伯氏疟原虫氯喹抗性逆转的实验观察

Objective To search for reverser of chloroquine-resistance in Plasmodium berghei (P.berghei) ANKA. Methods Seventy-two healthy Kunming mice were each infected with chloroquine sensitive (CS) or chloroquine resistance (CR) P. berghei ANKA respectively, then treated with various schedule of reversal agents C-2832 、D-6182 or ketotifen(Ket) respectively with or without co-administration of low dose (5%ED90) of chloroquine (CQ). Schedule 1: mice infected with CS were randomly distributed into 4 groups, 6 mice in each group, 30 min after infection,then treated i.g. with D-6182, C-2832, Ket or 0. 1% gum tragacanth(control) respectively for 5 consecutive days. The parasitemia of each experiment group was then determined by routine microscopic examination on blood smears from the tail blood of each mouse from D1 to D7.Schedule 2:mice infected with CR were randomly distriduted into 8 groups, 6 mice in each group, 3 d after infection, then treated i.g. with D-6182, C-2832, Ket, chloroquine or 0. 1% gum tragacanth (control) with or without co-administration of 12 mg/(kg · d) chloroquine (5% ED90) 2 h after the first administration for 5 consecutive days. The parasitemia of each experiment group was then determined microscopically by examination on blood smears from the tail blood of each mouse from D4 to D7. The reduction rates of each group were calculated and compared between the groups with or without treatment of reverser. Results 1. The parasitemia of mice infected with CS was going up daily from D1 to D4 and reached the peak on D4 in all groups administered with 80 mg/(kg · d) D-6182, 120 mg/(kg · d) C-2832 or 10 mg/(kg · d) Ket for5 d. From D5 the parasitemia kept going up in control group while it kept at the level of D4 in all treated groups. 2. Chloroquine 12 mg/(kg · d) administered i.g. 2 h after administration of C-2832 or D-6182 or Ket for 5 d(D3-D7) could reach 97.77%, 99.28% or 96.73% of reduction rate of parasitemia on D4 and 99.81%, 98.87% or 100.00% on D7 respectively in CR P. berghei infected mice. Conclusion Two compounds of C-2832 and D-6182 exhibited the reversal activity on CQ resistance in CR P. berghei infected mice at the same range of that of well-recognized reverser Ket.     

咯萘啶、阿莫地喹、甲氟喹及青蒿素对伯氏疟原虫的疗效

用4天抑制试验法给感染伯氏疟原虫ANKA株小鼠灌胃咯萘啶12.5mg/kg·d或阿莫地喹25mg/kg·d均能完全杀灭原虫;甲氟喹25mg/kg·d或青蒿素100mg/kg·d虽有抑制作用但治愈率仅为50％及0％。咯萘啶12.5mg/kg·d对中度抗氯喹的NS系原虫亦有明显作用,洽愈率70％,而阿莫地喹100mg/kg·d、甲氟喹100mg/kg·d及青蒿素200mg/kg·d均无治愈鼠,示后3种药与氯喹有不同程度的交叉抗性。上述剂量的阿莫地喹、甲氟喹和青蒿素对高度抗咯萘啶的伯氏疟原虫无明显抑制作用,示它们与咯萘啶有交叉抗性。     

咯萘啶对鼠疟原虫红内期裂殖体的持效作用

小鼠在腹腔注射(D_0)约氏鼠疟原虫前15天(D_(-15))肌注或灌胃咯萘啶100mg/kg,可使大部分小鼠不出现原虫血症。D_(-10)肌注或灌胃10mg/kg,D_(-5)肌注5mg/kg或D_(-4)灌胃5mg/kg亦有抑制原虫血症的作用。因该株原虫对氯喹具固有的抗药性,故即使在D_(-3)肌注或灌胃400mg/kg氯喹(>(2/3)LD_(50))亦不能保护小鼠不受感染。 应用伯氏鼠疟原虫的实验证明:咯萘啶杀裂殖体的持效作用明显比氯喹的为长。咯萘啶剂量为10mg/kg(ED_(50)的1.47倍,或LD_(50)的0.7％)时,D_(-6)灌胃一剂即能抑制原虫血症的出现,但相应剂量的氯喹67mg/kg(ED_(50)的1.47倍,或LD_(50)的10％),仅于D_(-2)给药有微效。     

