复方萘酚阿奇对延缓恶性疟原虫抗性的实验性研究

发布时间：2018-01-03 07:21

本文关键词：复方萘酚阿奇对延缓恶性疟原虫抗性的实验性研究　出处：《大理学院》2011年硕士论文　论文类型：学位论文

【摘要】：1.研究目的 1.1观察对照组(对照组单一用萘酚喹)与实验组(萘酚喹与阿奇霉素联合用药)恶性疟原虫在体外药物刺激(压力)下,疟原虫对其敏感性的变化情况或产生速度。 1.2用WHO推荐的Rieckmann体外测定不同阶段对照组与实验组恶性疟原虫对萘酚喹、阿奇霉素、复方萘酚阿奇的敏感性,计算半数抑制量(IC50)。 1.3阐述两药物联用体外抗疟作用的特点；评价两药物联合有或无延缓抗性的作用或潜力。为疟疾的联合用药的临床治疗方案提供科学依据。 2.研究方法 2.1虫株与培养：恶性疟原虫FccSM/YN株,该株采自位于中老、中缅边境地区云南思茅,按Trager等蜡烛缸培养法,通过实验室驯化,建立体外传代培养,实验室长期保种虫株。 2.2药物作用观察：用体外剂量递增间隔接触法进行抗性培育 2.3体外测定：测试采用Rieckmann体外微量测定法。将同步化处理过的疟原虫稀释到20,000-60,000个/μl血,加5倍培养基稀释,混匀后加入测定板各药井,从低浓度到高浓度每井加50μl,每个浓度同时测定两组(2行)。37℃培养20-24h后收获1个对照井,视其疟原虫发育情况决定收获时间。用ICEstimator software计算IC50。 2.4判别标准接触药物后的恶性疟原虫的IC50较亲代上升5倍以上,可认为对该药的敏感性下降。在相同的抗性培育时间内,接触萘酚喹/阿奇霉素的恶性疟原虫的IC50分别较亲代上升5倍,可认为对该药的敏感性下降；萘酚喹/阿奇霉素对恶性疟原虫的IC50分别与亲代相比,无明显上升或者虽然有所上升,但明显低于对照组,可认为该联合用药有延缓抗性作用。 3.结果 3.1药物接触的次数、浓度及结果对照组恶性疟原虫接触萘酚喹120天,共接触药物15次,药物浓度分别为2.56nmol/L5次,160nmol/L2次,5、10、20、40、80、320、625、1280nmol/L各1次。接触药物前,敏感株(亲代系)的IC50为2.11nmol/L,药物刺激15次后,其IC50增至65.47nmol/L。为亲代原虫的31倍。实验组恶性疟原虫接触萘酚喹/阿奇霉素共120天,共接触药物19次,药物浓度分别为0.31/0.256nmol/L 8次,2.49/2.135 nmol/L 5次,0.625/0.53、4.98/4.27、9.735/8.34、19.47/16.685、38.94/33.375、77.88/66.755nmol/L各一次。接触药物前,敏感株(亲代系)的IC50为0.18 nmol/L、0.15nmol/L,药物刺激19次后,其IC50增至2.89 nmol/L、2.68 nmol/L,分别为亲代原虫的16.1倍、17.9倍。 3.2用药前恶性疟原虫的敏感性测定结果在恶性疟原虫未接触药物前,在萘酚喹测定板、阿奇霉素测定板及萘酚喹/阿奇霉素复合测定板中测定疟原虫的敏感性,在单一萘酚喹测定板中测得IC50为2.11nmol/L;在单一的阿奇霉素测定板中测得IC50为3.21 nmol/L;在萘酚喹/阿奇霉素复合测定板中测得萘酚喹与阿奇霉素的IC50分别为0.18nmol/L、0.15 nmol/L。单一测定板中萘酚喹的IC50是复合测定板的11.7(2.11/0.18)倍；阿奇霉素的IC50是复合测定板的21.4(3.21/0.15)倍。 3.3对照组恶性疟原虫接触萘酚喹前后(药物刺激15次,历时120天)的敏感性测定结果对照组恶性疟原虫在单一接触萘酚喹后的IC50为单一接触萘酚喹前的31.0(65.47/2.11)倍。对照组疟原虫在单一接触萘酚喹后对阿奇霉素的敏感性是单一接触萘酚喹前的21.4(68.58/3.21)倍。 3.4实验组恶性疟原虫接触萘酚喹/阿奇霉素前后(药物刺激19次,历时120天)的敏感性测定结果实验组疟原虫在接触萘酚喹/阿奇霉素后对萘酚喹的IC50为接触药物前的16.8(35.47/2.11)倍。实验组疟原虫在接触萘酚喹/阿奇霉素后对阿奇霉素的IC50为接触药物前的15.6(50.04/3.21)倍。 3.5萘酚喹/阿奇霉素联用与萘酚喹单用对恶性疟原虫作用的比较对照组在接触药物120天后在单一萘酚喹测定板中的IC50为65.47nmol/L,在单一阿奇霉素测定板中测得IC5。为68.58nmol/L,在萘酚喹/阿奇霉素复合测定板中的IC50分别为10.34nmol/L/8.46nmol/L。实验组在接触药物120天后在单一萘酚喹测定板中的IC50为35.47nmol/L,在单一阿奇霉素测定板中测得IC50为50.04nmol/L,在萘酚喹/阿奇霉素复合测定板中的IC50分别为2.89nmol/L、2.68 nmol/L。 4.结论 4.1恶性疟原虫在单一接触萘酚喹后一段时间敏感性有明显的下降,其IC50远远高于亲代的5倍以上,进一步证实用人工培养的方法可以培育出恶性疟原虫抗萘酚喹耐药株。 4.2萘酚喹与阿奇霉素配伍对恶性疟原虫的作用观察显示,无论对敏感株(对照组)还是抗药株在萘酚喹/阿奇霉素复合测定板中测的IC50显著低于单一的萘酚喹测定板和阿奇霉素测定板,提示萘酚喹与阿奇霉素配伍对恶性疟原虫的作用明显大于单一用药,两种药物联用有明显增效作用。 4.3实验组恶性疟原虫对药物耐受性的增长远远慢于对照组,前者在药物刺激120天后萘酚喹、阿奇霉素、萘酚喹/阿奇霉素的IC50分别上升16.8倍、15.6倍、16.1/17.9倍,后者在药物刺激120天后萘酚喹、阿奇霉素、萘酚喹/阿奇霉素的IC50分别上升31.0倍、21.4倍、57.4/56.4倍,提示萘酚喹与阿奇霉素两种药物联用有明显延缓抗性作用。
