动物造模,非人灵长类疾病模型 z-bo 2021-01-08 589

　　1 Non-human primates

　　Primates are the highest group in the animal kingdom, with a total of 11 families, 51 genera, and 180 species, including monkeys, apes, etc., as well as the last human beings. Because they have the most developed brain in the animal kingdom, in addition to their high intelligence and complex social behavior, in terms of movement, their thumbs are flexible and can hold other fingers. This finger-to-hand grip is a characteristic of primate species, giving them a highly flexible and delicate finger grip function. In terms of perception, when other mammals rely heavily on smell and touch, primates have developed advanced visual systems. They are one of the few species with trichromatic vision (the ability to see three colors), so they have rich visual perception and Powerful object resolution ability. As the name implies, non-human primates are primates other than humans. They are very important experimental animals due to their many biological characteristics similar to humans. In the research work, the most used are the Old World monkeys, among which rhesus monkeys and cynomolgus monkeys are the main ones. Due to the similarity in evolution, 93% of the rhesus monkey gene sequence is the same as that of humans, compared with 84% of mice. Therefore, compared with other experimental animals, non-human primates have unique advantages in solving human problems, especially brain-related problems: in addition to being the key experimental animals for studying the normal advanced functions of the human brain, they are also excellent for studying brain disease mechanisms And treatment methods.

　　2 Brain diseases require non-human primate disease models

　　At present, there are nearly 1 billion patients with brain diseases in the world, and the economic burden brought by them is about 1 trillion US dollars per year. In my country, in terms of mental illness, there are currently 1.64 million children with autism; there are about 16 million people with schizophrenia and more than 26 million people with depression. And the neurodegenerative diseases closely related to the rapid development of aging in my country, such as: the number of patients with Alzheimer’s reaches 9 million, and it is increasing at a rate of doubling every 10 years; the number of patients with Parkinson’s disease in my country has exceeded 3 million, It has become the country with the most Parkinson's disease patients. Currently, there is no effective treatment for most brain diseases. Therefore, brain disease research and new drug development are very urgent.

　　Due to social ethics and moral constraints, human health and medical research are inseparable from animal models. The lack of ideal animal models is an important bottleneck in the study of brain disease mechanisms and drug development, because the currently widely used murine animal models have great limitations: in terms of evolution, compared with humans and monkeys 20 million years ago Separation, humans and mice were separated 100 million years ago. The consequence is that there is a huge difference in brain structure and function between mice and humans. When modeling with mice, the complex symptoms of human brain diseases are often difficult to faithfully reflect. For example, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease have similar cognitive phenotypes in transgenic mice carrying human gene mutations, but very few neuronal death symptoms in the brains of human patients are seen. Therefore, it is impossible to screen related drugs in mouse disease models to alleviate symptoms such as neuronal death. Because of this difference, a large number of drugs that were effective on mice failed in clinical trials. Among them, the most influential is the recent anti-Aβ monoclonal antibody Bapineuzumab of two famous US pharmaceutical companies, Pfizer and Johnson & Johnson, which has no obvious therapeutic effect after entering the phase III clinical trial, and there is angio cerebral edema and brain sulcus. Adverse reactions such as fluid effusion have failed; another new drug for Alzheimer's disease, Solanezumab, an anti-Aβ monoclonal antibody developed by Eli Lilly, a well-known American pharmaceutical company, has failed because of no obvious therapeutic effect. This phenomenon has attracted widespread attention and reflection. In 2014, some foreign scholars wrote an article on Nature "Misleading mouse research wasted medical resources".

　　In short, an embarrassing fact is that humans have spent a lot of money and energy on brain disease mechanism research and new drug development, but the brain mechanism and drugs of rats are out, which has caused major international pharmaceutical companies to withdraw from this field. How to break through this "rat poison" situation?

　　Due to the similarity in evolution, the brain structure and physiological activities of non-human primates are highly similar to humans in many aspects, making them ideal modeling animals for brain disease models. The human disease model created by it can better replicate the characteristics of human diseases, especially neurological diseases, than other model animals, and can better simulate the mechanism and pathological process of the disease. More importantly, due to its developed central system, non-human primates have complex cognition and social behavior, sophisticated motor control, and can complete memory tasks similar to humans. This is useful for objective evaluation of emotion and social cognition. Core behavior indicators related to brain diseases such as memory ability and fine motor are particularly important. Therefore, the use of non-human primates to establish animal models of brain diseases will greatly reduce the risk of failure in the development of new drugs. It is a necessary and sometimes the only bridge between basic and clinical research.