白细胞介素-2伍用氯喹对伯氏疟原虫的作用

由于恶性疟原虫对氯喹(CQ)产生了抗药性,在许多流行区不再用氯喹治疗恶性疟。不过对半免疫力患者,氯喹治疗抗性恶性疟仍然有效。感染伯氏疟原虫的小鼠,可因脑疟在7d内死亡;但如若渡过危象期,小鼠体内的原虫数可下降[1]。Kremsner等以γ干扰素(IFN-γ)作为免疫佐剂合并氯喹治疗鼠疟有效[2]。本文采用氯喹和白细胞介素-2(IL-2)联合应用治疗感染伯氏疟原虫的小鼠,使之渡过危象期,取得了显著的治疗效果。　　材料和方法1　疟原虫　伯氏疟原虫系1985年由北京医学院惠赠,本室液氮保存已10年。2　实验动物　昆明株小鼠20g±1g。3　白细胞介素-2…     

百日咳菌苗配伍抗疟药治疗伯氏鼠疟的增效作用

Budge(1981)报告百日咳杆菌配伍氯喹治疗感染夏氏疟原虫(Plasmodium Chabaudi)免疫小鼠未发生原虫复燃,提示百日咳杆菌有增效作用。我们用百日咳菌苗分别配伍氯喹及蒿甲醚进行了试验。 材料和方法 疟原虫苗制备:取感染伯氏疟原虫小鼠心脏血.按Gwadz法制备虫苗,-20℃保存,于1～2周内     

伯氏疟原虫感染红细胞中的游离氨基酸和多胺生成的研究

伯氏疟原虫氯喹敏感株(CS)和抗氯喹株(CR)感染红细胞中的游离氨基酸量和鸟氨酸脱羧酶活力均相接近,用氯喹10mg/kg im两个株的感染小鼠,20h后对游离氨基酸无抑制作用.氯喹剂量为5mg/kg时,抑制CS和CR感染红细胞的鸟氨酸脱羧酶活力为79.6和55.7％。 CS和CR感染红细胞中的精脒量为139±27和528±140nmol/10~9感染红细胞。环亮氨酸剂量为80mg/kg时,能抑制CS和CR感染红细胞的精脒生成,其抑制率分别为44和57％,加喂甲硫氨酸(100mg/kg)后,精脒量分别上升至294和657nmol/10~9感染红细胞。但是与两者仍有一定差距,故认为在S-腺苷甲硫氨酸合成酶及其后的代谢环节仍有差异.     

瑞香素杀疟原虫裂殖体的作用

[目的 ]研究中药瑞香素在体外和体内的杀裂殖体作用。 [方法 ]在恶性疟原虫FCCl株常规体外培养中测试瑞香素杀裂殖体活性 ,并按“四天抑制性试验”在感染伯氏疟原虫ANKA株的小鼠中测定不同剂量瑞香素的体内抗疟活性。 [结果 ]体外试验中瑞香素在 1～ 10 μmol L剂量范围内有明显杀灭裂殖体作用 ,而体内试验中按D4减虫率与感染鼠在 30d内的平均生存天数评价 ,5 0或 10 0mg kg .d- 1 × 4d瑞香素灌胃以及 10 ,5 0或 10 0mg kg.d- 1 × 4d瑞香素腹腔注射给药在伯氏鼠疟原虫ANKA株感染鼠中的抗疟作用与 10mg kg .d- 1 × 4d氯喹 (CQ)灌胃的疗效相似。 [结论 ]瑞香素在体外和体内均有一定的杀裂殖体作用。     

