Huang Xian-xi, Zhou Li-min, Yi Guo-hui, Wu Jin-yan, Pan Zai-yong, Xue Wei-ling, Guo Hong
Scientific Experiment Center, Hainan Medical University, Haikou 571101, China.
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2012 Feb 29;30(1):12-6, 19.
To analyze the immunological characteristics of murine model of piperaquine sensitive (PQS) line and resistant (PQR) line of Plasmodium berghei (Pb) ANKA strain.
64 Kunming mice were divided into three groups, 16 in each of groups A and C, 32 in group B (16 of 32 were used for observing survival days). Each mouse in groups A and B was infected with 1 x 10(7) erythrocytic stage parasites of PbPQS and PbPQR, respectively. Mice in group C were injected with the same volume of normal saline. On days 4, 8, 12 and 16 after inoculation, 4 mice from each group were sacrificed. Blood samples were collected for thin blood smear examination, and parasitemia rate calculated. Spleens were removed and spleen lymphocytes suspension prepared. Spleen lymphocytes were stimulated with ConA, and cell proliferation was measured by Cell Counting Kit-8 (CCK-8) assay. Nitrogen oxide (NO) and IFN-gamma level of spleen cell culture supernatants were detected by the Griess reagent and ELISA methods, respectively. Another 10 mice were each inoculated with 1 x 10(7) parasites of PbPQR line, and the mice were then challenged with lethal PQS line when the parasites turned into blue stained cells. The parasitemia and survival days were recorded.
The average survival time of group A was (9.0 +/- 3.0) d, the parasitemia rate was over 50% at 6-12 days post- infection with severe anemia. On 16th day post-infection, no death was recorded in group B with a parasitemia rate of (2666 +/- 254) %. After ConA stimulation, the proliferation of spleen lymphocytes in groups A (0.65 +/- 0.08) and B (0.86 +/- 0.20) at 12 days after infection was significantly higher than that of group C (0.18 +/- 0.03) (P < 00.01). NO level in spleen cell culture supernatant increased with prolonged infection time. On 12th day post-infection, NO level of groups A [(48.80 +/- 3.49) micromol/L] and B [(54.80 +/- 2.17) micromol/L] was higher than that of group C [(7.80 +/- 0.71) micromol/L] (P < 0.01). IFN-gamma concentration in spleen lymphocytes culture supernatant increased with prolonged infection time. The highest IFN-gamma level of group A was (752.20 +/- 39.49) pg/ml on 12th day post-infection, while in group B it was (855.80 +/- 33.65) pg/ml on 8th day after infection, then decreased on 12th day [(620.20 +/- 27.11) pg/ml]. IFN-gamma level showed a significant difference between groups A and B (P < 0.01). In 10 days after challenge, the parasitemia rate in PQR group was up to (2.44 +/- 2.07)%, and gradually disappeared. No parasite was detected on 40th day after challenge and no mice died.
The proliferation of spleen cells, NO and IFN-gamma levels of spleen lymphocytes culture supernatant in PbANKA strain PQR line are significantly higher than that of PQS line. PbPQR line can induce certain protective immunoreaction.
分析伯氏疟原虫（Pb）ANKA株哌喹敏感（PQS）系和耐药（PQR）系小鼠模型的免疫学特征。
64只昆明小鼠分为三组，A组和C组每组16只，B组32只（其中16只用于观察存活天数）。A组和B组小鼠分别感染1×10⁷个PbPQS和PbPQR红细胞期疟原虫。C组小鼠注射相同体积的生理盐水。接种后第4、8、12和16天，每组处死4只小鼠。采集血样进行薄血涂片检查并计算疟原虫血症率。取出脾脏并制备脾淋巴细胞悬液。用刀豆蛋白A刺激脾淋巴细胞，采用细胞计数试剂盒-8（CCK-8）法检测细胞增殖。分别用格里斯试剂和ELISA法检测脾细胞培养上清液中的一氧化氮（NO）和干扰素-γ水平。另取10只小鼠各接种1×10⁷个PbPQR系疟原虫，当疟原虫变为蓝色染色细胞时，用致死剂量的PQS系疟原虫攻击这些小鼠。记录疟原虫血症和存活天数。
A组平均存活时间为（9.0±3.0）天，感染后6 - 12天疟原虫血症率超过50%，伴有严重贫血。感染后第16天，B组无死亡，疟原虫血症率为（2666±254）%。感染后第12天，经刀豆蛋白A刺激后，A组（0.65±0.08）和B组（0.86±0.20）脾淋巴细胞增殖明显高于C组（0.18±0.03）（P<0.01）。脾细胞培养上清液中NO水平随感染时间延长而升高。感染后第12天，A组[（48.80±3.49）μmol/L]和B组[（54.80±2.17）μmol/L]的NO水平高于C组[（7.80±0.71）μmol/L]（P<0.01）。脾淋巴细胞培养上清液中干扰素-γ浓度随感染时间延长而升高。A组感染后第12天干扰素-γ最高水平为（752.20±39.49）pg/ml，而B组感染后第8天为（855.80±33.65）pg/ml，第12天下降[（620.20±27.11）pg/ml]。A组和B组干扰素-γ水平差异有统计学意义（P<0.01）。攻击后10天内，PQR组疟原虫血症率高达（2.44±2.07）%，并逐渐消失。攻击后第40天未检测到疟原虫，无小鼠死亡。
PbANKA株PQR系脾细胞增殖、脾淋巴细胞培养上清液中NO和干扰素-γ水平明显高于PQS系。PbPQR系可诱导一定的保护性免疫反应。
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