CN101048497A - Method for acquiring and applying needed raw material for reccogniting self antigen immunity - Google Patents

Method for acquiring and applying needed raw material for reccogniting self antigen immunity Download PDF

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CN101048497A
CN101048497A CNA2005800370506A CN200580037050A CN101048497A CN 101048497 A CN101048497 A CN 101048497A CN A2005800370506 A CNA2005800370506 A CN A2005800370506A CN 200580037050 A CN200580037050 A CN 200580037050A CN 101048497 A CN101048497 A CN 101048497A
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immune
autoantigen
cell
identification
stem cell
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程滋颐
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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

Method for acquiring and applying needed raw material for reccogniting self antigen immunity
Re-start and re-started Autoantigen Immune after birth with rejection and disease of immune system is treated the present invention relates to a kind of from raw material acquisition and methods for using them technical field needed for antigen immune identification and recognized required raw material acquisition methods and methods for using them.It is particularly a kind of to generate and obtain the early immune identification cell of hematopoiesis thousand and/or late period Immune discrimination candidate stem cell re-started required for Autoantigen Immune identification, the histoorgan relevant with Immune discrimination in period is recognized with acquisition Autoantigen Immune, including：The cell relevant with Autoantigen Immune identification and the factor etc. in thymus gland and marrow.And each in body occurs in order to treat rejection and disease of immune system the histoorgan and graft antigen of autoantigen variation, re-start the treatment method of Autoantigen Immune identification.The rejection after a variety of diseases related to disease of immune system and organ transplant can not only be treated using the method, and a variety of Different Organs of receptor, different types of disease can be treated.The present invention can be not only used for people, can be also used for animal.Noun used in the background technology present invention is explained, and required raw material acquisition and methods for using them explanation-
1. immune system in people or animal autoantigen is carried out Immune discrimination period be divided into-
1) from embryonated egg to embryo or prefoetal period, this section of period immune system does not carry out Immune discrimination also to autoantigen, and this period is " Autoantigen Immune recognizes early stage ".
2) it is " Autoantigen Immune recognizes the phase " in the period of embryonic period, embryonic phase Immune discrimination is carried out to autoantigen.
3) it is autoantigen to be carried out in the period of Immune discrimination terminates being " Autoantigen Immune end of identification phase " in foetal period and postnatal infancy and adulthood.
Therefore early stage Autoantigen Immune identification candidate stem cell is to recognize that early stage and Autoantigen Immune recognize the Immune discrimination candidate stem cell of phase in Autoantigen Immune.And late period Immune discrimination candidate stem cell is the Immune discrimination candidate stem cell of Autoantigen Immune end of identification phase.
4) early immune identification candidate stem cell can include：Autoantigen Immune recognize early stage and/or Autoantigen Immune other phase with Immune discrimination, and/or the candidate stem cell relevant with Immune discrimination, and/or group cell etc., and/or oneself cell through differentiation, and/or all kinds of ripe blood system cells broken up relevant with being immunized, the early immune identification cell of hematopoiesis thousand is can to carry out the cell of Autoantigen Immune identification in certain circumstances.For example：Hematopoietic pluripotential stem cell（Including candidate stem cell（Haematopoietic Stem Cells)), and/or the various progenitor cells relevant with immune system（Immune multipotential stem cell）Such as：Myeloid stem cell, lymphatic stem cells, and/or the various single energy committed stem cells relevant with being immunized, such as：Grain, monokaryon lineage stem cells, forerunner's B cell, forerunner's T cell, macronucleus stem cell, and the cell of secretion Autoantigen Immune recognition factor, and/or secretion promotees cell of Autoantigen Immune recognition factor etc., various and immune relevant cell（Note：When practical application, it should determine it is the candidate stem cell using embryo or fetus or each para-immunity stem cell relevant with being immunized according to actual conditions, from that type, the cell in which stage）.The cell of these species in this manual referred to as：Early immune recognizes the cell of hematopoiesis thousand.
5) late period Immune discrimination candidate stem cell can include：Be the Autoantigen Immune end of identification phase with Immune discrimination and/or the cell of the stem cell relevant with Immune discrimination or the undeveloped mature broken up, or with relevant all kinds of stem cells, and/or undifferentiated ripe cell is immunized, late II Immune discriminations candidate stem cell can not carry out Autoantigen Immune identification in general, and what only having, " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " was participated in particular cases can just carry out Autoantigen Immune identification.For example：Hematopoietic pluripotential stem cell（Including candidate stem cell (Haematopoietic Stem Cells)), and/or the various progenitor cells relevant with being immunized（Immune multipotential stem cell）Such as：Myeloid stem cell, lymphatic stem cells, and/or the various single energy committed stem cells relevant with being immunized, such as：Grain, monokaryon lineage stem cells, forerunner's T cell（That is proT and early stage proT cell）, ' forerunner's B cell（That is proB and early stage proB cell）, monocyte（DC) the various and immune relevant cells of macronucleus stem cell etc.（Note：When practical application, It should determine it is to use that type, the cell in which stage according to actual conditions）.The cell of these species in this manual referred to as：" late period Immune discrimination candidate stem cell ".
Immune discrimination candidate stem cell includes：Early immune recognizes candidate stem cell, and/or late period Immune discrimination candidate stem cell.Immune discrimination candidate stem cell is also referred to as without Immune discrimination stem cell in this manual, or without Immune discrimination candidate stem cell.
2. " the tissue relevant with Autoantigen Immune identification, organ, cell, the factor " is " Autoantigen Immune identification early stage ", and/or embryo and the fetal state of the human or animal of " Autoantigen Immune recognizes the phase ", early immune identification candidate stem cell can carry out Autoantigen Immune identification period, participate in related organization's organ that early immune identification candidate stem cell carries out Immune discrimination to autoantigen in body, and/or tissue, and/or cell, and/or the hormone relevant with Immune discrimination, various cell factors, polypeptide, protein etc..Although be not up to the present fully apparent from also it is all participation Autoantigen Immunes identification histoorgans, at present by medical experiment find just like：Various cells, hormone, various cell factors, polypeptide protein, tissue, tissue fluid, cell included in the organs such as thymus gland, spleen, lymph node and marrow, and these organs etc..（See " cell and molecular immunology " chapter 6, chapter 7 and chapter 9, " cell and molecular immunology " second edition Jin Baiquan chief editor Chinese science publishing houses first printing of in September, 2001）.After the Autoantigen Immune end of identification of " Autoantigen Immune recognizes the phase " of fetus, " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " no longer carries out Autoantigen Immune identification, only under specific circumstances, be such as possible to re-start Autoantigen Immune in the presence of " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " of " Autoantigen Immune recognize phase " and/or " Autoantigen Immune recognizes early stage " and recognize.