感染抗氯喹NS系小鼠血清对氯喹抗疟药效的拮抗作用

本文首次报道感染抗氯喹‘NS’系小鼠血清对氯喹抗疟药效具有拮抗作用。将抗氯喹‘NS’系(抗性为20～30倍)和正常伯氏疟原虫(Plasmodium berghei)株分别接种两组昆明株小鼠,另设一组无原虫感染的小鼠作对照。于接种后第5d自三组小鼠采血清,采血前检查前两组原虫感染率,分别为13和22％,按无菌操作,自心脏抽血,将血液置于37℃使其充分凝固,析出的血清经1500～2000rpm离心5min后置于4℃保存备用。用感染‘NS’系小鼠血清(SMNS)、感染正     

伯氏疟原虫氯喹抗性株红内期虫体的凋亡

研究伯氏疟原虫 (Ｐｌａｓｍｏｄｉｕｍｂｅｒｇｈｅｉ)氯喹抗性 (ＲＣ)株红内期虫体细胞内死亡或危机状态的性质。应用光学显微镜和透射电镜及基因组ＤＮＡ琼脂糖电泳技术 ,观察感染伯氏疟原虫氯喹ＲＣ株或氯喹敏感(Ｎ)株ＩＣＲ小鼠 ,在原虫血症上升期和感染ＲＣ株小鼠自愈期虫体的显微及超微结构和基因组ＤＮＡ琼脂糖电泳图谱。结果表明 ,在光镜下 ,原虫血症上升期的ＲＣ株和Ｎ株血液涂片 ,可见感染红细胞和血浆内有大量不同发育期的虫体 ,结构无异常 ,而ＲＣ株感染小鼠自愈期血液涂片 ,虫体密度下降 ,大部分虫体固缩 ,周围呈空泡状。在电…     

瑞香素抗红外期疟原虫作用的研究

目的　研究瑞香素 (DPNT)抗红外期疟原虫的作用。方法　于ICR小鼠腹腔注射约氏疟原虫子孢子后 0 5h灌胃给药 ,连续 4d。不同剂量的DPNT及DPNT伍用伯氨喹 (PQ)的抗疟作用 ,分别以d7ICR小鼠阴性率及d1 1 或d1 2 ICR小鼠每千个红细胞被原虫感染数作评价 ,并观察DPNT对ICR小鼠血红蛋白浓度的影响。结果　DPNT的剂量范围为每天 10～ 10 0mg/kg ,连服 4d ,d7原虫阴性小鼠数及d1 1 红细胞被感染程度与对照组相比其差异均无显著性 ;DPNT每天 5 0mg/kg和每天PQ5mg/kg配伍组的d7小鼠阴性率与PQ每天 10mg/kg组相当。ICR小鼠DPNT每天 5 0mg/kg组与对照组血红蛋白浓度在d8天有差异。结论　DPNT单独用药 ,无明显抗红外期疟原虫作用 ,但DPNT每天 5 0mg/kg与PQ每天 5mg/kg伍用的抗疟效果与PQ每天 10mg/kg相当。DPNT在短期内可致小鼠贫血。     

三氟乙酰伯氨喹对鼠疟红内期原虫作用

参照ＷＨＯ推荐的“４ｄ抑制性试验法”给药，并采用延长疗效的观察期和取血再转种的方法证明：三氟乙酰伯氨喹２０ｍｇ／ｋｇ·ｄ×４ｄ能完全清除血内的伯氏疟原虫ＡＮＫＡ株；对抗氯喹伯氏疟原虫ＮＳ系和抗咯萘啶伯氏疟原虫ＲＰ系亦显示有不同程度的抑制作用，但疗效明显较差，与用伯氨喹相似。提示，８－氨基喹啉类药物与氯喹、咯萘啶等红内期杀灭剂有一定的交叉抗性。     

咯萘啶,阿莫地喹,甲氟喹及青蒿素对伯氏疟原虫..

