本文關(guān)鍵詞：惡性瘧原蟲(chóng)二氫乳清酸脫氫酶抑制劑體外誘導(dǎo)惡性瘧原蟲(chóng)耐藥的實(shí)驗(yàn)研究　出處：《中國(guó)寄生蟲(chóng)學(xué)與寄生蟲(chóng)病雜志》2017年04期 　論文類型：期刊論文

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【摘要】：目的分析惡性瘧原蟲(chóng)二氫乳清酸脫氫酶(Plasmodium falciparum dihydroorotate dehydrogenase,PfDHODH)抑制劑(二氫噻吩酮類化合物,編號(hào)50,以下簡(jiǎn)稱PfDHODH抑制劑50)對(duì)體外培養(yǎng)惡性瘧原蟲(chóng)的作用特點(diǎn)及其誘導(dǎo)耐藥的可能機(jī)制。方法惡性瘧原蟲(chóng)氯喹敏感株(3D7株)和氯喹抗性株(Dd2株)同步化培養(yǎng)后分為不加藥對(duì)照組、環(huán)狀體期加藥組和大滋養(yǎng)體期加藥組,藥物終濃度為80 nmol/L。分別在同步化后0 h(環(huán)狀體期)、24 h(大滋養(yǎng)體期)、42 h涂薄血膜片鏡檢;通過(guò)逐步加大藥物濃度的方法,體外誘導(dǎo)產(chǎn)生耐藥蟲(chóng)株,3個(gè)月后經(jīng)有限稀釋培養(yǎng),獲得單克隆耐藥蟲(chóng)株。采用SYBR GreenⅠ染料法檢測(cè)各耐藥蟲(chóng)株對(duì)PfDHODH抑制劑50、氯喹和青蒿素的半數(shù)抑制濃度(IC_(50));PCR擴(kuò)增各耐藥蟲(chóng)株P(guān)fdhodh基因并測(cè)序,分析其突變情況。結(jié)果與不加藥對(duì)照組相比,環(huán)狀體期加藥組惡性瘧原蟲(chóng)從滋養(yǎng)體到裂殖體的發(fā)育受到明顯抑制,大滋養(yǎng)體期加藥組惡性瘧原蟲(chóng)呈現(xiàn)明顯的空泡化,核質(zhì)密度大大降低。通過(guò)體外誘導(dǎo)并經(jīng)有限稀釋培養(yǎng),獲得44株P(guān)fDHODH抑制劑50的單克隆耐藥蟲(chóng)株,其中,母本為Dd2、3D7的耐藥蟲(chóng)株分別為24和20株,它們對(duì)PfDHODH抑制劑50的IC_(50)分別為(2.284±0.096)和(0.678±0.018)μmol/L,較母本蟲(chóng)株的(0.018±0.002)和(0.015±0.002)μmol/L分別提高了近130倍和50倍;對(duì)氯喹和青蒿素的IC_(50)分別為(0.011±0.002)、(0.014±0.004)和(0.013±0.003)、(0.012±0.001)μmol/L;與母本Dd2蟲(chóng)株相比,Dd2耐藥蟲(chóng)株對(duì)氯喹的IC_(50)從(0.072±0.002)μmol/L下降為(0.011±0.002)μmol/L。測(cè)序分析結(jié)果顯示,23株Dd2來(lái)源的耐藥蟲(chóng)株P(guān)fDHODH蛋白氨基酸序列發(fā)生了G181D的點(diǎn)突變,另有1株除G181D的點(diǎn)突變外,還產(chǎn)生了K32N的點(diǎn)突變;3D7來(lái)源的耐藥蟲(chóng)株未發(fā)現(xiàn)相應(yīng)突變。結(jié)論體外誘導(dǎo)獲得PfDHODH抑制劑50的單克隆耐藥蟲(chóng)株,G181D的點(diǎn)突變可能是導(dǎo)致惡性瘧原蟲(chóng)高水平耐受PfDHODH抑制劑50的重要分子機(jī)制。
[Abstract]:Objective to analyze Plasmodium falciparum dihydroorotate dehydrogenase of Plasmodium falciparum dihydroorotate. PfDHODH) inhibitor (dihydrothiophenone compounds, No. 50). The effect of PfDHODH inhibitor 50 on in vitro culture of Plasmodium falciparum and its possible mechanism of inducing drug resistance. Methods Plasmodium falciparum chloroquine sensitive strain 3D7 and chloroquine resistant strain (chloroquine resistant strain). Dd2 strain was divided into control group after synchronous culture. The final concentration of the drug was 80 nmol / L in the cyclomeric and trophoblastic groups, respectively, at 0 h (24 h) after synchronization (macrotrophoblastic phase). 42 h thin blood membrane examination; By increasing drug concentration step by step, drug-resistant strains were induced in vitro and cultured in limited dilution after 3 months. SYBR Green 鈪，

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