动物实验 z-bo 2020-08-27 472

　　The development of science and technology has gradually overcome the difficulties of various xenogeneic organ transplants. If one day your organs are exhausted and can no longer support your life, you don't have to wait for the 1% organ transplant opportunity. The (gene-edited) pig organs tailored for you will bring you new life.

　　1. Pigs are the most ideal donor for human organs

　　Since the application of immunosuppressive drugs in the 1980s, clinical organ transplantation has become the treatment of choice for advanced organ failure. However, there has been a serious shortage of organ sources all over the world, and people thought of finding relevant organ sources from animals. Regardless of ethics, reproductive characteristics, infectious disease risk, organ size and physiology, pigs are the ideal donors for human organs.

　　However, one of the biggest obstacles to the implantation of pig organs into the human body is hyperacute rejection. Therefore, avoiding this reaction is the key to xenotransplantation.

　　There is an antigen called α-1,3-galactose (α-Gal) on the cell surface of non-primates. In the long-term evolution of life, α-Gal has disappeared in primates, including humans. But there are natural antibodies against this epitope. Therefore, if the organs and tissues of other animals are transplanted into primates, the combination of the intrinsic antibodies and the α-Gal on the surface of the cells of the foreign tissues and organs will trigger the chain activation of the complement system, which can take tens of minutes to several hours. Kill the foreign graft within. This has produced a "hyperacute rejection".

　　Initially, people tried to produce transgenic pigs that could reduce or conceal the activity of galactosyltransferase through general transgenic technology, but the effect was not obvious. Only by breeding genetically modified pigs completely free of α-Gal can this rejection reaction be completely eliminated. Since 2002, Lai Liangxue’s American research team has overcome the two major technical obstacles of somatic gene targeting technology and somatic cell nuclear transfer technology, and has successfully obtained the world’s first "galactosyltransferase gene knockout cloned pig", Harvard University Medical School, USA The hospital also used the organs of the "double-gene knockout cloned pig with galactosyltransferase" to transplant them into baboons. The baboon survived for 83 days after the kidney transplantation, and the baboon survived for up to 6 months after the heart transplantation (the results were published in Nature Medicine). In the following ten years, scientists have carried out multi-gene modification on pigs based on the "double-galactosidase transferase knockout pig" to reduce the immune rejection of pig organs. Pig organs are in non-human primates. The survival time in the animals is also getting longer and longer.

　　In April this year, "Nature Communications" published a breakthrough in xenogeneic pig heart transplantation from the National Institutes of Health. The research team obtained a "three-gene modified transgenic pig" on the basis of the "galactosidase gene knockout homozygous pig". Scientists transplanted the heart of the genetically modified pig into baboons, assisted in antibody drug treatment, and finally allowed the xenogeneic pig heart to survive in the baboon for an average of 298 days. One of them survived for 945 days, setting a new record for xenogeneic organ transplantation.

　　There is a consensus in the field of xenotransplantation that if pig organs can survive for one year in baboons, human transplantation experiments of pig organs can be carried out. Therefore, clinical trials of pig heart transplantation into humans are just around the corner.

　　2, xenogeneic islet transplantation or the first clinical application

　　As early as the 1920s, the medical profession discovered the function of insulin. At the beginning, scientists discovered insulin and insulin-producing pancreatic islets in dogs. They thought it was very interesting-usually inactive, but once the glucose concentration in the surrounding environment increases, the pancreatic islets will secrete insulin to regulate various organs and tissues in the body Metabolize and decompose glucose. With the support of this theory, the biomedical and chemical circles have been looking for the most suitable source of insulin for diabetic patients.

　　Pancreatic islets are hidden in the pancreas, and they are only 150-400 microns in size. The human pancreas contains 750,000 to 2 million islets, and generally only 200,000 to 500,000 islets can be retained after purification.

　　In the 1950s, British scientists first determined the entire amino acid sequence of bovine insulin, which opened the way for humans to understand the chemical structure of protein molecules. On September 17, 1965, Chinese scientists artificially synthesized crystalline bovine insulin with full biological activity. It was the first protein synthesized artificially in the laboratory. However, there is a three amino acid difference between bovine insulin and human insulin, so the potency is lower, and larger doses are required for treatment, and it is more prone to drug dependence (the more the insulin is, the more it is).

