外源性一氧化氮体外杀伤红内期间日疟原虫的实验研究

In Vitro Lethal Effect of Exogenous Nitric Oxide on Blood-Stage Plasmodium vivax

目的探讨外源性一氧化氮(NO)对红内期间日疟原虫的杀伤作用。方法采集以早期滋养体期为主,原虫密度>0.5%的患者血样,配成含2%红细胞的虫血混悬液。96孔板中每孔加100μl虫血混悬液,再加入各组试剂,亚硝基铁氰化钠(SNP,分别为0.02、0.05、0.10、0.20、0.50、1.00mmol/L),SNP+血红蛋白(Hb)0.15mmol/L,SNP+硫酸亚铁(FeSO4)0.15mmol/L,SNP+L-半胱氨酸(L-cyst)1.00mmol/L,SNP+FeSO40.15mmol/L+L-cyst1.00mmol/L,后4组SNP浓度均为1.00mmol/L,设对照组,每组重复3次,置37℃5%CO2培养箱中培养。根据虫龄,估计发育至裂殖体时间,提前3h取空白对照孔涂片镜检,确定终止培养时间,培养终止后吉氏染色,镜检计数成熟裂殖体,计算NO对红内期间日疟原虫的抑制率,统计分析各组对间日疟原虫抑制率的差异。结果SNP0.02mmol/L组,间日疟原虫抑制率为(0.84±1.69)%,对培养的红内期间日疟原虫无杀伤作用(P>0.05);SNP0.05mmol/L组,间日疟原虫抑制率为(12.26±3.04)%,对培养的红内期间日疟原虫存在杀伤作用(P<0.01)。且抑制率随SNP浓度上升而升高。在含有1.00mmol/LSNP的孔中分别加入Hb、L-cyst、FeSO4和FeSO4+L-cyst与只添加SNP1.00mmol/L孔相比,红内期间日疟原虫抑制率自(85.40±2.90)%下降为(5.90±2.90)%、(25.86±4.02)%、(30.16±2.75)%和(16.71±2.30)%。结论外源性NO对体外培养的红内期间日疟原虫有杀伤作用,而Hb、L-cyst和FeSO4可以逆转这种杀伤作用。

Objective To investigate the lethal effect of exogenous nitric oxide on blood-stage Plasmodium vivax in vitro. Methods The immature trophozoites from patient were diluted with human RBC to be a suspension of P. vivax-human RBC at 2% hematocrit and over 0.5% parasitemia. Sodium nitroprusside （SNP）, hemoglobin （Hb）, L-cysteine and FeSO4 were added to the parasite-blood suspension and made the drug final concentration to SNP 0, 0.02, 0.05, 0.10, 0.20, 0.50, and 1.00 mmol/L, SNP 1.00 mmol/L＋Hb 0.15 mmol/L, SNP 1.00 mmol/L＋FeSO4 0.15 mmol/L, SNP 1.00 mmol/L ＋L-cysteine 1.00 mmol/L, and SNP 1.00 mmol/L ＋ FeSO4 0.15 mmol/L ＋ L-cysteine 1.00 mmol/L, respectively. After at least 12 h incubation, the parasites developed to mature schizonts. Parasite maturation was observed in culture by Giemsa staining of samples. The mature schizonts were counted, and the inhibition ratio of exogenous nitric oxide to blood-stage P. vivax was computed. The differences of inhibition ratio in the groups were compared. Results SNP （0.02 mmol/L） was not cytotoxic to blood-stage P. vivax parasites with an inhibition of （0.84±1.69）%. When the concentration of SNP increased to 0.05 mmol/L, the inhibition ratio was （12.26±3.04）% which showed that exogenous nitric oxide released from SNP （≥0.05 mmol/L） killed the blood-stage P. vivax parasites, and the higher the SNP concentration, the larger the inhibition. Addition of hemoglobin, L-cysteine, FeSO4 and L-cysteine with SNP led to a decrease of the inhibition from （85.40±2.90）% to （5.90±2.90）%, （25.86±4.02）%, （30.16±2.75）%, （16.71±2.30）%, respectively. Conclusion Exogenous nitric oxide released from SNP kills blood-stage P. vivax parasites in vitro. However, hemoglobin, L-cysteine, and FeSO4 can reverse the lethal effect of the parasites.