" tissue relevant with Autoantigen Immune identification, organ, cell, the factor " can include：
1) " tissue relevant with Autoantigen Immune identification, organ, cell, include because of ^：The embryo of human or animal and the fetal state, early immune identification candidate stem cell is participated in the related organization in autoantigen progress Immune discrimination period, organ, cell, the factor in body, its complete tissue, organ, or the incomplete tissue in part, organ.It provides early immune and recognizes that candidate stem cell carries out the microenvironment required for Immune discrimination to autoantigen in body, there is provided promoting early immune to recognize, cell of the candidate stem cell to the secretion " Autoantigen Immune recognition factor " required for autoantigen progress Immune discrimination in body, and/or secretion " promote（Make secretion）The cell of Autoantigen Immune recognition factor "." Autoantigen Immune recognition factor " can include：Startup factor, promote recognition factor, terminate recognition factor.And autoantigen is provided carries out the various microenvironments such as the tissue fluid of various confactors etc. required for Immune discrimination.
2) " the tissue relevant with Autoantigen Immune identification, organ, cell, the factor " can be included whole cells of above-mentioned histoorgan, and/or part cell, and/or one or more of which special cells, input is internal, part cell can be applied to by going back to the nest up to target organ, grown in target organ, grown that can also be beyond target organ is developed, early immune identification candidate stem cell is provided to the microenvironment required for autoantigen progress Immune discrimination in body, there is provided and promote early immune to recognize cell of the candidate stem cell to the secretion " Autoantigen Immune recognition factor " required for autoantigen progress Immune discrimination in body, and/or secretion " promotees（Make secretion）The cell of Autoantigen Immune recognition factor ".And secretion autoantigen carries out the cell of the various cell factors of the secretion such as the cell of various confactors required for Immune discrimination, and the various auxiliary cells for participating in Autoantigen Immune identification, and the various microenvironments required for autoantigen progress Immune discrimination are provided.
Cell in these histoorgans relevant with Autoantigen Immune identification is extracted in the method using the extraction specific cells being previously mentioned in density gradient centrifugation method and description of the invention.Extract such as：Early immune is promoted to recognize cell of the cell of hematopoiesis thousand to the secretion " Autoantigen Immune recognition factor " required for autoantigen progress Immune discrimination in body, " rush (makes secretion for secretion）The cell of Autoantigen Immune recognition factor ".The cell of the various confactors for participating in Autoantigen Immune identification of secretion, and the various auxiliary cells for participating in Autoantigen Immune identification.
3) " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " can include by providing secreted by above-mentioned histoorgan promote early immune recognize candidate stem cell Immune discrimination is carried out to autoantigen in body required for " Autoantigen Immune recognition factor ", and/or " promote（Make secretion）Various confactors required for Autoantigen Immune recognition factor ", and/or participation autoantigen progress Immune discrimination etc..Above-mentioned several organizers can be used as needed One or more in the several types of materials such as official, cell, the factor.The used various factors can be extracted using the various methods that the used factor is extracted at present, and a variety of methods manufacturings can be cloned using such as transgenic cell.
" tissue relevant with Autoantigen Immune identification, organ, cell, the factor " is also referred to as in this manual：" Immune discrimination organ " or " starting recognition factor " or " startup factor " or " stopping recognition factor " or " stopping the factor ".
The cell of secretion " Autoantigen Immune recognition factor " is extracted, secretion " promotees（Make secretion）The cell of Autoantigen Immune recognition factor ".The method of the cell of the various confactors for participating in Autoantigen Immune identification of secretion and the cell of various participation Autoantigen Immunes identification.And " Autoantigen Immune recognition factor " is extracted, and/or " promote（Make secretion）Various confactors required for Autoantigen Immune recognition factor ", and/or participation autoantigen progress Immune discrimination etc., used method.
4) find, extract " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " and there are a variety of methods, including：
A), which finds the tissue relevant with Autoantigen Immune identification, organ method, a variety of, including：Utilize the method for embodiment in the present invention 9, single cell suspension is made in the tissue and/or organ that carry out Autoantigen Immune identification period, or use a part for histoorgan, or use complete histoorgan, individually or different Grafting assemblies are carried out in animal body, carry out Autoantigen Immune identification experiment, if any tissue, organ or those tissues, the combination of organ can cause Autoantigen Immune to recognize Success in Experiment, so this is organized, included tissue in organ or this combination, organ is exactly the " tissue relevant with Autoantigen Immune identification, organ ".
B) finding " cell relevant with Autoantigen Immune identification " method has a variety of, including：1) single cell suspension is made in the tissue or organ that find in, the method centrifuged using gradient of continuous density isolates each cell.Utilize the method for embodiment in the present invention 9, individually or different Grafting assemblies are carried out in experimental animal body, carry out Autoantigen Immune identification experiment, if the combination of any cell or those cells can cause Autoantigen Immune to recognize that cell included in Success in Experiment, Hao's this cell of end or this combination is exactly " cell relevant with Autoantigen Immune identification ".
C) finding the method for " factor relevant with Autoantigen Immune identification " has many kinds, including：A. by B) in find carry out Autoantigen Immune identification period have Autoantigen Immune identification activity cell and have passed through by carry out Autoantigen Immune identification period same type of cell, crushed using ultrasonic wave or other method, then find different albumen between two kinds of cells, amino acid peptide chain using chromatogram, mass spectrum（Polypeptide）.Using the method for embodiment in the present invention 9, the independent or different combinations of progress are expelled in experimental animal body, Autoantigen Immune identification experiment are carried out, if any albumen, amino acid peptide chain（Polypeptide）Or those albumen, amino acid peptide chain（Polypeptide）Combination can cause Autoantigen Immune recognize Success in Experiment, then this albumen, amino acid peptide chain（Polypeptide）Or albumen, amino acid peptide chain included by this combination（Polypeptide）It is exactly " factor relevant with Autoantigen Immune identification ".
