WO2006047954A1

WO2006047954A1 - The method of obtaining and applying the materials for autoantigen immunological recognition

Info

Publication number: WO2006047954A1
Application number: PCT/CN2005/001842
Authority: WO
Inventors: Ziyi Cheng
Original assignee: Ziyi Cheng
Priority date: 2004-11-04
Filing date: 2005-11-04
Publication date: 2006-05-11

Abstract

A method of obtaining and applying the materials required for autoantigen immunological recognition after birth to treat rejection and immune system diseases. Especially, the method of making and obtaining early immunological recognition hemopoietic stem cells and/or late immunological recognition hemopoietic stem cells required for autoantigen immunological recognition, and obtaining the immune relating tissues and organs of the autoantigen immunological recognition period, comprising: the autoantigen immunological recognition relating cells and factors etc. in the thymus and marrow. The method for treating rejection and immune system diseases to carry on the autoantigen immunological recognition to each tissue, organ and graft antigen with autoantigen variations. The present method could not only treat multiple diseases conditions related to the immune disorders and the rejection after organ graft, but also treat varieties of diseases for different organs of the receptors. The present invention could be applied not only to human beings, but also to animals.

Description

TECHNICAL FIELD The present invention relates to a method and method for obtaining raw materials required for re-immunization of self-antigens after birth by treating rejection and immune system diseases. In particular, an early immune recognition hematopoietic stem cell and/or a late immune recognition hematopoietic stem cell required for re-imaging the self-antigen immune recognition, and a tissue organ related to immune recognition at the time of obtaining an autoantigen immune recognition period, including: thymus Cells and factors related to immune recognition of autoantigens in the bone marrow. And a treatment method for re-imaging the autoantigen by recognizing the tissues and organs and the graft antigens of the autoantigens in the body for the treatment of rejection and immune system diseases. This method can not only treat a variety of diseases associated with diseases of the immune system and rejection reactions after organ transplantation, but also treat a variety of different organs and different types of diseases of the recipient. The invention can be used not only in humans but also in animals. BACKGROUND OF THE INVENTION Noun annotations used in the present invention, and methods for obtaining and applying materials required -

1. According to the immune system of human or animal, the immune recognition period of self antigen is divided into -

1) In the period from fertilized eggs to embryos, or pre-fetus, the immune system has not yet been immune-recognized by autoantigens. This period is "pre-immune identification of autoantigens."

2) The period during which the autoantigen is being immunologically recognized during the embryonic period is the "autoantigen immune recognition period".

3) The period in which the autoimmune recognition of autoantigens has ended in the infancy and postnatal infancy and in adulthood is "the end period of autoantigen immune recognition".

Therefore, early autoantigen immune recognition of hematopoietic stem cells is an immunologically recognized hematopoietic stem cell in the pre-immune recognition of autoantigens and in the autoimmune recognition phase of autoantigens. The late immune recognition hematopoietic stem cells are immunologically recognized hematopoietic stem cells at the end of the autoantigen immune recognition.

4) Early immune recognition Hematopoietic stem cells may include: hematopoietic stem cells associated with immune recognition, and/or immune recognition, and/or group cells, etc., and/or in autoantigen immune recognition pre- and/or autoantigen immunological phase, and/or The differentiated cells, and/or the various differentiated mature blood system cells associated with immunity, early immune recognition hematopoietic cells are cells that can be self-immunologically recognized under certain circumstances. For example: hematopoietic pluripotent stem cells (including Haematopoietic Stem Cells), and/or various immune system-related progenitor cells (immune pluripotent stem cells) such as: myeloid stem cells, lymphoid stem cells, and/or various Immune-related unipotent stem cells, such as: granules, monocyte stem cells, precursor B cells, precursor T cells, megakaryocyte stem cells, and cells secreting autoantigen immune recognition factors, and/or secreting autoantigen immune recognition factors Cells, etc., various immune-related cells (Note: In actual application, it should be decided to use embryos, or fetal hematopoietic stem cells or immune-related types of immune stem cells, depending on the actual situation, which type and which Stage cells). These types of cells are referred to in this specification as abbreviations: Early immune recognition of hematopoietic cells.

5) Late immune recognition Hematopoietic stem cells may include: stem cells that are associated with immune recognition and/or immune recognition, or differentiated immature cells, or various types of stem cells related to immunity, at the end of autoantigen immune recognition, And/or undifferentiated mature cells, late II immunoreactive hematopoietic stem cells are generally unable to carry out autoantigen immune recognition, only in special cases involving "organizations, organs, cells, factors related to autoantigen immune recognition" It is possible to carry out self-antigen immune recognition. For example: hematopoietic pluripotent stem cells (including Haematopoietic Stem Cells), and/or various immune-related progenitor cells (immune pluripotent stem cells) such as: myeloid stem cells, lymphoid stem cells, and/or various Immune-related unipotent stem cells, such as: granules, monocyte stem cells, precursor T cells (ie, proT and early proT cells), 'precursor B cells (ie, proB and early proB cells), monocytes (DC) megakaryocytes And so on and various immune-related cells (Note: In practical applications, The type and stage of the cell should be used according to the actual situation. These types of cells are referred to in this specification as "late immune recognition hematopoietic stem cells".

Immune recognition of hematopoietic stem cells includes: early immune recognition of hematopoietic stem cells, and/or late immune recognition of hematopoietic stem cells. In the present specification, immunologically recognized hematopoietic stem cells are also referred to as non-immunologically recognized stem cells, or immunologically recognized hematopoietic stem cells.

2. "The tissues, organs, cells, and factors related to the autoimmune recognition of autoantigens" are the "preimmune recognition of autoantigens", and/or the embryonic and fetal stages of human or animal immunization recognition period, early immune recognition Hematopoietic stem cells can perform self-antigen immune recognition period, participate in early immune recognition of hematopoietic stem cells, and/or tissues, and/or cells, and/or hormones related to immune recognition, which are immunologically recognized by autoantigens in the body. Cytokines, peptides, proteins, and more. Although all the tissues and organs involved in the recognition of autoantigens have not been fully understood so far, they have been discovered through medical experiments such as: organs such as thymus, spleen, lymph nodes and bone marrow, and various cells and hormones contained in these organs. , various cytokines, polypeptide proteins, tissues, tissue fluids, cells, and the like. (See "Cellular and Molecular Immunology" Chapter 6, Chapters VII and IX, "Cellular and Molecular Immunology", Second Edition, Jin Boquan, Editor-in-Chief, China Science Press, 2001, first print). After the end of the autoantigen immune recognition of the "autoantigen immune recognition phase" of the fetus, "organisms, organs, cells, and factors related to autoantigen immune recognition" are no longer subjected to autoantigen immune recognition, only under certain circumstances, such as in It is possible to re-examine autoantigens in the presence of "autoantigen-immune recognition phase" and/or "pre-autoimmune immune recognition pre-existing" "organisms, organs, cells, and factors related to autoantigen immune recognition".

"Microorganisms, organs, cells, factors related to immune recognition of autoantigens" may include:

1) "The tissues, organs, cells, and genes involved in the immune recognition of autoantigens include: human or animal embryos and fetal stages, and participate in early immune recognition of hematopoietic stem cells to the body, organs, organs, organs, organs, organs, A cell, a factor, a complete tissue, an organ, or a partially incomplete tissue or organ. It provides the microenvironment required for early immune recognition of hematopoietic stem cells to immunologically recognize autoantigens in the body, providing early immune recognition to recognize hematopoietic stem cells. A cell that secretes a "autoantigen immune recognition factor" required for immunological recognition of a self antigen in the body, and/or a cell that secretes a "promoting (producing) autoantigen immune recognition factor". "Self-antigen immune recognition factor" may include : a starter factor, a recognition factor, a termination recognition factor, and various microenvironments such as a tissue fluid that provides a self-antigen for various auxiliary factors required for immunological recognition.

2) "tissue, organ, cell, factor related to immune recognition of autoantigen" may include all cells of the above-mentioned tissue and organs, and / or part of cells, and / or one or more of these special cells, into the body, part Cells can reach the target organ through homing, grow in the target organ, and grow and develop outside the target organ, providing the microenvironment needed for early immune recognition of hematopoietic stem cells to identify the autoantigen in the body. Early immune recognition identifies cells that secrete "autoantigen immune recognition factor" required for hematopoietic stem cell immune recognition of autoantigens in the body, and/or cells that secrete "promoting (producing) autoantigen immune recognition factors". And cells that secrete various cytokines, such as cells that secrete autoantigens for various cofactors required for immunological recognition, and various helper cells involved in immune recognition of self-antigens, and various micro-requirements for providing self-antigen for immunological recognition surroundings.

These cells in tissues and organs associated with autoantigen immune recognition are extracted using a density gradient centrifugation method and a method of extracting specific cells as mentioned in the present specification. Extraction, such as: Promoting early immune recognition, hematopoietic cells, cells that secrete "autoantigen immune recognition factor" required for immunological recognition of autoantigens in the body, secrete cells that "promote (self-immunize) immune antigen recognition factors". Cells that secrete a variety of cofactors involved in the recognition of autoantigens, as well as a variety of helper cells involved in the recognition of autoantigens.

3) "tissue, organ, cell, factor related to autoantigen immune recognition" may include "self-antigen immunization required by the above-mentioned tissue and organ to provide early immune recognition for hematopoietic stem cells to recognize the autoantigen in the body. The recognition factor ", and/or "promotes (secrets) autoantigen immune recognition factor", and/or various cofactors required for immunological recognition of self-antigens, and the like. You can use the above several organizers as needed. One or more of several types of materials such as officials, cells, and factors. The various factors used can be extracted by various methods of factor extraction currently used, and can be produced by various methods such as transgenic cell cloning.

"Microorganisms, organs, cells, and factors related to autoantigen immune recognition" are also referred to in this specification as: "immune recognition organs" or "initiation recognition factors" or "activation factors" or "suspension recognition factors" or "stop factors" ".

Cells that secrete the "autoantigen immune recognition factor" are secreted, and cells that "promote (enact) secreted autoantigen immune recognition factor" are secreted. A method of secreting various cells involved in cofactors for self-antigen immune recognition and various cells involved in immune recognition of autoantigens. And methods for extracting "autoantigen immune recognition factor", and/or "promoting (producing) autoantigen immune recognition factor", and/or various cofactors required for immunological recognition of self antigens.

4) There are many ways to find and extract "tissue, organ, cell, and factor related to autoantigen immune recognition", including:

A) There are various methods for finding tissues and organs related to autoimmune antigen recognition, including: using the method of Example 9 of the present invention, making tissues and/or organs undergoing self-antigen immune recognition into single cells. Suspension, or use a part of tissue organs, or use intact tissue organs, transplanted into animals separately or in different combinations, to carry out autoantigen immune recognition experiments, if any tissue, organ or combination of those tissues and organs can The self-antigen immune recognition experiment is successful, and the tissues and organs included in the tissue, the organ or the combination are "organisms and organs related to the immune recognition of the self antigen".

B) There are various methods for finding "cells related to autoantigen immune recognition", including: making a single cell suspension of the tissue or organ found in 1), and separating each cell by continuous density gradient centrifugation . Using the method of Example 9 of the present invention, transplanted into the experimental animal alone or in different combinations, and performing autoantigen immunological recognition experiments, if any cell or combination of those cells can make the self-antigen immune recognition experiment successful, Hao Hao The cells included in this cell or this combination are "cells involved in the immune recognition of autoantigens".

C) There are many methods for finding "factors related to autoimmune recognition of autoantigens", including: a. Cells with autoantigen immunoreactivity that are found in B) in the process of autoimmune recognition of autoantigens have passed. The same type of cells in the period of self-antigen immune recognition are disrupted by ultrasonic or other methods, and then different proteins and amino acid peptide chains (polypeptides) between the two cells are found by chromatography and mass spectrometry. Using the method of Example 9 of the present invention, injection into a test animal alone or in a different combination, and performing an autoantigen immunoreactivity assay, if any protein, amino acid peptide chain (polypeptide) or those proteins, amino acid peptide chains (polypeptides) The combination of these can make the self-antigen immune recognition experiment successful, and the protein, amino acid peptide chain (polypeptide) or the amino acid peptide chain (polypeptide) included in this combination is "a factor related to the immune recognition of the self antigen".

b. The cells of the same type that have been found to have their own antigen-immunological recognition activity in the period of self-antigen-immunization recognition in B) and the same type of cells that have undergone the self-antigen-immunization recognition period, and culture the two cells separately And allowing the cells having autoantigen immunoreactivity to retain the activity of immunologically recognizing hematopoietic stem cells for self-antigen immunological recognition, and purifying the culture medium of the two cells, removing or retaining the immunity to the body or the autoantigen in the culture solution Identify the relevant ingredients. By using the method of Example 9 of the present invention, it is injected into an experimental animal to perform an autoantigen immune recognition experiment. If the autoantigen immune recognition experiment can be successfully performed, the culture fluid input into the body contains "immunity recognition related to autoantigen". Factor". Then, using chromatography and mass spectrometry, different proteins and amino acid peptide chains (polypeptides) between the two cells were found. Using the method of Example 9 of the present invention, it is injected into an experimental animal to perform an autoantigen immune recognition experiment. If the autoantigen immunological recognition experiment is successful, the protein and amino acid peptide chain (peptide) input into the body is "with Self-antigen immune recognition related factors".

3. Preparing transplants of organs, tissues, cells, etc. that have been transplanted into the body, and/or cells, tissues, organs, proteins, polypeptides, etc. of the antigenic family carrying the immune recognition, etc., in the present specification Abbreviation: Graft. 4. Since the graft is derived from organs, tissues, cells, etc. in the donor's body, the antigenicity of the graft and the antigenicity of the donor are the same, and the graft may be used, or other donors may be used. An antigenic organ, tissue, cell, or the like, such as a tissue, is used as a donor antigen donor for early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells for self-antigen recognition. These are referred to in the present specification as: graft antigens.

5. Through medical practice, it is found that people start from the fertilized egg to the embryo for about 3 months. During this period, the embryo body does not react to the graft, so this period is not a period of immune recognition of the autoantigen. From about 3 months to about 5 months, the immune system in the body begins to recognize the antigen itself. Before this time, or within this time, allogeneic grafts in the body can be recognized by the immune system. For its own organization, it is added to the self-antigen immune recognition library together with various autoantigens in the fetal body, and will not be recognized as foreign matter in the future, and a rejection reaction will occur. Although it has not been specifically found to initiate, maintain, and terminate the immune organs in the body, the autoantigen immune recognition factor "initiates, promotes, and terminates the self-immunological immune recognition factor", and/or "promotes, promotes, terminates Self-antigen immune recognition factors" and cells that secrete these factors, but they can be found through medical practice, and the presence of these factors and the tissues and organs of the cells secreting these factors are "related to the autoimmune recognition of autoantigens." Tissues, organs, cells, factors", including: embryos from 3 months to 5 months or so in the thymus tissue of the autoantigen immune recognition phase, and/or bone marrow tissue, etc., may exist in these tissues and organs A cell that secretes a "autoantigen immune recognition factor", and/or a cell that secretes a "promoting (producing) autoantigen immune recognition factor". Each of these factors may be a single factor or a composite factor composed of a plurality of factors. The activation of the autoantigen immune recognition factor, the promotion of the autoantigen immune recognition factor, and the termination of the autoantigen immune recognition factor may be a plurality of different factors, or the same factor may play different roles at different stages, and these factors may be: Hormones, cytokines, peptides, proteins, tissues, tissue fluids, cells, organs, and more. It has been discovered through medical practice that these factors can sometimes transform late-stage immune recognition hematopoietic stem cells to re-identify their own antigens. It is currently possible to input an immune recognition microenvironment that provides input and promotes autoantigen immune recognition by inputting organs and tissues (such as thymus tissue, thymus cells, bone marrow tissue, bone marrow cells, etc.) that provide an immune recognition microenvironment that promotes autoantigen immune recognition. These factors have been introduced to promote the recognition of autoantigens.

