嗜人按蚊细胞系的建立及其特征

Establishment of a Cell Line from Anopheles anthropophagus and Its Characterization

嗜人按蚊细胞系——Ana 104是取材于其Ⅰ龄幼虫培养成功的。培养液以7C199培养基,另加0.4%水解乳白蛋白,0.03%谷氨酰胺、10～20%胎牛血清及适量的青、链霉素和非必需氨基酸配制而成。Ⅰ龄幼虫组织片段在培养液中缓慢地、有节律地收缩,并不断长出生发囊和细胞,达二个半月之久。经传代培养后,约7～10天可长满单层,能维持22天以上。至今已传27代。能适应在L-15、DMEM等培养基中生长。从第9代起,将部分细胞进行液氮冻存,经复苏试验复活成功,生长良好。Ana 104细胞系以上皮型细胞为主,也有梭型细胞,染色体2n=6。动态观察其对氨基酸的需求,发现对谷氨酸、酪氨酸、鸟氨酸、胱氨酸和精氨酸需量较大,并可合成天门冬氨酸等。经电镜观察,未见有病毒和支原体污染。还测试了酯酶同功酶带为3条和羧酸酯酶、乙酰胆碱酯酶的比活力。嗜人按蚊嗜吸人血,是我国重要的媒介蚊种。Ana 104细胞系是继冈比亚按蚊、斯氏按蚊和中华按蚊细胞系建立后,又新增添的1种按蚊细胞系。它将有助于从细胞分子角度对嗜人按蚊进行深入的生物学、营养代谢、细胞遗传、与病原体的关系、杀虫机理及生物工程等方面的研究。

A cell line from Anopheles anthrophagus-Ana 104 is established from 1st instar larvae. The medium is TC199, supplemented with 0.4% Lactabumin Hydrolysate (LH), 0.03% glutamine, 10-20% FBS and adequate amount of penicillin and streptomycin as well as non essential amigo acids. The fragment of the larvae remained active for 2.5 months and continuously produced cells. After 7-10 days of subculture, the cells formed a moaolayer which could be maintained for more than 22 days. The cell line is on its 27 passage leveal now. The cell is adapt to the medium L-15, DMEM etc. From Oth passage on some cells were stored in liquid nitrogen and successfully resuscitated. Ana 104 mainly contains epitherial type cells, spindal like cells also exist. The chromosome number is 2n=6, The observation with electron microscope showed no contamination of virus or micoplasma. The dynamic study of amino acids requirement indicated that the cells consumed relatively large amount of Glu. , Tyr. , Orn. , Arg. , Cys. , etc, but increased the concentration of Asp. in the medium. The activity of carboxylesterase and cholinesterase were detected. Esterase Zymograms form polyacrylamide gel electrophoresis revealed 3 bands.The Anopheles anthropophagus is affidicted to human blood, it is important vecor in China. Ana 104 was the 4th cell line established from Anopholine mosquito, The other three was established from An. gambia, An. stephensi, An. sinensis. The cell line Ana 104 would be helpful in the study of virology, parasitology, cell and molecular biology and its management.