中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2011年
8期
707-712
,共6页
毛艳%陈佳%许东%邢铭友%王丽丽%谢旭华%龚凤云%夏超%申爱霞%宋莹
毛豔%陳佳%許東%邢銘友%王麗麗%謝旭華%龔鳳雲%夏超%申愛霞%宋瑩
모염%진가%허동%형명우%왕려려%사욱화%공봉운%하초%신애하%송형
siHybrids技术%RNAi%铜绿假单胞菌%外排泵%Real-time PCR
siHybrids技術%RNAi%銅綠假單胞菌%外排泵%Real-time PCR
siHybrids기술%RNAi%동록가단포균%외배빙%Real-time PCR
siHybrids technique%RNA interference%Pseudomonas aeruginosa%Effiux pump%Real-time PCR
目的 初步研究siHybrids技术对铜绿假单胞菌野生菌PAO1外排泵mexB基因体外沉默效应。方法 针对铜绿假单胞菌野生株PAO1外排泵mexB基因设计并合成3条特异性siHybrids分子和1条阴性对照siHybrids分子。在分子浓度为50 nmol/L下，分别以合成的siHybrids分子干扰铜绿假单胞菌野生菌PAO1，并设实验组为铜绿假单胞菌野生菌PAO1空白对照组，阴性对照组scamble(sc)-001，干预组siHybrids( si) -001、siHybrids( si) -002及siHybrids( si) -003，分别在干预12h及24h后采用real-time PCR法检测各实验组中靶基因mexB基因mRNA的表达水平。进一步采用Mueller-Hinton倍比稀释法检测50 nmol/L浓度下，siHy brids分子干预铜绿假单胞菌野生菌PAO1前后氯霉素(CP)、红霉素(EM)、左氧氟沙星(L-OrLX)、头孢他啶(CAZ)、美洛培南(MER)的最小抑菌浓度(MIC)值。结果 不同siHybrids分子干预PAO1 12 h后，mexB基因mRNA表达量无明显差异性；但干预24 h后，mexB基因mRNA表达量：干预组（si-001，si-002，si-003）比空白对照组、阴性对照组（sc-001）有明显下降。对比干预12h、24h后mexB基因mRNA表达量，可以发现空白对照组、阴性对照组（sc-001）mRNA的表达量成上升趋势，而干预组（si-001，si-002，si-003）mexB基因mRNA表达量均呈下降趋势。siHybrids分子在干预铜绿假单胞菌野生菌24h前后的氯霉素(CP)、红霉素(EM)、左氧氟沙星( L-OFLX)、头孢他啶(CAZ)、美洛培南(MER) MIC无明显差异性。结论 在mRNA表达水平上，siHybrids分子能体外干预铜绿假单胞菌PAO1 mexB基因mRNA表达，此种沉默作用呈现时间依赖性，且在24h能有效地发挥干预作用。
目的 初步研究siHybrids技術對銅綠假單胞菌野生菌PAO1外排泵mexB基因體外沉默效應。方法 針對銅綠假單胞菌野生株PAO1外排泵mexB基因設計併閤成3條特異性siHybrids分子和1條陰性對照siHybrids分子。在分子濃度為50 nmol/L下，分彆以閤成的siHybrids分子榦擾銅綠假單胞菌野生菌PAO1，併設實驗組為銅綠假單胞菌野生菌PAO1空白對照組，陰性對照組scamble(sc)-001，榦預組siHybrids( si) -001、siHybrids( si) -002及siHybrids( si) -003，分彆在榦預12h及24h後採用real-time PCR法檢測各實驗組中靶基因mexB基因mRNA的錶達水平。進一步採用Mueller-Hinton倍比稀釋法檢測50 nmol/L濃度下，siHy brids分子榦預銅綠假單胞菌野生菌PAO1前後氯黴素(CP)、紅黴素(EM)、左氧氟沙星(L-OrLX)、頭孢他啶(CAZ)、美洛培南(MER)的最小抑菌濃度(MIC)值。結果 不同siHybrids分子榦預PAO1 12 h後，mexB基因mRNA錶達量無明顯差異性；但榦預24 h後，mexB基因mRNA錶達量：榦預組（si-001，si-002，si-003）比空白對照組、陰性對照組（sc-001）有明顯下降。對比榦預12h、24h後mexB基因mRNA錶達量，可以髮現空白對照組、陰性對照組（sc-001）mRNA的錶達量成上升趨勢，而榦預組（si-001，si-002，si-003）mexB基因mRNA錶達量均呈下降趨勢。siHybrids分子在榦預銅綠假單胞菌野生菌24h前後的氯黴素(CP)、紅黴素(EM)、左氧氟沙星( L-OFLX)、頭孢他啶(CAZ)、美洛培南(MER) MIC無明顯差異性。結論 在mRNA錶達水平上，siHybrids分子能體外榦預銅綠假單胞菌PAO1 mexB基因mRNA錶達，此種沉默作用呈現時間依賴性，且在24h能有效地髮揮榦預作用。
목적 초보연구siHybrids기술대동록가단포균야생균PAO1외배빙mexB기인체외침묵효응。방법 침대동록가단포균야생주PAO1외배빙mexB기인설계병합성3조특이성siHybrids분자화1조음성대조siHybrids분자。재분자농도위50 nmol/L하，분별이합성적siHybrids분자간우동록가단포균야생균PAO1，병설실험조위동록가단포균야생균PAO1공백대조조，음성대조조scamble(sc)-001，간예조siHybrids( si) -001、siHybrids( si) -002급siHybrids( si) -003，분별재간예12h급24h후채용real-time PCR법검측각실험조중파기인mexB기인mRNA적표체수평。