In the present specification: a factor related to initiation, promotion of recognition of autoantigen immune recognition by an immune-free recognition cell, and termination of recognition, and an organ and tissue (eg, thymus tissue) providing an immune recognition microenvironment for promoting autoantigen immune recognition , thymocyte and bone marrow tissue, bone marrow cells, etc.) Abbreviation: initiation factor, motivating recognition factor, termination recognition factor. However, these factors are collectively referred to in this specification as: Startup recognition factor.

The relevant factors for terminating the non-immunologically recognized stem cells for self-antigen immune recognition may also be referred to in the present specification as a termination identifier.

In practice, since it has not been isolated yet, and the non-immunologically recognized stem cells are involved in the self-antigen immune recognition in the immune recognition organ, the factors that recognize, promote, and/or terminate the autoantigen immune recognition are activated, but they do exist. The "regulatory effects of transcription factors on immune cells" (see "Cell and Molecular Immunology" Chapter 6, Chapters VII and IX, "Cell and Molecular Immunology", Second Edition, Jin Boquan, Editor-in-Chief, China Science Press, 2001 First print in September). Specifically, which are organs or cells that promote the secretion of a recognition factor, a recognition factor, and a termination recognition factor, and/or the presence or absence of a secretory factor that secretes these factors, to be demonstrated later and/or isolated.

6. Immune system diseases, which may include, for example, systemic lupus erythematosus, rheumatism, rheumatoid arthritis, glomerulonephritis, scleroderma, etc., and may also include antigenic antigenic changes due to various causes of the disease. The induced immune disease can cause changes in autoantigens, and the mutated autoantigens can be organs, tissues, cells, proteins, polypeptides, etc., which are referred to in the present specification as: mutated autoantigens.

7. Bone marrow transplantation: The more popular word is "hematopoietic stem cell transplantation".

The bone marrow transplantation treatment used in the present invention is an immunologically recognized hematopoietic stem cell transplantation by performing clearance of immune-related cells in the present invention, and/or performing "organisms, organs, cells, factors related to autoantigen immune recognition". "Transplantation, the treatment process for secondary self-antigen immune recognition. The method of bone marrow transplantation is -

1) Methods for clearing immune-related cells, and/or "self-antigen immune recognition libraries" and cells containing the autoantigen immune recognition library are clear or semi-myeloablative:

a. Non-specific clearance of immune-related cells

Including: the use of drugs, such as: cyclophosphamide, and / or radiation, such as: cobalt ⁶ °, medical linear accelerator, high-energy X-ray systemic irradiation, etc., the original, have been immune to their own antigens The identified immune-related cells in the body, such as stem cells that have been immunologically recognized, are killed.

b. Specific clearance of immune-related cells, and/or removal of the "autoantigen immune recognition library" and cells storing the autoantigen immune recognition library.

This is the use of drugs to kill one or more immune-related cells in the body, or to inactivate immune-related cytokines with drugs, or to kill cells that store a specific library of immune-clearing antigens. Death, immunological methods can also be used to remove specific cells, and/or inactivate cytokines, such as T lymphocytes and other immune cells, such as the use of CD3 monoclonal antibodies combined with cytotoxins to form cytotoxic drugs plus complement input. The body kills T lymphocytes in the body and anti-lymphocyte globulin (ALG) and other drugs that kill immune cells in the body's immune system. This is a specific therapy for a certain cell or a certain type of cells. .

This is done by high-dose radiation therapy, chemotherapy or other immunosuppressive preconditioning to remove tumor cells, abnormal clones, original hematopoietic stem cells and various immune progenitor cells, immune stem cells, and/or various immune cells. Such cells, blocking the pathogenesis, this is called myeloablative treatment, can also use partial myeloablative or non-myeloablative treatment. Then, hematopoietic stem cell transplantation is performed on the recipient, and the recipient is reconstructed from normal hematopoiesis and immunity, thereby achieving a therapeutic treatment purpose.

2) Perform immunological recognition of hematopoietic stem cell transplantation, and / or carry out "transplantation of tissues, organs, cells, and factors related to autoantigen immune recognition". If partial myeloablative or non-myeloablative methods are used, hematopoietic stem cell transplantation can also be performed without immunological recognition, and the original immune recognition hematopoietic stem cells can be directly used for secondary self-antigen immune recognition.

Find out a variety of cells storing the autoantigen immune recognition library, including: making a single cell suspension of the found tissue or organ containing cells storing the autoantigen immune recognition library, using continuous density gradient centrifugation Each cell was isolated. The prepared immunological method is used to remove specific cellular agents of these cells, clear the body alone or perform different combinations of cell clearance, and then transplant the graft into the experimental animal to observe whether a rejection reaction occurs. If one or a different combination of cells is removed without rejection, then the cells of this or different combinations are cells that store the autoantigen immune recognition pool.

8. Use immunological methods to remove specific cells:

It is a specific cell-specific method that selectively binds specific cell-specific antibodies to specific cells for clearance. There are many methods that can be used, such as the commonly used ones:

1) Complement-dependent cytotoxicity method: After the monoclonal antibody binds to the corresponding target cell, the complement is added, and the target cell is dissolved and destroyed by activating the complement pathway to achieve purification. For example, a variety of B cell monoclonal antibodies (such as: CD9, CD10, CD19, CD20) or T cell antibodies (such as: CD2, CD3, CD4, CD8, etc.) are combined with complement to purify bone marrow T cells and sputum cells in vitro. In addition, AML-223 (anti-CD14 mAb) plus immune complement purification can be used to treat acute myeloid leukemia, which is similar to Alio-bone marrow transplantation.

2) Immunotoxin-mediated cytotoxicity method, which combines specific antibodies with cytotoxin (ricin toxin) to form an immunotoxin, which has a powerful effect of killing specific cells and is specific.

3) Immunophysical method, coating the microspheres or metal particles with a monoclonal antibody, labeling the target cells to increase the density or magnetic properties of the cells, and then removing the target cells by precipitation or magnetic field adsorption.

4) Clear or disable specific cytokines, such as the loss of IL2 R y , and the loss of immune activity of NK cells. And the method of making a particular cell lose its function.

Each of the above methods has its own advantages and disadvantages, and is currently mainly used for purification of specific cells in the clinic.

9. Patients receiving grafts or patients with immune system diseases, referred to in this specification: recipients. This recipient can be either a human or an animal. 10. Provide donor or graft antigen, referred to in this specification: donor. This donor can be either a human or an animal.

11. The early immunological recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells of the present invention are performed twice in vitro, or in the donor, or in the recipient, on the donor's graft, and/or the variant autoantigen. Immune recognition. Because this autoantigen immune recognition differs from the immune recognition of the autoantigen by the immune system in the embryonic stage in which the human or animal naturally grows, it is an autoantigen including a donor's graft, and/or a variant self antigen. The immunological recognition is to identify the graft and/or the self-antigen that causes mutation of the immune system disease as a normal autoantigen, and therefore, in the present specification, it is referred to as: secondary immune recognition. In this specification, secondary immune recognition is also referred to as: re-existing autoantigen immune recognition, or secondary self-antigen immune recognition, or self-antigen secondary immune recognition.

12. In the present invention, from egg cells to obtaining early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic cells and/or grafts, to intermediate stages of obtaining stem cells and/or incubated grafts that have undergone immunological recognition, Sometimes, the same species, the same species, or different species, different species of animals or humans, and the development, culture, identification of their own antigens, etc., are used in the body of these animals or humans. These animals or persons are referred to in this specification as abbreviations: intermediaries.

13. In the present invention, from egg cells to obtaining early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells and/or grafts, to intermediate stages of obtaining thousands of cells and/or incubated grafts that have undergone immunological recognition, In vitro culture tools such as cell culture flasks, culture dishes, and cultures are used to develop, culture, and self-antigen identification in in vitro culture tools such as culture flasks, culture dishes, and culture instruments. Abbreviation in the manual: test tube.

14. Method of extracting specific cells:

1) An immunomagnetic bead separation method can be performed using a specific antibody of a certain cell.

2) A cell suspension containing a certain tissue of a particular cell can be used. Of course, depending on the difference in the extracted cells, there are more ways to adopt them. Here are just a few typical extraction methods. Because of the different extraction methods used and the different chemical components used, various cell extraction methods can be very different, but the purpose of extracting cells is consistent.

a. Immunological separation of specific cell culture methods, such as:

Isolation of cells such as dendritic cells (DC): Various methods can be used, which can include the following protocols: The thymus tissue is ground into a single cell suspension by a single cell grinder, containing 6 X 10 ⁸ PBMC suspension 9 ml containing 1 sleep ol / L EDTA calcium-free magnesium PBS), add anti-CD32 monoclonal antibody blocking solution 0. 4ml, 37 °C 5% C02 culture for 10min, then add StemSepTM human DC enriched mixed antibody magnetic beads lml, fully mixed, 37 Incubate at 5% C02 for 30 min, pass through a sterile magnetic sieve column, collect unlabeled cells of interest, and wash 3 times with PBS. IX 105 cells were taken, and the cell phenotype was analyzed by fluorescent antibody labeling and flow cytometry, and the remaining cells were cultured in an IMDM medium containing 10 ng/ml of GM-CSF and 10 ng/ml of IL-4 in 10% FCS.

Isolation of NK cells 9 ml of 4×108PBMC calcium-free magnesium PBS suspension was isolated and assayed in the same way as DC, and the remaining cells were cultured in IMDM medium containing 10% FCS.

Isolation of T cells 9 ml of 4×108PBMC calcium-free magnesium PBS suspension was isolated and detected by DC separation. The remaining cells were cultured in IMDM medium containing 10% FCS.

b. Using a method of adding a specific cell culture medium, various methods may be employed, which may include the following protocols: isolation and culture of pancreatic stem cells: (Wen Jin, Zhang Yuhai et al. Journal of Jiangxi Medical College, 2002, Volume 42, Issue 3)

The pancreas was added to 4 CHank's solution and cut into tissue pieces of 1 mm in diameter. After repeated washing, 0.5 rag/ml of collagenase was added, and the solution was shaken at 37 ° C for 15 minutes. The reaction was stopped by adding 4 CHank's solution. Centrifuge three times at 800 rpm, and add the supernatant to 4 CHank's solution without calf serum. Pass through a 150 mesh sieve and add 4 CHank's solution containing 10% calf serum to the resulting cell pellet. After repeated pipetting, the cell suspension was added to a culture solution of 0.1 mg/ml ConA to remove non-islet cells such as fibroblasts, and 5% C0 ₂ 37 ° C pzyh 24 hours later, ⁵ ×10 ⁵ /piece was added. 20 ng/ml BFGF (basic fibroblast growth factor) 11. 1 mmpl/1 glucose 10% calf serum culture flask was continued to be cultured, and after 7-10 days, EDTA was diluted with 0. 05% trypsin, digested and passaged. cell. After five weeks, the culture medium was changed to low-sugar (2.5 mmol/1) 4 CHank's solution containing 2% calf serum, and BFGF was removed. 10 mM nicotinamide was added to induce cell differentiation to form islet cell-like cell colonies. The cells therein are islet stem cells.

15. Several issues related to the re-initiation of autoantigen immunological recognition treatment for rejection and autoimmune diseases:

1) Cells storing their own antigen recognition library: How many kinds of cells currently store their own antigen recognition libraries, and the specific locations stored in that cells, stored in which way, have not been fully understood. However, it is currently known that some of the cortical epithelial cells and medullary dendritic cells present in the thymus, as well as the corresponding immunologically relevant cells in the bone marrow.

Treatment: a Knock out the self-antigen immune recognition library or the cells that store the self-antigen recognition library. And/or after knocking out these cells, the same type of cells into which the autoantigen immune recognition library is stored are not included, including cells such as stem cells and/or progenitor cells that can be differentiated into these cells, and stored for self-antigen immune recognition. After the new autoantigen immune recognition library.

b. Adding an antigen to the original autoantigen immune recognition library after re-identification of the self-antigen, and making the antigen from the self-immunization recognition of the autoantigen become an autoantigen.

2) Immunological cells such as T cells and B cells that have been negatively selected and positively selected in the thymus and bone marrow in peripheral blood and immune tissues.

Treatment: a. Knock out immune cells such as T lymphocytes, B cells, etc. in the thymus, bone marrow, peripheral blood, and immune tissues.

b. In peripheral blood and immune tissues, immune cells such as T cells and B cells that have been negatively selected and positively selected in the thymus and bone marrow after re-immunization with autoantigen are recognized.

3) Self-antigen recognition factors, as well as self-antigen recognition factors, and pro-antigen recognition factors, which may be factors or cells. Medical practice indicates that it may be present in tissues, organs, cells, and factors related to immune recognition of autoantigens such as thymus and/or bone marrow during the period of self-antigen immune recognition.

Treatment: a. Look for cells that secrete these factors, do cell culture and input into the body for treatment.

b. Look for the structure and function of the factors, and use bioengineering to make commodities for clinical use.

c. Implantation using organ tissues containing cells or secretory factor cells such as thymus and/or bone marrow, and/or intooculation of these organ tissues into a single cell suspension.

4) Thymus and/or bone marrow cells undergoing self-antigen immune recognition.

Treatment: a. Use the original thymus and bone marrow cells in the body.

b. Implant thymus tissue and/or bone marrow cells undergoing self-antigen immune recognition, or enter cultured thymocytes. Enter the cultured bone marrow cells.

5) Hematopoietic stem cells, thymic T progenitor cells, and/or B 袓 cells in the bone marrow undergoing self-antigen immune recognition.

Treatment: a. Input early non-immune recognition of hematopoietic stem cells, thymic T progenitor cells and / or B progenitor cells in the bone marrow.

b. Use original hematopoietic stem cells, thymus T progenitor cells and/or B progenitor cells in the bone marrow.

6) Hematopoietic stem cells undergoing self-antigen immune recognition.

Treatment: a. Enter early non-immune recognition of hematopoietic stem cells.

b. Use the original hematopoietic cells in the body.

The present invention can be applied not only to humans but also to animals, but in the description of the present invention, it is mostly represented by humans, including various embodiments in the specification. However, the present specification and various embodiments can also be applied to various animals. Organ transplantation can be performed between humans or animals of the same species, or organ transplantation can be performed between humans or animals of different species. Or transplanting between the immune systems of different species of humans or animals, using the animals of the transplanted immune system for immune system diseases or organ transplant treatment trials. Not only can you carry out various animal experiments, but you can also Organs are transplanted into animals, and the organs of the animals grow on the animals. When they grow up, they can be transplanted to the human body, and then transplanted from the animals to the human body and other diseases.

The principle and significance of the invention:

Because the human body is roughly between three months and six months between the fetuses, the fetal non-immune recognition stem cells perform an immune recognition on the autoantigens of various tissues and organs in the body, and store the results of this recognition into the autoantigen. Lifetime memory in the immune recognition library. After the immune system compares various antigens entering the body or antigens that are self-mutating, this memory is compared with the memory of the identification, and the antigens that are different from the autoantigens of various tissues and organs in the body are found. Generate an immune attack.