　　3 Current status of domestic and foreign applications of non-human primates The international community has attached great importance to non-human primates. The United States, Germany, Japan, New Zealand, South Korea, Thailand and India have established primate breeding and experiment centers for life medicine research, totaling 70 More than one. However, due to the scarcity of animal resources and the opposition of animal protectionists, progress has been slow. In addition, in the current economic downturn, the high cost of use of macaques (the purchase and breeding cost of macaques in the United States is 4-6 times that of China) has also made research on non-human primates in Europe and America worse. . According to statistics, between 2008 and 2011, European non-human primate research dropped by 28%. In May 2015, Harvard Medical School closed its primate center.

　　On the contrary, my country is a country with relatively rich distribution of wild primates in the world, with 24 species of primates, accounting for about 10% of the global primate species. In recent years, with the rapid development of life science research and translational medicine research, primate breeding and breeding have developed rapidly. There are more than 20 monkey breeding farms in Yunnan, Guangdong, Guangxi and Suzhou alone, and the number of artificial breeding stocks There are more than 300,000 pieces. The country also attaches great importance to the significance of non-human primates in the study of brain and related diseases. In the forthcoming "China Brain Project", research on brain functions and diseases using macaques as model animals will occupy a prominent position. The Animal Model Project of the National Natural Science Foundation of China has also favored the construction of non-human primate disease models in recent years. In 2016, the state approved a large-scale non-human primate scientific device. It will establish a large-scale, standardized management system, advanced equipment, primate model animal breeding, phenotypic and genetic analysis and research in Kunming, Yunnan. , Intelligent, precise, integrated and automated large-scale research facilities.

　　Among the many research institutions in China, the Chinese Academy of Sciences has always attached great importance to non-human primates, which has traditions and advantages. As early as 1959, the Chinese Academy of Sciences established my country’s first artificial domestication and breeding center for primates at the Kunming Institute of Zoology, and passed the certification of the international authority AAALAC (International Laboratory Animal Evaluation and Certification Committee) in October 2008. The use has reached the international level. It is one of the institutions with the longest research history and the largest variety of primates in China. On this basis, in recent years, in view of the increasing importance of non-human primates in the field of biomedicine, the Chinese Academy of Sciences has established multiple bases in Shanghai, Beijing and Shenzhen.

　　Combining with the country's strategic layout needs, after years of construction and development, my country's non-human primate research has formed a good situation with distinctive characteristics and local international leadership, and has formed a wide range of influences at home and abroad. Among them, our research on primate experimental animals has received special reports from internationally renowned journals such as Nature and Cell.

　　Facing such a situation, foreign scholars have worried that if such development continues, China may form a monopoly in the world of drug screening scientific research in the future, and then the pharmacodynamic evaluation of global drugs will be from China [1]. This concern from another aspect proves that the improvement of primate research capabilities will greatly enhance my country's independent innovation capabilities in the life sciences and biomedical industry technologies in the health field.

　　4 Current status of domestic brain disease non-human primate disease models

　　my country has abundant primate resources, but only after they are transformed into corresponding brain disease models can this huge potential be transformed into ability and strength to benefit patients. The commonly used brain disease models can be divided into three categories: spontaneous models, transgenic models and induced models. As there is no recognized spontaneous brain disease model at home and abroad, this article only introduces the development of domestic transgenic models and induced models.