진일보채용Mueller-Hinton배비희석법검측50 nmol/L농도하，siHy brids분자간예동록가단포균야생균PAO1전후록매소(CP)、홍매소(EM)、좌양불사성(L-OrLX)、두포타정(CAZ)、미락배남(MER)적최소억균농도(MIC)치。결과 불동siHybrids분자간예PAO1 12 h후，mexB기인mRNA표체량무명현차이성；단간예24 h후，mexB기인mRNA표체량：간예조（si-001，si-002，si-003）비공백대조조、음성대조조（sc-001）유명현하강。대비간예12h、24h후mexB기인mRNA표체량，가이발현공백대조조、음성대조조（sc-001）mRNA적표체량성상승추세，이간예조（si-001，si-002，si-003）mexB기인mRNA표체량균정하강추세。siHybrids분자재간예동록가단포균야생균24h전후적록매소(CP)、홍매소(EM)、좌양불사성( L-OFLX)、두포타정(CAZ)、미락배남(MER) MIC무명현차이성。결론 재mRNA표체수평상，siHybrids분자능체외간예동록가단포균PAO1 mexB기인mRNA표체，차충침묵작용정현시간의뢰성，차재24h능유효지발휘간예작용。
Objective To investigate the effect and mechanism of siHybrids technique on inhibiring the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 in vitro. Methods Targeting the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 ,we designed and synthesized three siHybrids molecule and one negative scamble siHybrids molecule. Pseudomonas aeruginosa PAO1 were intervened by the siHybrids molecules in 50 nmol/L, respectively. And the experiments were made of control groups[blank and scamble (sc) -001]and intervened groups[siHybrids( si ) -001, siHybrids( si ) -002 and siHybrids(si) -003]of Pseudomonas aeruginosa PAO1. The targeting efflux pump gene mexB mRNA expressions of Pseudomonas aeruginosa PAO1, including all groups, were measured by real-time PCR in 12 h and 24 h after interference in vitro. Further, the minimal inhibitory concentration (MIC) of chlormycetinCP, erythrocin( EM ), levofloxacin ( L-OFLX), ceftazidime ( CAZ), meropenem (MER) to those groups were detected by using Mueller-Hinton broth dilution before and after interference. Results The relative mexB mRNA amounts of Pseudomonas aeruginosa PAO1 intervened by different siHybrids were not much more different from each other after 12 h,but the expression of mexB mRNA of the intervened group ( si-001 ,si-002 ,si-003 ) was much lower than control groups after interference for 24 h. The relative mexB mRNA amounts, comparing 12 h with 24 h, we would find the blank control and negative control submit escalating trend. And the intervened control ,three different siHybrids were all with a downward tendency. However, in presence of 50 nmol/L siHybrids, the minimal inhibitory concentration(MIC) of CP, EM, L-OFLX, CAZ, MER to those controls were not much more different before and after interference. Conclusion On level of the mRNA expressions, siHybrids could inhibit the efflux pump gene mexB of Pseudomonas aeruginosa PAO1 in vitro, and within 24 h would be able to function effectively. Further, the effect was time-dependent.
