中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2012年
6期
464-467
,共4页
孙丽娟%李宏云%付群%秦超%冯永海
孫麗娟%李宏雲%付群%秦超%馮永海
손려연%리굉운%부군%진초%풍영해
哮喘%微RNAs%白细胞介素10%气道炎症
哮喘%微RNAs%白細胞介素10%氣道炎癥
효천%미RNAs%백세포개소10%기도염증
Asthma%MicroRNAs%Interleukin-10%Airway inflammation
目的 观察let-7家族微RNA抑制剂(anti-let-7)对支气管哮喘(简称哮喘)小鼠气道炎性反应的影响,并探讨let-7参与哮喘形成的机制.方法 32只小鼠按随机数字法分为4组(n=8),即正常对照组(A组)、哮喘组(B组)、干预对照组(C组)和干预组(D组).其中B、C和D组用鸡卵蛋白(OVA)免疫,建立哮喘模型,A组以生理盐水替代OVA处理.D组小鼠激发前注射anti-let-7以抑制内源性let-7表达,C组小鼠注射乱序siRNA对照.比较各组小鼠支气管肺泡灌洗液(BALF)的细胞计数,肺组织中let-7e以及BALF中白细胞介素-10(IL-10)含量；体外用anti-let-7转染肺癌细胞A549,并检测细胞中let-7e表达和细胞培养上清中IL-10的含量；荧光素酶报告法检测let-7e是否直接靶向IL-10.结果 与A组相比,B组和C组小鼠BALF中细胞总数和嗜酸粒细胞数显著增加[(20.32±5.33)×109/L和(24.74±6.69)×109/L比(7.12± 1.88)×109/L,(6.45±2.5)×109/L和(7.12±2.66)×109/L比(0.04±0.01)×109/L,均P＜0.01]；肺组织中let-7e水平明显升高(分别为3.83倍和3.27倍,均P＜0.01).与C组比较,D组BALF中细胞总数和嗜酸粒细胞数明显减少[(13.85±3.74)× 109/L比(24.74±6.69)×109/L,(2.15±1.13)×109/L比(7.12±2.66)×109/L,均P＜0.05]；肺组织中let-7e显著降低[(0.45±0.22)比(3.28±0.45),P＜0.01],同时BALF中IL-10水平明显升高[(4.68±0.85)比(1.70±0.29),P＜0.01].此外,在肺癌细胞A549 中转染anti-let-7,let-7e表达显著下降[(0.22±0.03)比(1.00±0.11),P＜0.01],同时培养上清中IL-10明显上升[(2.58±0.35)比(1.00±0.15),P＜0.01].体外let-7e过表达显著降低IL-10报告载体的荧光素酶活性[(0.59±0.06)比(1.00±0.03),P＜0.01],而对突变的IL-10报告载体没有抑制作用.结论 anti-let-7对哮喘小鼠气道炎性反应具有明显的抑制作用,其作用机制可能与let-7直接靶向并抑制IL-10有关.
目的 觀察let-7傢族微RNA抑製劑(anti-let-7)對支氣管哮喘(簡稱哮喘)小鼠氣道炎性反應的影響,併探討let-7參與哮喘形成的機製.方法 32隻小鼠按隨機數字法分為4組(n=8),即正常對照組(A組)、哮喘組(B組)、榦預對照組(C組)和榦預組(D組).其中B、C和D組用鷄卵蛋白(OVA)免疫,建立哮喘模型,A組以生理鹽水替代OVA處理.D組小鼠激髮前註射anti-let-7以抑製內源性let-7錶達,C組小鼠註射亂序siRNA對照.比較各組小鼠支氣管肺泡灌洗液(BALF)的細胞計數,肺組織中let-7e以及BALF中白細胞介素-10(IL-10)含量；體外用anti-let-7轉染肺癌細胞A549,併檢測細胞中let-7e錶達和細胞培養上清中IL-10的含量；熒光素酶報告法檢測let-7e是否直接靶嚮IL-10.結果 與A組相比,B組和C組小鼠BALF中細胞總數和嗜痠粒細胞數顯著增加[(20.32±5.33)×109/L和(24.74±6.69)×109/L比(7.12± 1.88)×109/L,(6.45±2.5)×109/L和(7.12±2.66)×109/L比(0.04±0.01)×109/L,均P＜0.01]；肺組織中let-7e水平明顯升高(分彆為3.83倍和3.27倍,均P＜0.01).與C組比較,D組BALF中細胞總數和嗜痠粒細胞數明顯減少[(13.85±3.74)× 109/L比(24.74±6.69)×109/L,(2.15±1.13)×109/L比(7.12±2.66)×109/L,均P＜0.05]；肺組織中let-7e顯著降低[(0.45±0.22)比(3.28±0.45),P＜0.01],同時BALF中IL-10水平明顯升高[(4.68±0.85)比(1.70±0.29),P＜0.01].此外,在肺癌細胞A549 中轉染anti-let-7,let-7e錶達顯著下降[(0.22±0.03)比(1.00±0.11),P＜0.01],同時培養上清中IL-10明顯上升[(2.58±0.35)比(1.00±0.15),P＜0.01].體外let-7e過錶達顯著降低IL-10報告載體的熒光素酶活性[(0.59±0.06)比(1.00±0.03),P＜0.01],而對突變的IL-10報告載體沒有抑製作用.結論 anti-let-7對哮喘小鼠氣道炎性反應具有明顯的抑製作用,其作用機製可能與let-7直接靶嚮併抑製IL-10有關.
