Differentiated cells often lose the ability to redifferentiate completely because of high differentiation, and such cells will eventually age and die. However, in the process of animal development, some undifferentiated cells will remain in the body, which are stem cells. The aging of stem cells is an important factor in the aging of the body or human beings. Therefore, transplanting (or injecting) human stem cells is of great significance to prevent human aging. Stem cells, also known as origin cells and universal cells, are a kind of cells with self-renewal and differentiation potential. It can be said that the animal body realizes cell renewal through the division of stem cells, thus ensuring the sustainable growth and development of the animal body.
Stem cells can be divided into two types according to their differentiation potential: totipotent stem cells and tissue stem cells. The former can differentiate into a complete animal individual, and the latter is the origin cell of one or more tissues and organs. Human embryonic stem cells can develop into complete human beings, so they belong to totipotent stem cells.
As early as19th century, developmental biologists knew that an egg would divide quickly after fertilization. First, 1 fertilized egg divides into two cells, and then continues to divide until it divides into a cell mass with 16 to 32 cells, which is called a morula. At this time, if the cells that make up the morula are separated and implanted into the mother's uterus, each cell can develop into a complete embryo. This kind of cell is embryonic stem cell, which belongs to totipotent stem cell. Tissue stem cells can be obtained from bone marrow, umbilical cord, placenta and fat. Everyone has some stem cells in his body that accompany him all his life. However, the older people get, the less stem cells there are. In order to make up for the shortage of stem cells, some scientists suggest obtaining stem cells from embryos or fetuses and other animals. Conduct training and research. Stem cells are widely used in many fields of medicine. Scientists have been able to identify, isolate, purify, expand and culture human embryonic stem cells in vitro, and use these stem cells as "seeds" to cultivate some human tissues and organs. The extensive clinical application of stem cells and their derived tissues and organs will produce a brand-new medical technology, that is, to reconstruct normal or even young tissues and organs of human body, so that people can use their own or others' stem cells or stem cell-derived new tissues and organs to replace their sick or aging tissues and organs. If an elderly person can use stem cells and their derived tissues and organs preserved by himself or others in infancy or adolescence, his life span can be significantly prolonged. 1999, American Science magazine listed stem cell research as one of the top ten scientific achievements in the world, ahead of human genome sequencing and cloning technology.
The National University Hospital of Singapore and Central Hospital cured a boy suffering from severe thalassemia due to family inheritance through umbilical cord blood stem cell transplantation, which is the first operation in the world to transplant unrelated umbilical cord blood stem cells and make the patient recover. Doctors believe that umbilical cord blood stem cell transplantation is not complicated, just like giving blood to patients. Because of the inherent characteristics of umbilical cord blood, stem cell transplantation from umbilical cord blood is more effective than bone marrow transplantation. Hematopoietic stem cell transplantation has gradually become an important means to treat diseases such as hematopoietic system and immune system dysfunction caused by leukemia and various malignant tumors after radiotherapy and chemotherapy. Scientists predict that replacing damaged nerve cells with neural stem cells is expected to make patients with spinal cord injury and paralysis stand up again; In the near future, patients with blindness, Parkinson's disease, AIDS, Alzheimer's disease, myocardial infarction and diabetes are expected to recover with the help of stem cell transplantation.
Compared with embryonic stem cells, adult stem cells can only develop into more than 20 kinds of tissues and organs, while embryonic stem cells can develop into almost all tissues and organs. However, if stem cells are extracted from embryos, the embryos will die. Therefore, ethical issues have become one of the biggest problems in embryonic stem cell research. The U.S. government explicitly opposes destroying new embryos to obtain embryonic stem cells, and the U.S. House of Representatives even proposed a bill to completely ban embryonic stem cell cloning research. Some scientists in the United States have sharply criticized this. They believe that the use of stem cells in medical research has great potential in alleviating the suffering of patients. If such a good opportunity is wasted, the result will be tragic.