　　4.1 Transgenic model

　　Humans have used non-human primates to study brain function and brain diseases for many years. There have been reports of successful transgenic macaques in the past, but why has it attracted great attention at home and abroad in recent years? For example, the brain project that Japan has launched is designed and implemented on the basis of marmoset monkeys. The main reason is that the transgenic technology of macaque has made a major breakthrough. In recent years, efficient and rapid gene targeting methods have emerged one after another. A series of efficient gene knockout systems such as TALEN and CRISPR/Cas9 methods have emerged at the level of fertilized eggs, making it possible to precisely edit rhesus monkey genes. This important prospect has attracted the attention and competition of many teams at home and abroad. Through close collaboration, a team of Chinese scientists has successfully applied these two technologies to macaques for the first time [2, 3, 4]. The success of this technology has made my country one of the few countries in the world that can develop non-human primate transgenic animal models for major human diseases, and has raised my country's non-human primate gene editing and genetic engineering work to the world's leading level. On this basis, in recent years, domestic non-human primate brain disease models have made rapid progress. In 2014, two independent domestic teams successfully constructed the first gene knockout of the neurodevelopmental disease Reiter syndrome related gene MECP2 in the world. The cynomolgus monkey provides an important animal model for further research on the neural mechanism of neurodevelopmental diseases. In 2015, another team used the CRISPR/Cas9 gene editing method to construct a non-human primate model of DMD gene mutation, which provided the possibility for further research on clinical intervention methods for DMD gene mutation. They also used lentiviral transgenic methods to construct a transgenic non-human primate model of Parkinson's disease. This transgenic model expresses a mutation in the human Parkinson's disease gene alphasynuclein. Transgenic monkeys carrying this mutation show a certain degree of cognitive dysfunction. Transgenic monkeys also have brain lesions similar to human Parkinson's disease patients. In January 2016, Nature published an online research paper entitled "MECP2 Transgenic Monkey's Autism-like Behavior Characterization and Germline Transmission", which was completed by the neuroscience research team of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. The study constructed a transgenic macaque model carrying the human autism gene MECP2 and conducted molecular genetics and behavioral analysis on the transgenic monkeys. It was found that the MECP2 transgenic macaque exhibited behaviors such as stereotyped behaviors and social disorders similar to human autism. The research team also successfully obtained the second generation of transgenic macaques carrying the human MECP2 gene through the method of testis allotransplantation, and found that they showed the same autism-like phenotype as their parents in terms of social behavior. This study is the world's first non-human primate model of autism, and provides an important foundation for in-depth study of the pathology of autism and exploration of possible therapeutic intervention methods. The successful establishment of the above series of models has further expanded my country's world leading position in transgenic non-human primate transgenic models.

　　4.2 Induction model

　　Thanks to breakthroughs in key technologies, transgenic models of non-human primate brain diseases have ushered in a golden age, and will shine in future neuropsychiatric research. However, since every living individual is the result of the interaction between environment and genes, with the exception of a few diseases where genetic factors dominate, most diseases are often caused by both environment and genes. For example, Alzheimer's disease and Parkinson's disease, which rank first and second in neurodegenerative diseases, account for less than 10% of genetic abnormalities, and the rest may be related to environmental factors. In depression, no clear risk genes have even been found. Therefore, as the relationship between genes and diseases has become clearer, finding the environmental predisposing factors of these diseases, and then using them to induce animal modeling methods for related diseases has also attracted the attention of scientists at home and abroad. Similarly, in this modeling strategy, non-human primates also have huge advantages: Because the living environment, metabolism and physiological processes of rats and mice are very different from those of humans, there is evidence that human risk factors may not Corresponding diseases are induced in rodents. Our research team has been committed to this work for a long time, and has made important progress in the induction of Alzheimer's disease and depression macaque models by environmental factors.

　　4.2.1 Alzheimer's disease model

　　Epidemiological surveys show that increasing age is the most important risk factor for Alzheimer’s disease. However, there are many factors that change with age in the body. What factors play a major role? Formaldehyde is a normal metabolite in the brain. Its concentration in the brain increases with age. The concentration of formaldehyde in patients with Alzheimer's disease is higher than that of controls of the same age. In vitro experiments have shown that it can cause Tau protein hyperphosphorylation and aβ aggregation, which is the pre-phenotype of the two typical pathological symptoms of Alzheimer's disease, neurofibrillary tangles and senile plaques. Therefore, formaldehyde may be a natural inducer of Alzheimer's disease. Through long-term low-dose intracranial injections to increase the formaldehyde in the brain to simulate the phenomenon of formaldehyde changes with age, macaques have all the key symptoms of Alzheimer’s: functionally, memory is steadily decreased for a long time; pathologically, neurofibrillary tangles, Age spots. In other words, regardless of exogenous or endogenous increase in brain formaldehyde, the core clinical symptoms and core pathological symptoms of Alzheimer’s can be induced in the macaque brain, suggesting that formaldehyde plays an important role in the occurrence and development of Alzheimer’s. Roles. This result provides a new perspective and means for the prevention and treatment of Alzheimer's disease, and it is also the first rhesus monkey model with all core symptoms of Alzheimer's disease at home and abroad.

