【作者】 王建国；

【导师】 李兰娟；

【作者基本信息】 浙江大学 ， 内科学， 2006， 博士

【摘要】 无菌动物是不携带包括细菌、病毒、真菌和寄生虫等任何外源生命形式的动物。其研究起源于19世纪的法国科学家巴斯德,他研究了宿主和肠道菌群之间的关系后认为:肠道菌群对于宿主的生存是极为重要的,如果没有肠道菌群,宿主的生命维持将是不可能的。他实质上依据的是“适者生存”的进化论观点。 无菌动物的研究和建立,经历了一段漫长的过程。1895年Kijanizin在无菌代谢试验笼内饲养家兔,饲喂无菌饲料和水,但兔的体重却逐渐下降最后死亡。同年Nuttal和Thierfelder将剖腹产获得的无菌豚鼠饲养于玻璃罩内,人工哺乳灭菌牛奶和纯动物性饲料共计8天,动物外观健康,其肠内容物没有检测出细菌。直到20世纪40年代美国圣母大学Lobund实验室的Reyniers等人培育出无菌大鼠和无菌鸡,并能连续传代,证实高等动物能够在没有外源性生物的情况下正常生存。 第一个无菌大白鼠群是美国Lobund小组于1951年建立的。我国由王荫槐等人于1991年也曾培育成无菌大鼠,但是未见建立种群的报导。国内现在没有培育无菌大鼠。 普通的环境中存在大量的微生物,无菌动物必须饲养在一个完全不含有微生物的空间内。这样的空间在自然界内是不存在的,必须人为建造。这种人自己创造的并能够保证无菌的装置命名为隔离器。随着无菌动物研究的开展,无菌动物的饲养装置也不断地改进。1928年美国圣母大学细菌学实验室的Reyniers研制出更多还原

