本文關(guān)鍵詞：復(fù)方萘酚阿奇對延緩惡性瘧原蟲抗性的實(shí)驗(yàn)性研究　出處：《大理學(xué)院》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 惡性瘧原蟲 萘酚喹 阿奇霉素 抗藥性 體外培養(yǎng)

【摘要】：1.研究目的 1.1觀察對照組(對照組單一用萘酚喹)與實(shí)驗(yàn)組(萘酚喹與阿奇霉素聯(lián)合用藥)惡性瘧原蟲在體外藥物刺激(壓力)下,瘧原蟲對其敏感性的變化情況或產(chǎn)生速度。 1.2用WHO推薦的Rieckmann體外測定不同階段對照組與實(shí)驗(yàn)組惡性瘧原蟲對萘酚喹、阿奇霉素、復(fù)方萘酚阿奇的敏感性,計(jì)算半數(shù)抑制量(IC50)。 1.3闡述兩藥物聯(lián)用體外抗瘧作用的特點(diǎn)；評價(jià)兩藥物聯(lián)合有或無延緩抗性的作用或潛力。為瘧疾的聯(lián)合用藥的臨床治療方案提供科學(xué)依據(jù)。 2.研究方法 2.1蟲株與培養(yǎng)：惡性瘧原蟲FccSM/YN株,該株采自位于中老、中緬邊境地區(qū)云南思茅,按Trager等蠟燭缸培養(yǎng)法,通過實(shí)驗(yàn)室馴化,建立體外傳代培養(yǎng),實(shí)驗(yàn)室長期保種蟲株。 2.2藥物作用觀察：用體外劑量遞增間隔接觸法進(jìn)行抗性培育 2.3體外測定：測試采用Rieckmann體外微量測定法。將同步化處理過的瘧原蟲稀釋到20,000-60,000個/μl血,加5倍培養(yǎng)基稀釋,混勻后加入測定板各藥井,從低濃度到高濃度每井加50μl,每個濃度同時測定兩組(2行)。37℃培養(yǎng)20-24h后收獲1個對照井,視其瘧原蟲發(fā)育情況決定收獲時間。用ICEstimator software計(jì)算IC50。 2.4判別標(biāo)準(zhǔn)接觸藥物后的惡性瘧原蟲的IC50較親代上升5倍以上,可認(rèn)為對該藥的敏感性下降。在相同的抗性培育時間內(nèi),接觸萘酚喹/阿奇霉素的惡性瘧原蟲的IC50分別較親代上升5倍,可認(rèn)為對該藥的敏感性下降；萘酚喹/阿奇霉素對惡性瘧原蟲的IC50分別與親代相比,無明顯上升或者雖然有所上升,但明顯低于對照組,可認(rèn)為該聯(lián)合用藥有延緩抗性作用。 3.結(jié)果 3.1藥物接觸的次數(shù)、濃度及結(jié)果對照組惡性瘧原蟲接觸萘酚喹120天,共接觸藥物15次,藥物濃度分別為2.56nmol/L5次,160nmol/L2次,5、10、20、40、80、320、625、1280nmol/L各1次。接觸藥物前,敏感株(親代系)的IC50為2.11nmol/L,藥物刺激15次后,其IC50增至65.47nmol/L。為親代原蟲的31倍。實(shí)驗(yàn)組惡性瘧原蟲接觸萘酚喹/阿奇霉素共120天,共接觸藥物19次,藥物濃度分別為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。接觸藥物前,敏感株(親代系)的IC50為0.18 nmol/L、0.15nmol/L,藥物刺激19次后,其IC50增至2.89 nmol/L、2.68 nmol/L,分別為親代原蟲的16.1倍、17.9倍。 