　　4.2.2 Depression model

　　(1) The macaque depression model induced by social stress.

　　Depression is a multiple mood disorder, with an incidence of about 10%-15% in the population. There is no clear risk gene yet. Among environmental factors, stress and depression have a great correlation. An important feature of higher non-human primates is their strict and complex social structure. In this structure, low-status individuals have to bear greater social pressure, so it is a good animal to establish a social stress-related depression model. In foreign countries, researchers have established social stress-induced depression models by using cynomolgus monkeys living in groups. For the first time, a Chinese research team used macaques to establish a similar model, expanding the types of modeling animals, and laying a foundation for future research on depression in non-human primates. (2) Biological rhythm-induced depression model in rhesus monkeys.

　　 This is the first macaque model in my country. As we all know, depression is closely related to biological rhythms. In Northern Europe, the incidence of winter depression (also known as seasonal affective disorder) is about 30%. For the first time, a Chinese research team tried to establish a winter depression model in macaques, and found that short light can cause macaques to exhibit depression-like curling behavior, decrease in voluntary activity, and decrease in reactive activity. In addition to these depression-related behavioral abnormalities, these macaques also showed physiological abnormalities similar to those of winter depression patients, including weight loss, anhedonia, and increased cortisol. Further studies have found that antidepressant treatment can alleviate all depression-related symptoms exhibited by macaques, including depression-like curling behavior, decreased voluntary activity, decreased reactive activity, weight loss, reduced sugar consumption, and cortisol increase. In summary, this study observed the symptoms of winter depression in humans for the first time in rhesus monkeys, which can provide a good platform for future winter depression pathological research and drug development.

　　 (3) Early adversity-induced juvenile macaque depression model.

　　, etc.), adolescence and adulthood are prone to emotional disorders such as violent behavior and depression. At present, its formation mechanism is unclear. Using juvenile macaques to simulate early adversity by separating mother and baby, the results of our research team showed for the first time that the early adverse environment can cause long-term negative effects on young macaques, but no depressive symptoms were found in macaques. The team added chronic stress, an environmental factor closely related to depression, to the study of adolescent macaques separated from mother and child for the first time. Compared with the control group, the macaques of the mother-infant separation group experienced a decrease in free activity after chronic stress. Depressive behaviors such as depressive curling behavior and increased stereotyped behavior. The macaques of the mother-infant separation group also found an increase in cortisone secretion and depression symptoms of weight loss. These results show for the first time that early adverse environment and chronic stress can indeed cause depression in juvenile rhesus monkeys, laying a foundation for using rhesus monkeys to study the mechanism of early adversity. This is also the first macaque model in my country [13].

　　4.2.3 Establishment of two new models of macaque Parkinson's disease

　　 Pathological studies have shown that a large number of deaths of dopaminergic neurons in the brain are the cause of Parkinson's disease. MPTP is a compound that can specifically and efficiently kill dopaminergic neurons in the primate brain. By injecting it into rhesus monkeys, a classic MPTP rhesus Parkinson's disease model can be established. Several domestic units have been built and used. However, this model also has several serious flaws, which affect its widespread use. By injecting MPTP's metabolite MPP+ into the lateral ventricle and unilateral substantia nigra of macaques, the scope of drug action is limited to the central nervous system. For the first time in the world, the domestic team overcomes the large individual differences in the classical MPTP induction model, poor health and There are three major defects in the recovery of symptoms after drug withdrawal. Two new models of rhesus Parkinson’s disease, chronic and acute, have been established with good practical value.

　　4.2.4 Non-human primate model of advanced cognitive functions of the brain

　　 In addition to disease research, the biggest advantage of non-human primates is to study human advanced brain functions. At present, we know little about the evolutionary origin of a series of human advanced cognitive behaviors represented by self-awareness and language, especially the explosive development of cognitive behaviors during the evolution from monkeys to great apes and humans. At the same time, the lack of effective and suitable animal models has greatly hindered the in-depth study of the neural mechanisms of advanced cognitive functions such as self-awareness and language. Therefore, it is particularly important to establish a paradigm that uses macaques as a model to study human advanced cognitive functions.