[Abstract]:1. purpose of research
1.1 observe the control group (control group single use naphthol) and the experimental group (naphthol Quine and azithromycin combination) Plasmodium falciparum in vitro drug stimulation (pressure), the sensitivity of malaria parasite to change or speed.
The different stages of the experimental group and the control group of naphthoquine falciparum, azithromycin determination of in vitro Rieckmann 1.2 recommended by WHO, the sensitivity of compound, naphthol half inhibition, the calculation amount (IC50).
1.3, we elaborated the characteristics of the antimalarial effect of two drug combination in vitro, evaluated the role or potential of two drugs combination with or without delayed resistance, and provided scientific basis for the clinical treatment of malaria combined drugs.
2. research methods
2.1 worm and culture: Plasmodium falciparum FccSM/YN strain, which is located in the middle and old Burmese border area of Simao, Yunnan. According to Trager and other candle culture methods, it was constructed by three-dimensional domestication and laboratory culture.
2.2 drug effect observation: resistance cultivation by in vitro dose increasing interval contact method
2.3 in vitro test: determination by Rieckmann in vitro. The synchronization of the treated parasites diluted to 20000-60000 / L blood, with 5 times dilution medium plate, determination of each drug well mixed evenly, from low to high density per well and 50 mu L, simultaneous determination of two groups of each concentration (2).37 C after 20-24h culture harvest 1 control wells, the development of the parasite decided to harvest time. IC50. ICEstimator software calculation
The 2.4 criterion of contact drugs of Plasmodium falciparum IC50 compared with parental rise by more than 5 times, that decreased sensitivity to the drug. In the same time cultivating resistance, contact naphthoquine / azithromycin of Plasmodium falciparum IC50 respectively compared with the parental 5 fold increase in sensitivity to the drug that can decrease the naphthoquine /; IC50 of azithromycin on Plasmodium falciparum were compared with parental species, no significant increase or although increased, but significantly lower than the control group, can be considered that the combination effect of delaying resistance.
