Development and Application of Insect Cell Culture Techniques

以桉树Antheraea eucalyptus细胞系为研究对象，研究了鳞翅目昆虫病原微孢子虫（Nosema bombycis NIS 001、nsp . NIS M11、nsp . F-30、nsp . mesnili AH、Thelohania sp. K-25）的侵染和发育。用0.1 N氢氧化钾溶液将微孢子虫孢子与桉树桉树细胞培养物混合，培养物保持18个月以上。研究了低成本昆虫细胞培养基（MM）及其在昆虫细胞纺丝培养中的应用。比较了两种鳞翅目和一种双翅目细胞系在静止和旋转培养条件下的生长情况。显然，当使用较小的培养容器时，在塑料培养瓶中培养的昆虫细胞具有更高的生长速率。同时，研制了一种用于昆虫（鳞翅目、双翅目、半翅目、鞘翅目）细胞的通用培养基MGM-450。利用家蚕细胞系（SES-BoMo-15AII）克隆昆虫细胞，未成功建立单细胞家蚕细胞系。认为群体定殖是目前昆虫细胞克隆的一种实用方法。furgiperda Spodoptera Sf21AEII细胞对几种苏云金芽孢杆菌血清型产生的热不稳定细胞病变因子(s)高度敏感。然而，用类似的系统对苏云金芽孢杆菌热稳定外毒素（蝇毒素）进行体外测定，结果为阴性。在体外实验中，向家蚕感染核型多角体病毒的BM-N细胞中添加昆虫血淋巴因子，可显著提高病毒结构蛋白和包涵蛋白的产量。研究了鳞翅目细胞表面抗原的特异性和共性，并对多种昆虫细胞系进行了血清学鉴定。

Infection and development of microsporidians (Nosema bombycis NIS 001, N. sp. NIS M11, N. sp. F-30, N. mesnili AH, Thelohania sp. K-25) pathogenic to lepidopterous insects were studied in a cell line of Antheraea eucalypti. Microsporidian spores primed with 0.1 N potassium hydroxide solution were mixed with the A. eucalypti cell cultures and infected cultures were maintained for more than 18 months.Low cost insect cell culture media (MM) and its application to spinner culture of insect cells were investigated. Growth of two lepidopteran and one dipteran cell lines was compared under still and spinner culture conditions. Apparently, the insect cells cultured in plastic culture flasks showed higher growth rates when smaller culture vessels were employed.Also, a general purpose culture medium (MGM-450) for insect (Lepidopera, Diptera, Hemiptera and Coleoptera) cells was newly formulated.Cloning of insect cells using Bombyx mori cell line (SES-BoMo-15AII) was studied and establishment of the B. mori cell line resulted from a single cell was unsuccessful. It was suggested that a colonial coloning was practical method for cloning of insect cells at the present time.Spodoptera furgiperda Sf21AEII cells were highly sensitive to heat labile cytopathic factor (s) produced by several serovars of Bacillus thuringiensis. However, in vitro assay of the heat stable exotoxin (fly toxin) of B. thuringiensis using the similar system showed negative results.Addition of insect hemolymph factor (s) to Bombyx mori BM-N cells infected with a nuclear polyhedrosis virus, markedly enhanced the production of viral structural and inclusion proteins in vitro.Specific and common cell surface antigens of cultured lepidopteran cells were demonstrated and serological identification of insect cell lines was applicable for several insect cell lines.