实验动物 z-bo 2020-11-05 338

　　In 2004, approved by the Sichuan Provincial Department of Forestry, the Sichuan Provincial Department of Science and Technology established a project-the standardization of Tibetan chief monkey laboratory animals. Sichuan Academy of Medical Sciences·Sichuan Provincial People’s Hospital Laboratory Animal Research Institute became the only unit engaged in Tibetan chief monkey research and research. Since 2004, the institute has invested nearly 6 million yuan in various aspects of research on Tibetan chief monkeys as follows.

　　1. Research on domestication environment and facilities

　　The Tibetan chief monkey taming farm has been established, with sufficient indoor living and outdoor venues, sturdy and capable of preventing escape, taking into account the living habits of Tibetan chief monkeys in the wild, and setting up corresponding places for playing, eating and sleeping.

　　2. Research on artificial domestication and breeding

　　173 wild Tibetan chief monkeys (named sub-Macaca thibetana thibetana Milne-Edwards) were introduced from Mabian County through the Wildlife Rescue Center of the Forestry Department. The imported wild Tibetan chief monkeys were tested for internal and external parasites and dewormed, resulting in zoonotic diseases. For the detection of tuberculosis bacteria, the quarantine (tuberculosis and internal and external parasites) procedures and technical specifications for entering wild monkeys have been compiled; the manual domestication and management procedures for wild monkeys;

　　In the early stage, some wild Tibetan chief monkeys with strong physique and good physical and morphological characteristics after quarantine were selected to establish a breeding herd.

　　At present, there are nearly 120 baby Tibetan chief monkeys during pregnancy and delivery, and nearly 100 survived. The method of artificial breastfeeding of Tibetan chief monkey babies has been explored.

　　After years of research and observation, the understanding of the pregnancy and childbirth laws of Tibetan chief monkeys has been updated. According to the data, the wild Tibetan chief monkeys mate non-seasonally and reproduce seasonally. The calving time is from January to August. During the breeding process, we found that the wild Tibetan chief monkeys also gave birth in September and December 2006 in our domestication and breeding farm. This shows that with the improvement of the breeding environment in the wild, Tibetan chief monkeys can be bred under artificial domestication conditions. Transform into non-seasonal mating and non-seasonal reproduction.

　　3. The design of feed formula and the research of food preparation technology and feeding method.

　　According to the national primate laboratory animal feed nutrition standard, combined with the characteristics of Tibetan chief monkeys, we have carried out research on the design of feed formula, food preparation technology and feeding method.

　　4. Research on disease prevention and treatment technologies and methods.

　　Establish daily sanitation and disinfection, disease quarantine of new monkeys, epidemic prevention management measures and systems, and establishment of prevention and treatment methods for main common diseases, which can effectively control the prevalence and prevention and treatment of frequently-occurring diseases of Tibetan monkeys. Diarrhea is a common disease in Tibetan monkeys. We have some experience and experience. Its prevalence has dropped from 90% in 2005 to 10% in 2012, and the cure rate of the disease has increased significantly.

　　5. Individual identification of Tibetan chief monkeys, establishment and management of individual archives

　　can easily, accurately and effectively identify the individual, and build the individual file data and family genealogy genetic data accordingly. With individual identification, we can record the growth and reproduction, genetic background, disease prevention and treatment of each monkey from birth to adulthood, laying a foundation for future research work.

　　6. Microbial control in accordance with the national microbial and parasite control standards for primate laboratory animals

　　Establish microbial detection methods. The content includes: monkey ectoparasites, intestinal worms, tuberculosis, intestinal pathogens (Salmon, Shigella), skin pathogenic fungi, monkey B virus (simian herpes virus type I), monkey Toxoplasma, etc.

　　7. Basic laboratory research

　　7.1 Tibetan Chief Monkey Genetics DNA (RNA) Research

　　In order to analyze and establish the genetic background of individuals in the breeding herd, a method and technology for extracting DNA (RNA) from monkey blood was established.

　　Lymphocytes were separated from the peripheral blood of Tibetan monkeys by gradient density centrifugation, cultured in 1640 + 10% fetal bovine serum culture medium, and activated by PHA for division. The total DNA and RNA were extracted from the cells, and the primers of human and rhesus monkey gamma interferon and interleukin-6 genes were used to amplify the cDNA of the two Tibetan monkey genes by TR-PCR and PCR methods, and the purified cDNA was connected to The PMD16-T cloning vector was transfected into E. coli cells and amplified and purified to obtain a large amount of DNA of the above two genes. Then use the ABI3730 sequence analyzer for nucleotide sequence analysis. The sequence results obtained were compared with other primates (including 17 primates including rhesus monkeys, southern pig-tailed monkeys, cynomolgus monkeys, guinea baboons, white-collar monkeys) interferon and interleukin-6 genes. The study of science and phylogeny. The nucleotide sequences of the interferon and interleukin-6 genes of the Tibetan chief monkey were determined for the first time, and the evolutionary status of the old world monkeys of the Tibetan chief monkey was determined at the genetic level, which is highly consistent with the two gene sequences of the rhesus monkey. It reaches 99.4-99.8%, which is very close to the basis of human infection medicine, confirming its application value in biomedical research. The in-depth understanding and analysis of the genetic background of the Tibetan chieftain monkey as a new experimental animal and the genetic management of the experimental animal population provide a large amount of guiding information and theoretical basis.