　　In the 21st century, biomedical technology has been able to directly industrialize the production of human insulin. But even if a sufficient dose of insulin is produced, there are still many problems. For example, short-acting insulin requires one injection after eating; long-acting insulin requires two injections a day, and so on. And the longer the patient gets sick, the larger the dose often used. Therefore, human islets obtained from donors for transplantation have been tried as early as 1977. As of 2012, a total of 1,400 people worldwide have received human islet allotransplantation, but the success rate of transplantation is only 58%. Islet transplantation itself is not technically difficult. Compared with other organ transplants such as liver transplantation and kidney transplantation, islet transplantation is a cell transplantation, and the risk of transplantation is relatively small. But the source of pancreatic islet organs is very limited.

　　Pancreatic islet donations derived from humans are too scarce, and the medical profession has turned its attention to other animals. For example, pigs and cattle, which are also mammals. They are very similar to humans at the genetic level, exceeding 80%. Specific to the level of islets and insulin, the difference is even smaller. Doctors from China and New Zealand have been conducting research on animal-derived islet transplantation. For example, Wang Wei's research team from the Third Xiangya Hospital of Central South University conducted a review of 22 diabetic patients undergoing "pig-human" islet transplantation. The results showed that the insulin consumption of 20 patients after the operation was reduced by more than 30%. Effective"; 6 cases were reduced by more than 50%, achieving "significant curative effect", and one of them was "temporarily cured" without insulin for a week. The key to this research is that the bio-safety problems of direct transplantation of islets from pigs did not arise. Patients who have used porcine pancreatic islets are not more susceptible to swine flu or Streptococcus suis. But the effect of this treatment for diabetes is still not as good as human islet transplantation, after all, there is an amino acid difference between the two.

　　Recently, the relevant team of Xiangya Hospital has applied the new technology of inducing immune tolerance to the xenogeneic treatment of diabetes in porcine islets, achieving a clinical effect that is very close to that of human pancreatic islet transplantation. The "homozygous humanized insulin genetically modified pig" obtained in this study will provide human insulin for the treatment of diabetes, and will also provide a more ideal source of donors for the treatment of clinical xenogeneic islet transplantation. At present, Lai Liangxue’s research team is also speeding up the reproduction of the “humanized insulin gene-modified pig” through cloning technology and normal animal breeding technology. It is trying to modify the pig insulin coding gene to directly encode and produce human insulin. -After 3 years of non-human primate porcine pancreatic islet transplantation, the clinical trial will be officially carried out.

　　3. The offspring of "genetically modified pigs" are "relative pigs"

　　A few days ago, the Lai Liangxue research group of the Guangzhou Institute of Biomedicine and Health of the Chinese Academy of Sciences, after precise modification of specific genes of the piglet, the piglet and its offspring and grandchildren born into this kind of human secretion The "kind pig" of insulin.

　　After the 30-35-day-old piglet fetus is processed, the researchers can obtain the piglet fibroblasts in advance. After precise modification of the specific genes of the obtained piglet fibroblasts, the gene-edited fibroblasts are used to replace the nucleus of the pig eggs by cloning technology, so that new pig cloned embryonic cells can be generated and then replanted. Into the sow's fallopian tube. The rest of the pregnancy and childbirth work is done by the piglet's mother.

　　In order to meet the needs of medical clinical trials, it is necessary to expand the breeding of this type of pig herd. After all, a type I diabetic islet transplantation may require about 20 piglets to provide it. Only after expansion, whether it is to use these pigs to produce human insulin, or just to purify the islets of pigs for islet transplantation, it will be more convenient.

　　This technology is still under follow-up research. In the next step, the researchers hope to be able to transplant the gene-edited porcine islets in monkeys, the primate. Then it is expected to enter clinical trials within 5 years and then enter clinical applications. At present, the relevant research of this group has been published online in the internationally renowned Journal of Molecular Cell Biology.

　　In fact, modern medicine has discovered that most of all genetic diseases in humans belong to clear genetic point mutation diseases. After clarifying the pathogenesis, scientists have been able to simulate human diseases through pigs and other organisms. Through gene editing technology, pigs can even suffer from diseases such as thalassemia, Parkinsonism, aging, and then use these "human disease" pigs to test drugs for others, and finally select effective treatment drugs and treatment strategies, and in turn Cure people's diseases. This is the huge role played by "model organisms" in the process of selecting drugs for modern medicine.