B. by B) in find carry out Autoantigen Immune identification period have Autoantigen Immune identification activity cell and have passed through by carry out Autoantigen Immune identification period same type of cell, both cells are cultivated respectively, and cause the cell for having Autoantigen Immune identification activity to retain the activity for enabling to Immune discrimination candidate stem cell to carry out Autoantigen Immune identification, and the nutrient solution of two kinds of cells is purified, relevant composition is recognized to body or to Autoantigen Immune in removal or reservation nutrient solution.Utilize the method for embodiment in the present invention 9, it is expelled in experimental animal body, Autoantigen Immune identification experiment is carried out, if Autoantigen Immune can be caused to recognize Success in Experiment, is input in the nutrient solution in body and just contains " factor relevant with Autoantigen Immune identification ".Then different albumen between two kinds of cells, amino acid peptide chain are found using chromatogram, mass spectrum（Polypeptide）.The method of embodiment 9 in the present invention is utilized respectively, is expelled in experimental animal body, Autoantigen Immune identification experiment is carried out, if Autoantigen Immune can be caused to recognize Success in Experiment, is input to albumen, amino acid peptide chain in body（Polypeptide）It is exactly " factor relevant with Autoantigen Immune identification ".
3. the grafts such as internal organ, tissue, cell have been transplanted in preparation, and/or the cell for carrying the antigen family relevant with Immune discrimination of graft, tissue, organ, protein, polypeptide etc., these are in this manual referred to as：Graft. 4. because graft derives from organ, tissue, cell in donor body etc., therefore antigenic and donor the antigenicity of graft is identical, graft can be used, other graft antigen suppliers that there is antigenic organ, tissue, the cells such as donor tissue's organ etc., carry out during autoantigen identification as early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell in donor body can also be used.These in this manual referred to as：Graft antigen.
5. by medical practice finder since embryonated egg to embryo 3 months or so, to graft rejection will not occur for this section of period embryo's body, therefore this period is the period for not carrying out Immune discrimination to autoantigen also, from embryo 3 months or so to 5 months or so, immune system in body starts to carry out Immune discrimination to autoantigen, before this period, or until the graft of internal allosome can be identified as the tissue of itself by immune system within this period, it is added to Autoantigen Immune identification storehouse together with original various autoantigens in fetal body to suffer, foreign matter will not be identified as later, generation rejection.Although up to the present not finding startup specifically also, keep, Autoantigen Immune recognition factor is carried out with the immune organ in body when terminating " to start, promote, terminate the factor of Autoantigen Immune identification ", and/or " promote to start, promote, terminate the factor of Autoantigen Immune identification " and secrete the cell of these factors, but it can be found that they are implicitly present in by medical practice, and there are these factors and secrete the histoorgan of the cell of these factors, these histoorgans are the " tissues relevant with Autoantigen Immune identification, organ, cell, the factor ", including:In the thymic tissue, and/or myeloid tissue of 3 months or so to 5 months or so Autoantigen Immune identification phases of embryo etc. histoorgan, the cell of secretion " Autoantigen Immune recognition factor " is there may be in these histoorgans, and/or secretion " promotees（Make secretion）The cell of Autoantigen Immune recognition factor ".Every kind of factor in this several factor can be the composite factor that the single factor or a variety of factors are constituted.Start Autoantigen Immune recognition factor, promote Autoantigen Immune recognition factor and terminate Autoantigen Immune recognition factor, can be a variety of different factors, can also be that the same factor acts on the different effects of different stages performances, these factors can be：Hormone, cell factor, polypeptide, protein, tissue, tissue fluid, cell, organ etc..Find that these factors can convert late period Immune discrimination candidate stem cell and carry out Immune discrimination to carrying out autoantigen again sometimes by medical practice.The organ and tissue for the Immune discrimination microenvironment for promoting Autoantigen Immune to recognize can be provided by inputting at present（Such as thymic tissue, thymocyte and myeloid tissue, bone marrow cell）, promote the Immune discrimination microenvironment of Autoantigen Immune identification to have the factor that have input these promotion Autoantigen Immune identifications to realize both to input to provide.
It is right in this manual：With starting, promoting the no cell of Immune discrimination thousand to carry out the relevant factor of Autoantigen Immune identification and terminate the factor of identification, and provide the organ and tissue of the Immune discrimination microenvironment for promoting Autoantigen Immune identification（Such as thymic tissue, thymocyte and myeloid tissue, bone marrow cell）Referred to as：Startup factor, promote recognition factor, terminate recognition factor.But these factors are collectively referred in this manual：Start recognition factor.
And termination in this manual can also in addition referred to as without the relevant factor that Immune discrimination stem cell carries out Autoantigen Immune identification：Terminate recognition factor.
Because there is presently no isolate during practical operation, it is relevant with Autoantigen Immune identification is carried out in Immune discrimination organ without Immune discrimination stem cell, start identification, promote identification, and/or terminate the factor of Autoantigen Immune identification, but be implicitly present in, see each " adjustment effect of transcription factor to immunocyte "（See " cell and molecular immunology " chapter 6, chapter 7 and chapter 9, " cell and molecular immunology " second edition Jin Baiquan chief editors Chinese science publishing house in September, 2001 first printing）.Specifically which is to promote secretion to start recognition factor, promote recognition factor and terminate the organ or cell of recognition factor, and/or with the presence or absence of the rush secretion factor for secreting these factors, tests and/or separate just can prove that after needing.
6. disease of immune system, can be included such as：Systemic loupus erythematosus, rheumatism, rheumatoid arthritis, glomerulonephritis, chorionitis etc., it can also include because a variety of causes disease causes the immunity disease caused by autoantigen antigenic change, the change of autoantigen can just be caused, its autoantigen made a variation can be organ, tissue, cell, protein, polypeptide etc., and these are in this manual referred to as：The autoantigen of variation.
7. bone-marrow transplantation：The word for being more prone to use is " HSCT ".
Used bone marrow transplantation therapy in the present invention, it is by being purged and immune relevant cell in the present invention, carry out Immune discrimination HSCT, and/or carry out " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " and transplant, to carry out the therapeutic process that secondary Autoantigen Immune identification is carried out. The method of bone-marrow transplantation is-
1) remove has with immune relevant cell, and/or " Autoantigen Immune recognizes storehouse " with the clear marrow for the cell for storing Autoantigen Immune identification storehouse or the method for half clear marrow：
A. it is non-specific to remove and immune relevant cell
Including：Using medicine, such as：Endoxan, and/or radioactive ray irradiation, such as：Cobalt6°, clinac, the method such as high-energy X-ray full-body exposure, by it is original in human body, oneself through autoantigen was carried out Immune discrimination progress the stem cell of Immune discrimination etc. is original in vivo is killed with immune relevant cell.