　　 Self-awareness is one of the most important high-level cognitive functions of human beings, and one of its manifestations is to recognize the self in the mirror. In terms of development, human children do not have the ability to recognize themselves in the mirror until they are about two years old. In terms of evolution, the previously accepted view is that only humans and a few apes (such as chimpanzees) can recognize themselves. A large number of studies have tried Proving or training the monkey to recognize the self in the mirror ended in failure. The research team of the Institute of Neuroscience, Shanghai Academy of Biological Sciences, Chinese Academy of Sciences pioneered a visual sensory coupling training method, which proved for the first time that rhesus monkeys can learn to recognize the self in the mirror. This research fills a gap in the evolution of animal cognition and provides an ideal animal model and new research approach for studying self-recognition and self-awareness. At the same time, in certain neurological diseases such as intellectual disability, autism, schizophrenia, and Alzheimer's, patients will show impairment of self-awareness and cannot recognize themselves in the mirror. The research and its training program provide useful clues for clinical treatment of this lack of self-consciousness.

　　5 Suggestions for the development of non-human primate models in my country

　　 Through the above summary, it can be seen that my country not only has abundant non-human primate resources, but also has accumulated a good accumulation in the modeling of major brain diseases, especially in transgenic macaque models, we are already in a leading position in the world. Although scientists have established more efficient gene manipulation methods in non-human primates (such as lentiviral transfection and CRISPR/Cas9 gene editing), the transgenic individuals obtained by these two methods still have serious mosaicism. . Chimerism means that each first animal has a different genotype, which makes it almost impossible to use the first monkey for scientific research and large-scale drug screening applications. In theory, obtaining a large number of F1 generations of the same mutant individual can solve this problem, but the non-human primate experimental animals rhesus monkeys and cynomolgus monkeys have a long time of sexual maturity (4-6 years). Therefore, it can be said that the current non-human primate transgenic technology has not yet reached perfection, and needs to be improved and optimized. In the next step, we should actively carry out the research and application of new non-human primate transgenic technology and the accelerated passage of non-human primate models. On this basis, we should increase investment and step up the establishment of important non-human primate models, including transgenic and drug-induced models of brain diseases, to consolidate our leading advantages and seize the commanding heights. In addition, stepping up the development of tool monkeys is also a top priority. Tool monkeys refer to the use of modern and mature optogenetics technology to make optogenetic tool monkeys that can be applied to the study of cognitive behavioral functions of non-human primates, including those that express optogenetic tool proteins throughout the brain Transgenic monkeys can avoid the current shortcomings of low transfection efficiency of acute viruses on non-human primates, so as to study the causal relationship between high-level cognitive activities of the brain and neurons in non-human primates. Providing effective means will greatly promote the understanding of the working mechanism of the human brain. In addition, tool monkeys also include transgenic monkeys specifically expressed in specific types of neurons, such as Cre recombinase, which will also provide effective tools for the study of brain network connectivity maps.

　　 Another issue that should be highly valued is the development of phenotypic analysis techniques for non-human primate model animals. Phenotype, also known as trait, refers to the performance of individual morphology, function, etc., which is the result of the joint effect of genotype and environment. Therefore, various behavioral, physiological and pathological characteristics of the disease are also phenotypes. Although we can establish disease models through induction of genetic modification and environmental factors, phenotypic analysis is the basis for using these models for related disease mechanism research and drug screening. In other words, without effective phenotypic analysis methods, the model loses its meaning. Because mice and humans differ greatly in the complexity of individuals and behaviors, it is difficult to transplant existing rodent analysis systems to non-human primates. Due to historical reasons, non-human primate modeling has not been used on a large scale to study the history of brain diseases at home and abroad. Most non-human primate research at home and abroad is in a "small workshop" situation, lacking an integrated and systematic table. Type analysis system to study pathogenesis and drug evaluation. An efficient phenotyping analysis system should cover multiple disciplines such as behavior and neurobiology, genetics and molecular biology, genomics, reproduction and developmental biology, immunology, imaging, etc. The research level extends from molecules, cells, and tissues. To individual biological research. Whether a breakthrough can be made in the study of human disease primate models depends to a large extent on the establishment of a multidisciplinary, multi-domain, multi-level, automated and integrated comprehensive phenotypic analysis and evaluation system.