本文關(guān)鍵詞： siHybrids技術(shù) RNAi 銅綠假單胞菌 外排泵 real-timePCR MIC　出處：《華中科技大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:研究siHybrids技術(shù)對(duì)銅綠假單胞菌PAO1外排泵mexB基因體外沉默效應(yīng)。 方法:1.針對(duì)銅綠假單胞菌野生株P(guān)AO1外排泵mexB基因設(shè)計(jì)并合成3條特異性siHybrids分子和一條陰性對(duì)照siHybrids分子。 2.在分子濃度為50nM下,分別以合成設(shè)計(jì)的siHybrids分子干擾銅綠假單胞菌PAO1,并設(shè)實(shí)驗(yàn)組為銅綠假單胞菌PAO1空白對(duì)照組,陰性對(duì)照組scambl(esc)-001,干預(yù)組siHybrids(si)-001,siHybrids(si)-002及siHybrids(si)-003。 3.分別在干預(yù)12小時(shí)后及24小時(shí)后采用實(shí)時(shí)熒光定量PCR檢測(cè)各實(shí)驗(yàn)組中外排泵靶基因mexB[1]基因mRNA表達(dá)水平。 4.進(jìn)一步采用Mueller-Hinton倍比稀釋法檢測(cè)50nM濃度的siHybrids分子對(duì)銅綠假單胞菌PAO1與氯霉素(CP),紅霉素(EM),左氧氟沙星(L-OFLX)干預(yù)作用前后的MIC(最小抑菌濃度). 結(jié)果:1.不同siHybrids分子干預(yù)PAO1 12小時(shí)后,mexB基因mRNA的表達(dá)量無明顯差異性。 2.干預(yù)24小時(shí)后,mexB基因mRNA的表達(dá)量干預(yù)組(si-001,si-002,si-003)比空白對(duì)照組,陰性對(duì)照組(sc-001)有明顯下降。 3.對(duì)比干預(yù)12小時(shí),24小時(shí)后mexB基因mRNA表達(dá)量,可以發(fā)現(xiàn)空白對(duì)照組,陰性對(duì)照組(sc-001)mRNA的表達(dá)量成上升趨勢(shì),而干預(yù)組(si-001,si-002,si-003)mexB基因mRNA表達(dá)量均呈下降趨勢(shì)。 4.siHybrids分子在干預(yù)24小時(shí)前后氯霉素(CP),紅霉素(EM),左氧氟沙星(L-OFLX)的MIC無明顯差異性 結(jié)論:在mRNA的表達(dá)水平上, siHybrids分子能體外干預(yù)銅綠假單胞菌PAO1 mexB基因mRNA的表達(dá),此種沉默作用呈現(xiàn)時(shí)間依賴性,且在24小時(shí)能有效地發(fā)揮干預(yù)作用。
[Abstract]:Aim: to study the silencing effect of siHybrids on mexB gene of Pseudomonas aeruginosa PAO1 efflux pump in vitro. Methods 1. Three specific siHybrids molecules and one negative control siHybrids molecule were designed and synthesized from the mexB gene of the wild strain of Pseudomonas aeruginosa PAO1 efflux pump. 2. At the molecular concentration of 50 nm, the designed siHybrids molecule was used to interfere with Pseudomonas aeruginosa PAO1, and the experimental group was divided into two groups: PAO1 blank control group, negative control group scamblesc-001, intervention group siHybridssi-001 and siHybridges sisi-002 and siHybridssisi-003. 3. After 12 hours and 24 hours of intervention, real-time quantitative PCR was used to detect the expression of mexB [1] gene in each experimental group. 4. Mueller-Hinton dilution method was used to detect the minimal inhibitory concentration (MEC) of 50nM siHybrids on Pseudomonas aeruginosa PAO1 and chloramphenicol, erythromycin Erythromycin and levofloxacin L-OFLX before and after the intervention. Results 1. There was no significant difference in the expression of mRNA of mexB gene in PAO1 treated with different siHybrids molecules for 12 hours. 2.After 24 hours of intervention, the mRNA expression of mexB gene in the intervention group was significantly lower than that in the blank control group and the negative control group. 3. Comparing the mRNA expression of mexB gene after 12 hours and 24 hours of intervention, it was found that in blank control group, the expression of sc-001 mRNA in negative control group showed an upward trend, while in intervention group, the mRNA expression of si-001si-002si-003mexB gene showed a downward trend. 4. The MIC of hybrids molecules before and after 24 hours of intervention were not significantly different from those of chloramphenicol, erythromycin and levofloxacin L-OFLX. Conclusion: at the level of mRNA expression, siHybrids molecule can interfere with the expression of PAO1 mexB gene mRNA of Pseudomonas aeruginosa in vitro, and the silencing effect is time dependent, and it can play an effective role in 24 hours.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R378

【參考文獻(xiàn)】

相關(guān)期刊論文 前8條

1 李學(xué)如,孟濤,王艷;銅綠假單胞菌耐藥機(jī)理研究進(jìn)展[J];國外醫(yī)藥(抗生素分冊(cè));2004年03期

2 ;Dose-dependent Inhibition of Gynecophoral Canal Protein Gene Expression in Vitro in the Schistosome(Schistosomajaponicum)by RNA Interference[J];Acta Biochimica et Biophysica Sinica;2005年06期

3 李耘,李家泰,王進(jìn);中國重癥監(jiān)護(hù)病房細(xì)菌耐藥性監(jiān)測(cè)研究[J];中華檢驗(yàn)醫(yī)學(xué)雜志;2004年11期

4 李家泰,李耘,齊慧敏;2002—2003年中國革蘭陰性細(xì)菌耐藥性監(jiān)測(cè)研究[J];中華檢驗(yàn)醫(yī)學(xué)雜志;2005年01期

5 李家泰,李耘,王進(jìn),中國細(xì)菌耐藥監(jiān)測(cè)研究組;我國醫(yī)院和社區(qū)獲得性感染革蘭陰性桿菌耐藥性監(jiān)測(cè)研究[J];中華醫(yī)學(xué)雜志;2003年12期

6 吳春明;李洪濤;覃慧敏;李國軍;宋建新;;建立SYBR Green實(shí)時(shí)RT-PCR定量方法檢測(cè)銅綠假單胞菌MexAB-OprM mRNA水平[J];中華醫(yī)院感染學(xué)雜志;2007年11期

7 邵圣文;張志智;顧紅光;王文琴;;RNA干擾銅綠假單胞菌群體感應(yīng)系統(tǒng)對(duì)生物膜影響的研究[J];中國抗生素雜志;2009年03期

8 嚴(yán)燕國;詹文華;趙剛;馬晉平;蔡世榮;;SiRNA特異性抑制幽門螺桿菌CagA基因表達(dá)的實(shí)驗(yàn)研究[J];中國人獸共患病學(xué)報(bào);2006年04期

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