关键词: 湖北钉螺, 肝脏细胞, 细胞培养, 细胞大小, 琥珀酸脱氢酶, 乳酸脱氢酶

Abstract: Objective To develop a method for primary culture of cells from Oncomelania hupensis liver， and to observe the distribution of succinate dehydrogenase （SDH） and lactate dehydrogenase （LDH） in the cultured cells. Methods O.hupensis was anatomized to separate the liver. Livers were soaked in 0.2% benzalkonium bromide and washed by physiological saline containing antibiotics in turns. Cells from the liver were harvested by mechanical mulling and filtering. The isolated cells were then incubated with methods of the combination culture and standing suspension culture， respectively. The culture medium for the cells was a mixture of Medium 199 （50 ml）， 0.3% lactoalbumin hydrolysate dissolved in a balanced salt solution （BBS，30 ml）， and fetal calf serum （FCS，20 ml） containing a moderate amount of antibiotics （100 IU/ml penicillin， 100 μg/ml streptomycin and 50 μg/ml kanamycin） at pH 7.2-7.4 under the temperature of 26.5 ℃. The cells were stained by using Giemsa and Pearson methods （for SDH and LDH respectively） to observe the shape of cultured cells and enzyme distribution in cells. The living and stained cells were microscopically observed. Results Under microscope， the attached cells incubated with method of the combination culture showed round， elliptic， triangular and irregular shapes, with more round and elliptic cells. The size was approximately （4-16）μm×（6-20）μm in average. The clustered cells with an unclear nucleus and abundant and lucid cytoplasm were smaller than diffused cells with a large， obvious nuclei and less cytoplasm. Degeneration was observed after culturing for 5-7 days. The cultured cells could be divided into two types based on the color shown after Giemsa staining. The first type cells showed blue cytoplasm and mauve nuclei while the second type cells were opposite. There were blue granules in different sizes and shade in the cytoplasm after SDH and LDH staining. It was difficult for the cells to attach the wall of the culture flask using method of the standing suspension culture. The shape of the cultured cells were almost round with unclear nuclei， and the size was about （4-6）μm×（6-8）μm in average. The cells incubated with the standing suspension method were found to be contaminated after culturing for 3 days. Conclusion The combination culture method is suitable for primary culture of the cells from O.hupensis liver and the cells show activities of both SDH and LDH in cytoplasm.

Key words: Oncomelania hupensis, Liver, Cell culture, Cell size, Succinate dehydrogenase （SDH）, Lactate dehydrogenase （LDH）