Life science is one of the fastest developing disciplines in the 20th century, and it has become the most striking field in natural science. 1957 donar Thomas of the University of Washington, USA, found that transplantation of normal bone marrow into patients can treat hematopoietic dysfunction. The discovery of this technology won Donna Thomas the Nobel Prize.
This technology was quickly recognized by the whole world and became the main means to treat leukemia and other diseases. The discovery and application of hematopoietic stem cell transplantation technology has brought new hope for human beings to overcome diseases.
1999 Petersen et al. found that liver stem cells and some hepatocytes may be partly derived from or related to bone marrow. They verified this idea through the following experiments: (1) Transplanting the bone marrow of a male rat into a homologous female rat irradiated with lethal dose, and using DNA probe to detect whether there is a male Y chromosome in the liver of the recipient rat. ⑵ Lewis rats expressing histocompatibility antigen ⅱ antigen L2 1-6 were used as recipients, and Brown-Norway rats not expressing L2 1-6 were used as total liver transplantation donors to determine whether L2 1-6 positive cells from extrahepatic sources could be located in the transplanted liver. They found that 13 days after bone marrow transplantation, Y chromosome signal was detected in the liver, and oval cells began to differentiate into hepatocytes at this time. If the oval cells differentiated into hepatocytes come from the liver, no hepatocytes will express positive Y chromosome signals, but the results show that some hepatocytes express obvious Y chromosome signals, indicating that they come from bone marrow donor cells. Similarly, after total liver transplantation, there were obvious L2 1-6 positive cells in the transplanted liver, which indicated that some oval cells came from outside the liver, while L2 1-6 from inside the liver was negative. Experiments show that bone marrow contains stem cells that can differentiate into hepatocytes, and some oval cells may come from bone marrow.
Prehepatic cells in bone marrow can be used for transplantation treatment of liver failure, without considering the matching problem of histocompatibility antigen, because the patient's own bone marrow cells can be used for transplantation. Bone marrow cells have the following advantages: ① Bone marrow cells rich in stem cells can be prepared. ⑵ Promoting gene transduction can increase liver cells derived from bone marrow. ③ Bone marrow-derived hepatocytes can be used for bioartificial liver; In addition, HGF can also be used to treat liver cirrhosis by promoting the differentiation of pre-hepatocytes including bone marrow stem cells. Autologous bone marrow stem cell transplantation for liver injury will provide a new way for the treatment of liver diseases. The basic principle of stem cell therapy is to repair damaged tissue cells, replace the function of damaged cells and stimulate the regeneration function of the body's own cells.
Respiratory diseases
Autologous stem cell immunotherapy for asthma, tracheitis, emphysema and cor pulmonale.
Stem cell immunotherapy is to repair damaged tissue cells by regulating cytokines, and then inhibit the proliferation and immune response of damaged cells through the interaction between cells, thus playing the role of immune reconstruction. Fundamentally eliminate the pathogenesis of asthma. These treatments are completely different from traditional treatments in concept, and mainly emphasize the treatment of respiratory diseases such as asthma by repairing human immune cells. Beijing Jinghua Friendship Hospital has been clinically proved by modern medicine that stem cell immunotherapy has obvious therapeutic effect on asthma symptoms such as cough, excessive phlegm and chest tightness. It has the advantages of quick curative effect, short course of treatment and difficult recurrence, and breaks through the disadvantages of "effective treatment-recurrence after drug withdrawal" in the past. The antiasthmatic stem cells specially cultured in the cell culture laboratory can enhance patients' immunity, relax smooth muscle, promote metabolism in vivo, repair respiratory system injury, activate lung cell regeneration, comprehensively regulate spleen, lung and kidney, activate lung cell regeneration to repair lung ventilation function, enhance lung function, provide sufficient oxygen supply for lungs, completely repair lung and airway mucosa, and restore cilia's ability to discharge pollutants. After 100 clinical cases, the cure rate can reach 98%. It is the most ideal and standardized treatment method for asthma and tracheitis at present, and it can control the disease for a long time with the help of traditional Chinese medicine in the later stage.