At present, the transplantation of various organs in the body includes: liver transplantation, kidney transplantation, islet transplantation of diabetes, etc., because the consistent organs and / or tissues are foreign antigens, and the autoantigens in the self-antigen recognition memory in the body are different, so Inevitably, a rejection reaction occurs. HLA matching is required before surgery, preferably all matches, at least semi-match is required, so that the antigen of the graft or graft antigen is as much as possible in the memory of the memory recognized by the autoantigen in the body, and at the same time After surgery, it is necessary to take anti-rejection drugs for life, such as cyclosporin (CSA), etc., taking anti-rejection drugs, it costs about 5,000 yuan a month, and the long-term cost is very expensive. At the same time, many immune system diseases such as: systemic lupus erythematosus, rheumatism, rheumatoid arthritis, glomerulonephritis, scleroderma, etc., including immune diseases caused by self-antigenic changes caused by various causes of diseases, For example, cirrhosis caused by viral hepatitis is an immune attack caused by the immune system's own immune recognition error, causing physical damage. These immune system diseases are still treated by immunosuppressive agents, but the cytotoxic and immunosuppressive treatment of these diseases can only relieve symptoms, such as taking: sterols, methotrexate, azathioprine, other related drugs, but These drugs may occur during use, such as: side effects such as liver inflammation caused by immunosuppressive agents such as cyclosporin A. Moreover, these medications can only relieve symptoms and prevent them from being cured. Many patients suffer from the pain of the disease, spend a lot of money on treatment, and eventually die from these diseases. At present, many diseases including congenital hereditary factors are still incurable. For example, diseases such as congenital chain-like erythrocyte anemia have not been cured. It is the primary object of the present invention to find a way to treat these diseases.

The invention can alleviate or cure the inevitable rejection reaction of transplantation of various organs, so that organ transplantation and tissue cell transplantation can be used to treat many diseases which are currently untreated, such as: Diabetes can be treated by islet cell transplantation. It can also alleviate or cure various immune system diseases (AIDs) such as systemic lupus erythematosus, etc. These diseases are diseases in which the immune system causes immune recognition errors in its own tissues and causes physical damage. It can also treat a variety of other diseases that can be treated by this method, such as: congenital genetic diseases such as hematopoietic diseases.

The team led by Esmail Zanjani of the University of Nevada in the United States has been able to "plant" human organs into intermediaries and transplant them into the human body after maturity, replacing the corresponding Research results of diseased organs. Of course, this theory will take a long time to apply to clinical practice, but the first phase of research has been completed.

Zhagani's mesenchymal cells, extracted from human bone marrow, are injected into the fetus of the sheep's uterus, and human cells grow into part of the sheep's heart, skin, muscles, fat and other tissues, but the number of human cells at that time very few. Now, the team he led has made further progress on the basis of the original, that is, surprisingly greatly increased the content of human cells in some sheep organs. According to the demonstration at a meeting in early December, the human cell content in the liver of the tested sheep has reached 7-15%.

The key to this technology is that human cells must be injected in the mid-pregnancy of the sheep, that is, before the fetus has been formed, but its immune system has not yet learned to recognize different cells, so that there is no rejection. This scientific experiment is a powerful proof: In the fetal stage of human or animal, non-immunological recognition of stem cells not only recognizes the autoantigens of various tissues and organs in the body as autoantigens, but also recognizes allogeneic antigens such as graft antigens and recognizes them as themselves. The antigen, and the result of this recognition is stored in the "self antigen immune recognition library" lifetime memory. It has also been demonstrated that "self-antigen immunological recognition library", and cells storing "self-antigen immune recognition library" are present in the body. A recent study by Israeli biologists found that allogeneic embryonic stem cells could not escape the "eyes of the immune system." In the online edition of the Proceedings of the National Academy of Sciences, the research team of the Hebrew University of Jerusalem, led by Nissim Benvenisti, published a paper saying that because the human immune system uses an MHC molecule to identify "invaders", they In the three stages of development of the Human Embryonic Stem Cell, fluorescently labeled antibodies were used to measure the number of MHC molecules in the various stages of allogeneic embryonic stem cells. As a reference, they also measured the response of HeLa cells simultaneously. The measurement results show that the MHC content in the undivided embryonic stem cells is low, but stable. Embryonic stem cells, the embryos, have not yet differentiated into "raw" cells of various tissues. According to early human embryo research, scientists have suggested that embryonic stem cells may not be discovered by the body's mechanism of defense against foreign cells. Hugh O'Kinkos of Harvard Medical School in the United States said that the new results prove that embryonic stem cells cannot escape the detection of the immune system.

Prof. Kuharchuk, the discoverer of the Ukrainian "Kulhalchuk-Radechenko-Silman effect", said: "We hypothesized and proved that: injecting a certain amount of embryonic stem cells into the body can lead to its existing immunity. Systematic update. The essence of this effect is to "replace" a problematic or damaged immune system into a new, healthy system. "But embryonic stem cells cannot escape the detection of the immune system, so even if it is input to the body. Embryonic stem cells require HLA (tissue-associated antigen) matching before input. Otherwise embryonic stem cells are difficult to transplant successfully. However, the success rate of HLA (tissue-associated antigen) matching is quite low, and a large number of fetal stem cells of different human origins are required for successful matching, because the influence of moral factors on embryonic stem cell sources is limited, so if it is actually used for treatment The possibility of widespread use of diseases is quite low. At the same time, this update is not complete, because only a certain amount of embryonic stem cells are injected into the body, and the original hematopoietic stem cells and immune progenitor cells that have been immunologically recognized in the body are still alive, and there is still a certain maintenance. Immunity, rejection, but this feature is reduced, but will not disappear.

St. Jude Children's Research Hospital published an article in the Journal of Immunology P6540-6545 on May 15, 2005, describing them at NOD/LtSz -scid IL2R Y " ^un Hematopoietic stem cells transplanted from mice have completed the experimental animal model of the human immune system. This is the method used in the experiment and the passage of the invention patent filed in China on October 10, 2004. The transplantation of human hematopoietic stem cells in NOD-scid mice makes the purpose of the human immune system of NOD-scid mice consistent with the general direction, except that NOD/LtSz-scid IL2R Y " ^u11 mice are NK cells that are non-functional. It is impossible to kill the transplanted human hematopoietic stem cells, so the mouse does not have any immune function and is completely receptive to the graft. It is completely acceptable. While the present invention uses a mouse having NK cell function (having a certain immune function), the successful transplantation of the test examples of the present invention proves that the present invention does overcome the rejection reaction, and proves the theory and the theory of the present invention. The correctness of practice.

These experiments demonstrate that allogeneic antigens entering the fetus prior to or during recognition of the fetal antigen can be recognized as fetal autoantigens without rejection, and that rejection is more than just rejection. It is determined by the autologous tissue antigen in the animal, and the foreign antigen that enters the body can also be recognized as an autoantigen before or during the period of recognition of the autoantigen. Therefore, it is proved that there may be a self-antigen immune recognition in the animal. "self-antigen immune recognition library" (although it is not yet fully known about the specific storage location of this "self-antigen immune recognition library", in which cell, in which organelle of the cell, and the specific location, but at least It is known that some cells harboring autoantigen immune recognition libraries, such as: cortical epithelial cells and medullary dendritic cells associated with immune recognition in the thymus, and/or corresponding cells associated with immune recognition of bone marrow cells (see " Cellular and Molecular Immunology" Chapter 6, Section Chapters 7 and 9), this "self-antigen immune recognition library" determines the self-antigen reference standard for immunological recognition after birth. For normal pregnant fetuses, because the fetus is performing itself in the early stage of growth and development in the mother Before the antigen is recognized by the antigen or in the middle of recognition, no external allogeneic antigen is implanted into the fetus. Therefore, when the fetus is identified by the autoantigen, only the fetus's own tissue antigen can be recognized, so the allogeneic organ transplantation is performed after birth. Or when the autoimmune disease is caused by self-antigen mutation, because the allogeneic organ or the mutated tissue antigen does not enter the fetus when the fetus performs autoantigen immunological recognition, the "self antigen" established after autoantigen immune recognition The antigenic gene of this transplanted organ is not recognized as its own antigen, so it will be issued. Give birth to an immune response.

At present, British scientists have used a somatic cell nucleus of a sheep to transplant into the nucleus of the nucleus, and then transplanted it into the uterus of the sheep.

Scientists in Shanghai, China have transplanted the nuclei of human foreskin cells into rabbit egg cells that have been removed from the nucleus and have been cultured in vitro.

At present, humans have successfully differentiated embryonic stem cells cultured in test tubes into hematopoietic stem cells.

Taiwan Regeneration Biotechnology Co., Ltd. has developed "Umbilical Cord Blood Stem Cell-Free Medium", which enables umbilical cord blood stem cells to increase value by 30 times within five to seven days, increase to seven thousand times within five weeks, and maintain differentiation. The characteristics and potential of stem cells. SUMMARY OF THE INVENTION According to one aspect of the present invention, a method for obtaining and applying stem cells for treating diseases of the immune system and rejection is provided, characterized in that the self-antigen is used for treating diseases of the immune system and rejection diseases. Early immune recognition of secondary immune recognition The source of hematopoietic stem cells and/or late immune recognition hematopoietic stem cells is obtained from the following pathways:

A. Source of clonal cells or fertilized eggs:

a clonal cell formed from the graft recipient or the somatic cell nucleus of the immune system and the diseased patient to the human egg from which the nucleus is removed;

b. clonal cells formed from the recipient's somatic cell nucleus to the animal's egg from which the nucleus is removed; c. The fertilized egg is formed from the sperm or egg of the recipient and the donor or other human or animal sperm or egg.

B. Early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cell sources:

a. These clonal cells or fertilized eggs of different origins develop into an early immune recognition hematopoietic stem cell and/or embryos of late immune recognition hematopoietic stem cell stage, and early immune recognition hematopoietic stem cells and/or late immunity are extracted from the embryo. Identifying hematopoietic stem cells;

b. Differentiating into early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells in a test tube; c extracting early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells from aborted stillbirth;

d. Extract late-stage immunoreactive hematopoietic stem cells from human bone marrow or blood after birth. In particular: including early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells are expanded in vitro or transplanted into an intermediary for amplification. In particular: Including early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells for secondary immune recognition is carried out in an in vitro test tube, either in an intermediary or in a recipient. In particular: including early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells in a test tube, or in an intermediary, secondary immunization of the donor's graft, and/or variant autoantigen. In particular: including transplanting the graft into the recipient, and then transplanting the early immune recognition hematopoietic stem cells and/or the late immune recognition hematopoietic stem cells into the recipient using a myeloablative, partially clear or non-myeloablative bone marrow transplantation method. The early immune recognition hematopoietic stem cells and/or the late immune recognition hematopoietic stem cells are subjected to secondary immune recognition in the body. In particular: including bone marrow transplantation using myeloablative, partial clear pulp or non-myeloablative, transplanting stem cells that have undergone immunological recognition by graft or graft antigen secondary immune recognition in recipients to recipients Then transplant the graft into the recipient. In particular: including mediators using immunodeficient animals, or non-immune-deficient animals, but after myeloablative treatment, early immunological identification of hematopoietic stem cells and/or advanced immune recognition of hematopoietic cell bone marrow transplanted animals: transplant donors and / Or recipient's graft or graft antigen into the body of the intermediary: Transplantation of non-immunologically recognized stem cells for self-antigen recognition: The animal is made into an animal with a human or an immune system that has been subjected to graft or graft antigen recognition. In particular: including mediators using immunodeficient animals, or non-immune-deficient animals, but with myeloablative treatment, early immunological identification of hematopoietic stem cells and/or advanced immune recognition Blood stem cell bone marrow transplanted animals: Transplantation without immunological recognition of stem cells for self-antigen recognition: Animals are made an animal with an immune system recognized by humans or affected antigens. In particular: including transplantation of immune organs into the intermediary: in particular: including input initiation factors, and/or termination factors. The principle of the present invention is based on a research team led by Esmai l Zanjani of the University of Nevada, USA, which demonstrates that human organs can be planted in intermediaries. And is recognized by the animal's immune system as its own tissue, self-antigen, without rejection.

Those involved in immune recognition in the human body are:

A. "Self-antigen immune recognition library". The T cells in the thymus pass through the thymocyte precursor (prothymocyte) in the early thymus as CD4XD8-double-negative cells, which then develop into CD4 ⁺ C8 ⁺ TCR ⁺ double positive cells, which pass through the MHC class I with the surface of the thymic cortex epithelial cells or A positive selection for binding of MHC class II molecules/self peptides, and dendritic cells that express high levels of MHC class I or MHO class II molecules and autoantigens at the junction of the thymic cortex and medulla with cell surface ( DC) and macrophage (ΜΦ) for negative selection of high affinity binding, through self tolerance or clonal deletion, become CD4+CD8 that can recognize both foreign antigens and non-antigens. Or CD4-CD8+ single positive sputum cells. (Cell and Molecular Immunology, Second Edition, Jin Boquan, ed., Science Press, September 2001, Chapter 6, Major histocompatibility antigen, MHC, Section II, MHC, in immune response, III. MHC and T cell thymus selection) c. In the bone marrow, progenitor B cells undergo developmental stages of early progenitor B cells, late progenitor B cells, pre-B cells, and immature B cells in the bone marrow, and are cloned and deleted to develop autoimmune-tolerant mature B cells ( Chapter IX B lymphocytes). According to the "Separation of Cellular and Molecular Immunology," edited by Kim Bo-chun, "Self-antigen immune recognition library" seems to be in the thymic thymic cortex epithelial cells and dendritic cells (DC) and macrophages (ΜΦ) and In the bone marrow, but whether it is in these two organs is still inconclusive.

B. Single positive T in the body that has undergone self tolerance or clonal deletion in the thymus or bone marrow to recognize both foreign antigens and CD4+CD8- or CD4XD8+ Cells and B cells.

Therefore, when dealing with immune system diseases and rejection reactions, there are two aspects that need to be addressed:

1. (1) Delete the original "autoantigen immune recognition library" in the body, and then input the tissues, organs, cells, and factors related to the immune recognition of the autoantigen in vivo, including: "self antigen immune recognition factor", and / Or a tissue organ that secretes the "autoantigen immune recognition factor", or all cells of the tissue or organ, or certain types of cells that secrete "autoantigen immune recognition factor", and/or secrete "immunize" (self-immunized) antigen The cells such as the recognition factor secrete cells that cause the immune-recognizing hematopoietic stem cells to carry out the self-antigen immune recognition factor, or directly input these factors. These include: thymus, and/or bone marrow organs in the period of self-antigen immune recognition, and/or certain tissue cells in these organs. And/or thymocytes, bone marrow cells, early immune recognition, hematopoietic stem cells, and/or advanced immune recognition of hematopoietic stem cells, and/or use their own late-stage immune recognition to identify hematopoietic stem cells, re-existing autoantigen immune recognition, and mutating autoantigens and / or the antigen of the transplanted tissue organ is recognized as an autoantigen, and a new "autoantigen immune recognition library" including a self-antigen recognizing the graft antigen or mutation is established, thereby avoiding a rejection reaction or an immune reaction.