【Abstract】 The term germfree animal which is free from all demonstrable associated forms of life including bacteria, viruses, fungi, protozoa and other saprophytic or parasitic forms. In the 19th century, Pasteur speculated that on elimination of microbial associates, as is the case in the germ-free experiment, life of the animal host would become impossible. Apparently, he based this idea on the concept of the "survival of the fittest" in evolution.It was very difficult to establish germfree animals. In 1895, Kijanizin tried to rear rabbit under germfree conditions, but failed. It was not successful until Reyniers established germfree rats and chicken at University of Notre Dame in the 1940’s and reared to successive generations.The first colony of germfree rats was developed by Lobund Laboratory in 1951. Wang et al also established germfree rats in China in 1991. But there was no report on colony establishment of germfree rats. There are no germfree rats in China.The closed system isolators containing the sterile environment have been constructed of rigid materials until Trexler invented the flexible film apparatus for the rearing and use of germfree animals in 1957. Plastic isolators have two advantages over the stainless steel equipment. First of all, many delicate manipulations such as the canulation of blood vessels in experimental animals can be performed in the flexible film apparatus. Secondly, potential manufacturers estimate the cost of the plastic isolator to be less than one-tenth that of the stainless steel units. The cost of installation and operation is reduced because no special facilities or personnel are required.Germfree animals were widely used in the field of biological and medical sciences.Our hospital has been funded by the National Basic Research Program (973) of China (No. 2003CB515506) to study the micro ecology of liver transplantation in rats. To promote the further investigation, we developed the germfree rats by ourselves.Part one Development and optimization of environment for germfree ratsAims: To investigate the effects of controlling of amounts of microbes in the environment on the contamination of germfree rats in the isolators. To apply the new apparatus for artificially reared rats under sterile conditions.Methods:1. After the sterilization and run of barriers facilities, sampling was inoculated in the media to test the bacterial and fungus contamination.2. After the sterilization and run of barriers facilities, without regular disinfection, record the entry times and colonies of bacteria or fungus in the media.3. After the sterilization and run of barriers facilities and with regular disinfection, record colonies of bacteria or fungus in the media after entering 1,10, 50, 100 times.4. Put the isolators in the barrier facilities and common environments. These isolators were run at the same conditions. Note the length of time when the isolators were kept free from germs.5. Compare former instrument with our new design instrument for artificially reared rats under axenic conditions.Results:1. There were no bacteria and fungi in the media.2. When the room in the facilities was not regularly disinfected, the colonies of the bacteria were beyond the national standard on the fourth time of entering. There was a direct correlation between the times of entering the barrier facilities and the count of colony of bacteria in the air(R=0.884, PO.05).3. Even after 100 times entering the facilities being regularly disinfected, the counts of colony of bacteria in the air were in the range of the national standard.4. The length of time (54, 43. 76, 43. 76. 58, 87, 104, 91, 134 and 153 days) when the isolators were kept free from germs in the barrier system is longer than the length of time (25, 36, 34, 16, 53, 35, 42, 54, 46, 66 days) when the isolators kept free from germs in the common environment by statistical analysis(P<0.01).5. Note the average feeding time with new style apparatus: 11.1 ±2.1, 8.6± 1.6, 7.3±1.4, 6.6±1.3fP6.3±1.2min, the average feeding time with old style apparatus: 13.2±3.3, 10.7±2.5, 9.6±2.2, 8.6±1.9#J8.2±1.5min. The average feeding time with new apparatus is shorter than old apparatus (P<0.05).Part two Establishment of germfree rats with new techniqueAims: To investigate the key on establishment of germfree rats, the accurate rate of the methods of judgement for the circumnatal period, abdominal delivery under germfree conditions, dynamic accommodation of viscosity and nutritional ingredients of the artificial formula for the rats. Microbiological detection for the germfree rats.Methods1. Judgement of circumnatal period with calculation, palpation, observation method and synthetic method. The intrapartum and the anticipation are within one hour.2. Abdominal delivery under germfree conditions. Put the womb and placenta in the media.3. New methods for feeding neonatal rats. Dynamic accommodation of viscosity and nutritional ingredients of the artificial formula for the rats.?Preparation of artificial milk for different rats. Measurements of nutrient compositions and physical properties of rat’s milk and LWF.?Artificial feeding (AR) of rats. Germfree mother-reared (MR) pups were as controls. 3 litters of 30 pups were fed for each group. When these rats were over 100 days old, one male rat was mated with a female rat randomly. 8 couples from three different litters of artificially reared rats were mated. Feed the pups six times a day.The milk formula volume of feeding everyday was about 20% body weight of the pups. Survival of the AR rats was 73.3%, while no MR rats died.?The development of pups after weaning.Note the growth of 9 female rats and 9 male rats till they were 14 weeks old. 10 SPF mother-reared male rats and 8 female rats were as controls.4. Microbiological detectAccording to the national standard, to test the bacterial, virus, fungi, parasite and mycoplasma.Results:1. Calculation 20% palpation76%, observation method 70% and synthetic method 84%.2. No bacteria and fungi.3. New methods for feeding neonatal rats. Dynamic accommodation of viscosity and nutritional ingredients of the artificial formula for the rats.(DLWF was closed to rat’s milk in nutrient composition and physical properties, with a growing viscosity and nutrient ingredients for growing pups.(2)Cesarean-derived pups were hairless, blind, with closed ears. Infant rats suckling LWF were in normal development. The ears opened at 3 days;incisors erupted between 8 and 10 days;and eyes opened at age of 13 days. They were fully haired at about 10. Survival of the AR rats was 73.3%, while no MR rats died. Germfree AR rats fed on LWF grew normally and had fecundity, although the body weight gain of these rats was not as good as MR rats.?After weaning, at the 4 week of age, AR rats were not as good as MR rats(P<0.05);But at the 6 week of age, there were no different (.P>0.05).@A11 mated rats had fertility. Pregnancy could be achieved in a few days. Gestation was 21—23 days. Litter size was 6~12 pups. Pups birth weights were from 4.2-6.8 g.4. No germs were found. Conclusion:1. We have established the germfree rats.2. Dynamic accommodation of viscosity and nutritional ingredient of the artificial formula for the rats is suitable for the germfree rats.3. The new apparatus we design may contribute to shorten the length of the time when feeding rats.4. Controlling the count of the microbe may help prolong the time of germfree conditions.更多还原