Treat nephropathy
The principle of stem cell transplantation in the treatment of nephropathy: Because stem cells have unlimited proliferation and multi-directional differentiation potential, they have the characteristics of hematopoietic support, immune regulation and self-replication. It can be used as an ideal "seed" cell to repair tissue and organ damage caused by pathological changes. Basic research has found that stem cells can differentiate into renal intrinsic cells and renal parenchymal cells, so stem cell transplantation has a good repair and reconstruction effect on renal function.
Characteristics and advantages of stem cells in treating nephropathy Stem cells have strong proliferation ability and multi-directional differentiation potential, and can proliferate and differentiate to produce a large number of offspring. Low immunogenicity. Because the cells are in the primitive state, they are not easy to be recognized, so there is no immune rejection and there is no blood type matching problem. Long-term passage does not change biological characteristics. The ability to differentiate into renal proper cells, muscle cells, hepatocytes, osteoblasts, chondrocytes and other cells. It is precisely because of these immunological characteristics and advantages that stem cells have broad clinical application prospects in the treatment of nephropathy.
Treat cerebral palsy
Stem cell transplantation for children with cerebral palsy is gradually known. Stem cell transplantation is based on the potential of self-renewal and differentiation of cells into neurons, astrocytes and oligodendrocytes. The differentiated neurons after cell transplantation can supplement the defective neurons, promote the differentiation and function of nerve cells in children's brain tissue, restore the normal growth and development of cranial nerves, improve the cognitive dysfunction of the brain, and provide more opportunities for the further rehabilitation of children's cerebral palsy. It has always been the most advanced and effective treatment method. Moreover, the younger the age, the stronger the compensatory ability and the greater the possibility of treatment. Early intervention and treatment is the only way to prevent children with cerebral palsy from being disabled.
1. Self-renewal: Stem cells can divide symmetrically and asymmetrically, thus maintaining the stability of the stem cell bank.
Multi-directional differentiation potential: Stem cells can differentiate into neurons, astrocytes and oligodendrocytes. Low immunogenicity: Stem cells are undifferentiated primitive cells that do not express mature cell antigens and are not recognized by the immune system.
3. Good tissue fusion: it can be well fused with the nerve tissue of the host and survive in the host for a long time.
Treat autism
Umbilical cord blood stem cells and umbilical cord mesenchymal stem cells have the functions of immunomodulation and improvement of cerebral microcirculation. Stem cells can regulate the immune function of the body, stimulate the formation of new blood vessels through their own differentiation and secretion of cytokines and neuropeptides, improve the ischemic and anoxic state of the brain, and activate and repair damaged nerve cells in the brain. Combined transplantation of umbilical cord blood mononuclear cells and umbilical cord mesenchymal stem cells is helpful to improve children's language communication ability and social communication ability. Autoimmune liver disease is a special type of chronic liver disease caused by autoimmune reaction. In the past, autoimmune liver disease was considered rare. Due to the deepening understanding of this kind of disease and the introduction and improvement of related immunological examination methods, the number of patients with autoimmune liver disease in China is increasing. Clinical common autoimmune liver diseases include autoimmune hepatitis, primary biliary cirrhosis and primary sclerosing cholangitis. Many patients with autoimmune liver diseases are accompanied by Sjogren's syndrome, rheumatoid arthritis and other autoimmune diseases.
The research team led by the director of the Liver Disease Center of Beijing 304 Hospital conducted in-depth research on the pathogenesis, pathogenesis and immunotherapy countermeasures of autoimmune liver disease. International conference identified autoimmune liver disease as non-viral infectious autoimmune disease. The patient's immune regulation function is defective, which leads to the body's reaction to its own hepatocyte antigen. Traditional treatments are mainly immune preparations and hormones, but immunosuppression therapy and hormone shock therapy have certain effects in the early stage. At the stage of liver cirrhosis, not only the curative effect is not obvious, but also the adverse reaction of hormones is obviously aggravated.