The asexual stages of P. berghei ANKA were completely eliminated as revealed in a "4-day suppressive test" with the daily dose of pyronaridine 12.5 mg base/kg or amodiaquine 25 mg base/kg. Mefloquine 25 mg base/kg and qinghaosu 100 mg/kg though exerted obvious suppressive effect, the cure rates were only 50% and 0%, respectively. In treating chloroquine-sensitive P. berghei ANKA strain pyronaridine exhibited the best therapeutic activity, which was followed by amodiaquine, mefloquine and quinghaosu. In treating moderately chloroquine-resistant P. berghei NS line the cure rate of pyronaridine 12.5 mg/kg.d x 4 was 70%, but none of the 10 infected mice from any group was cured by amodiaquine 100 mg/kg.d, mefloquine 100 mg/kg.d or qinghaosu 200 mg/kg.d. Though the latter 3 drugs showed prominent suppressive effects, parasitemia remained positive or recrudesced after dosing. We demonstrate that parasites resistant to chloroquine had cross resistance to amodiaquine, mefloquine and inghaosu at various degrees. Amodiaquine 100 mg/kg.d, mefloquine 100 mg/kg.d and qinghaosu 200 mg/kg.d exhibited no obvious suppressive activity on highly pyronaridine-resistant line of P. berghei, indicating the existence of cross resistance to pyronaridine.     

蒿甲醚与瑞香素伍用对感染伯氏疟原虫小鼠的治疗作用

目的　研究蒿甲醚与瑞香素伍用(A+D)对感染伯氏疟原虫ANKA株小鼠的疗效及其联合作用方式。　方法　按“四天抑制法”d0 感染,d0 ～d3 给药,每天1次,d4涂薄血膜检查,并计算d4减虫率及各药丰数有效置(ED50),用等效应图解法分析A+D的合并作用。　结果　蒿甲醚0.4mg/(kg·d)× 4d的d4减虫率与对照组相比差异无显著性 ;[A0 .4mg/(kg·d) +D7.7mg/(kg·d) ]× 4d的抗疟效果提高 ,与对照组相比差异有显著性(P<0.05)。A+D各组的ED50均低于单用药组;不同配伍比例的R值皆大于0.4,小于2.7(0.4     

青蒿明胶丸的抗疟作用研究

明胶丸对小鼠的LD_(50)剂量为162.5±10.1g(生药)/kg,对伯氏敏感株鼠疟原虫的半数转阴剂量ED_(50)为11.9±2.4g(生药)/kg,化疗指数为13.6,明胶丸的抗疟效价比青蒿素大3.5倍;对鼠疟、人间日疟的治愈率为100％,与青蒿浸膏、氯喹相同;退热时间及转阴时间均快于氯喹,但近期复燃仍较高,当延长服药天数至6d,增加日服药次数至4次或与伯氨喹伍用,可显著降低近期复燃率。该制剂生产成本大大低于青蒿素,服药量及毒副反应小于青蒿浸膏片,易于推广,病人乐于接受。     

Chloroquine efficacy in Plasmodium berghei NK65-infected ICR mice, with reference to the influence of initial parasite load and starting day of drug administration on the outcome of treatment

We examined whether the initial number of parasites inoculated and the starting day of medication post-infection influenced the antimalarial efficacy of chloroquine (CQ) against Plasmodium berghei NK65 infection in ICR mice. Male ICR mice were inoculated intraperitoneally with 1 x 10(5), 1x10(6), 1 x 10(7), 1 x 10(8) P. berghei NK65-parasitized erythrocytes (pRBC). In the treated group, all mice received an oral dose of 20 mg/kg of CQ base for 4 days starting on day 0 after infection. From day 3, Giemsa-stained thin blood smears from tail vein blood were used to assess parasitemia. Mice in the untreated control in each group showed a progressive increase in parasitemia leading to death. Treatment of mice, inoculated with 1 x 10(5), 1 x 10(6) and 1 x 10(7) pRBC, with CQ showed a marked effect. All the mice survived during the experiment. During the observation period, malaria parasites could not be detected on microscopic examination. Conversely, mice inoculated with 1 x 10(8) pRBC showed little response to CQ treatment, and all mice showed a progressive increase in parasitemia and ultimately died. In another experiment, mice infected with 1 x 10(3) and 1x 10(5) pRBC were treated with an oral four-day dosage of 20 mg/kg of CQ base from days 2, 3 or 4 post-infection. Treatment of mice, inoculated with 1 x 10(3) pRBC, with CQ from days 2 and 3 showed a marked effect. All mice survived during the experiment. However, treatment from day 4 showed a limited derease in parasitemia and all the mice ultimately died. On the other hand, treatment from day 2 showed a marked effect against 1 x 10(5) P. berghei NK65-infected mice, but treatment from days 3 or 4 was only slightly effective and all the mice died with an increasing parasitemia. The present results indicate that in in vivo antimalarial drug-assay systems, several factors, sush as initial parasite load and starting time of treatment may influence the drug response in the host.     

