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目的分析恶性疟原虫二氢乳清酸脱氢酶(Plasmodium falciparum dihydroorotate dehydrogenase,PfDHODH)抑制剂(二氢噻吩酮类化合物,编号50,以下简称PfDHODH抑制剂50)对体外培养恶性疟原虫的作用特点及其诱导耐药的可能机制。方法恶性疟原虫氯喹敏感株(3D7株)和氯喹抗性株(Dd2株)同步化培养后分为不加药对照组、环状体期加药组和大滋养体期加药组,药物终浓度为80 nmol/L。分别在同步化后0 h(环状体期)、24 h(大滋养体期)、42 h涂薄血膜片镜检;通过逐步加大药物浓度的方法,体外诱导产生耐药虫株,3个月后经有限稀释培养,获得单克隆耐药虫株。采用SYBR GreenⅠ染料法检测各耐药虫株对PfDHODH抑制剂50、氯喹和青蒿素的半数抑制浓度(IC_(50));PCR扩增各耐药虫株Pfdhodh基因并测序,分析其突变情况。结果与不加药对照组相比,环状体期加药组恶性疟原虫从滋养体到裂殖体的发育受到明显抑制,大滋养体期加药组恶性疟原虫呈现明显的空泡化,核质密度大大降低。通过体外诱导并经有限稀释培养,获得44株PfDHODH抑制剂50的单克隆耐药虫株,其中,母本为Dd2、3D7的耐药虫株分别为24和20株,它们对PfDHODH抑制剂50的IC_(50)分别为(2.284±0.096)和(0.678±0.018)μmol/L,较母本虫株的(0.018±0.002)和(0.015±0.002)μmol/L分别提高了近130倍和50倍;对氯喹和青蒿素的IC_(50)分别为(0.011±0.002)、(0.014±0.004)和(0.013±0.003)、(0.012±0.001)μmol/L;与母本Dd2虫株相比,Dd2耐药虫株对氯喹的IC_(50)从(0.072±0.002)μmol/L下降为(0.011±0.002)μmol/L。测序分析结果显示,23株Dd2来源的耐药虫株PfDHODH蛋白氨基酸序列发生了G181D的点突变,另有1株除G181D的点突变外,还产生了K32N的点突变;3D7来源的耐药虫株未发现相应突变。结论体外诱导获得PfDHODH抑制剂50的单克隆耐药虫株,G181D的点突变可能是导致恶性疟原虫高水平耐受PfDHODH抑制剂50的重要分子机制。
Objective To analyze the effect of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitor (dihydrothiophenone compound No. 50, hereinafter referred to as PfDHODH inhibitor 50) on Plasmodium falciparum in vitro And its possible mechanism of inducing resistance. Methods The synchronized challenge of Plasmodium falciparum chloroquine sensitive strains (3D7 strain) and chloroquine resistant strains (Dd2 strain) were divided into four groups: drug-free control group, The concentration of 80 nmol / L. They were microscopically examined at 0 h (annular phase), 24 h (maximal nocturnal phase), and 42 h after synchrotronization. The drug-resistant strains were induced in vitro by gradually increasing the drug concentration. Three months after limited dilution culture, access to monoclonal resistant strains of insects. The half inhibitory concentration (P50) of PfDHODH inhibitor 50, chloroquine and artemisinin was detected by SYBR GreenⅠ dye method. The Pfdhodh gene was amplified by PCR and sequenced, and its mutation status was analyzed . Results Compared with the control group, the development of Plasmodium falciparum from trophozoites to schizonts was significantly inhibited in the ring-dosed period. Plasmodium falciparum was significantly vacuolized in the dauer-feeding period, Nuclear density is greatly reduced. By in vitro induction and limited dilution culture, 44 strains of PfDHODH inhibitor 50 monoclonal resistant strains were obtained, of which 24 and 20 strains were resistant to Dd2 and 3D7, respectively, and their inhibitory effect on PfDHODH inhibitor 50 IC50 of 2.284 ± 0.096 and 0.678 ± 0.018 μmol / L were (0.18 ± 0.002) and (0.015 ± 0.002) μmol / L, respectively, which were nearly 130 times and 50 The IC 50 of chloroquine and artemisinin were (0.011 ± 0.002), (0.014 ± 0.004) and (0.013 ± 0.003) and (0.012 ± 0.001) μmol / L, respectively. The IC 50 of chloroquine decreased from (0.072 ± 0.002) μmol / L to (0.011 ± 0.002) μmol / L for Dd2 resistant strains. Sequencing analysis showed that the point mutation of G181D was found in the amino acid sequence of PfDHODH in 23 Dd2-derived drug-resistant strains, and another point mutation of K32N was found in addition to the point mutation of G181D. The resistant strain of 3D7 The strain did not find the corresponding mutation. CONCLUSION: The point mutation of G181D induced by PfDHODH inhibitor 50 in vitro can be an important molecular mechanism of high level of PfDHODH inhibitor 50 in Plasmodium falciparum.