Since it is an autoimmune disease with similar pathogenesis, can stem cells be used for treatment? After communicating with rheumatologists, Director Gong decided to adopt umbilical cord mesenchymal stem cell transplantation scheme. Director Gong said that umbilical cord mesenchymal stem cells have immunomodulatory effect, and can repair and immunomodulate autoimmune diseases, so as to achieve the purpose of treating diseases, such as systemic lupus erythematosus, pemphigus, rheumatoid arthritis, scleroderma, dermatomyositis, etc., which have been carried out in Rheumatology Immunology Department, and achieved very good results.
Application prospect of embryonic stem cells, animal cloning and human therapeutic cloning, application in transgenic animals and preparation of chimera animals.
The research of embryonic stem cells faces a difficult problem. In vitro culture of ES cells, it is necessary to choose suitable culture conditions and balance the contradiction between proliferation and differentiation. They are highly undifferentiated and may form teratomas. Organ cloning and transplantation still need technical breakthroughs.
Separation method
From 65438 to 0998, two groups in the United States cultivated human pluripotent stem cells:
A research team led by James A. Thomson of the University of Wisconsin has cultivated stem cell lines from human embryonic tissues. Cell strain. The cell surface markers and enzyme activities of these cell lines were tested, and it was confirmed that they were pluripotent stem cells.
At present, the main sources of embryonic stem cells are cell groups in blastocysts and primordial germ cells in reproductive ridges.
Immunological methods: There are many special marks on the surface of stem cells. Using these markers, stem cells were separated and purified from single cell suspension by fluorescent cell separator.
Immunosurgery: the basic principle of this method is to use the impermeability of blastocyst cavity to antibody, remove trophoblast cells through the toxic killing effect of antibody combined with complement, and keep CIM for culture.
Tissue culture: 4-6-day-old embryos are taken out for culture, and the trophoblast grows flat on the bottom of the Petri dish, while CIM forms an oval columnar structure. This columnar structure was picked out with a glass needle under a microscope and digested for passage.
Microsurgical methods: CIM was directly sucked out of blastocysts for culture under microscope.
Ethical debate
Although human embryonic stem cells have great potential for medical application, ethical issues surrounding this research have also emerged. These problems mainly include whether the source of human embryonic stem cells is legal and moral, and whether the application potential will cause ethical and legal problems. Can ES cells obtained from human embryos fertilized in vitro develop into adults under appropriate conditions? What if stem cells come from pregnant women who voluntarily terminate pregnancy? Is it moral to kill human embryos to obtain ES cells? Can good wishes justify evil means? Is it appropriate to use cells from spontaneous abortion or accidental abortion embryos? Some people think that it is immoral to collect embryonic stem cells from human embryos, because human life is not valued and human embryos are also a form of life. No matter how noble the purpose is, it is unthinkable to destroy human embryos. Some people think this is moral, because scientists didn't kill cells, they just changed their fate. Some people worry that in order to obtain more cell lines, the company will fund in vitro fertilization to obtain blastocysts and abortion to obtain fetal tissues. They suggested that adult stem cell research should be encouraged and embryonic stem cell research should be abandoned.
If embryonic stem cells and embryonic germ cells can be bought and sold as cell lines, is it ethical for scientists to use them? What kind of research is acceptable? Can scientists be allowed to cultivate individual tissues and organs for studying the development process or establishing medical transplant tissues? Since it has been accepted that human genes can be implanted into animal cells, is it moral to implant human embryonic stem cells into livestock embryos to make chimeras to obtain transplanted human organs? Is it ethical to change the genes of es cells from embryos with genetic defects so that they can continue to develop into healthy individuals for treatment? If people's alternative organizations are easy to obtain, will more people live irresponsibly and engage in high-risk activities? These questions are difficult to answer simply, so we must seriously study the ethical, social, legal, medical, theological and moral issues involved in the research of human embryonic stem cells.