(2) The original "autoantigen immune recognition library" in the body is not deleted, but the tissues and organs and cytokines related to the immune recognition of the autoantigen are input into the body, including: "autoantigen immune recognition factor", and/or secretion. The organ of the "autoantigen immune recognition factor", or all cells of the tissue or organ, or certain types of cells secreting "autoantigen immune recognition factor", and/or secretory "promoting" autoantigen immune recognition factor The secretion of cells such as cells promotes the identification of hematopoietic stem cells by cells that carry out their own antigenic immune recognition factors, or directly inputs these factors. These include: thymus, and/or bone marrow organs in the period of self-antigen immune recognition, and/or certain tissue cells in these organs. And/or thymocytes, bone marrow cells, early immune recognition, hematopoietic stem cells, and/or advanced immune recognition of hematopoietic stem cells, and/or use their own late-stage immune recognition to identify hematopoietic stem cells, re-existing autoantigen immune recognition, allowing the body to re-identify the transplant The antigen or the mutated autoantigen, and the re-identification of the graft antigen or the variant autoantigen is added to the original "autoantigen immune recognition library", so that the newly generated one can recognize the foreign antigen and not react with the autoantigen. CD4+CD8- or CD4 D8+ single positive T cells and B cells The immune system does not immunoreact with the graft antigen or the variant autoantigen.

2. Removal of CD4+CD8- or CD4 D8+ single-positive T-cell nuclear B cells already existing in the body and capable of recognizing foreign antigens and not reacting with autoantigens by resolving the autoantigens by clearing the marrow and semi-myeloablative The cells of the immune system remove these cells that are immune to the graft antigen or the variant autoantigen and the antibodies produced by them. It has been reported in the literature that a variety of B cell monoclonal antibodies (CD9, CD10, CD19, CD20) or T cell antibodies (CD2, CD3, CD4, CD5, etc.) are combined with complement, and/or antibodies bind to cytotoxic drugs such as castor bean toxin. Perform myeloablative treatment of these cells.

Currently used for target cell-specific clearance therapy, a variety of therapies can be used, such as:

1. The monoclonal antibody binds to the corresponding target cell and then adds complement, and the target cell is lysed and broken by activating the classical pathway of complement to achieve purification.

2. Immunotoxin-mediated cytotoxicity method, which combines specific antibodies with cytotoxin (ricin toxin) to form an immunotoxin, which has a strong tumor killing effect and specificity.

3. Immunophysical methods, coating microspheres or metal particles with a monoclonal antibody, labeling the target cells to increase or increase the density of the cells, and then removing the target cells by precipitation or magnetic field adsorption. Each of the above methods has its advantages and disadvantages. For example, anti-CD32 mAb against dendritic cells, anti-CD11 mAb anti-CD79 mAb, anti-CD82 mAb, and other monoclonal antibodies may be used in combination with complement, and/or antibody-binding cytotoxic drugs, and/or antibody binding. Coating microspheres or metal particles with a monoclonal antibody, labeling the target cells to increase the density of the cells or generating magnetic properties, and then removing the target cells by precipitation or magnetic field adsorption, and performing anti-dendritic cell demyelination treatment of the cells, or Other monoclonal antibodies that complement the cells of the autoantigen immune recognition library, combined with complement, and/or antibody binding to cytotoxic drugs, perform myeloablative treatment of these cells.

Using multiple B-cell monoclonal antibodies (CD9, CD10, CD19, CD20) or T-cell antibodies (CD2, CD3, CD4, CD5, etc.) in combination with complement, and/or antibodies binding to cytotoxic drugs such as castor bean toxin, and/or antibodies In combination with the monoclonal antibody or the metal particles coated with the monoclonal antibody, the target cells are labeled to increase the density or magnetic properties, and then the cells are subjected to myeloablative treatment by precipitation or magnetic field adsorption.

The present invention is a cell for stem cells, progenitor cells, etc., which is immunologically recognized and/or immunologically associated with autoantigens, which have not yet been and/or have been immunologically recognized for autoantigens of various tissues and organs in the body, for example, from fertilized eggs. Stem cells associated with autoantigen immunological recognition and/or immunity, such as hematopoietic stem cells, and/or various types of immune-related hematopoietic progenitor cells, are produced at various developmental stages of embryos of gestational age below 7 months of age. After the cells are cultured and expanded, the original, mature, hematopoietic stem cells with immune recognition function are killed, scored or killed by the bone marrow transplantation of the marrow, part of the marrow, or the unclear marrow. Killing, and then inputting expanded stem cells that have not been immunologically recognized by autoantigens of various tissues and organs in the body, and are associated with immune recognition, such as hematopoietic stem cells and/or various types of Early immune recognition of hematopoietic stem cells such as immune-related hematopoietic stem cells and/or advanced immune recognition of hematopoietic stem cells Cells, through these early identification with hematopoietic stem cells and/or late immune recognition hematopoietic cells, re-identify the autoantigens of various tissues and organs (which may include grafts or graft antigens) in the body. And/or stem cells that have been immunologically recognized by early immune recognition of hematopoietic stem cells and/or advanced immune recognition hematopoietic stem cells in vitro for graft or graft antigen, variant tissue immune recognition, by bone marrow transplantation or directly into recipient blood A method in the system that treats a variety of diseases associated with diseases of the immune system, and uses this method to treat a variety of different organs and different types of diseases.

"organisms, organs, cells, and factors related to immune recognition of autoantigens" include: organs such as thymus, spleen, lymph nodes, and bone marrow, and various cells, hormones, and various cells involved in the immune recognition of self antigens contained in these organs. Cytokines, peptides, proteins, tissues, tissue fluids, cells, and more.

The immunological recognition of hematopoietic stem cells required for re-immunization of autoantigens is obtained from the following pathways:

1. Source of cloned cells or fertilized eggs:

1) clonal cells formed from the graft recipient or the somatic cell nucleus of the immune system and the diseased patient to the human egg from which the nucleus is taken out;

2) clonal cells formed from the somatic cell nuclei of the recipient to the animal eggs from which the nuclei are removed;

3) Forming fertilization from the sperm or egg of the recipient and the donor or other human or animal sperm or egg Egg

4) Form the fertilized egg from the sperm or egg of the volunteer donor and the sperm or egg of the recipient or other human or animal.

2. Sources of stem cells without immune recognition -

1) cloning cells or fertilized eggs of different origins are developed into a non-immune-recognized stem cell stage embryo in the intermediary, and the immune-free stem cells are extracted from the embryo;

2) culturing stem cells that are differentiated into non-immune recognition in test tubes;

3) From the embryonic and fetal stages of human or animal, early immune recognition of hematopoietic stem cells, self-antigen immune recognition period, early abortion of hematopoietic stem cells extracted from stillbirths;

4) From the embryo and the embryo stage of the human or animal, and after the birth, the immune recognition hematopoietic cells are subjected to the period of self-antigen immune recognition, and the latent immune recognition hematopoietic stem cells extracted from the stillbirth are aborted;

5) Directly extract late-stage immunoreactive hematopoietic stem cells from the blood system of the recipient or donor or donor.

These clonal cells or fertilized eggs of different origins are developed into an early immune recognition hematopoietic stem cell and/or an embryo of the late immune recognition hematopoietic stem cell stage, and early immune recognition hematopoietic stem cells and/or late immune recognition hematopoiesis are extracted from the embryo. Stem cells, or cultured in vitro, differentiate into early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells.

The extracted early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells can be used not only to treat immune system diseases, but also to treat a variety of other diseases, such as: hematopoietic diseases caused by congenital inheritance, and the like.

Note: The UK Parliament and many governments have agreed to conduct therapeutic research on human cell cloning by researchers.

Early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells for culture expansion is:

1. Amplification is carried out in a test tube.

2. Transplant into the mediator for amplification, and then extract the expanded early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells.

However, most of them are currently expanded in vitro.

Early immune recognition of hematopoietic stem cells and/or advanced immune recognition of hematopoietic stem cells for secondary immune recognition is as follows:

1. carried out in vitro in vitro;

2. Conducted in the intermediary;

3. In the recipient's body.

Early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells is performed in vitro in vitro, or in an intermediary, for secondary immunization of donor grafts, and/or variant autoantigens.

1. Early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells obtained from several pathways in a. in vitro test tubes, b. mediators, cultured, expanded, expanded to a suitable number for transplantation in vivo , performing immunological recognition of the donor's graft antigen and/or the variant autologous antigen in the recipient. Whether amplification is needed can determine the number of hematopoietic stem cells and/or late immune recognition hematopoietic stem cells according to the required early immune identification, such as: The amount required for secondary immune recognition in the transplanted mediator has been met, perhaps No amplification is required.

The early immune recognition hematopoietic stem cells and/or the latent immune recognition hematopoietic stem cells are transplanted into the intermediary, and the intermediary used may be: an immunodeficient animal or an immunodeficient animal.

When using non-immune-deficient animals, it is necessary to perform myeloablative methods to clear the cells involved in the immune system, including the "autoantigen immune recognition library" and the cells storing the autoantigen immune recognition library. An early immunologically recognized hematopoietic cell and/or a latent immune recognition hematopoietic stem cell transplanter is then performed.

2. The early immune recognition hematopoietic stem cells and/or the late immune recognition hematopoietic stem cells are transplanted into the intermediary to identify the secondary immune recognition. 1) The graft or graft antigen and/or the variant internal antigen in the recipient is transferred to the intermediary.

2) Early transplantation of immune recognition hematopoietic stem cells and / or late immune recognition of hematopoietic stem cells into the intermediary.

3) Transplanting "tissue, organ, cell, and factor related to autoantigen immune recognition", including autoantigen immune recognition initiation recognition factor, and/or autoantigen immune recognition termination recognition factor, activation of secondary immune recognition, termination after recognition The factor it identifies.

4) After identification, extract cells such as stem cells that have been subjected to immunological recognition, and perform amplification (may not be amplified according to the situation), transplant them into the recipient after amplification, and transplant the donor's graft into the recipient. In the body. In the case of secondary immune recognition in the recipient, the graft or graft antigen of the transplant donor is transplanted into the recipient, and then the recipient is recognized by secondary immunization.

3. Early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells transplanted into vitro for identification of secondary immune recognition.

1) Transplant recipients and/or donor grafts or graft antigens and/or variants of autologous antigens in recipients into vitro in vitro (the graft or graft antigen and/or the variant autoantigen in the recipient may be It is a tissue, a cell, or an organ, etc.).

2) Re-implantation of early immune recognition hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells.

3) transplantation of "organisms, organs, cells, factors related to autoantigen immune recognition", including autoantigen immune recognition initiation recognition factor, and/or autoantigen immune recognition termination recognition factor into a test tube, which may be an organ It can also organize, cells, factors, and more.

4) Transplantation of autoantigen immune recognition promoters, and/or immune recognition arrest factors, and tissues and organs for self-antigen immune recognition, and/or cells of these tissues and organs that are immunologically recognized by autoantigens are transplanted into vitro tubes. After self-antigen immunological recognition, stem cells that have been immunologically recognized are extracted.

5) Determine whether amplification is required depending on the number of cells, and transplant to the recipient after amplification.

6) The graft of the transplant donor is transplanted into the recipient.

4. Early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells are transplanted into the recipient for recognition of secondary immune recognition.

1) Transplantation of graft or graft antigen into recipients requires whether the transplant can be determined according to the condition to be treated. For example, treatment of immune system disease does not require transplantation.

2) transplanting early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic cells into the recipient, and depending on the condition, whether to transplant the autoantigen immune recognition initiation factor, and/or the autoantigen immune recognition arrest factor, and/or Cells of tissues, organs, and/or tissues and organs that are self-immunologically recognized are transplanted into the body, secondary immune recognition is activated, and recognition is terminated after recognition.

Note: The purpose of early immune recognition of hematopoietic stem cells and/or late immune recognition hematopoietic stem cells for secondary immune recognition is to replace the original self-antigen identification library in the recipient with a new self-antigen identification library. There are two ways to replace it. Pathway:

1. The original self-antigen identification library is completely destroyed, that is, using bone marrow transplantation methods, such as: using radiation, and / or using cytotoxic drugs, and / or using monoclonal antibodies combined with cytotoxic drugs will be the original All cells in the body that store their own antigen recognition library are killed.

1) Input early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells, and / or "organisms, organs, cells, and factors related to autoantigen immune recognition", and perform secondary immune recognition in the recipient.

2) Alternatively, enter a stem cell that has been immunologically recognized by a secondary immune recognition outside the recipient and/or a new "autoantigen immune recognition library" that has been subjected to secondary immune recognition and/or store a new "self" The antigen-immune recognition library "cells, using these two methods to establish a new "self-antigen immune recognition library".

2. On the basis of the original "autoantigen immune recognition library", add new library contents, use partial myeloablative or non-myeloablative methods, and do not remove or partially remove cells with autoantigen immune recognition library stored in the body. The primary immune recognition hematopoietic stem cells and/or the late immune recognition hematopoietic stem cells, and/or the "organisms, organs, cells, and factors associated with autoantigen immune recognition" are subjected to secondary immune recognition in the recipient. Or input through the recipient Stem cells that have been subjected to secondary immunological recognition in vitro and/or cells that store new "autoantigen immune recognition library" cells, and add new library contents. The specific method used, the results of the experiment, can be selected according to the specific needs of the disease.

At present, humans have not isolated factors that initiate, motivate, and stop the identification of autoimmune immune cells by non-immunologically recognized stem cells, but are conducted by a group led by Esmail Zanjani of the University of Nevada, USA. It has been proved in the experiment that it exists. The possible place is "organisms, organs, cells, and factors related to the immune recognition of autoantigens", including: Each organ that allows non-immunologically recognized stem cells to recognize their own antigens, such as T lymphocytes. Immune recognition in the thymus, and B lymphocytes in the bone marrow for immune recognition and other organs and tissues related to autoimmune recognition, while nude mice with thymic function loss have no immune function of T lymphocytes, such as BALB/ cA-nude nude mice, CW Friis of Denmark in 1973 found spontaneously mutated hairless mice in BALB/cA inbred mice. The mutant mouse has thymic dysplasia, immune T cell loss, and has been cultured as BALB. /cA-nude, while C. B-17 SCID mice have no mature B and T lymphocytes, so the thymus, spleen, and lymph nodes The weight is less than 30% of normal, and histologically manifested as a significant defect in lymphocytes. The thymus is surrounded by multiple adipose tissue, no cortical structure, only the remaining medulla, mainly composed of epithelial-like synthetic fibroblasts, and occasionally focal follicular lymphocytes. The spleen white pulp is not obvious, the red pulp is normal, and the spleen has no lymphocyte aggregation, mainly composed of reticular cells. There is no obvious cortical area in the lymph nodes, the cortical area is missing, and there are reticulocytes. Submucosal and bronchial lymph nodes are rare in the small intestine, and there is no lymphoid accumulation in the structure. Both thymus and spleen function are absent and have neither T lymphocyte function nor B lymphocyte function. There were no mature B and T lymphocytes in N0D-SCID mice.

