中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2015年
42期
3464-3467
,共4页
白丽云%张雅娴%王占黎%王英%于慧
白麗雲%張雅嫻%王佔黎%王英%于慧
백려운%장아한%왕점려%왕영%우혜
布鲁杆菌,马尔他%基因外重复回文序列%酶联免疫吸附测定%Toll样受体9
佈魯桿菌,馬爾他%基因外重複迴文序列%酶聯免疫吸附測定%Toll樣受體9
포로간균,마이타%기인외중복회문서렬%매련면역흡부측정%Toll양수체9
Brucella melitensis%Repetitive extragenic palindromic sequences%Enzyme-linked immunosorbent assay%Toll-like receptor 9
目的 筛选具有活化Toll样受体9(TLR9)活性的羊种布氏杆菌DNA中基因外重复回文序列(REPs),检测其经TLR9诱导的干扰素-α(IFN-α)表达,为羊种布氏杆菌病的防治提供新思路.方法 针对羊种布氏杆菌Brucella melitensis NI基因组序列,利用生物信息学技术识别其REPs后,合成序列.将合成的天然骨架脱氧寡核苷酸(ODNs)转染小鼠单核巨噬细胞株RAW264.7,酶联免疫吸附测定法(ELISA)检测IFN-α的分泌水平.采用TLR9-siRNA沉默RAW264.7中的TLR9,将上述诱导IFN-α分泌增加的阳性ODN序列转染RAW264.7,ELISA方法检测IFN-α的分泌变化.结果 筛选获得2 200个羊种布氏杆菌基因组REPs,选择二级茎环结构较好的12条ODNs序列进行合成,ELISA结果显示,ODNs M2、M3、M4、M5、M6、M7、M9、M12介导IFN-α分泌量分别为(26.944±1.868)、(46.461±2.562)、(34.980±2.055)、(43.016±2.162)、(62.533±4.031)、(67.125±5.069)、(18.908±1.633)、(39.572±2.465)pg/ml,显著高于阴性对照组的(12.594±1.338)pg/ml,差异有统计学意义(t=10.817、20.295、15.812、20.724、20.365、18.016、5.180、16.660,均P＜0.05),且阳性ODN M6所介导的IFN-α分泌可以被TLR9-siRNA显著抑制.结论 羊种布氏杆菌基因组REPs可经TLR9信号通路引起炎性因子IFN-α分泌增加,参与介导羊种布氏杆菌早期感染后的固有免疫应答.
目的 篩選具有活化Toll樣受體9(TLR9)活性的羊種佈氏桿菌DNA中基因外重複迴文序列(REPs),檢測其經TLR9誘導的榦擾素-α(IFN-α)錶達,為羊種佈氏桿菌病的防治提供新思路.方法 針對羊種佈氏桿菌Brucella melitensis NI基因組序列,利用生物信息學技術識彆其REPs後,閤成序列.將閤成的天然骨架脫氧寡覈苷痠(ODNs)轉染小鼠單覈巨噬細胞株RAW264.7,酶聯免疫吸附測定法(ELISA)檢測IFN-α的分泌水平.採用TLR9-siRNA沉默RAW264.7中的TLR9,將上述誘導IFN-α分泌增加的暘性ODN序列轉染RAW264.7,ELISA方法檢測IFN-α的分泌變化.結果 篩選穫得2 200箇羊種佈氏桿菌基因組REPs,選擇二級莖環結構較好的12條ODNs序列進行閤成,ELISA結果顯示,ODNs M2、M3、M4、M5、M6、M7、M9、M12介導IFN-α分泌量分彆為(26.944±1.868)、(46.461±2.562)、(34.980±2.055)、(43.016±2.162)、(62.533±4.031)、(67.125±5.069)、(18.908±1.633)、(39.572±2.465)pg/ml,顯著高于陰性對照組的(12.594±1.338)pg/ml,差異有統計學意義(t=10.817、20.295、15.812、20.724、20.365、18.016、5.180、16.660,均P＜0.05),且暘性ODN M6所介導的IFN-α分泌可以被TLR9-siRNA顯著抑製.結論 羊種佈氏桿菌基因組REPs可經TLR9信號通路引起炎性因子IFN-α分泌增加,參與介導羊種佈氏桿菌早期感染後的固有免疫應答.
목적 사선구유활화Toll양수체9(TLR9)활성적양충포씨간균DNA중기인외중복회문서렬(REPs),검측기경TLR9유도적간우소-α(IFN-α)표체,위양충포씨간균병적방치제공신사로.방법 침대양충포씨간균Brucella melitensis NI기인조서렬,이용생물신식학기술식별기REPs후,합성서렬.장합성적천연골가탈양과핵감산(ODNs)전염소서단핵거서세포주RAW264.7,매련면역흡부측정법(ELISA)검측IFN-α적분비수평.채용TLR9-siRNA침묵RAW264.7중적TLR9,장상술유도IFN-α분비증가적양성ODN서렬전염RAW264.7,ELISA방법검측IFN-α적분비변화.결과 사선획득2 200개양충포씨간균기인조REPs,선택이급경배결구교호적12조ODNs서렬진행합성,ELISA결과현시,ODNs M2、M3、M4、M5、M6、M7、M9、M12개도IFN-α분비량분별위(26.944±1.868)、(46.461±2.562)、(34.980±2.055)、(43.016±2.162)、(62.533±4.031)、(67.125±5.069)、(18.908±1.633)、(39.572±2.465)pg/ml,현저고우음성대조조적(12.594±1.338)pg/ml,차이유통계학의의(t=10.817、20.295、15.812、20.724、20.365、18.016、5.180、16.660,균P＜0.05),차양성ODN M6소개도적IFN-α분비가이피TLR9-siRNA현저억제.결론 양충포씨간균기인조REPs가경TLR9신호통로인기염성인자IFN-α분비증가,삼여개도양충포씨간균조기감염후적고유면역응답.
Objective To screen the repetitive extragenic palindromic sequences with activation of toll-like receptor 9(TLR9) activity from Brucella melitensis DNA,providing new ideas and new targets for prevention and treatment of brucellosis.Methods Bioinformatics methods were used to detect repetitive extragenic palindromic(REP) sequences from Brucella melitensis DNA.The studied REPs were selected and synthesized.RAW264.7 was cultured and transfected with REPs mediated by lipofectamine 3000.Additionally,TLR9-siRNA was used to downregulate TLR9 expression.The content of interferon-α(IFN-α) in the supernatant was then measured by ELISA.Results A total of 2 200 REP sequences in Brucella melitensis DNA were identified.Twelve REP sequences were synthesized for further detecting of the TLR9 agonistic activity.IFN-α expression in RAW264.7 treated with M2,M3,M4,M5,M6,M7,M9,M12 were (26.944 ± 1.868),(46.461 ± 2.562),(34.980 ± 2.055),(43.016 ± 2.162),(62.533 ± 4.031),(67.125 ± 5.069),(18.908 ± 1.633),(39.572 ± 2.465) pg/ml respectively,which significantly increased when compared with the negative control group [(12.594 ± 1.338) pg/ml,t =10.817,20.295,15.812,20.724,20.365,18.016,5.180,16.660,all P ＜ 0.05].Additionally,TLR9-siRNA can significantly decrease the levels of IFN-α in RAW264.7 treated with M6.Conclusion REP sequences presented in Brucella melitensis DNA are able to induce IFN-α expression through TLR9,which can be helpful for the understanding of pathogenesis and immunity of Brucella melitensis.