伯氏疟原虫氯喹结合蛋白的分离与定量

目的： 分离伯氏疟原虫敏感株（ Ｃ Ｓ） 与抗氯喹株的氯喹（ｃｈｌｏｒｏｑｕｉｎｅ Ｃ Ｑ） 结合蛋白。方法： 给两株感染鼠ｉｇ Ｃ Ｑ４００ ｍｇ／ｋｇ , ３ ｈ 后, 收集伯氏疟原虫用（ Ｕｌｔｒｏｇｅｌ Ｒ） Ａｃ Ａ３４ 凝胶柱分离蛋白, 按 Ｂｅｒｇｑｖｉｓｔ 法抽提与蛋白结合的 Ｃ Ｑ, 然后用 Ｈ Ｐ Ｌ Ｃ （ 外标法） 定量。结果： Ｃ Ｓ 有５８ 个蛋白峰, 均与 Ｃ Ｑ 结合, 其中以 Ｎｏ６４ ～８６部分（ 峰９） 结合的 Ｃ Ｑ 量最多, 占总结合量的１６４ ％ 。抗氯喹伯氏疟原虫（ Ｃ Ｒ） 有４０ 个蛋白峰, 除８ 个峰外,均与 Ｃ Ｑ 结合, 结合量最多的蛋白部分与 Ｃ Ｓ相同, 在 Ｎｏ６０ ～８３ 部分（ 峰６ 及峰７） , 但是它与 Ｃ Ｑ 的结合能力（ Ｃ Ｑｎｍｏｌ／ ｍｇ 蛋白） 显著高于 Ｃ Ｓ。结论： 分离与测定了 Ｃ Ｓ和 Ｃ Ｒ 两株疟原虫各个蛋白峰的分布与定量, 及其与 Ｃ Ｑ 结合的能力。在同一株疟原虫中, 各蛋白与 Ｃ Ｑ 的结合能力存有差异, 在 Ｃ Ｓ与 Ｃ Ｒ 间此差异更大。     