B. specificity removes the cell relevant with being immunized, and/or removes " Autoantigen Immune recognizes storehouse " and store the cell that Autoantigen Immune recognizes storehouse.
This is to be killed original one or more cells relevant with being immunized in vivo using medicine, or will be inactivated using medicine with immune relevant cell factor, or the cell kill in specificity removing Autoantigen Immune identification storehouse will be stored, specific cells can also be removed using immunological method, and/or the killed cells factor, such as T lymphocytes immunocyte is killed, the cell toxicity medicament for example after being combined using CD3 monoclonal antibodies with cytotoxin formed kills internal T lymphocytes and antilymphocyte globulin (ALG) in vivo plus complement input（) etc. ALG a variety of medicines for killing immunocyte in vivo immuning systems, this is a kind of for a certain cell or the specific therapy of a certain class cell.
So pre-processed through Large Dose Irradiation, chemotherapy or other immunosupress, remove the tumour cell in recipient's body, in vivo abnormal clone, original candidate stem cell and various immune progenitor cells, the cell such as stem cell, and/or the immune cell of various participations is immunized, blocking pathogenesis, this makes clear marrow handle, and can also use the clear marrow in part or non-clear marrow processing.Then HSCT is carried out to receptor, receptor is rebuild normal hematopoiesis and immune, so as to reach a kind for the treatment of means of therapeutic purposes.
2) Immune discrimination HSCT is carried out, and/or carries out " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " transplanting.If using the method for the clear marrow in part or non-clear marrow, secondary Autoantigen Immune identification directly can also be carried out using original Immune discrimination candidate stem cell in vivo without Immune discrimination HSCT.
Find out and store the cell in Autoantigen Immune identification storehouse and have a variety of, including：Single cell suspension is made in the tissue containing the cell for storing Autoantigen Immune identification storehouse or organ that find, the method centrifuged using gradient of continuous density isolates each cell.Remove the specific cells medicament of these cells using the immunological method being made, remove independent in body or carry out different combination cell clearances, then transplant graft in experimental animal body, see whether rejection occur.If the cell for removing certain or various combination occurs without rejection, then this or various combination cell is exactly to store the cell that Autoantigen Immune recognizes storehouse.
8. remove specific cells using immunological method：
It is the method for specific cells, by specific cells specific antibody selective binding on specific cells, is removed.The method that can be used has many kinds, such as conventional has：
1) the cell toxicant method of Complement Dependent：Monoclonal antibody adds complement after being combined with corresponding target cell, and target cell lysis is destroyed by activating complement classical pathway, reaches purification purpose.Such as, using a variety of B cell monoclonal antibodies（Such as：CD9, CD10, CD19, CD20) or T cell antibody is (such as:CD2, CD3, CD4, CD8 etc.) combine complement extracorporeal marrow purification T cell, Β cells.Further, it is also possible to using (the anti-CD14 monoclonal antibodies of AML- 223）Plus immune complement purification treatment acute myeloid leukaemia, achieve the curative effect similar to Alio- bone-marrow transplantations.
2) the cell toxicant method of immunotoxin mediation, this method is by specific antibody and cytotoxin（Ricin (WA)）With reference to immunotoxin is made, that is, there is the effect of powerful killing specific cells, there is specificity again.
3) Physical is immunized, with monoclonal antibody coating microsphere or metallic particles, marking makes cell density increase or produce magnetic, again by precipitation or magnetic field adsorption method removing target cell after target cell.
4) remove or the specific cells factor is lost function, for example, allow IL2 R y to lack, and NK cells is lost immunocompetence.And the method for making specific cells lose proper function.
The above method cuts both ways, and is clinically mainly used in the purification of specific cells at present.
9. receive the patient or disease of immune system patient of graft, in this manual referred to as：Receptor.This receptor can be that people can also be animal. 10. graft or graft antigen person are provided, in this manual referred to as：Donor.This donor can be that people can also be animal.
11. the early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell of the present invention are in vitro in test tube, or carry out secondary immunity identification to the graft of donor, and/or the autoantigen of variation in tertium quid's body, or in recipient's body.Because this time Autoantigen Immune identification is different with the Immune discrimination that the embryo stage immune system grown naturally in human body or animal is carried out to autoantigen, it is that Immune discrimination is carried out to the autoantigen including the autoantigen to the graft of donor, and/or variation, it is in order to which the autoantigen of graft and/or the variation for causing disease of immune system is identified as into normal autoantigen, therefore in this manual referred to as：Secondary immunity is recognized.Secondary immunity identification in this manual is also referred to as：Autoantigen Immune identification is re-started, either secondary Autoantigen Immune identification or the identification of autoantigen secondary immunity.
12. recognize candidate stem cell and/or the cell of late period Immune discrimination hematopoiesis thousand and/or graft from egg cell to early immune is obtained in the present invention, each stage of the centre of graft for obtaining the stem cell for carrying out Immune discrimination and/or cultivation is arrived again, use sometimes and donor, the same kind of receptor, same to species, or different genera, different types of other animal or people, developed, cultivated in the body of these animals or people, autoantigen identification etc. process, these animals or people are in this manual referred to as：Tertium quid.
13. recognize candidate stem cell and/or late period Immune discrimination candidate stem cell and/or graft from egg cell to early immune is obtained in the present invention, each stage of the centre of graft for obtaining thousand cells for carrying out Immune discrimination and/or cultivation is arrived again, sometimes using Tissue Culture Flask, culture dish etc. and the in vitro culture instrument such as culture, developed, cultivated in the in vitro culture instrument such as these blake bottles, culture dish, culture apparatus, autoantigen identification etc. process, these in vitro culture instruments in this manual referred to as：Test tube.
14. extract the method for specific cells：
1) can use the specific antibodies of a certain cell to carry out immuno magnetic cell separation method.
2) can use the cell suspension of some tissue containing a certain specific cells.Certainly according to the difference for extracting cell, also more methods can be used.Merely just provide several than more typical extracting method, because the extracting method used is different when specifically used, the medicine liquid ingredient used is different, and various cell extraction methods can make a big difference, but the purpose for extracting cell is consistent.
