摘要
目的 筛选雄虫抽提物对日本血吸虫卵黄培养细胞影响的最适作用浓度。方法 灌注法获取28d虫龄的日本血吸虫虫体，无菌处理后分离雌雄虫体。将雄虫匀浆，反复冻融3次，离心取上清液，考马斯亮蓝法测定蛋白后置于-20℃下保存备用。切下雌虫卵黄腺段虫体，冷消化法制备细胞悬液，调整细胞数为1×10^9／L后，将卵黄细胞接种于小盖玻片上，RPMI-1640含20％小牛血清培养，根据加入不同质量浓度雄虫抽提物分为6组（0、10、50、100、150、200mg／L组）。培养第7天，以核仁组织区相关嗜银蛋白（argyrophilic nucleolar organizer region associated proteins，AgNORs）及乳酸脱氢酶（lactate dehydrogenase，LDH）为评价指标，对卵黄细胞进行AgNORs及LDH染色，Olympus-BH2显微镜观察，输入HPIAS-2000图像分析仪测定培养细胞的灰度值。结果 随着雄虫抽提物质量浓度的增加，培养细胞的AgNORs与LDH着色均逐渐加深，至100mg／L时最深,随后又逐渐变浅。图像分析显示，雄虫抽提物处理的各组细胞AgNORs及LDH均明显高于对照组（q10=14．2189、q50=18．3358、q100=35．6491、q150=13．1849、q200=13．8604，P＜0．01；g10=10．3377、q50=11．7532、q100=23．1883、q150=10．2792、q200=9．3052，P＜0．01）。雄虫抽提物100mg／L组与其他各组比较，AgNORs及LDH明显升高（q100=21．4302、q50=17．3132、q150=22．4642、q200=21．7887，P＜0．01；g10=12．8506、q50=11．4351、q150=12．8896、q200=13．8831，P＜0．01）。结论 雄虫抽提物可显著提高日本血吸虫卵黄细胞的增殖能力与代谢活性，其作用的最适质量浓度为100mg/L。
Objective To study the effects of different concentrations of male worm extraction on the proliferation and metabolic activity of cultured vitelline cells from Schistosomajaponicum （S. japonicum）, in order to select the optimal concentration of male worm extraction. Methods The 28 day adult ＆ japonicum were harvested via perfusion. The male and female were isolated after asepsis treatment. The male worms were homogenized, frozen and thawed three times, respectively. After centrifugation, the supernatant protein concentration was measured by Coomassies Brilliant Blue method, and then the supernatant was stored at temperature of-20℃ . The vitelline glands of female worms were isolated. The vitelline cells were harvested by cold digestion, then were diluted to a concentration of 1×10^9 cells/L, inoculated using the moist system method on the small glass cover slips, and cultured in RPMI-1640 containing 20% calf serum and a moderate amount of antibiotics. Then, the cultured cells were divided into six groups according to male worm extraction concentrations of 0, 10,50,100,150,200 mg/L, respectively. The cells were stained to measured the activities of argyrophilic nucleolar organizer region associated proteins （AgNORs） and lactate dehydrogenase （LDH） when they were cultured for 7 days. The contents of AgNORs and LDH in the cultured cells of each group were measured by the photometer-image analytical instrument （HPIAS-2000）. Results The staining of the AgNORs and LDH in the cultured cells became deeper along with the increase of the concentration of male worm extraction by degrees. The stain was the deepest with the concentration of 100 mg/L, and then became lighter gradually when the concentration continued to increase. The contents of the AgNORs and LDH in the cells treated by the concentration of male worm extraction were higher than the control group （q10 = 14.2189,q50 = 18.3358 ,q100 = 35.6491,q150 = 13.1849,q200 = 13.8604,P〈0.01 ;q10 = 10.3377,q50 = 11.7532,q100 = 23.1883,q150 = 10.2792,q200 = 9.3052, P〈 0.01）. The contents were the strongest in the concentrations of 100 mg/L （q10 = 21.4302,q50 = 17.3132,q150 = 22.4642,q200 = 21.7887,P〈0.01;q10 = 12.8506,q50 = 11.4351,q150 = 12.8896,q200 = 13.8831,P 〈 0.01）. Conclusions Male worm extraction promotes the proliferation and metabolic activity of the vitelline cells from S. japonicum, with the concentration of 100 mg/L being the most effective.
出处
《中国地方病学杂志》
CAS
CSCD
北大核心
2006年第5期482-486,共5页
Chinese Jouranl of Endemiology
基金
国家自然科学基金(30570243)
湖北省卫生厅资助项目(JX1B067)
武汉大学杰出青年基金(20030515)
关键词
雄虫抽提物
血吸虫日本
卵黄细胞
核仁组织区相关嗜银蛋白
乳酸脱氢酶
Male worm extraction
Schistosoma japonicum
Cultured vitelline cell
Argyrophilicnucleolar organizer region associated proteins
Lactate dehydrogenase