小鼠疟原虫感染经氯喹治疗痊愈后再感染免疫特征分析

目的　探讨疟原虫感染小鼠在原虫血症高峰期经氯喹治愈后,再感染同/异种疟原虫的疾病进程和免疫保护特征。方法　用非致死型约氏疟原虫17XNL株（P. y 17XNL株）感染C57BL/6小鼠,感染率达高峰时（第9天）半数小鼠以氯喹治疗,其余小鼠自然痊愈。痊愈后小鼠于初次感染90 d后分别采用等量致死型约氏疟原虫17XL株（P. y 17XL株）或伯氏疟原虫ANKA株（P. b ANKA株）再次感染,采用吉姆萨薄血膜染色法观察原虫血症水平变化,并应用酶联免疫吸附试验和流式细胞术分别检测再感染前后小鼠血清中IgG抗体水平和脾细胞中记忆性T细胞亚群比例。结果　初次感染P. y 17XNL株后,自愈与氯喹治愈组小鼠血清IgG抗体水平分别为（5.047 ± 0.924）、（4.429 ± 0.624） pg/mL,差异无统计学意义（t = 0.437,P > 0.05）;但均显著高于无感染正常组小鼠的（1.624 ± 0.280） pg/mL（F = 22.522,P < 0.01）。自愈和氯喹治愈组小鼠再感染P. y 17XL株或P. b ANKA株并痊愈后,各组小鼠血清IgG抗体水平分别为（15.487 ± 1.173）、（14.644 ± 1.523）、（15.965 ± 1.150） pg/mL和（15.185 ± 1.333） pg/mL,差异无统计学意义（F = 0.542,P > 0.05）;但均高于初次感染组,差异有统计学意义（F = 67.383,P < 0.01）。感染P. y 17XNL株后自愈及氯喹治愈组小鼠再感染P. y 17XL株或P. b ANKA株并痊愈后,各组小鼠CD4+记忆性 T细胞比例分别为（34.023 ± 2.289）%、（35.608 ± 1.779）%、（34.208 ± 2.106）%和（32.820 ± 1.930）%, CD8+记忆性T细胞比例分别为（17.103 ± 1.627）%、（17.873 ± 1.425）%、（17.935 ± 2.092）%和（18.918 ± 2.823）%,组间差异均无统计学意义（F = 0.944、0.390,P均> 0.05）;但均高于初次感染后小鼠,且差异有统计学意义（CD4+记忆性T细胞,F = 50.532,P < 0.01;CD8+记忆性T细胞,F = 21.751,P < 0.01）。结论　小鼠疟疾经氯喹治疗痊愈不影响宿主在再感染时产生有效免疫保护力。初次感染后,小鼠对同种和异种疟原虫再感染均具有一定保护性且对同种疟原虫再感染的抵抗力强于异种疟原虫。     

伯氏疟原虫氯喹敏感株和抗性株中的氯喹积聚和排出

目的探测进入抗氯喹疟原虫中的氯喹量较敏感株低的原因是受阻于感染的红细胞，还是疟原虫自身，以及抗氯喹疟原虫是否具有快速排出氯喹的特点。方法：给两株感染小鼠ｉｇ氯喹，用高压液相（外标法）检测感染红细胞和疟原虫中的氯喹量。结果：ｉｇ氯喹４．０６ ｍｇ／ｋｇ与４００ｍｇ／ｋｇ氯喹３ｈ后，敏感株感染红细胞和抗氯喹株感染红细胞中的氯喹量无明显差异，而抗氯喹疟原虫中的氯喹量，两个剂量组分别较敏感的低５４．０％与４２．１％（Ｐ＜０．００１）。比较ｉｇ氯喹３ｈ与７ｈ后疟原虫积聚的氯喹量，敏感株两者相同，抗性株７ｈ后不仅无明显减少，相反地上升（０．４５２±０．０７９ｎｍｏｌ／ｍｇ蛋白与０．５５９±０．１２４ｎｍｏｌ／ｍｇ）蛋白（Ｐ＜０．０２）。结论：进入抗氯喹疟原虫中的氯喹量低，其受阻部位是在疟原虫本身而不是在感染的红细胞，未发现抗氯喹疟原虫快速排出氯喹。     

实验观察伯氏疟原虫再感染对免疫记忆形成的影响

目的探讨疟疾再感染对免疫记忆形成的影响。方法用伯氏疟原虫感染DBA/2小鼠,感染后3 d进行根治性治疗,并于初次感染后90 d进行再感染。通过姬姆萨薄血膜染色法计数红细胞感染率,流式细胞术检测再感染前(0 d)和再感染后(1、3、5 d)不同时间点脾细胞中记忆性T细胞和记忆性B细胞百分率。结果再感染前小鼠脾细胞中记忆性T、B细胞百分率均略高于正常鼠;再感染后,小鼠仅出现短暂的低水平原虫血症,记忆性T、B细胞百分率分别于再感染后第1 d和第3 d较对照组显著升高(P<0.01),但再感染后第5出现一定程度的下降。结论疟疾再感染可促进免疫记忆的产生,但高水平的免疫记忆不能持久存在。