箇；SH 、SL、JH、JM、JL組錶達下調的基因均包括在SM組的錶達譜中,錶明中劑量疏風宣肺方調控NK細胞毒性作用最顯著.熒光定量PCR和Western blotting測定結果顯示,與M組比較,疏風宣肺方與解錶清裏方各劑量組對腫瘤壞死因子-α (TNF-α)的mRNA及蛋白錶達均有顯著抑製作用,疏風宣肺方和解錶清裏方均以中劑量組最低(TNF-α mRNA:1.07±0.19、1.19±0.14比3.20±0.56,均P＜0.01).結論 流感病毒感染後在宿主細胞內增殖即成為帶有病毒抗原的靶細胞,併激活NK細胞毒性信號轉導通路的相關基因錶達增加,被NK細胞識彆併殺傷；兩種方藥可下調NK細胞中錶達上調的差異基因,下調機製與其能明確減少流感病毒性肺炎體內靶抗原、減弱NK細胞識彆活化作用有關.
목적 관찰소풍선폐방화해표청리방대류감병독성폐염소서자연살상세포(NK세포)독성상관신호전도통로차이기인표체적조공작용.방법 장90지ICR소서안수궤수자표법분위대조(N)조、모형(M)조、오사타위(C)조이급중약소풍선폐방고、중、저제량(SH、SM、SL)조화해표청리방고、중、저제량(JH、JM、JL)조,매조10지.채용류감병독아갑형서폐괄응주FM1적비감염방법제비류감병독성폐염모형；N조이0.05 ml등삼염수적비.우제모후2h,C조급여오사타위11.375 mg· kg-1·d-1관위；SH、SM、SL조급여소풍선폐방(주요약물금은화、련교、판람근등)3.76、1.88、0.94 g· kg-1·d-1관위；JH、JM、JL조급여해표청리방(주요약물마황、석고、생감초등)4.36、2.18、1.09 g·kg-1·d-1관위；N조급M조칙관위등량생리염수；각조균0.2 ml/d,련용4d.제취폐조직총RNA,채용기인심편사선출NK세포독성신호전도통로상관적차이표체기인,이I표시신호강도；병채용형광정량취합매련반응(PCR)화단백질면역인적시험(Western blotting)대부분기인진행험증.결과 재NK세포독성상관신호전도통로중,M조교N조상조차이기인43개.용약치료후,SM조하조차이표체기인36개,JM조하조차이표체기인29개,SL조하조차이표체기인22개,JH조하조차이표체기인21개,SH조하조차이표체기인20개,JL조하조차이표체기인10개；SH 、SL、JH、JM、JL조표체하조적기인균포괄재SM조적표체보중,표명중제량소풍선폐방조공NK세포독성작용최현저.형광정량PCR화Western blotting측정결과현시,여M조비교,소풍선폐방여해표청리방각제량조대종류배사인자-α (TNF-α)적mRNA급단백표체균유현저억제작용,소풍선폐방화해표청리방균이중제량조최저(TNF-α mRNA:1.07±0.19、1.19±0.14비3.20±0.56,균P＜0.01).결론 류감병독감염후재숙주세포내증식즉성위대유병독항원적파세포,병격활NK세포독성신호전도통로적상관기인표체증가,피NK세포식별병살상；량충방약가하조NK세포중표체상조적차이기인,하조궤제여기능명학감소류감병독성폐염체내파항원、감약NK세포식별활화작용유관.
Objective To investigate the regulation of two herbal anti-virus formulas on gene expression profile associated with natural killer cell (NK cell) mediated cytotoxicity in pneumonia mice infected with influenza virus.Methods According to random number table,90 ICR mice were divided into nine groups with 10 mice in each group:normal group (N),model group (M),osehamivir group (control group,C),low-dose,medium-dose and high-dose Shufeng Xuanfei formula groups (SL,SM,SH groups),and low-dose,medium-dose and high-dose Jiebiao Qingli formula groups (JL,JM,JH groups).The model of pneumonia was reproduced by nasal dropping influenza virus A (FM1) in mice.N group was given isotonic saline 0.05 ml in nasal drops.After 2 hours of model-building,C group was received 11.375 mg· kg-1· d-1 osehamivir phosphate.Shufeng Xuanfei formula (mainly honeysuckle,forsythia and radix isatidis,etc.) with 3.76,1.88 and 0.94 g ·kg-1 ·d-1 were administrated to SH,SM and SL groups by gastric irrigation respectively.Jiebiao Qingli formula (mainly ephedra,gypsum,glycyrrhiza glabra,etc.) with 4.36,2.18 and 1.09 g ·kg-1 ·d-1 were administrated to JH,JM and JL groups by gastric irrigation respectively.In N and M groups,normal saline was administrated with gastric perfusion.Each group was in equal dose of 0.2 ml daily over a 4-day period.Total RNA in lung tissue of mice were extracted in each group,thengene chips were used to screen these RNA samples.Some genes involved NK cell mediated cytotoxicity were select,with "I" representing of signal intensity.These candidate genes were verified by real-time fluorescent quantitation polymerase chain reaction (PCR) and Western blotting.Results In the pathway of NK cell mediated cytotoxicity,M group up-regulated 43 genes expression,and 36,29,22,21,20 and 10 genes showed down-regulation in SM,JM,SL,JH,SH and JL groups,respectively.Apart from gene co-expression network in SH,SL,JH,JM and JL,SM also expressed other differential genes which SH,SL,JH,JM and JL did not.So medium-does Shufeng Xuanfei formula had the most significant regulation in gene expression of NK cell mediated cytotoxicity.By real-time PCR and Western blotting experiments showed that compared with the M group,mRNA and protein expression of tumor necrosis factor-α (TNF-α) in these two formula groups were significantly down-regulated,especially prominent in SM group and JM group (TNF-α mRNA:1.07 ±0.19,1.19 ±0.14 vs.3.20 ± 0.56,both P＜0.01).Conclusions Influenza viral replication in host cell,which means influenza antigens exposure in infected cells as target cells.NK cells recognize and exert cell mediated cytotoxic function against influenza antigens.Genes associated with NK cell mediated cytotoxicity in influenza infection were up-regulated.Shufeng Xuanfei and Jiebiao Qingli formulas could down-regulate these genes.The mechanism of down-regulated genes is that the number of influenza infected cells and NK cells activation decreases in treatment with two formulas.