A. immunology separates specific cells cultural method, such as：
Isolating dendritic cells（The cell such as DC)：Various methods can be used, wherein following several schemes can be included:Thymic tissue is ground to form into single cell suspension by unicellular mill, containing 6 X 108PBMC suspensions 9ml sleeps ol/L EDTA without calcium and magnesium PBS containing 1), add the 4ml of anti-CD32 monoclonal antibodies confining liquid 0., 37 °C of 5% C02 cultivates 10min, add StemSepTM people DC enrichment mixed antibody magnetic beads lml, it is sufficiently mixed, 37 °C of 5% C02 cultivates 30min, excessively sterile Magnetic screen post, unlabelled aim cell is collected, PBS is washed 3 times.105 cells of I X are taken, are marked with fluorescence antibody, flow cytometry analysis cell phenotype, remaining cell adds GM-CSF containing 10ng/ml, 10ng/ml IL-4 10% FCS IMDM nutrient solution cultures.
Separation NK cells are taken containing 4 X 108PBMC without calcium and magnesium PBS suspension 9ml, and separation and detection method adds the IMDM nutrient solution cultures containing 10% FCS with DC, remaining cell is separated.
Separation T cell is taken containing 4 X 108PBMC without calcium and magnesium PBS suspension 9ml, and separation and detection method adds the IMDM nutrient solution cultures containing 10% FCS with DC, remaining cell is separated.
B. using by adding specific cells nutrient solution cultural method, various methods can be used, wherein following scheme can be included：Pancreatic stem cells are separately cultured and identified：（The Jiangxi Medical College such as Wen Jin, Zhang Yuhai journal third phases of volume 42 in 2002）
Pancreas is taken to add the tissue block for being cut into 1 millimeter of diameter in 4 CHank's liquid, after cyclic washing, add 0. 5rag/ml clostridiopetidase A, 37 °C shake water-soluble 15 minutes, add 4 CHank's liquid stopped reactions, centrifuged three times with 800 turns/min, its supernatant, add the 4 CHank's liquid without calf serum, cross 150 mesh sieves, the 4 CHank's liquid containing 10% calf serum are added in gained cell mass, after suction pipe is blown and beaten repeatedly, cultivated in the nutrient solution that cell suspension is added to 0. lmg/ml ConA, to remove the nonislet cells such as fibroblast, 5%C0237 °C of pzyh are after 24 hours, with 5X105/ addition 20 ng/ml BFGF (Basic Fibroblast Growth Factors）11. continuing to cultivate in the calf serum blake bottle of 1 mmpl/1 glucose 10%, later 7-10 days 0. 05% pancreatin using EDTA digest, and digest and passage cell.After five weeks, change nutrient solution into low sugar (2. 5 mmol/1) and contain 4 CHank's liquid of 2% calf serum, while removing BFGF, add 10mM niacinamide inducing cell directed differentiations, islet cells like cell colony is formed, cell therein is exactly pancreatic stem cell.
15. the Railway Project relevant with autoimmune disease with Autoantigen Immune identification treatment rejection is re-started：
1) cell that autoantigen recognizes storehouse is stored：How many, which plants cell storage, at present has autoantigen to recognize storehouse, and is stored in that cell wherein, and the particular location of storage is not made clear also completely with what any mode was stored.But know that part is present in cortical epithelial cells and the medullary substance BMDC of thymus gland, and the corresponding cell relevant with Immune discrimination in marrow at present.
Processing：A knocks out Autoantigen Immune identification storehouse or storage autoantigen recognizes the cell in storehouse.And/or after these cells are knocked out, the cell of the identical type for not storing Autoantigen Immune identification storehouse also of input, the cells such as stem cell and/or progenitor cells including that can be divided into these cells, storage re-starts the new Autoantigen Immune identification storehouse after Autoantigen Immune identification.
B. the antigen after addition during original Autoantigen Immune recognizes storehouse re-starts Autoantigen Immune identification, makes to turn into autoantigen from the antigen re-started after Autoantigen Immune identification.
2) it is original in peripheral blood and immuning tissue that the immunocyte such as Solid phase and the T cells and B cell of positive selection was carried out in thymus gland and marrow.
Processing：The immunocytes such as the T lymph B cells in a. knocking out in thymus gland, marrow, peripheral blood and immuning tissue.
B. the addition in peripheral blood and immuning tissue carried out the immunocytes such as the T cell and B cell of Solid phase and positive selection after re-starting Autoantigen Immune identification in thymus gland and marrow.
3) autoantigen recognition factor, and promote autoantigen recognition factor, and promote autoantigen recognition factor, it may be possible to the factor is also likely to be cell.Medical practice shows to be likely to be present in the thymus gland for carrying out Autoantigen Immune identification period and/or marrow etc. " tissue relevant with Autoantigen Immune identification, organ, cell, the factor ".
Processing：A. the cell for secreting these factors is found, cell culture is done and is input in human body and treated.
B. the 26S Proteasome Structure and Function of the factor is found, commodity, which are made, by bioengineering is applied to clinic.
C. transplanted using the organ-tissue containing the factor or secretion factor cell such as thymus gland and/or marrow, and/or single cell suspension is made in these organ-tissues and be input in vivo.
4) thymus gland and/or bone marrow cell that Autoantigen Immune recognizes period are being carried out.
Processing：A. using internal original thymus gland and bone marrow cell.
B. it is implanted into and carries out thymic tissue and/or bone marrow cell that Autoantigen Immune recognizes period, or thymocyte of the input by culture.Input the bone marrow cell of culture.
5) the B Cho cells in candidate stem cell, thymus gland T progenitor cells and/or the marrow in Autoantigen Immune identification period are being carried out.
Processing：A. input early stage is without B progenitor cells in Immune discrimination candidate stem cell, thymus gland T progenitor cells and/or marrow etc..
B. using B progenitor cells in internal original candidate stem cell, thymus gland T progenitor cells and/or marrow etc..
6) candidate stem cell that Autoantigen Immune recognizes period is being carried out.
Processing：A. input early stage is without Immune discrimination candidate stem cell.