　　(Source: Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences)

　　Research on gradual freezing disease——gradual freezing pig

　　The popular "Ice Bucket Challenge" earlier was launched to call the world to pay attention to "Frozen People". Amyotrophic lateral sclerosis, commonly known as "gradual freezing syndrome", is an incurable and fatal neurodegenerative disease. Clinically, the patient showed mixed damage of upper and lower motor neurons, resulting in muscle stiffness, twitching and gradual atrophy of the ball, limbs, trunk, chest and abdomen. Eventually, the patient died due to difficulty in swallowing and breathing.

　　The currently known pathogenic genes of gradual freezing are mainly SOD1, TDP-43, FUS, etc. However, research on transgenic mouse models has been difficult to simulate this typical pathological change in patients. The Lai Liangxue research group of the Guangzhou Institute of Biomedicine and Health of the Chinese Academy of Sciences and the Li Xiaojiang research group of the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences have successfully established the "transgenic Huntington's disease pig" and the "transformed mutant SOD1" using the method of somatic cell nuclear transfer. Genetically frozen pig model.

　　Later, they cooperated to establish a "transgenic TDP-43 Tibet minipig" model. The transgenic pig showed the symptoms of "gradual freezing of humans", and there was a phenomenon similar to the accumulation of neurocytoplasmic TDP-43 in patients with human diseases. This study not only reveals the new pathogenic mechanism of TDP-43, but also suggests that large animal models can show characteristics closer to the pathological changes of patients.

　　Relevant human experiments have not yet been approved

　　The latest news from the Wall Street Times stated that in the United States, more than 120,000 people are waiting for organ transplants, but there are not enough donors. This severe shortage puts researchers on the agenda for an unusual solution: using genetically modified pigs to provide organs for human transplantation.

　　In fact, this xenotransplantation research has been going on for decades. Last year, a group of researchers from Harvard University in the United States published a paper saying that they have used a new gene editing technology CRISPR-Cas9 to basically knock out the virus residues in pig genes. Therefore, there is even news that the United States may start conducting related experiments on humans next year.

　　But the International Xenotransplantation Association updated its statement this year, stating that xenogenes that have obtained modified islets of genetically modified pigs must survive at least six months or more before conducting human clinical trials. They certainly hope that the first patient to participate in the experiment will benefit, but after all, we don’t know whether the pig gene still carries some unknown virus, which may be harmless to pigs, or harmless to other animals, but may be infected. Humanity. At present, the US Food and Drug Administration's attitude towards the application for initiating human clinical trials is still conservative: this kind of xenotransplantation can only be limited to those who have certain diseases that are severe to life-threatening, and the premise should be other treatments. It is not enough to treat it.

　　However, Dr. Tector, who participated in the relevant research, said that for some older patients, they may not be able to wait for a matching organ for transplantation before they die, so trying to transplant a transgenic pig organ is not a problem. Good thing.

　　The birth of the first cloned pig

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　　Because the physiological characteristics and tissue cell structure of pigs are very similar to those of humans, research on gene modification and induced pluripotent stem cells (iPS) of pigs and other large animals has attracted the attention of scientists from all over the world.

　　In 2002, Lai Liangxue’s American research team successfully obtained the world’s first "galactosyltransferase gene knockout cloned pig". This is the world's first "gene knockout pig" (or "gene targeted pig"). In 2011, with the joint efforts of Dr. Lai Liangxue, who worked at the Guangzhou Institute of Biomedicine and Health of the Chinese Academy of Sciences, Dr. Xiao Lei from Zhejiang University, and Dr. Du Yutao from the BGI Institute of Genetics, the research team of Lai Liangxue took the lead in the world’s first survival "IPS clone pig".

　　After obtaining gene-targeted iPS cells, on the one hand, germline chimeric animals can be obtained through chimera technology, and then “genetically modified animals” can be obtained through mating; on the other hand, these gene-targeted iPS cells can be used as nuclear donors. Obtain "genetically modified cloned animals" through nuclear transfer technology. Through long-term research on the isolation and culture of human and animal embryonic stem cells, the establishment of transgenic animals, animal cloning and human therapeutic cloning, Lai Liangxue has published more than 100 related papers and successfully established more than 30 species in the fields of biomedicine and agriculture. Transgenic pigs with important uses.