Therefore, it is inferred that multiple organs in tissues, organs, cells, and factors related to immune recognition of autoantigens are important organs for early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells for immune recognition, in which multiple organs are In blood, tissue fluids, various cells, or entire organs, there should be activating recognition factors that promote, promote, and/or halt early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells for self-antigen recognition, motivating recognition factors, and The recognition factors are terminated, which can be: polypeptides, proteins, tissues, cells, organs, and the like. Before the humans are not isolated, cells associated with immune recognition, cells in various organs, which are in the initial stage of embryonic autoantigen immune recognition and/or in the recognition phase and/or termination phase, may be present in the blood and The composition of the tissue fluid, etc., is added to the culture medium, and/or imported into the body of the human or intermediary to initiate early identification of hematopoietic stem cells and/or late immune recognition in a test tube or in a human or intermediary body. Hematopoietic cells are recognized for their own antigens. Once humans have discovered that activating and terminating non-immunologically recognized stem cells for self-antigen immune recognition can be created, it is not necessary to use these organ tissues to initiate and/or terminate non-immune recognition of stem cells for autoantigen immune recognition.

Aborted, from a few days pregnant to about four months, in human embryos, preferably in the body of a dead embryo that is less than three months pregnant, if the embryo has early immunity in organs such as bone marrow or liver By identifying hematopoietic stem cells and/or latent immune recognition hematopoietic stem cells, early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells can be extracted from these organs. If not, early immune recognition hematopoiesis can be directly extracted from the corpse of the fetus. Stem cells and/or late immune recognition of hematopoietic stem cells. And "organisms, organs, cells, and factors related to immune recognition of autoantigens" include: organs such as thymus, spleen, lymph nodes, and bone marrow. The non-immune recognition stem cells of these fetuses are not immunologically recognized by the autoantigens of various tissues and organs in the fetal body. (It can also be obtained from various other sources, such as other kinds of stem cells, such as single cells such as adipose stem cells. The non-immune-recognized stem cells obtained by the transformation, and the non-immunologically recognized stem cells obtained by the differentiation of the embryonic stem cells, culture and amplify the non-immunologically recognized stem cells, such as hematopoietic stem cells and/or hematopoietic progenitor cells, to a certain amount, which is about 1 (The number of Γ ⁶ is preferably l (the number of TV _kB . Patients who have undergone organ transplantation can be taken immediately after surgery according to the condition of the disease, or they can continue taking anti-immune rejection drugs after surgery, and wait until the body recovers to a certain extent. The bone marrow transplantation method is used to remove immune-related cells, including "autoantigen immune recognition library" and cells storing an autoantigen immune recognition library. For example, using drugs such as: cyclophosphamide, and/or radiation irradiation, such as : cobalt ⁶ °, medical linear accelerators, high-energy X-ray whole body irradiation The human body's original method, has been autoantigen immune recognition of hematopoietic stem cells and / or hematopoietic progenitor cells in vivo hematopoietic stem cells to kill the original, the original use of drugs in vivo T lymphocytes Immune cells are killed, for example: Cytotoxic drugs formed by binding CD3 monoclonal antibodies to cytotoxins plus complement input to kill T lymphocytes in the body, etc., killing immune cells in the body's immune system, and / or "self antigen immune recognition library" and drugs that store cells with their own antigen immune recognition library. Re-entering the cultured, expanded non-immune-recognizing stem cells, and/or inputting the early immune recognition hematopoietic stem cells and/or the late-stage immune recognition hematopoietic stem cells to immunologically recognize the autoantigens of various tissues and organs in the fetal body and/or The termination factor, its own antigen immune recognition initiation and/or termination factor, may be one or more polypeptides, one or more proteins, or one or more tissue cells, or a Or multiple tissues and organs.

Simultaneous input (other cells can be included in the input cells, and the original hematopoietic stem cells in the body can be killed without using drugs and/or radiation according to different diseases and different case needs. No immune recognition of stem cells entered into the body). After inputting such amplified non-immune-recognized stem cells, hormones ("autoantigen-immuno-recognition factors", and/or "promoting" ("autoantigen-immuno-recognition factors", and/or "promoting" in the body "immunity-recognition-associated tissues, organs, cells, and factors" Under the action of the secretion of the self-antigen immune recognition factor, and various auxiliary factors required for the immune recognition of the autoantigen, the input amplified non-immune recognition cells will immediately perform a new immune recognition. It recognizes the transplanted organs and the self-organizing tissue that produces the mutation as its normal tissue and organ, which greatly reduces the intensity of the immune attack, and does not even generate new immune attacks. Thereby treating the disease, it can greatly reduce the medical expenses after transplantation of various organs. By using this method, various immune system diseases can be cured or alleviated, and since the transplanted organs, tissues or cells can be re-identified, various diseases such as: islet transplantation for islet transplantation, and the like can be treated by organ or tissue transplantation. Because the input is a healthy fetal amplified non-immune recognition stem cell, it can produce normal blood cells, thus curing various diseases of the hematopoietic system caused by congenital inheritance such as: congenital chain-like erythrocyte anemia. It is also possible to treat other types of diseases by this method as long as the treatment is beneficial to the recovery of the patient. After inputting this new immune recognition with the amplified non-immune recognition cells, the foreign tissue entering the human body or the self-organizing tissue that produces the mutation is regarded as a foreign body, which generates an immune attack and therefore does not occur at present. The use of immunosuppressive agents in the treatment of the problem of reduced immune function.

Because family planning is a basic national policy of the Chinese government, abortion surgery is widespread and legal in all parts of China. At the same time, abortion must be performed within three months of gestational age, which is related to immunity in the embryonic fetus. Stem cells, such as hematopoietic stem cells and/or various types of hematopoietic progenitor cells, perform an immunological recognition of the autoantigens of various tissues and organs in the body. After the fetus is a fragmented stillbirth. Therefore, the extraction of stem cells from a fragmented stillbirth, such as hematopoietic stem cells, has no moral problems, and the diversity of HLA sources of fetal immune-related stem cells, such as hematopoietic stem cells, can be guaranteed. Therefore, the hematopoietic stem cells extracted from the stillbirth obtained by abortion curettage are very suitable for the treatment of adult autoantigen re-identification and immune system diseases.

The non-immune-recognizing stem cells used in the present invention, such stem cells: stem cells derived from abortion of gestational fetuses having a gestational age of less than three months, which are immunologically recognized and/or immune-related, such as hematopoietic stem cells and/or Various types of immune-related hematopoietic progenitor cells, raw embryonic stem cells obtained from various sources in vivo or in vitro, and natural breeding or artificial culture or artificial intervention in embryonic bodies or culture flasks, and original embryos Stem cells or various stem cells obtained from various sources differentiate or transform into various stages of self-antigen immune recognition and/or immune-related stem cells, which may include: totipotent stem cells (embryonic stem cells), pluripotent stem cells ( For example, hematopoietic stem cells and/or various types of stem cells, such as hematopoietic stem cells, and/or various types of stem cells, such as hematopoietic stem cells, and/or immune-related stem cells, such as various hematopoietic progenitor cells, are transformed into various stages. Hematopoietic progenitor cells associated with immunity, However, according to the treatment requirements of the various stages may be used more stem cells, stem cells, also called single multipotent stem cells. Embryonic stem cells, that is, "primitive" cells (ie, pluripotent stem cells) that have not yet differentiated into various tissues in the embryo can differentiate into all cells possessed by the adult body. More than 200 kinds of cells in the human body can be produced by reproduction. Finally, the cells can produce tissues, tissues and synthetic organs, and become human bodies. Embryonic stem cells are mainly found in early embryos. There is a process in embryonic development. The combination of sperm and egg produces fertilized eggs. The fertilized eggs are further divided into morula. The morula is further divided into blastocysts. The blastocysts produce inner cells. At this stage, only one is extracted. Cells can develop into adult bodies, so they are omnipotent stem cells. Multi-functional The cell has been developed late. There are also many stem cells in the bone marrow that are immunologically recognized and/or immune to autoantigens, such as hematopoietic cells, which are pluripotent stem cells. Hematopoietic stem cells are tissue-specific stem cells derived from the mesoderm cells of the embryonic yolk sac and successively migrated to the hematopoietic organs, liver, spleen and bone marrow in the embryo, maintaining the self-determination by asymmetrical mitosis. On the other hand, the progenitor cells of each line are continuously produced to maintain the normal hematopoietic function of the body. Hematopoietic cells (HSC) are key factors in the reconstruction of long-term hematopoiesis after hematopoietic tissue transplantation.

Can differentiate into a wide variety of progenitor cells. It can be differentiated into white blood cells, red blood cells, platelets, etc. in the bone marrow, which is pluripotent. The other is unipotent or multi-functional, can only differentiate into a kind of cell, or differentiate into two closely related cells, called unipotent stem cells (such as multiple hematopoietic progenitors). As long as the various stages and types of stem cells can be induced to differentiate into hematopoietic stem cells by various stem cell lines and transformed into non-immune-recognized stem cells, they can be used for the re-identification of adult autoantigens and the treatment of immune system diseases.

In the case of irradiation and drug killing of the original hematopoietic cells, since only the stem cells of the active phase can be killed, and the stem cells that are not in the active phase cannot be killed, it is necessary to kill the stem cells in the stationary phase, and there are various methods -

1. Use drugs that allow stem cells in quiescent phase to enter the active phase, such as: 5-FU, which inhibits hematopoietic stem cell division by inhibiting DNA cleavage, thus stimulating the body to let the hematopoietic stem cells in quiescent phase enter the active phase, and then proceed Radiation exposure or drugs kill active hematopoietic stem cells; various hematopoietic stimulating factors can also be used to stimulate the quiescent hematopoietic stem cells in the body to enter the active phase, and then irradiate or kill the active hematopoiesis stem cell.

2. Using antibody-mediated chemotherapy for stem cells that have undergone immunological recognition, using monoclonal antibodies against hematopoietic stem cells, and/or various immune progenitor cells, and/or immune cells at various developmental stages or Monoclonal antibody fragments, such as: monoclonal antibody 3A5 Fab' fragment, bispecific antibody, trispecific antibody, antibody cytokine fusion protein, and the like. Novel genetically engineered antibodies continue to emerge, such as humanized antibodies, monovalent small molecule antibodies (Fab, single-chain antibodies, single-domain antibodies, hypervariable region polypeptides, etc.), multivalent small molecule antibodies (double-chain antibodies, triple-chain antibodies, mini Antibodies, etc., certain types of antibodies (bispecific antibodies, trispecific antibodies, antibody cytokine fusion proteins, antigenized antibodies, intracellular antibodies, catalytic antibodies, immunoliposomes, etc.) and antibody fusion proteins (immunotoxin, immunity) Adhesin) A combination of various cytotoxins (such as ricin, etc.). An immunoconjugate comprising a chemically cross-linked monoclonal antibody can be used to produce hematopoietic stem cells, and/or various immune progenitor cells, and/or various immune cells. 1) Antibody-directed chemotherapy (antibody- Mediated chemotherapy), a combination of a killing active cell drug and a monoclonal antibody, an immunologically-killing chemical, a specific killing activity against hematopoietic stem cells, a cytotoxic chemical such as arsenic, cisplatin, fluorouracil, vincristine , doxorubicin, etc.; 2) . Immunotoxin therapy, the toxin is linked to a monoclonal antibody, and the prepared immunotoxin is a stem cell that has been immunologically recognized in the recipient, or one of them, Several cells have specific strong killing activity. There are two types of commonly used toxins: one is phytotoxin, including castor bean toxin (RT), acacia toxin (abrin), and bitter melon toxin (MD). The other type is cytotoxins, including diphtheria toxin (DT), Pseudomonas aeruginosa exotoxin (PE), and the like. Specific antibodies such as hematopoietic stem cells and/or various immune progenitor cells can be used, such as monoclonal antibodies such as CD34+ or A133, such as hematopoietic stem cell-specific antibodies, combined with cytotoxic drugs, such as cytotoxic drugs such as ricin and diphtheria toxin. A specific missile that kills hematopoietic stem cells, kills residual hematopoietic stem cells, and/or various immune progenitor cells, and/or various immune cells. Brief Description of the Drawings Fig. 1 is a flow chart showing a method for extracting, culturing and amplifying hematopoietic stem cells and/or late immune recognition hematopoietic stem cells of the present invention;

Figure 2 is a flow chart showing the treatment of adult autoantigen re-identification and immune system diseases by organ transplant patients and patients with immune system diseases of the present invention;

Figure 3. Is a method for obtaining, culturing, amplifying, and performing secondary immune recognition of a hematopoietic stem cell and/or a late immune recognition hematopoietic stem cell in vitro, in vitro, for treating a graft rejection reaction, and/or Immunology Flow chart of the systemic disease method;

Figure 4 is a diagram of the present invention for early immune recognition of hematopoietic stem cells and/or advanced immune recognition of hematopoietic stem cells in vitro, extraction, expansion, and secondary immune recognition in recipients to treat graft rejection, And/or a flow chart of the immune system disease method;

Figure 5 is a flow chart showing an experimental method for performing secondary immune recognition by an early immune recognition hematopoietic stem cell and/or a late immune recognition hematopoietic stem cell in a mediator;

Figure 6. is a flow chart showing another method for extracting, culturing and amplifying an early immune recognition hematopoietic stem cell and/or a late immune recognition hematopoietic stem cell for bone marrow transplantation of the present invention;

Figure 7 is a flow chart showing the treatment of adult autoantigen re-identification and immune system diseases in another organ transplant patient and immune system disease patient of the present invention;

Figure 8 is a flow chart showing the extraction of hematopoietic stem cells and thymocytes and bone marrow cells of one embodiment of the present invention; and Figure 9 is an illustration of an autoantigen re-identification and immune system disease of an organ transplant in an animal of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, embodiments will be further described with reference to the accompanying drawings. Example 1

Fig. 1. A flow chart of a method for extracting, culturing and amplifying hematopoietic stem cells and/or late immune recognition hematopoietic stem cells according to the present invention. Stem cells without immune recognition are obtained, and the following is an example of the acquisition of hematopoietic stem cells.

1. The stillbirth from abortion: The liver and bone marrow of the stillbirth are peeled off under the microscope. Because the bone marrow is relatively easy to obtain, it is best to obtain stem cells without immune recognition from the bone marrow, but because the fetus is liver hematopoietic, it can also be Stem cells that are not immune-recognized are obtained in the liver, or stem cells that are not immune-recognized from other organs or tissues.

2. Extract nucleated cells.

3. Immunomagnetic beads-flow cytometry two-step sorting method can be used to sort out non-immunologically recognized stem cells from bone marrow, for example, monoclonal antibodies and packages using stem cell antigen (Stemcellantigen, Sea) specifically expressed on the surface of stem cells. Hematopoietic stem cells which are not immunologically recognized in the bone marrow are separated by a magnetic adsorption cell sorting method (MACS) by a second antibody on the surface of the magnetic particle. Other methods can also be used to sort out stem cells that are not immunologically recognized.

Restriction-dilution tests showed that: cells with long-term continuous implantation were KDR+, and 20% of CD34+ KDR ⁺ cells in bone marrow were hematopoietic stem cells; hematopoietic stem cells with long-term implantation ability were CD34 + KDR+ subpopulations, so CD34 could be used. ⁺ , KDR+ monoclonal antibodies are sorted. Other markers for selecting hematopoietic stem cells can also be used for sorting, such as: AC133, CD50 ^+, and the like.