B. using internal original cell of hematopoiesis thousand.
The present invention, which can be not only used for people, can be used for animal, but in the description of the invention mostly taking human as each embodiment in representative, including specification.But this specification and each embodiment can be used for various animals.Organ transplant can be carried out between the human or animal of identical kind, organ transplant can also be carried out between the human or animal of different genera.Or mutually transplanted between the immune system of the human or animal of different genera, carry out disease of immune system or organ transplant therapeutic test using the animal for being transplanted immune system.Various zooperies can be not only carried out, and can be by people's Organ transplant allows the organ of people to grow in animals to animal, when growing up to after being transplanted on the body of people, then takes out from animal and to be transplanted on the body of people etc. various disease treatments.
The principle and meaning of the present invention：
Because human body is generally during this section between fetus three months to six months, the autoantigen progress primary immune response identification without Immune discrimination stem cell to each histoorgan in body of fetus, and the result storage specifically recognized is remembered all the life into Autoantigen Immune identification storehouse, later immune system is compared to the data base that the antigen into the various antigens in body or itself generation variation all and is specifically recognized, it is found that the antigens different from the autoantigen of the histoorgan of each in body will produce immune attack.
The transplanting of each organ in current body, including：Liver transfer operation, kidney transplant, diabetes patient's pancreatic islets transplantation etc., it is different with the autoantigen in autoantigen recognition memory storehouse in body because consistent organ and/or tissue is exotic antigen, therefore all can inevitably produce rejection.Preoperative progress HLA matchings are needed, preferably all matchings, at least require it is half matching（Half match) such that the antigen of graft or graft antigen is as far as possible identical with the autoantigen in the data base that autoantigen in body is recognized, to need to take anti-rejection medication throughout one's life after surgery simultaneously, such as cyclosporine (ciclosporin) cyclosporine A (CSA), take anti-rejection medication, about 5000 yuan or so of RMB need to be spent within one month, it is costly that long-term taking, which is spent,.While many disease of immune system are such as：Systemic loupus erythematosus, rheumatism, rheumatoid arthritis, glomerulonephritis, chorionitis etc., including because a variety of causes disease causes autoantigen to sexually revise caused immunity disease, such as：Hepatic sclerosis caused by virus hepatitis, is all that immune system generates immune attack to autologous tissue's Immune discrimination mistake, causes physical impairment.These disease of immune system are treated at present or by immunodepressant, but the cell toxicant of these diseases and immunosuppressant treatment can only relief of symptoms, such as take：Steroid, methotrexate (MTX), imuran, other related drugs, but occur during these medicine uses as：The immunodepressant such as cyclosporin A cause the side effects such as hepatic failure.And these drug therapies can only relief of symptoms can not effect a radical cure, cause many patients to endure the pain that disease is brought to the fullest extent, spend mint of money to be treated, finally still die from these diseases.Some a variety of diseases including congenital hereditary sexual factor including can not also be cured at present, such as disease of hematopoietic system therein such as：The diseases such as congenital chain globular anemia also have no idea to cure.Searching is a kind of can to treat the method for these diseases, be the main object of the present invention.
The inevitable rejection of transplanting of various organs can be alleviated or be cured to the present invention, can thus utilize organ transplant, many diseases that can not be treated at present of histocyte transplantation treatment, such as：Diabetes can be treated using islet cell transplantation.And can equally alleviate or cure various disease of immune system（Antoimmunedisease, AID) such as：Systemic loupus erythematosus etc., these diseases are due to that immune system generates Immune discrimination mistake to autologous tissue and carries out the disease that immune attack causes physical impairment.It can also treat other various by the treatable disease of the method, such as：The a variety of diseases of such as disease of hematopoietic system that congenital heredity is caused.
Nevada, USA university, the group of Ai Si Mels-Zha Jiani (Esmail Zanjani) leader achieves, it can be transplanted to after maturation to be grown in human body by human organ's " plantation " in tertium quid's body, substitute the achievement in research of corresponding diseased organ.Certainly, this theory also needs to long time applied to clinical practice, but the research of first stage has been completed.
Zha Jiani by the mesenchymal cell extracted from human marrow, it is expelled in the fetus body in the uterus of sheep, human cell is just grown to serve as sheep heart, skin, muscle, a part for fatty and other tissues, but the quantity of human cell at that time is considerably less.Now, the team that he is led achieves further progress on the basis of original, i.e., surprisingly largely improve the content of the human cell in the sheep organ of part.Demonstration according at the beginning of at 12 months once in meeting, in the liver of experiment sheep, human cell's content has reached 7- 15%.
This technology it is crucial that human cell must complete to inject in sheep mid pregnancy, i.e., must be not in just only in this way rejection before in fetus, oneself does not learn the different cells of identification also through shaping but its immune system.This scientific experimentation demonstrating effectively：In the fetal state of human or animal, not only the autoantigen of the histoorgan of each in body is identified as autoantigen without Immune discrimination stem cell, it is autoantigen that the alloantigen such as graft antigen Immune discrimination, which can also be recognized, and the result storage specifically recognized to " Autoantigen Immune is recognized in storehouse " lifelong memory.Also demonstrate " Autoantigen Immune identification storehouse in ", and the cell of storage " in Autoantigen Immune identification storehouse " is present in body. The current research of Israel biologist finds that the embryonic stem cell of allosome can not escape from " piercing eye " of immune system.In the U.S.《NAS's journal》On the network edition, published thesis by the research group of Hebrew University of Jerusalem of Buddhist nun's Lyceum this Wen Nisidi leaders, because human immune system confirms " invader " by a kind of MHC molecule, they are in the three phases that the cell of human embryo thousand (Human Embryonic Stem Cell) is developed, and the antibody crossed respectively with fluorescence labeling measures the quantity of MHC molecule in each stage allosome embryonic stem cell.As object of reference, they go back the reaction caused by synchro measure HeLa cell.Measurement result shows that MHC content is low in the embryonic stem cell not divided, but very stable.Embryonic stem cell is " original " cell for not being divided into various tissues in embryo also.Once scientific research personnel is thought according to the human embryo study result of early stage, perhaps embryonic stem cell will not be found by the mechanism of body defenses foreign cell.The Auchincloss of stopping of medical college of Harvard University says that new result proves that embryonic stem cell can not escape the detecting of immune system.