목적 관찰let-7가족미RNA억제제(anti-let-7)대지기관효천(간칭효천)소서기도염성반응적영향,병탐토let-7삼여효천형성적궤제.방법 32지소서안수궤수자법분위4조(n=8),즉정상대조조(A조)、효천조(B조)、간예대조조(C조)화간예조(D조).기중B、C화D조용계란단백(OVA)면역,건립효천모형,A조이생리염수체대OVA처리.D조소서격발전주사anti-let-7이억제내원성let-7표체,C조소서주사란서siRNA대조.비교각조소서지기관폐포관세액(BALF)적세포계수,폐조직중let-7e이급BALF중백세포개소-10(IL-10)함량；체외용anti-let-7전염폐암세포A549,병검측세포중let-7e표체화세포배양상청중IL-10적함량；형광소매보고법검측let-7e시부직접파향IL-10.결과 여A조상비,B조화C조소서BALF중세포총수화기산립세포수현저증가[(20.32±5.33)×109/L화(24.74±6.69)×109/L비(7.12± 1.88)×109/L,(6.45±2.5)×109/L화(7.12±2.66)×109/L비(0.04±0.01)×109/L,균P＜0.01]；폐조직중let-7e수평명현승고(분별위3.83배화3.27배,균P＜0.01).여C조비교,D조BALF중세포총수화기산립세포수명현감소[(13.85±3.74)× 109/L비(24.74±6.69)×109/L,(2.15±1.13)×109/L비(7.12±2.66)×109/L,균P＜0.05]；폐조직중let-7e현저강저[(0.45±0.22)비(3.28±0.45),P＜0.01],동시BALF중IL-10수평명현승고[(4.68±0.85)비(1.70±0.29),P＜0.01].차외,재폐암세포A549 중전염anti-let-7,let-7e표체현저하강[(0.22±0.03)비(1.00±0.11),P＜0.01],동시배양상청중IL-10명현상승[(2.58±0.35)비(1.00±0.15),P＜0.01].체외let-7e과표체현저강저IL-10보고재체적형광소매활성[(0.59±0.06)비(1.00±0.03),P＜0.01],이대돌변적IL-10보고재체몰유억제작용.결론 anti-let-7대효천소서기도염성반응구유명현적억제작용,기작용궤제가능여let-7직접파향병억제IL-10유관.
Objective To explore the effects of let-7 inhibitor(anti-let-7) on airway inflammation in rats with asthma,and to investigate the mechanisms of let-7 in the development of asthma.Methods A total of 32 mice were randomly assigned to control group (group A,n=8),asthma group (group B,n=8),treatment control group(group C,n=8) and treatment group (group D,n=8),respectively.Groups B,C and D were sensitized with ovalbumin(OVA) for establishment of mouse asthma model.Group A was treated with normal saline orally.Group D was administered with anti-let-7 prior to challenge,for suppression of endogenous let-7e expression,while scrambled-siRNA was injecled in group C for treatment control.The cell count,let-7e in pulmonary tissues and interleukin-10 (11-10) in bronchoalveolar lavage fluid (BALF) were detected.A549 cells,the lung cancer cells,were transfected with anti-let-7 in vitro followed by measurement of let-7e expression and IL-IO in cell culture supernatants.Luciferase report assay was employed to assess if let-7e targeted at IL-10.Results Compared with group A,groups B and C significantly increased in the total cell count [(20.32±5.33) x 109/L and (24.74±6.69) x 109/L vs (7.12± 1.88) x 109/L,both P＜O.01] and eosinophil count [(6.45±2.5) x109/L and (7.12±2.66) x109/L vs (0.04±0.01) x109/L.both P＜O.01] in BALF as well as let-7e of Iung tissues (by 3.83-fold and 3.27-fold,both P＜O.01).When compared with group C,group D significantly reduced in total cell count [(13.85±3.74) x109/L vs (24.74±6.69) x109/L,P＜0.05] and eosinophil count[(2.15±1.13) x109/L vs (7.12±2.66) x109/L,P＜0.05] in BALF,and in let-7e of lung; tissues [(0.45±0.22) vs (3.28±0.45),P＜0.05],while markedly increased the level of IL-IO in BALF [(4.68±0.85) vs (1.70±0.29),P＜O.O1].Additionally,transfection of A549 cells using anti-let-7 resulted in reduction of let-7e expression [(0.22±0.03) vs (1.00±0.11),P＜O.O1] and elevation of 11-10 in the supernatant [(2.58±0.35) vs (1.00± 0.15),P＜O.01].Moreover.over-expression of let-7e led to significantly suppressed luciferase activity of IL-10 reporter vector [(0.59±0.06) vs (1.00±0.03),P＜O.01],but did not affect the mutant IL-10 reporter vector.Conclusion The anti-let-7 can markedly inhibit airway inflammation in mice with asthma,possibly by targeting at let-7 and inhibition of IL-10.