Considering that American law prohibits the use of government funds to fund human embryo research, Professor Womas, director of the National Institutes of Health (NIH), has consulted the US Department of Health and Welfare (DHHS), the government department in charge of NIH. DHHS decided in 1998 12: "The bill of the United States Congress prohibiting human embryo research does not apply to embryonic stem cell research, because according to the current definition, embryonic stem cells are not equal to embryos." In addition, "embryonic stem cells cannot be regarded as human embryos because they do not have the ability to develop into individuals by themselves after being implanted in the uterus." Therefore, DHHS can fund the research of embryonic pluripotent stem cells. As for human embryonic germ cells, because embryonic germ cells come from an inactive fetus, the acquisition and use of such cells meet the requirements of federal law on fetal tissue research, so they can also receive DHHS funding. People have different reactions to this decision. Seventy-three famous American scientists (including 67 Nobel Prize winners) immediately jointly expressed their support, saying that this decision is worthy of appreciation and foresight (Science, 1999, vol. 283: 1849). A certain kind of research has attracted the attention of so many Nobel Prize winners, which is unique in the history of science, which also reflects the importance and arduousness of embryonic stem cell research from one side. Several influential academic groups in the United States, such as the American Federation of Experimental Biology Societies, the American Society of Cell Biology and the American Society of Developmental Biology, also support the decision that federal funds can fund human embryonic stem cell research. Democratic Senator Tom. Harkin said that this decision will pave the way for scientific discovery of new treatments for many diseases, and stressed that the government should not ban medical research. Womas, director of NIH, said that the prospect of this research work will be brilliant, but he reminded researchers that it is still illegal to use federal funds to obtain new embryonic stem cell lines, but scientists can use federal funds to study human embryonic stem cell lines obtained by Thomson and Gearhart.
However, DHHS's regulations on embryonic stem cell research have been opposed by some people in Congress, churches and human rights organizations. Catholic Doyle Foehlinger accused this regulation of seriously violating the spirit of the law: "They will destroy embryos with private funds and conduct embryo experiments with federal funds." 1In February, 1999, 70 members of the House of Representatives wrote to the Minister of Health and Welfare asking for the abolition of this provision, saying that it "violated the letter and spirit of the federal law of the United States government prohibiting the funding of experimental research that destroys human embryos". Judy Brown, president of the American Life Alliance Human Rights Organization, protested the use of stem cells because they come from embryos that can develop into adults and should be protected by American law. Congressman Jay Dickey strongly opposed the law and even wanted to take DHHS to court. He believes that the law does not allow federal funds to be used for embryonic stem cell research, and there is no need to make any changes. He emphasized that "science should serve people, not human science". Anti-abortion activists demanded that Congress intervene and stop such research. On the basis of widely listening to opinions from all sides, NIH finally published the Guiding Principles of Embryonic Stem Cell Research in1June 5th, 999+June 5th+February.
It can be seen from the table that it is illegal to obtain new embryonic stem cell lines from human embryos by Thomson method, but it is allowed to study the cell lines obtained from human embryos. It is relatively tolerant to obtain, use and study cell lines from fetal tissues by Gearhart method. Although the regulations are still harsh, it opens the door for the research of human embryonic stem cells.
It is worth mentioning that shinya yamanaka, the Nobel Prize winner in 20 12, made it possible for us to reconvert completely differentiated cells such as skin cells into stem cells without obtaining stem cells from human embryonic cells, which became the morphological detection of IPS: small size, large nucleus, high nucleoli-cytoplasm ratio, one or more prominent nucleoli, few euchromatin and cytoplasm, and simple structure. In vitro culture: Cells are arranged closely to form colonies. Alkaline phosphatase staining showed that the cells were reddish brown and the surrounding fibroblasts were pale yellow. The boundaries between cell clones and their surroundings are obvious, and the boundaries between cell clones are unclear and diverse, mostly island-like or nested. Detection of alkaline phosphatase activity-in vivo differentiation experiment of dyed deep blue purple: in vitro differentiation experiment of teratoma: Karyotype analysis of cystic simple embryoid or embryoid and common types of cells mixed together: OCT activity detection of diploid normal karyotype: pluripotent gene marker. OCT gene expression products were detected by OCT antiserum and indirect immunofluorescence.