Finder professor Ku Haerchuke of Ukraine " Ku Haerchu gram-rads Qin Ke-western Germania effect " claims：" we assume that and demonstrating：The renewal of its existing immune system can be caused by injecting a certain amount of embryonic stem cell to body.The system that the essence of the effect is health the problematic or impaired immune system of people " replacing " Cheng Xin." but embryonic stem cell can not escape the detecting of immune system, even therefore input to body is embryonic stem cell, be also required to carry out HLA (tissue associated antigen) distribution type before the input.Otherwise embryonic stem cell is difficult to transplant successfully.But HLA (tissue associated antigens）The probability success rate of distribution type is at a fairly low, distribution type could be carried out successfully by needing the tire stem cell in substantial amounts of different human body source, because the influence derived from embryonic stem cells of moral element is by a definite limitation, so if practical application is used for treating disease, suitable low of the possibility of popularization and application.Simultaneously this update is also halfway, because simply injecting a certain amount of embryonic stem cell to body, and in body it is original oneself still survived through the candidate stem cell for carrying out Immune discrimination, immune progenitor cells etc., still there is the original immune, foreign substance removing function of certain maintenance, simply this function is reduced, but will not be disappeared.
Sage's Judas's child study hospital of the U.S.（St. Jude Children's Research Hospital) it is published on May 15th, 2005《Immunology》Article on magazine (The Journal of Immunology P6540-6545), describes them in NOD/LtSz-scid IL2R Y "unThe candidate stem cell that people is transplanted with mouse completes the experimental animal model of human immune system.This be method used in experiment with of the present invention patent of on the October 10th, 2004 in China's application by being consistent, simply NOD/LtSz-scid IL2R Y " with general orientation toward the purpose for human hematopoietic stem cell is transplanted with NOD-scid mouse making the immune system with people of NOD- scid mouseu11Mouse is that NK cells are non-functional, it is impossible to kill the human hematopoietic stem cell of transplanting, therefore this mouse is rejection for graft, received completely without any immunologic function.And used in the present invention is to have NK cell functions（With certain immunologic function）Mouse, therefore the transplanting success of the test example of the present invention, it was demonstrated that of the invention really to overcome rejection, it was demonstrated that the correctness of theory and practice of the invention.
These description of tests can be identified as the autoantigen of fetus into the alloantigen in fetus body before fetus carries out Autoantigen Immune identification or when being identified, without occurring rejection, illustrate what autologous tissue antigen of the rejection not exclusively in animal body was determined simultaneously, in the period of before Autoantigen Immune identification or in the middle of identification, it can also be identified as autoantigen into internal alloantigen, hence it is demonstrated that there may be " Autoantigen Immune recognizes storehouse " set up after Autoantigen Immune identification in animal body（Although at present can't fully known this " Autoantigen Immune recognize storehouse " specific restoration position, in which kind of cell, in which organelle of cell, and particular location, but be at least aware of part at present and preserve Autoantigen Immune identification storehouse cell, such as：The BMDC of the cortical epithelial cells relevant with Immune discrimination and medullary substance in thymus gland, and/or bone marrow cell the corresponding cell relevant with Immune discrimination（See " cell and molecular immunology " chapter 6, chapter 7 and chapter 9）, this " Autoantigen Immune recognizes storehouse " determines autoantigen control reference standard when Immune discrimination is carried out after birth.For the fetus of normal pregnancy, because early stage being grown in parent, in the period of fetus is before Autoantigen Immune identification is carried out or in the middle of identification, without in extraneous alloantigen implantation fetus body, therefore fetus is when Autoantigen Immune identification is carried out, only autologous tissue's antigen of fetus oneself can not recognized, therefore allosome organ transplant is carried out after birth, or due to when autoantigen variation causes autoimmune disease, because the organ of this allosome or the tissue antigen of variation are not entered into fetus body when fetus carries out Autoantigen Immune identification, therefore there is no the antigen gene of this transplant organ in " Autoantigen Immune recognizes storehouse " set up after Autoantigen Immune identification, autoantigen can not be identified as, therefore it will send out Raw immune response.
Then current British scientist is transplanted in the uterus of sheep, reproductive success clone sheep many cases using the body-cell neucleus transplanting of a sheep into the egg cell for taking out nucleus.
The scientist of Chinese Shanghai cultivates in test tube and survived by the nuclear transplantation of the foreskin cells of people to the rabbit egg cell for taking out nucleus.
The Oriented Differentiation of Embryonic Stem Cell cultivated in test tube has been successfully candidate stem cell by the current mankind.
Taiwan regeneration edge biotechnology company develops " Cord blood makes stem cell serum-free culture medium ", the culture medium can make Cord blood make stem cell in five to seven days to rise in value thirtyfold, increment is to 7,000 times in five weeks, and maintains the characteristic and potentiality of the stem cell of differentiation.The content of the invention is according to a technical scheme of the invention, and methods for using them is obtained there is provided a kind of stem cell for being used to treat disease of immune system and rejection, characterized in that, the early immune identification candidate stem cell of progress autoantigen secondary immunity identification and/or the source of late period Immune discrimination candidate stem cell for treating disease of immune system and rejection disease are obtained from following approach：
A. the source of clone cell or embryonated egg：
A. it is transplanted to from the somatic cells of graft receptor or immune system and Disease into the people's ovum for taking out nucleus, the clone cell of formation；
B. it is transplanted to from the somatic cells of receptor into the animal ovum for taking out nucleus, the clone cell of formation；C is combined from the sperm or ovum and donor of receptor or the sperm or ovum of other human or animals, forms embryonated egg.