3. results
3.1 times of drug exposure, concentration and results in the control group of Plasmodium falciparum contact naphthoquine for 120 days, a total of 15 times of exposure to drugs, drug concentration was 2.56nmol/L5 times, 160nmol/L2 times, 5,10,20,40,803206251280nmol/L 1 times each. Before contact with the drug sensitive strains (parental strains), IC50 2.11nmol/L, drug stimulation after 15 times, the IC50 to 65.47nmol/L. was 31 fold than protozoa. The experimental group of Plasmodium falciparum contact naphthoquine / azithromycin for 120 days, a total of 19 times of exposure to drugs, drug concentrations were 0.31/0.256nmol/L 8, 2.49/2.135 nmol/L 5, 0.625/ 0.53,4.98/4.27,9.735/8.34,19.47/16.685,38.94/33.375,77.88/66.755nmol/L each time. Contact with the drug, sensitive strains (parental lines) IC50 0.18 nmol/L, 0.15nmol/L. Drug stimulation after 19 IC50 to 2.89 nmol/L, 2.68 nmol/L, respectively 16.1 times, 17.9 times. The parental protozoa
Before the drug sensitivity of 3.2 Plasmodium falciparum results in P. falciparum not contact with drugs, in naphthoquine determination, determination of azithromycin and naphthoquine / azithromycin composite plate determination Plasmodium susceptibility plate, determination plate IC50 2.11nmol/L in a single naphthoquine; in a single azithromycin determination plate IC50 3.21 nmol/L; in naphthoquine / azithromycin composite determination plate naphthoquine and azithromycin IC50 were 0.18nmol/L, 0.15 nmol/L. single board in the determination of naphthoquine IC50 composite plate is measured 11.7 times (2.11/0.18); IC50 is a composite board Determination of azithromycin (3.21/0.15) 21.4 times.
3.3 control group contact naphthoquine falciparum (before and after drug stimulation 15 times, which lasted 120 days) the sensitivity test results in the control group IC50 of Plasmodium falciparum in a single contact naphthoquine for single contact naphthoquine 31 times (65.47/2.11). The control group in a single contact naphthoquine Plasmodium falciparum is sensitive to azithromycin single contact naphthoquine 21.4 (68.58/3.21) times.
Before and after the 3.4 experimental group of Plasmodium falciparum contact naphthoquine / azithromycin (drug stimulation 19 times, which lasted 120 days) the sensitivity test results in the experimental group of parasites in contact naphthoquine / azithromycin on naphthoquine IC50 for drug contact before 16.8 times (35.47/2.11). The experimental group Plasmodium in contact naphthoquine / azithromycin on the IC50 for azithromycin contact with drugs before 15.6 (50.04/3.21) times.
3.5 naphthoquine / Azithromycin combined with naphthoquine falciparum the control group in contact with the drug for 120 days in a single naphthoquine determination in IC50 65.47nmol/L determination plate IC5. 68.58nmol/L in single azithromycin, in naphthoquine / azithromycin composite determination in IC50 10.34nmol/L/8.46nmol/L. experiment respectively. Group in contact with the drug for 120 days in a single naphthoquine determination in IC50 35.47nmol/L determination plate IC50 50.04nmol/L in single azithromycin, in naphthoquine / azithromycin composite determination in IC50 were 2.89nmol/L, 2.68 nmol/L.
4. conclusion
4.1, after a period of exposure to naphthol, the sensitivity of Plasmodium falciparum decreased significantly. The IC50 of the Plasmodium falciparum was much higher than that of the parents. It is further proved that the resistance to falciparum resistant to Plasmodium falciparum can be made by using the method of artificial culture. 5
4.2 effect of naphthoquine and azithromycin combination on Plasmodium falciparum observation showed that both the sensitive strains (control group) or drug resistant strains in naphthoquine / azithromycin composite determination plate IC50 was significantly lower than that of single naphthoquine determination and determination of azithromycin in board, suggesting the role of compatibility of naphthoquine and azithromycin was significantly higher than that of Plasmodium falciparum a single drug, two kinds of drugs have obvious synergistic effect.
Growth of 4.3 in the experimental group of Plasmodium falciparum in drug resistance is much slower than the control group, the former in 120 days after drug stimulation naphthoquine, azithromycin, naphthoquine / azithromycin IC50 increased by 16.8 times, 15.6 times, 16.1/17.9 times, the latter in 120 days after azithromycin drug stimulation, naphthoquine, naphthoquine / azithromycin IC50 respectively. 31 times, 21.4 times, 57.4/56.4 times, suggesting that naphthoquine two drugs combined with azithromycin significantly delay the resistance effect.

【学位授予单位】：大理学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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