　　Completed the complete sequence determination of the mitochondrial DNA of the Tibetan chieftain monkey. The whole sequence is 16540bp long and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region. Combining information from other species gene databases and analyzing their genetic organization structure. Prove that its evolutionary status is similar to that of rhesus monkeys and is closely related to humans, and its genetic basis can replace the biomedical research work of rhesus monkeys; two new pseudogenes X23 (2003bp) and X9 (2180bp) have been discovered; and The genetic diversity of this population is high (h=0.9033, ∏=0.0155), inbreeding and genetic drift are small, which provides a theoretical basis for the genetic management and maintenance of genetic diversity of the population. The in-depth understanding and analysis of the genetic background of the Tibetan chieftain monkey as a new experimental animal and the genetic management of the experimental animal population provide a large amount of guiding information and theoretical basis.

　　7.2 Research on biological rhythms

　　Method: Using small implantable body temperature monitoring equipment and small collar type activity monitoring equipment, the body temperature and spontaneous activity rhythm changes of Tibetan chief monkeys were continuously monitored under natural and total black conditions, and the rhythm characteristics were analyzed by the halberg cosine method. At different time points (the first day of the experiment, six time points a day, four hours apart from each time point), the venous blood was collected, and the serum was separated to detect changes in serum corticosteroid levels by radioimmunoassay. The all-black living environment of Tibetan chief monkeys was placed in an environment with and without other monkey groups, and the changes in the spontaneous activity rhythm of Tibetan chief monkeys in their respective environments were analyzed. Obtain the blood samples of Tibetan monkeys to prepare cDNA, analyze the cDNA sequence homology of various animal rhythm genes, design PCR primers for the main rhythm genes with human templates, and sequence the PCR products to determine whether there are corresponding rhythm gene fragments in Tibetan monkeys . The circadian rhythm analysis method adopts a comprehensive analysis method based on the halberg cosine method.

　　Conclusion: Tibetan chief monkeys have obvious circadian rhythms. Their spontaneous activity rhythms adapt faster than humans and mice after entering the dark environment from natural light, and their free rhythm cycles are slightly shorter than that of humans, which may be related to their long-term wild life ; Tibetan chief monkeys have a rhythmic asynchronous phenomenon similar to human spontaneous endogenous asynchrony; there may be clock genes in Tibetan chief monkeys (homologous gene sequences have been found, but its function has not been confirmed); social factors may be caused by Tibetan chief monkeys Acting timing factor; in the rhythm analysis of Tibetan chief monkeys, no rhythm component was found in the past seven days.

　　7.3 Biological characteristics

　　With the increase of body weight and age, the lipid metabolism rate of Tibetan chieftain monkeys slows down, which easily induces similar hyperlipidemia in humans. It has been observed in breeding and experiments that compared with rhesus monkeys and cynomolgus monkeys, such as behavior, psychology, etc., the face, hand (skin print or fingerprint), tail (very short), skin characteristics, and spontaneous hair loss rate are higher. Male genitals are exposed, relatively quiet in character, docile, easy to domesticate, close to humans, middle-aged and elderly monkeys are prone to obesity, slow activity, etc., have biological characteristics such as widely used medicine and biological research, and have great potential scientific and application value high.

　　7.4 The test of normal blood picture and biochemical reference value is recommended

　　There are nearly 200 Tibetan chief monkeys (20 years old ∽ 1 year old) of various ages in our institute that have a lot of potential development value. The main ones are:

　　(1) The unique animal model of the intestinal bacteria of Tibetan chief monkey.

　　(2) Animal models of specific aspects of nutritional metabolism.

　　(3) Research on obesity animal models and other animal models caused by obesity.

　　(4) Time biology model.

　　(5) Animal models of reproductive physiology.

　　(6) Geriatric diseases.

　　For Tibetan chief monkeys, the main scientific research projects I can carry out are:

　　(1) Animal models of hyperlipidemia, hyperglycemia, hypertension, pathogenic bacteria Escherichia coli, Shiga, and Salmonella bacteria.

　　(2) Research on reproductive physiology

　　(3) Behavioral, psychiatric and neurobiological research

　　(4) The domestication of monkeys.

　　(5) Research on infectious diseases.

　　(6) Research on drug metabolism.

　　(7) Pharmacodynamic test research, acute toxicity test, long-term toxicity test.