VIRUS-INDUCED DIABETES-MELLITUS .1. HYPERGLYCEMIA AND HYPOINSULINEMIA IN MICE INFECTED WITH ENCEPHALOMYOCARDITIS VIRUS
VIRUS-INDUCED DIABETES-MELLITUS .1. HYPERGLYCEMIA AND HYPOINSULINEMIA IN MICE INFECTED WITH ENCEPHALOMYOCARDITIS VIRUS
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DOI：
10.1084/jem.137.5.1226
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发表时间：
1973-01-01
影响因子：
15.3
通讯作者：
NOTKINS, AL
中科院分区：
文献类型：
--
作者：
BOUCHER, DW;NOTKINS, AL
材料与方法Cells.通过胰蛋白酶消化切碎的17-19天龄CAF-1胚胎制备原代小鼠胚胎培养物。细胞用每ml补充有10%小牛血清、100 U青霉素、100 μ g链霉素和50 μ g新霉素的Eagle最小必需培养基（MEM）生长。DBA/2N和C57 BL/6 N小鼠获自国立卫生研究院的繁殖群。BALB/CJ、CBA/J和A/J小鼠购自缅因州巴尔港的杰克逊实验室。CD-1小鼠获自Charles River Breeding Laboratories，Inc.，马萨诸塞州威尔明顿将小鼠维持在含有5.0%脂肪和23.5%蛋白质的Purina NIH大鼠和小鼠日粮上。动物可自由进食和饮水，除非另有说明。除非另有说明，所有实验均使用雄性小鼠。Virus.--在小鼠中传代的EMC病毒M变体（MH 22）由J. Craighead博士友情提供（11，12）。用0.1 ml该材料的1：50稀释液腹膜内（ip）感染6周龄DBA/2N小鼠。5天后，通过在TenBroeck全玻璃组织研磨机中均质化，在含有5%小牛血清的Eagle's MEM中制备10%小鼠心脏混悬液。以相同的方式制备来自未感染小鼠的心脏的10%悬浮液。通过将0.2 ml该材料的适当稀释液滴加到15 mm塑料培养皿中的原代小鼠胚胎细胞的融合单层上，测定病毒滴度。在37 ℃吸附1小时后，将培养物用含有5%小牛血清和2%甲基纤维素的Eagle 'sMEM（Methocel，4000 cps; Fisher Scientific Co.，费尔劳恩，新泽西州）。4天后，用1：10，000稀释的中性红对单层进行染色，并在12小时内对噬菌斑进行计数。通过该方法得到的病毒储备池滴度为2.5 X 105空斑形成单位（PFU）/ml。
Materials and MethodsCells.--Primary mouse embryo cultures were prepared by trypsinization of minced 17-19-day old CAF-1 embryos. Cells were grown with Eagle's minimal essential medium (MEM) supplemented with 10% calf serum, 100 U of penicillin, 100~ g of streptomycin, and 50~ g of neomycin per ml.Animals.--DBA/2N and C57BL/6N mice were obtained from the breeding colony at the National Institutes of Health. BALB/CJ, CBA/J, and A/J mice were purchased from Jackson Laboratory, Bar Harbor, Maine. CD-1 mice were obtained from Charles River Breeding Laboratories, Inc., Wilmington, Mass. Mice were maintained on Purina NIH Rat and Mouse Ration containing 5.0% fat and 23.5% protein. Animals were allowed free access to food and water except where noted. Unless stated otherwise, male mice were used in all experiments. Virus.--The M variant of EMC virus passaged in mice (MH22) was kindly supplied by Dr. J. Craighead (11, 12). 6-wk old DBA/2N mice were infected intraperitoneally (ip) with 0.1 ml of a 1: 50 dilution of this material. 5 days later a 10% mouse heart suspension was prepared in Eagle's MEM with 5% calf serum by homogenization in a TenBroeck all glass tissue grinder. A 10% suspension of hearts from uninfected mice was prepared in the same way. The virus titer was determined by inoculating 0.2 ml of appropriate dilutions of this material onto confluent monolayers of primary mouse embryo cells in 15-mm plastic Petri dishes. After adsorption for 1 h at 37 C, cultures were overlayed with Eagle's MEM containing 5% calf serum and 2% methylcellulose (Methocel, 4000 cps; Fisher Scientific Co., Fairlawn, NJ). Monolayers were stained 4 days later with a 1: 10,000 dilution of neutral red and plaques were counted within 12 h. The titer of the stock virus pool by this method was 2.5 X 105 plaque-forming units (PFU)/ml.