3.2用藥前惡性瘧原蟲的敏感性測定結(jié)果在惡性瘧原蟲未接觸藥物前,在萘酚喹測定板、阿奇霉素測定板及萘酚喹/阿奇霉素復(fù)合測定板中測定瘧原蟲的敏感性,在單一萘酚喹測定板中測得IC50為2.11nmol/L;在單一的阿奇霉素測定板中測得IC50為3.21 nmol/L;在萘酚喹/阿奇霉素復(fù)合測定板中測得萘酚喹與阿奇霉素的IC50分別為0.18nmol/L、0.15 nmol/L。單一測定板中萘酚喹的IC50是復(fù)合測定板的11.7(2.11/0.18)倍；阿奇霉素的IC50是復(fù)合測定板的21.4(3.21/0.15)倍。 3.3對照組惡性瘧原蟲接觸萘酚喹前后(藥物刺激15次,歷時120天)的敏感性測定結(jié)果對照組惡性瘧原蟲在單一接觸萘酚喹后的IC50為單一接觸萘酚喹前的31.0(65.47/2.11)倍。對照組瘧原蟲在單一接觸萘酚喹后對阿奇霉素的敏感性是單一接觸萘酚喹前的21.4(68.58/3.21)倍。 3.4實(shí)驗(yàn)組惡性瘧原蟲接觸萘酚喹/阿奇霉素前后(藥物刺激19次,歷時120天)的敏感性測定結(jié)果實(shí)驗(yàn)組瘧原蟲在接觸萘酚喹/阿奇霉素后對萘酚喹的IC50為接觸藥物前的16.8(35.47/2.11)倍。實(shí)驗(yàn)組瘧原蟲在接觸萘酚喹/阿奇霉素后對阿奇霉素的IC50為接觸藥物前的15.6(50.04/3.21)倍。 3.5萘酚喹/阿奇霉素聯(lián)用與萘酚喹單用對惡性瘧原蟲作用的比較對照組在接觸藥物120天后在單一萘酚喹測定板中的IC50為65.47nmol/L,在單一阿奇霉素測定板中測得IC5。為68.58nmol/L,在萘酚喹/阿奇霉素復(fù)合測定板中的IC50分別為10.34nmol/L/8.46nmol/L。實(shí)驗(yàn)組在接觸藥物120天后在單一萘酚喹測定板中的IC50為35.47nmol/L,在單一阿奇霉素測定板中測得IC50為50.04nmol/L,在萘酚喹/阿奇霉素復(fù)合測定板中的IC50分別為2.89nmol/L、2.68 nmol/L。 4.結(jié)論 4.1惡性瘧原蟲在單一接觸萘酚喹后一段時間敏感性有明顯的下降,其IC50遠(yuǎn)遠(yuǎn)高于親代的5倍以上,進(jìn)一步證實(shí)用人工培養(yǎng)的方法可以培育出惡性瘧原蟲抗萘酚喹耐藥株。 4.2萘酚喹與阿奇霉素配伍對惡性瘧原蟲的作用觀察顯示,無論對敏感株(對照組)還是抗藥株在萘酚喹/阿奇霉素復(fù)合測定板中測的IC50顯著低于單一的萘酚喹測定板和阿奇霉素測定板,提示萘酚喹與阿奇霉素配伍對惡性瘧原蟲的作用明顯大于單一用藥,兩種藥物聯(lián)用有明顯增效作用。 4.3實(shí)驗(yàn)組惡性瘧原蟲對藥物耐受性的增長遠(yuǎn)遠(yuǎn)慢于對照組,前者在藥物刺激120天后萘酚喹、阿奇霉素、萘酚喹/阿奇霉素的IC50分別上升16.8倍、15.6倍、16.1/17.9倍,后者在藥物刺激120天后萘酚喹、阿奇霉素、萘酚喹/阿奇霉素的IC50分別上升31.0倍、21.4倍、57.4/56.4倍,提示萘酚喹與阿奇霉素兩種藥物聯(lián)用有明顯延緩抗性作用。
[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.

【學(xué)位授予單位】：大理學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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