4. The sorted non-immune-recognized stem cells are cultured in a culture medium containing a combination of different hematopoietic growth factors, and may be a serum-free medium. The currently identified stimulatory and inhibitory factors that act on the expansion (proliferation) of hematopoietic stem cells are quite numerous, but only a few cytokines can function alone. A variety of hematopoietic growth factors can be added to serum-free medium, such as SCF, IL-3, IL-6, TP0, Flt3-L, G-CSF, and Epo. In the expansion culture, SCF, IL-3 and GM-CSF mainly maintain the survival of the original hematopoietic cells, but can not induce their proliferation. The proliferation of the cells is required to induce proliferation. The bone marrow mesenchymal stem cells can be co-cultured with the hematopoietic stem cells. Hematopoietic stem cells have enhanced amplification. The number of nucleated cells (NC), surface antigens, and colony forming ability were monitored. The in vitro expansion culture is generally carried out for about 2 weeks. Of course, you can increase or decrease the number of days according to the actual situation.

5. When the time of in vitro expansion culture is reached, and/or the required number of non-immune-recognized stem cells for bone marrow transplantation is reached, it can be used immediately for bone marrow transplantation or cryopreservation. Example 2

Figure 2 is a flow chart showing the treatment of adult autoantigen re-identification and immune system diseases in patients with organ transplantation and patients with immune system diseases according to the present invention.

Can refer to the "Clinical Guide to Organ Transplantation" (Editor: Ye Qi, Beijing Science Press, First Edition, July 1999)

1. Organ transplantation stage:

1) First, prepare various preparations for donor organ and tissue cell transplantation.

2) HLA matching between the donor organ, the donor of the tissue cell, and the recipient patient to be transplanted, to prevent rejection, so that the transplanted organ can survive.

3) Perform an organ transplant operation.

4) Use anti-rejection drugs to prevent rejection.

Non-immune-recognized stem cell bone marrow transplantation is prepared after transplantation of the host patient or in the case of a patient such as an immune system disease.

2. Bone marrow transplantation, secondary immune antigen recognition stage:

1) The use of drugs such as: cyclophosphamide, and radiation exposure such as: cobalt ⁶ °, medical linear accelerator high-energy X-ray whole body irradiation (TBI) to kill the body's original, mature hematopoietic stem cells that have been immunologically recognized. Enter the sterile ward.

2) Enter the culture-amplified thousand cells that have not been immunologically recognized, such as hematopoietic stem cells. It is also possible to directly enter the body without the use of drugs and/or radiation to kill the original hematopoietic stem cells in accordance with different diseases and different case needs.

3) After inputting culture-amplified stem cells that have not been subjected to immunological recognition, drugs that promote hematopoietic stem cell homing may be used according to the condition (hematopoietic stem cell (HSC) homing refers to HSC passing through the peripheral blood circulation into the recipient after intravenous transplantation) Recognition and localization in the bone marrow mediated by complex intermolecular interactions. And in vivo stem cell expansion treatment (exvi _V0 ). The stem cell factor SCF can be used in combination with G-CSF to stimulate expansion of the chimeric/progenitor cells (PBPC) in vivo.

4) Prevention and treatment of complications after bone marrow transplantation: such as prevention of early rejection, use of anti-rejection drugs, prevention of infection treatment, and other treatments. Prevention of engraftment syndrome, using corticosteroids and other drugs. Because hematopoietic stem cell transplantation requires autoimmune recognition of the antigen and reconstitution of the immune system over a period of time, there is no anti-infective ability within this time period, requiring a strict aseptic environment, and antibacterial and antiviral infection treatment. .

5) The immune recognition of autoantigens is the time required for those factors to promote hematopoietic stem cells for autoimmune recognition and immune system reconstruction. At this time, it is necessary to track various immunological test indicators.

6) After the successful transplantation, leave the sterile ward, and then resume immune function treatment, such as injection of various vaccines.

The organ transplantation stage and bone marrow transplantation, and the secondary autoantigen immune recognition stage can be performed simultaneously, or the graft antigen can be transplanted first for bone marrow transplantation, secondary self antigen immune recognition, and the body's immune system recognizes the graft antigen as After the autoantigen, organ transplantation is performed, which can increase the survival rate of the graft. Example 3

Figure 3. It is a method for obtaining, culturing, amplifying and performing secondary immune recognition of a hematopoietic stem cell and/or a late immune recognition hematopoietic stem cell in vitro for human immunodiagnosis in vitro, and treating a graft rejection reaction, and/or Flow chart of the method of immune system disease.

1. Fertilized eggs (which can also be cloned egg cells) can be obtained in several ways: 1) After normal pregnancy, a miscarriage occurs, and no early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells, and/or transplants to be transplanted are extracted from stillborn fetuses. This graft may be organs, tissues, Cells, such as: liver, kidney, islet e cells, etc.

2) The somatic cell nucleus of the recipient or donor is cloned into the egg cell of the same species as the recipient or donor, and the formed clonal egg cell develops in the test tube, or in the human body, or in the intermediary. Immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cell stages, extraction of immunoglobulins, and/or graft cells requiring transplantation.

3) The somatic cell nuclei of the recipient or donor are cloned into the egg cell of the different species of the nucleus to form a cloned egg cell, which develops into an early immune recognition hematopoietic stem cell and/or late immunity in a test tube or in an intermediary or human body. The hematopoietic stem cell stage is identified, and early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells, and/or grafts requiring transplantation are extracted.

4) The fertilized egg obtained by fertilization of the sperm or egg of the recipient and the sperm or egg of the same species, including the fertilized egg of the recipient and/or the donor parent and the mother egg, in a test tube or in a mediator or The human body develops into an early immune recognition hematopoietic stem cell and/or a late immune recognition hematopoietic stem cell stage, extracting early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells, and/or transplants requiring transplantation.

5) Fertilized eggs can also be obtained from a variety of different pathways using different methods.

2. In vitro culture and/or in vivo development of fertilized eggs to obtain early immune recognition of hematopoietic stem cells and/or advanced immune recognition of hematopoietic stem cells.

1) In vitro culture is carried out by culturing a fertilized egg in a culture flask containing a medium, adding a hematopoietic stem cell-directed differentiation factor, and differentiating into an early immune recognition hematopoietic stem cell and/or a late immune recognition hematopoietic stem cell.

2) The fertilized egg is implanted in an intermediary or a human body, such as a human uterus, or an animal such as a mouse or a rabbit, and the development time can be determined according to the specific needs of the medical condition, for example, waiting for embryo development. Early appropriate identification of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells for any suitable period of time prior to autoimmune recognition. The early immunological recognition hematopoietic stem cells and/or the late immune recognition hematopoietic stem cells are extracted by the set method, for example, using a flow cytometer. It is also possible to perform amplification in a culture flask after extracting early detection of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells as needed.

3. There are multiple ways for early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells for self-regenic recognition and graft culture development. E.g:

1) In the intermediary, including 1. Transplantation of early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells into immunodeficient mediators, such as immunodeficient small BALB/cA-nude nude mice, SCID Mouse, NOD-SCID mice were performed in the body. Early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells and/or grafts can also be transplanted into the immunodeficient mediator body by bone marrow transplantation.

2) Perform in a culture flask.

3) In the recipient's body.

4) Hematopoietic stem cells and/or immune stem cells isolated and extracted from the recipient.

5) Non-immune recognition stem cells extracted from aborted fetuses, and/or isolated organs to be transplanted.

4. Specific methods for early immune recognition of hematopoietic stem cells and/or advanced immune recognition of hematopoietic stem cells in the mediator for self-antigen recognition (secondary immune recognition):

1) Secondary immunization recognition in the intermediary - a. Transplantation of the donor's or graft antigen or variant (recipient's) autoantigen transplanted into the human or animal body to no immune function (or Immune-deficient mediators, and/or through the use of bone marrow transplantation to kill stem cells and immune-related cells that have undergone immune recognition, and/or organs associated with immune recognition have lost their proper function after being irradiated Intermediary body. It is also possible to transplant donor hematopoietic stem cells and/or immune stem cells and immune recognition factors and/or thymocytes, bone marrow cells. Autoimmune recognition of the antigen is performed, followed by graft transplantation.

b. The transplanter or the intermediary is starting or undergoing immunization of the embryo at the stage of self-antigen immune recognition Identifying organs, and/or different types and types of tissues, cells, and/or internal organs associated with immune recognition that are selected for different types and types of organs that are transplanted according to design requirements, and/or different types of organs associated with immune recognition Contents such as tissue fluids are transferred to the intermediary. Deficiency of immune organ function in immunodeficiency mediators, and/or mediators undergoing radiation and/or drug therapy.

C. Transplantation early identification of hematopoietic stem cells and/or latent immune recognition of hematopoietic stem cells to the intermediary. d. The initiation factor can be input according to the design requirements to enable early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells to perform autoantigen immune recognition. It is also possible to use an immunologically-recognized organ of an embryo or an intermediary who is starting or performing an embryo at the stage of self-antigen immune recognition, and/or a different type, different type of cells selected from organs transplanted according to design requirements, and / or a starter factor, and/or a stop factor that already exists when the contents of different types, different types of tissue fluids inside the organ are in the intermediary.

e. Decide whether a need to enter a stop factor to enable early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells to stop the autoimmune immune recognition of the arrest factor.

f. Extract the stem cells that have been immunologically recognized from the intermediary using a flow cytometer or other device. g. Cellular expansion of early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells or stem cells that have been immunologically recognized.

h. Thousand cells that have been immunologically recognized are transplanted through the bone marrow and/or transplanted directly into the recipient.

i. Organs, tissues, and cells of the graft can be transplanted before, at the same time as, or after transplantation of the immunoreactive stem cells into the recipient. And / or treatment of immune system diseases.

2) Secondary immunization recognition in test tubes and culture flasks - a. Transplantation of grafts or graft antigens or variant autoantigens prepared for transplantation in humans into test tubes.

b. The transplanter or the intermediary is starting or undergoing an immune recognition organ of the embryo at the stage of self-antigen immune recognition, and/or is selected according to the design requirements to transplant the embryo in the selected different types, different types of immunity with the autoantigen Identify relevant tissues, cells, and/or contents of different types, different types of tissue fluids inside the organs associated with the immune recognition into the test tubes.

c. Transplantation of human early identification of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells into test tubes.

d. Input activating factor to enable early immune recognition of hematopoietic stem cells and/or late immune recognition hematopoietic stem cells for autoantigen immune recognition. And/or an immunologically recognized organ of an embryo or an intermediary who is starting or undergoing an embryo at the stage of self-antigen immune recognition, and/or selected different types, types of cells in an organ transplanted according to design requirements, and / or contents of different types, different types of tissue fluid inside the organ into the test tube.

e. The abort factor can be input as needed to enable early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells to stop autoantigen immune recognition.

f. Extract into stem cells that have been subjected to immunological recognition.

g. Amplification of stem cells that have been subjected to immunological recognition.

h. by the aforementioned method of bone marrow transplantation, and / or directly transplanted into the recipient.

i. Organs, tissues, and cells of the graft can be transplanted after the stem cells that have been subjected to immunological recognition are transplanted into the recipient. And / or treatment of immune system diseases. Since the immune system in the body has already recognized the graft as a self-antigen, immune rejection does not occur even if the HLA of the graft does not match the recipient. At the same time, the intermediary can be used to cultivate the organ to be transplanted. The source of the organ that needs to be transplanted is further expanded. Example 4

Figure 4 is a diagram of the present invention for early immune recognition of hematopoietic stem cells and/or advanced immune recognition of hematopoietic stem cells in vitro, extraction, expansion, and secondary immune recognition in recipients to treat graft rejection, And/or a flow chart of the immune system disease method.

1. Fertilized eggs (which can also be cloned egg cells) can be obtained in several ways - 1) After a normal pregnancy, a miscarriage occurs, and no early immune recognition of hematopoietic stem cells and/or late immune recognition is extracted from stillborn fetuses. Hematopoietic stem cells, and/or grafts that require transplantation, this graft can be an organ, Tissues, cells, such as: liver, kidney, islet beta cells, etc.

2) The somatic cell nucleus of the recipient or donor is cloned into the egg cell of the same species as the recipient or donor, and the formed clonal egg cell develops in the test tube, or in the human body, or in the intermediary. Immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cell stages, extraction of dry tissue without immunological recognition, and/or graft cells requiring transplantation.

4) The fertilized egg obtained by fertilization of the sperm or egg of the recipient and the sperm or egg of the same species, including the fertilized egg of the recipient and/or the donor parent and the mother egg, in a test tube or in a mediator or The human body develops into an early immune recognition hematopoietic stem cell and/or a late immune recognition hematopoietic stem cell stage, extracting early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells, and/or grafts requiring transplantation.

1) Perform in vitro culture of recipients, differentiate into early immune recognition hematopoietic stem cells and/or advanced immune recognition hematopoietic stem cells.

2) Implantation in the intermediary or human body, according to the design requirements, when the embryo develops to the early stage of immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells for autoimmune recognition. The embryos are extracted early to immunologically identify hematopoietic stem cells and/or late immune recognition hematopoietic stem cells, and/or various immune-related progenitor cells (pluripotent stem cells), and/or various immune-related pluripotent stem cells.

3) Hematopoietic stem cells and/or immune stem cells isolated and extracted from the recipient.

4) Non-immune recognition stem cells extracted from aborted fetuses, and/or isolated organs to be transplanted.

3. Cellular expansion of early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells in test tubes, culture flasks or culture vessels.

4. Transplant the graft or graft antigen to be transplanted into the human body into the human body. At the same time, anti-rejection therapy should be carried out. This step is not necessary if you are treating a variety of diseases such as your own immune disease.

5. Early immunization to identify hematopoietic stem cells and/or late immune recognition hematopoietic stem cells through bone marrow transplantation (may be the condition of the disease, using myeloablative bone marrow transplantation, partial myeloablative bone marrow transplantation, or nonmyeloablative bone marrow transplantation) Transplanted into the recipient.

6. The transplanted embryo is beginning or undergoing an immunological recognition organ at the stage of autoantigen immune recognition to the recipient. And/or an input promoter that allows early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells for autoantigen recognition. This step can be determined according to the actual situation at the time of treatment.

7. The abort factor can be input as needed to enable early immune recognition of hematopoietic stem cells and/or late immune recognition hematopoietic cells to stop autoantigen immune recognition.

Treatment of graft rejection and immune system diseases. This step can be determined according to the actual situation at the time of treatment. Example 5

Figure 5 is a flow chart showing an experimental method for early immune recognition of hematopoietic stem cells and/or late immune recognition hematopoietic stem cells of the present invention in a mediator.

Performing an early immunological identification of hematopoietic stem cells and/or a latent immune recognition hematopoietic stem cell extraction, culture, amplification, and mediator experiment for secondary immune recognition in a mediator can use the following method: The mediator is divided into two parts and cultured simultaneously. .

The first group: is the transplant donor's graft or graft antigen to the mediator's body group. 1. The intermediary used can use a. immunodeficient animals, such as: nude mice, SCID mice, NOD-SCID mice, etc., b. non-immune-deficient animals, for myeloablative, semi-myeloablative or non-myeloablative treatment, Early immune recognition of hematopoietic stem cells and/or advanced immune recognition of hematopoietic stem cell bone marrow transplanted animals can be performed.

2. Transplant donor or/or recipient graft or graft antigen into the body of the intermediary. In order to be immune-free, stem cells can recognize the donor and/or recipient's own antigen as self-antigens when performing self-antigen recognition. Since the recipient's non-immune recognition stem cells themselves also carry the recipient's own antigen and have strong expression, it is possible to experimentally determine whether or not to additionally transplant the recipient's graft antigen into the intermediary.

