关键词: 草鱼, 脑细胞, 原代培养, grass carp, brain cell, primary culture

Abstract: The establishment of in vitro cell culture system is an important approach in investigating the physiological metabolism. Meanwhile，the establishment of virus culture system based on stable cell is a necessary technique to isolate，identify and study fish viruses. As a kind of in vitro culture system, fish cell lines become significant experimental materials because of some advantages including low cost, high test repeatability and the experimental conditions can be controlled accurately. The cells in vitro from fish, as an significant research technology，has played a role in the theory and applied research of virology, immunology, fish resources protection，genetic breeding, oncology, environmental toxicology，fish physiology, endocrinology and so on. Although many fish cell lines have been established, relatively few are capable of viral propagation but producing the viruses slowly and giving low viral titers. In addition，its application is far less extensive than that of mammals. This study tried different methods to obtain grass carp primitive brain cells, including tissue block culture method, enzymatic digestion method and mechanical method. The brain cells collected by mechanical method were uniformly dispersed, and the cell formed cell monolayer after 24 h incubation in primary culture. The microscopy observation showed that cells were in polygon shape and adherent cells were mostly epithelial - like cells. The cell grew rapidly and was fairly homogenous in the L - 1 5 medium containing 10% fetal calf serum at the temperature 28℃. For mammalian cell culture,37℃ is usually chosen as the specified incubation temperature. However, fish culture cell temperature is different due to the differences among species. Fish are warm - blooded animals，which can maintain the integrity of cell function and physiology within a certain temperature range. Grass carp optimum growth temperature is 25 to 32℃, and the optimum culture temperature was consistent with the physiological and metabolic characteristics. MTT assay observed that the vitality of brain cells could maintain 3 days, and cells significantly degenerate after 3 days. The activity of cells on the 4th and 5th day was 11.7% and 34.8% lower than that on the 1st day, respectively, indicating that the 3d culture period is a suitable time for the in vitro experiment of primary brain cells of grass carp. The study indicates that the primary cells of grass carp brain can be used to analyze physiological mechanism in vitro. Fish cell culture techniques will improve future studies on viral infection，toxicological evaluation and the mechanism of nervous system.