3. Transplant immune organs into the intermediary, such as transplanted human thymus, spleen, human bone marrow tissue, organs and other organs, or cells in these tissues and organs related to autoimmune recognition. This step of transplanting the immune organ can be determined according to the test requirements.

4. Enter the initiation factor and/or termination factor ("tissue, organ, cell, factor associated with autoantigen immune recognition"). It is best to transplant or input the "organisms, organs, cells, and factors related to the autoimmune recognition of autoantigens" of the same species. It is also possible to use "species, organs, cells, and factors related to immune recognition of autoantigens" of different species. / This step of the transplant can be determined according to the experimental requirements.

5. Transplantation of non-immune recognition stem cells of the same species or of different species for self-antigen recognition. The graft antigen is also recognized as a self antigen at this time.

6. At this point the intermediary becomes: If the graft is human, such as a transplanted human hematopoietic stem cell, the animal at this time has a human or an animal that has undergone an immune system that has been identified by the graft or graft antigen. This animal with human immune system for graft or graft antigen recognition can be used not only for this experiment, but also for experiments of various types and purposes.

7. Immune recognition of human stem cells using immunomagnetic beads, and/or flow cytometry.

8. The extracted non-immune-recognized human stem cells are transplanted into a second group of intermediaries that have undergone autoantigen immune recognition.

Group 2: The graft or graft antigen of the donor is not transplanted into the body of the intermediary.

1. The mediator used can use a. immunodeficient animals, such as: nude mice, SCID mice or NOD-SCID mice, etc. b. Non-immune-deficient animals, but after clear marrow treatment, early immune recognition can be performed to hematopoiesis Stem cells and/or advanced immune recognition of hematopoietic stem cell bone marrow transplanted animals. The experiment can be carried out by taking SCID mice or NOD-SCID mice as an example.

2. Transplant the recipient's graft or graft antigen into the body of the intermediary. Since the recipient's non-immune recognition stem cells themselves carry the recipient's own antigen and have strong expression, it is possible to experimentally determine whether or not to additionally transplant the recipient's graft antigen into the intermediary. At this time, because the intermediary has no immune function, the graft is easy to transplant successfully. The intermediary can be used as a culture of the graft, allowing relatively small grafts to grow and develop in the intermediary's body, to reach a portable size. After that, the graft is taken out from the intermediary and transplanted into the recipient.

3. Immune organs are transplanted into the intermediary, such as transplanted homologous animals, or xenogeneic animals, or human thymus, spleen, human bone marrow tissue, and the like.

4. Enter the activation factor and/or termination factor.

5. Transplantation of non-immune recognition human cells for self-antigen recognition.

6. At this point the intermediary becomes: If the graft is human, then the animal has the human immune system at this time (the donor has not been graft-immunized). This animal with human immune system can be used not only for this experiment, but also for experiments of various types and purposes.

This intermediary with the human immune system is divided into two groups for experimentation - A. Group:

1. Transplantation of non-immune-recognized human stem cells extracted from the first group of mediators using myeloablative, partially clear pulp, or non-myeloablative bone marrow transplantation.

2. Transplant the graft or graft antigen to the body or body of the intermediary.

3. Observe the presence or absence of rejection. 4. The experiment is over.

This is to observe whether the graft rejection can be treated after the thousand-cell transplantation after immunological recognition, or to treat diseases of the immune system.

B. Group:

Because the immune system in the animal has a human immune system and is no longer an immunodeficiency animal, the transplantation of the graft is performed at this time for tissue-related matching, only HLA-matched grafts, or HLA half. Matching can be successfully transplanted. In this experiment, HLA semi-matched grafts were used for transplantation, and anti-rejection drugs were administered after transplantation.

1. Using a myeloablative method for bone marrow transplantation, a human bone marrow transplant is performed without immunologically recognized human stem cells.

2. Enter the start factor and / or termination factor.

3. Stop anti-rejection drugs.

4. Observe the presence or absence of graft rejection.

5. The experiment is over.

This is a method for observing hematopoietic stem cells and/or advanced immune recognition hematopoietic stem cell transplantation using partial myeloablative or whole myeloablative, to observe whether the autoantigen can be re-identified, the graft is recognized as an autoantigen, and the graft is treated. Different reactions, or can treat diseases of the immune system.

Note: Among them, early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells can be obtained using the method mentioned in the description of Fig. 3. The immune organs used for immune organ transplantation can be used either human or animal.

Methods for identifying hematopoietic stem cells and/or advanced immune recognition hematopoietic stem cells and performing stem cell identification using early immunization are:

1. Transplant the graft into the recipient, and then perform early immunological identification of hematopoietic stem cells and/or advanced immune recognition hematopoietic stem cell transplantation, so that early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells are twice in the body. Immune recognition, treatment of rejection.

2. The first thousand cells that have been immunologically recognized by the recipient's secondary immune recognition in vitro are transplanted into the recipient, and then the graft is transplanted into the recipient, and the graft is not caused to be excluded. reaction. Example 6

Figure 6. is a flow chart showing another method for extracting, culturing and amplifying hematopoietic stem cells and/or advanced immune recognition hematopoietic stem cells for early bone marrow transplantation in accordance with the present invention.

Early immune recognition of hematopoietic stem cells and/or advanced immune recognition of hematopoietic stem cells is obtained, exemplified by the acquisition of hematopoietic stem cells.

1. Stillbirth from abortion: Stripping can extract the organs needed for secondary immune recognition: thymus, bone, liver. Strip the liver, thymus and bone of the stillbirth, because the thymus and bones are easier to obtain, so

"The tissues, organs, cells, and factors involved in the immune recognition of autoantigens" are preferably obtained from the thymus and bones. Because the fetus is liver hematopoietic, early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells can be obtained from the liver, or early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells can be obtained from other organs or tissues. .

2. The fetal liver is made into a single cell suspension, and a nucleated cell containing a relatively high concentration of early immunologically recognized hematopoietic stem cells and/or a late immune recognition hematopoietic stem cell is extracted by density gradient centrifugation. The thymus and bone can be directly transplanted into the body, and can also be made into a thymus cell suspension, and a bone marrow cell suspension. It is also possible to extract a tissue related to autoantigen immune recognition by using a special cell separation method such as an immunomagnetic bead. The desired cells related to the immune recognition of the autoantigen in the organs, cells, and factors are further cultured or used directly.

3. Density gradient centrifugation can be used to extract nucleated cells containing high concentrations of early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells, or to further utilize immunomagnetic beads-flow cytometry The method selects early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells from fetal liver cell suspension, for example, monoclonal antibody using stem cell antigen (Stemcellantigen, Sea) specifically expressed on the surface of stem cells and coated with A second antibody on the surface of the magnetic particle is separated from the hematopoietic stem cell which is not immunologically recognized in the bone marrow by a magnetic adsorption cell sorting method (MACS:). Other methods can also be used to sort out early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells. Thymocytes and bone marrow cells required for immune recognition were extracted from thymus and bone marrow suspension.

Restriction-dilution tests showed that: cells with long-term continuous implantation were KDR+, and 20% of CD34+ KDR ⁺ cells in bone marrow were hematopoietic stem cells; hematopoietic stem cells with long-term implantation ability were CD34 ⁺ KDR ⁺ subpopulations, so they can be used. Monoclonal antibodies to CD34 ⁺ and KDR+ were sorted. Other markers for selecting hematopoietic stem cells can also be used for sorting, such as: AC133, CD50. ¹ and the like.

1. The sorted thymocytes, bone marrow cells, early immune recognition hematopoietic stem cells, and/or late immunorecognition hematopoietic stem cells are cultured in a culture medium containing a combination of different growth factors for amplification, and may be a serum-free culture solution. The currently identified stimuli and inhibitors that act on the expansion (proliferation) of hematopoietic stem cells are quite numerous, but only a few cytokines can function alone. Various hematopoietic stem cell growth factors can be added to serum-free medium, such as SCF, IL-3, IL-6, TP0, Flt3-L, G-CSF and Epo, and other hematopoietic growth factors are combined to expand in vitro. Increasing culture, in which SCF, IL-3 and GM-CSF mainly maintain the survival of primitive hematopoietic cells, but can not induce their proliferation. Inducing proliferation requires a synergistic effect between the factors. BMSCs can be co-cultured with hematopoietic stem cells. Hematopoietic stem cells have the effect of enhancing amplification. The nucleated cell number (NC), surface antigen, and colony forming ability were monitored. The in vitro expansion culture is generally carried out for about 2 weeks. Of course, you can increase or decrease the number of days according to the actual situation.

2. When the time of in vitro expansion culture is reached, and/or the number of early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells for bone marrow transplantation is reached, it can be used immediately for bone marrow transplantation or low temperature. Store frozen. Example 7

Figure 7 is a flow chart showing the treatment of adult autoantigen re-identification and immune system diseases in another organ transplant patient and immune system disease patient of the present invention.

1. Organ transplantation stage:

2) Perform tissue-associated antigen matching between the donor organ, tissue cells, and the trophy of the host patient to prevent rejection, so that the transplanted organ can survive.

3) Perform an organ transplant operation.

4) Use anti-rejection drugs to prevent rejection.

This organ transplant surgery can be performed before the secondary antigen recognition of the autoantigen, or the graft antigen can be transplanted into the body first, and the secondary antigen recognition of the autoantigen is first performed, and the graft antigen to be transplanted is recognized as the autoantigen, and the organ transplantation is performed. Surgery, which eliminates the need for tissue-associated antigen matching and prevents postoperative rejection.

2. Bone marrow transplantation stage -

1) Clearing immune-related cells, including the "autoantigen immune recognition library" and cells storing the autoantigen immune recognition library. Non-specific clearance, and / or specific immune blood method can be used to remove immune-related cells, including: "autoantigen immune recognition library" and cells storing the autoantigen immune recognition library, as well as peripheral blood and immune tissue. Immune cells, including: T cells and B cells that have undergone negative selection and positive selection in the thymus and bone marrow. 2) Input hematopoietic stem cells and/or late immune recognition hematopoietic stem cells through "immunity identification of tissues, organs, cells, factors related to autoantigen recognition" and/or early amplification by culture.

3) According to different diseases and different case needs, the original hematopoietic stem cells in the body can be killed without using drugs and/or radiation, and the early immune recognition hematopoietic stem cells and/or late immune recognition hematopoietic stem cells can be directly And/or "organisms, organs, cells, factors related to immune recognition of autoantigens" are entered into the body.

4) The immune recognition of autoantigens is a factor such as "autoantigen immune recognition factor" in "organizations, organs, cells, and factors related to autoimmune recognition of autoantigens" and/or various substances required for autoimmune recognition. The role of early immune recognition of hematopoietic stem cells and/or late immune recognition of hematopoietic stem cells. Perform self-antigen immune recognition. In the middle, it is necessary to observe the time required for the reconstruction of the immune system. At this time, it is necessary to track various immunological test indicators.

5) After bone marrow transplantation, complications can be prevented and treated according to the needs of the disease: such as prevention of early rejection, use of anti-rejection drugs, prevention of infection treatment, and other treatments. Prevention of engraftment syndrome, using corticosteroids and other drugs. Because hematopoietic stem cell transplantation requires autoimmune recognition of the antigen and reconstitution of the immune system over a period of time, there is no anti-infective ability within this time period, requiring a strict aseptic environment, and antibacterial and antiviral infection treatment. .

6) After successful transplantation or autoimmune disease treatment, immune function recovery treatment can be carried out as needed, such as injection of various vaccines. Example 8

Figure 8. Is an extraction process of hematopoietic stem cells, thymocytes, and bone marrow cells of one embodiment of the present invention: Fetus: 3 to 4 months old

Tissue / Cell Acquisition:

1. 75% ethanol soaked the fetus for 2 to 5 minutes, disinfected

2. Remove the thymus, liver, femur, tibia, and tibia, and place in the culture medium (containing 0.02% EDTA RPMI1640 medium)

3, extract thymus extract limbs

The thymus is ground into a single cell solution in a serum-free cell culture medium, and the thymus is gently ground by a tissue grinder to prepare a thymus cell suspension, and the bone marrow cells are washed out with the culture solution.

4, lysis of red blood cells, sieving and filtering impurities, specific cell extraction methods, secretion of "autoantigen immune recognition factor" cells, cell culture amplification, extraction: autoantigen immune recognition factor, transgenic cell clone, preparation: autoantigen immune recognition Factor

5, remove the liver

1) Cut into small pieces in a Petri dish (5 hidden left and right), and make a cell suspension in batches with a tissue grinder.

2) Centrifuge the supernatant, add the red blood cell lysate, and lyse for 10 minutes in the dark.

3) Centrifuge the supernatant and resuspend the pellet with 1640 medium.

4) 100~200 mesh cell sieve filtration hepatocyte suspension

5) Density gradient centrifugation

3 ml of a Percoll cell separation solution having a specific gravity of 1.110 was added to the bottom of the centrifuge tube, and then 3 ml of a Percoll cell separation solution having a specific gravity of 1.077 and 1.035 was slowly added thereto in the upper layer. The cell suspension-filtered liver cell suspension was added to the upper layer of the separation solution, centrifuged at 1500 rpm for 20 minutes.

6) Remove the upper layer of liquid and take the precipitate for 2 times with 11640 medium.

7) Purification of human CD34+ antibody immunomagnetic beads

The above three cells (thymus, bone marrow, liver) were adjusted to a concentration of 5× ^{10 6} with a culture solution containing 10% DMS0, and stored in liquid nitrogen.

If 3 ml of a cell separation liquid having a specific gravity of 1.077 was added to the bottom of the centrifuge tube, and then 3 ml of a cell separation liquid having a specific gravity of 1.035 was slowly added to the upper layer. The cell strain filtered hepatocyte suspension was added to the upper layer of the separation solution at 1500 rpm. Centrifuge for 20 minutes. The supernatant liquid was removed, and the precipitate was washed twice with 11640 culture solution. The isolated hepatocyte suspension was then labeled with human CD34 antibody and the proportion of hematopoietic stem cells was measured by flow cytometry. The purity of the human hematopoietic stem cells can be obtained from 13 to 30%. Centrifugation can also be carried out using continuous density gradient centrifugation to select the appropriate concentration.

Example 9

Figure 9. is an illustration of an autoantigen re-identification and immune system disease of an organ transplant in an animal of the present invention.

This example is an experiment for verifying the theory of the present invention, but this experiment also demonstrates that: tissues, organs, and cells related to immune recognition of autoantigens can be transplanted in the body of an animal having certain immune function. "Factors" include: thymus, thymocytes, bone marrow, bone marrow cells, and immune recognition of hematopoietic stem cells to obtain experimental animals with human immune system functions. This animal with human immune function, because of its human immune system, can Substituting the human body for various medical experiments, so that many scientific experiments that cannot be performed because of certain damage to the human body can be tested on such animals, and have the same effect as human experiments, because the St. Jude Children's Research Hospital of the United States ( St. Jude Children's Research Hospital, published in the May 15, 2005 issue of The journal of Immunology P6540-6545, describing them in

NOD/LtSz-scid IL2R Y ^nu11 transplanted human hematopoietic stem cells into an experimental animal model of the human immune system. Only NOD/LtSz-scid IL2R Y " ^u " mice are NK cells that are non-functional and cannot kill transplanted human hematopoietic stem cells, so this mouse does not have any immune function and is rejection of the graft. , is completely acceptable. In their experiments, NOD/LtSz-scid IL2R y ^nu mice with no immune function were used as experimental group and NOD-scid mice with certain immune function as control group, and NOD/LtSz- scid IL2R y ^{nu11 was} small. In the experimental group of rats, the human hematopoietic cell transplantation was successful, and the NOD/LtSz- scid IL2R y ^nu11 mice obtained human immune cells, which have human immune function, while the NOD-scid mice with certain immune function have hematopoiesis. The failure of stem cell transplantation, the result of hematopoietic stem cell transplantation failure of the NOD-scid mouse can be used as an experimental control group of the present invention, and it is proved that the method used in the present invention can be used in the animal having normal immune function by using the method of the present invention. Animals with human immune function are made.

The present invention uses a NOD-scid mouse having NK cell function (having a certain immune function), and usually requires human leukocyte antigen (HLA) matching when performing hematopoietic stem cell transplantation in a human body, and a semi-matched transplantation is required. Take anti-rejection drugs, otherwise the transplant will not be successful. In the experiment of this example, the cells of humans completely different from the experimental animals were used, and the major histocompatibility antigen complex (MHC) antigen matching was not possible at all because humans were HLA, and the mice were H-2 is two different MHC systems, so the success of transplantation is almost non-existent. In the experiments of this example, the "tissue, organ, cell, and factor related to autoantigen immune recognition" injected into mice include: Thymus, thymocytes, bone marrow, bone marrow cells, and immunologically recognized hematopoietic stem cells are a very strong antigenic graft, and no anti-rejection drugs are administered to mice during transplantation, and this transplantation method is equal to Graft antigen transplantation is performed simultaneously with hematopoietic cell transplantation. The successful transplantation of this example proves that the present invention can indeed carry out the immune system between different kinds of animals of the same species, and between humans and animals. The organs move to each other and overcome the rejection reaction. The correctness of the theory and practice of the present invention is demonstrated. At the same time, it is also demonstrated that the present invention can be used in a variety of animals having immune functions by transplanting human "organisms, organs, cells, and factors related to autoantigen immune recognition" including: thymus, thymus cells, bone marrow, bone marrow cells, and immunity. The experimental basis of this method is to identify hematopoietic stem cells and obtain human immune cells, experimental animals with human immune function. Of course, it is also possible to carry out "organisms, organs, cells, and factors related to autoantigen immune recognition" between animals of different kinds of animals, including: thymus, thymocytes, bone marrow, bone marrow cells, and immune recognition hematopoietic stem cell transplantation, The recipient has an animal that functions as a donor immune system. The practice of the present invention demonstrates that a human immune system can be established in an animal having immune function, a human immune system test can be performed in place of a human, and human tissues, cells, organs, and the like can be cultivated in the body of the animal. In the animal experiment of the present embodiment, the "organisms, organs, cells, and factors related to the autoimmune recognition of the autoantigens" of the same species should be transplanted, because the mice were only about 29 days from fertilization to birth, and because the mice were The period of self-antigen immune recognition is not clear in the first few days, and it is only necessary to transplant human "organisms, organs, cells, and factors related to autoantigen immune recognition", which has a great influence on the results of the experiment, but the test The results can also demonstrate the correctness of the principles of the present invention.

Animal experiment process:

1. The first stage test:

1) Nine N0D/SCID mice were irradiated, TBI 1. 0 cGy.

2) Transplantation of thymus and bone marrow in tissues, organs, cells, and factors related to autoantigen immune recognition, 6. 24 Preserved human fetal liver cells (human CD34+ antibody label, flow cytometry to detect hematopoietic stem cell ratio. 30%).

), resuscitation, wash the culture solution 2 times.

Counting results - fetal liver cells: 2. 3 xlO ⁸

9 N0D/SCID mice,

2克细胞悬浮, Hepatocytes 4. 6 xlO ⁷

Continue to raise in the laminar flow chamber for 2 to 3 months.

After 12 weeks, ocular venous blood was taken and anti-CD45 immunohistochemical pathology was performed. Anti-CD45-positive lymphocytes were observed in the thymus, spleen, bone marrow, liver and other organs. It is proved that hematopoietic stem cell transplantation is successful, and human immune cells are obtained in the body of the animal.

The present invention is an embodiment designed by using an early immune recognition hematopoietic stem cell and/or a late immune recognition hematopoietic stem cell obtained by various methods, and performing adult self antigen re-identification and treatment of an immune system disease, but it is also possible to utilize each Early stage of development, various types of stem cells obtained by transformation, early identification of hematopoietic stem cells and / or late immune recognition of hematopoietic stem cells, stem cells that have been immunologically recognized for various types of diseases.

As described above, the preferred embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the concept of the present invention should not be construed as being limited to the various embodiments described above. Those skilled in the art, through the enlightenment of the above embodiments, are not difficult to make various improvements, changes or replacements for the method of recognizing the adult self-antigen of the present invention and treating diseases of the immune system, and for the treatment of various diseases. Therefore, such modifications, changes and substitutions are not to be construed as a departure from the scope of the invention.

Claims

Rights request

A method for obtaining and applying a raw material required for re-immunization of an autoantigen, characterized in that the raw materials required for re-immunization of the self-antigen include: tissues, organs, cells, factors, and/or immunological recognition of autoantigens. Or autoantigen immune recognition of hematopoietic stem cells.

2 . The method for obtaining and applying raw materials required for re-imaging self-antigen immune recognition according to claim 1 , wherein the tissues, organs, cells and factors related to autoantigen immune recognition are involved in immune recognition and hematopoiesis Stem cells are hormones, cytokines, polypeptides, proteins involved in the immune recognition of autoantigens in the body, and/or tissues, and/or cells, and/or immune recognition of autoantigens.

The method for obtaining and applying raw materials required for re-imaging self-antigen immune recognition according to claim 1, wherein the tissues, organs, cells and factors related to autoantigen immune recognition include: human or animal The embryo and the fetal stage, involved in the immune recognition of hematopoietic stem cells for the immune recognition period of the body's own antigen. Providing a microenvironment required for immunological recognition of hematopoietic stem cells for self-identification of autoantigens in the body, and providing cells secreting "autoantigen immune recognition factors" required for immunological recognition of hematopoietic stem cells for self-identification of autoantigens in the body, and/or A cell that secretes "promoting (producing) a self-antigen immune recognition factor", and/or a tissue of a cell that secretes a self-antigen for various cofactors required for immunological recognition, using a complete tissue organ, or using a part of a tissue organ .

The method for obtaining and applying raw materials required for re-imaging self-antigen immune recognition according to claim 1, wherein the tissues, organs, cells and factors related to autoimmune immune recognition include: human or animal The embryo and the fetal stage, involved in the immune recognition of hematopoietic stem cells for the immune recognition period of the body's own antigen. Providing a microenvironment required for immunological recognition of hematopoietic stem cells for self-identification of autoantigens in the body, and providing cells secreting "autoantigen immune recognition factors" required for immunological recognition of hematopoietic stem cells for self-identification of autoantigens in the body, and/or a cell that secretes "promoting (producing) an autoantigen immune recognition factor", and all cells, and/or a part of cells, and/or of the relevant tissues and organs of cells that secrete autoantigens for various cofactors required for immunological recognition One or more special cells.

The method for obtaining and applying raw materials required for re-imaging self-antigen immune recognition according to claim 1, wherein the tissues, organs, cells and factors related to autoantigen immune recognition include: "self antigen The immune recognition factor ", and/or the "promoting (self-secreting) autoantigen immune recognition factor", and/or autoantigens are required for various functions of immune recognition.

The method for obtaining and applying raw materials required for re-imaging self-antigen immune recognition according to claim 1, wherein the tissues, organs, cells and factors related to autoantigen immune recognition include: thymus tissue, Thymocytes, and / or bone marrow tissue, bone marrow cells.

7. A method for obtaining and applying a raw material required for re-imaging self-antigen immune recognition according to claim 1, wherein the human and/or animal immune recognition hematopoietic stem cells required for re-immunization of the autoantigen is Obtained by the following ways:

A. Source of clonal cells or fertilized eggs:

a clonal cell formed from a transplant recipient or a somatic cell nucleus of an immune system and a disease patient to a human egg from which the nucleus is taken out;

b. from the recipient's somatic cell nucleus to the nucleus of the animal's egg, forming a clonal cell; c from the recipient's sperm or egg and donor or other human or animal sperm or egg combined to form fertilization Egg

d. Form the fertilized egg from the sperm or egg of the volunteer donor and the sperm or egg of the recipient or other human or animal.

B. Immune recognition of hematopoietic stem cell sources:

a. These cloned cells or fertilized eggs of different origins are developed into an embryo that is immunologically recognized in the hematopoietic stem cell stage, and the immune-recognizing hematopoietic stem cells are extracted from the embryo;

b. culturing in a test tube to differentiate into an immunologically recognized hematopoietic cell;

c. From the embryonic and fetal stages of human or animal, early immune recognition of hematopoietic stem cells for self-antigen immune recognition, abortion of early embryos extracted from stillbirth to identify hematopoietic stem cells;

d. From the embryonic and fetal stages of human or animal and after birth, the immune recognition hematopoietic stem cells undergo self-antigen immune recognition after the end of the period, abortion of the late fetal immune stem cells extracted from the stillbirth;

e. Direct extraction of latent immune recognition hematopoietic stem cells from the blood system of the recipient or donor or donor.

8. The method according to claim 1, wherein the immunologically recognized hematopoietic stem cells are subjected to density gradient centrifugation from a hepatocyte suspension of the liver of the fetus, and / or isolated by immunomagnetic beads separation method.

9. The method according to claim 1, wherein the immunologically recognized hematopoietic stem cells are expanded in a test tube or transplanted into an intermediary for amplification.

10. The method for obtaining and applying a raw material required for re-imaging self-antigen immune recognition according to claim 1, wherein the self-antigen immune recognition is input into the body, the tissue and the organ related to the autoimmune recognition of the autoantigen. Cells, factors, and/or enter the body to identify hematopoietic cells.

11. The method for obtaining and applying a raw material required for re-imaging self-antigen immune recognition according to claim 1, wherein the method for immunologically identifying hematopoietic stem cells in performing secondary immune recognition is: performing in vitro test tubes, or It is carried out in the intermediary or in the recipient.

12. A method for obtaining and applying a raw material required for re-immunization of autoantigens according to claim 1, wherein the donor and recipient of the graft or graft antigen are from the same species, and/ Or animals, or from different kinds of people, and/or animals.

13. The method according to claim 1, wherein the immunologically recognized hematopoietic stem cells are subjected to autoantigen immune recognition in an in vitro test tube or an intermediary. Alternatively, the donor's graft, and/or the variant self antigen are subjected to secondary immune recognition.

14. A method for obtaining and applying a raw material required for re-immunization of autoantigens according to claim 1, characterized in that non-specific therapy is used before secondary antigen recognition of human and/or animal autoantigens, and / Or specific therapy partially removes, partially removes, or does not clear cells associated with immunity.

15. A method for obtaining and applying a raw material for re-imaging self-antigen immune recognition according to claim 1, characterized in that non-specific therapy, and/or specific therapy removal is used before secondary antigen recognition of autoantigens. Partially cleared, or not cleared, immune-related cells, including the "self-antigen immune recognition library" and cells that store the autoantigen immune recognition library. And/or immune cells in the peripheral blood and immune tissues that are present in the immune system, including: negative selection and positive selection in the thymus and bone marrow, including: T cells and B cells.

16. A method for obtaining and applying a raw material for re-imaging self-antigen immune recognition according to claim 1, wherein the graft or graft antigen is transplanted into the recipient, and the marrow is removed, and the marrow is partially cleared. Or a non-myeloablative bone marrow transplantation method, which transplants immune-recognizing hematopoietic stem cells, and/or tissues, organs, cells, and factors related to autoimmune antigen recognition into a recipient, thereby allowing the immune-recognizing hematopoietic stem cells to undergo the body Secondary immune recognition.

17. A method for obtaining and applying a raw material required for re-imaging self-antigen immune recognition according to claim 1, wherein the transplantation of "organisms, organs, cells, factors related to autoantigen immune recognition", and/or Immune recognition of hematopoietic stem cells into the intermediary.

18. A method for obtaining and applying a raw material required for re-imaging self-antigen immune recognition according to claim 1, wherein an immunodeficient animal, or a non-immune-deficient animal, is used for immunologically identifying a hematopoietic stem cell bone marrow transplantation, such that the animal Become an animal with a human or an immune system.

19. A method for obtaining and applying a raw material for re-imaging self-antigen immune recognition according to claim 1, wherein the intermediary uses an immunodeficient animal, or a non-immune-deficient animal, a transplant donor and/or a recipient. The graft or graft antigen is introduced into the body of the intermediary: the transplant immunologically recognizes the hematopoietic cells, and/or the tissues, organs, cells, and factors involved in the immune recognition of the autoantigen for secondary self-antigen immune recognition: making the animal available And/or an animal that is exposed to the immune system that has been identified by the graft or graft antigen.

20. A method for obtaining and applying a raw material required for re-imaging self-antigen immune recognition according to claim 1, wherein the method of bone marrow transplantation using a myeloablative, partial myeloablative or non-myeloablative method is to be The immunologically recognized hematopoietic stem cells in which the graft or the graft antigen secondary autoantigen is immunologically recognized are transplanted into the recipient, and then the graft is transplanted into the recipient.

21. A method for obtaining and applying a raw material required for re-imaging self-antigen immune recognition according to claim 1, wherein the recipient's organ transplant operation is performed before or simultaneously with the secondary antigen recognition of the autoantigen, or The graft antigen is transplanted into the body, and the autoantigen is subjected to secondary immune recognition, and the transplanted graft antigen is recognized as an autoantigen, and then an organ transplant operation is performed.

22. A method for obtaining and applying a raw material for re-imaging self-antigen immune recognition according to claim 1, wherein the method comprises the following steps: clearing the marrow, partially clearing the marrow or non-myeloablative treatment, and then transplanting into the body "with itself" Antigen-immune recognition of tissues, organs, cells, factors, and/or input of in vivo immune recognition of hematopoietic cells for re-immunization of autoantigens.

A method of removing immune-related cells, and/or clearing the "self-antigen immune recognition library" and clearing the marrow or partially clearing the cells of the self-antigen immune recognition library, including:

1) Non-specific clearance of immune-related cells, including:

a. use of drugs;

b. Radiation exposure.

2) specifically clearing immune-related cells, and/or clearing the "autoantigen immune recognition library" and cells storing the autoantigen immune recognition library, including:

a. killing one or more immune-related cells in the body with a drug, or inactivating an immune-related cytokine using a drug;

b. Use immunological methods to remove specific cells, and/or and/or inactivate cytokines.

23. A method of obtaining and applying a raw material required for re-immunization of autoantigens according to claim 1, wherein the recipient or the donor is between the same species and/or different species of animals and animals. , and people and movements The immune system and organs move between objects, and the transplanted recipients or the "organisms, organs, cells, and factors involved in immune recognition with autoantigens" include: thymus, thymocytes, bone marrow, bone marrow cells, and immune recognition hematopoiesis Stem cells, which allow the recipient to obtain the function of the immune system.

24. A method for obtaining and applying a raw material required for re-imaging self-antigen immune recognition according to claim 1, characterized in that a human immune system is established in an animal having immune function, and a human is substituted for various human immunity. Systematic testing, as well as the cultivation of human tissues, cells and organs within the body of an animal.