B. early immune recognizes candidate stem cell and/or late period Immune discrimination derived from hematopoietic precursor cells：
A. the clone cell of these separate sources or embryonated egg are developed into early immune identification candidate stem cell and/or the embryo in Immune discrimination candidate stem cell stage in late period in tertium quid's body, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell is extracted from embryo；
B. culture is divided into early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell in test tube；C extracts early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell from stillborn foetus of miscarrying；
D. late period Immune discrimination candidate stem cell is extracted from postnatal human bone marrow or blood.Particularly：Recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell are in vitro expanded including early immune, or be implanted into tertium quid's body and expanded.Particularly：Recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell have in the approach for carrying out secondary immunity identification including early immune：Carry out, or carried out in tertium quid's body in test tube in vitro, or carried out in recipient's body.Particularly：Recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell are in vitro in test tube including early immune, or in tertium quid's body, the autoantigen of graft, and/or variation to donor carries out secondary immunity identification.Particularly：Including first by graft transplantation to recipient's body, then using the marrow transplant techniques of clear marrow, the clear marrow in part or non-clear marrow, recognize candidate stem cell and/or late period Immune discrimination HSCT in recipient's body, making early immune recognize candidate stem cell and/or late period Immune discrimination candidate stem cell by carrying out secondary immunity identification in body early immune.Particularly：Marrow transplant techniques including first using clear marrow, the clear marrow in part or non-clear marrow, to in vitro be carried out in receptor graft or the identification of graft antigen secondary immunity progress Immune discrimination stem cell transplantation in recipient's body, then again by graft transplantation to recipient's body.Particularly：Immunodeficient animals, or nonimmune deficient animals are used including tertium quid, but carried out clear marrow processing, the animal of early immune identification candidate stem cell and/or the cell bone-marrow transplantation of late period Immune discrimination hematopoiesis thousand can be carried out：The graft or graft antigen of transplantation donor and/or receptor are in tertium quid's body：Transplanting carries out autoantigen identification without Immune discrimination stem cell：So that animal turn into people or by progress graft or graft antigen recognizing immune system animal.Particularly：Immunodeficient animals, or nonimmune deficient animals are used including tertium quid, but carried out clear marrow processing, early immune identification candidate stem cell can be carried out and/or late period Immune discrimination is made The animal of hemocytoblast bone-marrow transplantation：Transplanting carries out autoantigen identification without Immune discrimination stem cell：So that animal turn into people or by antigen recognizing immune system animal.Particularly：Including in trnasplantion immunity organ to tertium quid's body：Particularly：Including input startup factor, and/or termination factor.The principle of the present invention is namely based on Nevada, USA university, Ai Si Mels-Zha Jiani（Esmai l Zanjani) group of leader obtains scientific achievement, and this research has shown that：Can be by human organ's " plantation " in tertium quid's body, and autologous tissue, autoantigen are identified as by the immune system of animal, without occurring rejection.
Having for Immune discrimination is participated in human body：
A. " Autoantigen Immune recognizes storehouse ".The thymocyte precursors (prothymocyte) passed through in the T cells of thymus gland in thymus gland early stage are CD4XD8-double negative cells, then develop into CD4+C8+TCR+Double positive cells, the positive selection combined by the MHC-I classes with thymic cortical epithelial cells surface or MHC- II quasi-molecules/itself peptide fragment, and with cell surface can express the BMDC of high-caliber MHC-I classes or MHO II quasi-molecules and autoantigen at thymic cortex and medullary junction（) and macrophage DC（Μ Φ) carry out high-affinity combination Solid phase, by self tolerance（Self tolerance) or clonal deletion（Clonal deletion), single positive Τ cells as the CD4+CD8-or CD4-CD8+ that can be recognized exotic antigen and not reacted with autoantigen.（The thymic selection of the effect of the chapter 6 of in September, 2001 major histocompatibility antigen MHC, second section MHC in immune response of cell and molecular immunology second edition Jin Baiquan chief editor Science Presses, three, MHC and T cell）C. ancestral's B cell is passed through and passes through early stage ancestral's B cell, late period ancestral's B cell, pre B cell, the stage of development of immature B cells in marrow in marrow, and by clonal deletion, development forms the mature B cell of autoimmune tolerance（Chapter 9, bone-marrow-derived lymphocyte）.Being seemed according to the description " Autoantigen Immune recognizes storehouse " in " cell and the molecular immunology second edition " that Jin Baiquan is edited should be in the thymic cortical epithelial cells of thymus gland and BMDC (DC) and macrophage（Μ Φ) and marrow in, but be whether that in the two organs there is presently no final conclusion.
B. passing through in body carried out self tolerance in thymus gland or marrow（Self tolerance) or clonal deletion（Clonal deletion) can recognize exotic antigen again not with autoantigen react CD4+CD8-or CD4XD8+ single positive T cell and B cell.
Therefore when treatment disease of immune system and rejection is carried out, two aspects need processing：
1. (1) original " Autoantigen Immune recognize storehouse " will delete in vivo, then by the input tissue relevant with Autoantigen Immune identification, organ, cell, the factor in vivo, including：" Autoantigen Immune recognition factor ", and/or the histoorgan of secretion " Autoantigen Immune recognition factor ", either whole cells of this histoorgan or the cell of some kinds of secretion " Autoantigen Immune recognition factor ", and/or secretion " rush（Make secretion）The secretion such as cell of Autoantigen Immune recognition factor " promotes Immune discrimination candidate stem cell to carry out the cell of Autoantigen Immune recognition factor, or directly inputs these factors.Including：Carrying out some of the thymus gland, and/or marrow organ, and/or these organs in Autoantigen Immune identification period histocyte.And/or thymocyte, bone marrow cell early immune identification candidate stem cell, and/or late period Immune discrimination candidate stem cell, and/or utilize itself original late period Immune discrimination candidate stem cell, re-start Autoantigen Immune identification, it is autoantigen by the antigen recognizing of the autoantigen of variation and/or the histoorgan of transplanting, setting up one includes identifying new " Autoantigen Immune recognizes storehouse " of graft antigen or the autoantigen of variation, and avoids occurring rejection or immune response.
(2) original in vivo " Autoantigen Immune recognizes storehouse " is not deleted, but by inputting histoorgan relevant with Autoantigen Immune identification in vivo, cell factor, including：" Autoantigen Immune recognition factor ", and/or the histoorgan of " Autoantigen Immune recognition factor " is secreted, either whole cells of this histoorgan or the cell of some kinds of secretion " Autoantigen Immune recognition factor ", and/or secretion " rush（Make secretion）The secretion such as cell of Autoantigen Immune recognition factor " promotes Immune discrimination candidate stem cell to carry out the cell of Autoantigen Immune recognition factor, or directly inputs these factors.Including：Carrying out some of the thymus gland, and/or marrow organ, and/or these organs in Autoantigen Immune identification period histocyte.And/or thymocyte, bone marrow cell early immune recognizes candidate stem cell, and/or late period Immune discrimination candidate stem cell, and/or utilize itself original late period Immune discrimination candidate stem cell, re-start Autoantigen Immune identification, so that graft antigen or the autoantigen of variation can be re-recognized in vivo, and the autoantigen for re-recognizing graft antigen or variation is added in original " Autoantigen Immune recognizes storehouse ", so that the cell such as the later newly-generated CD4+CD8-or CD4 D8+ single positive T cell and B cell that can recognize exotic antigen and not reacted with autoantigen Immune system will not occur immune response to graft antigen or the autoantigen of variation.
2. remove the immune system cells such as the single positive T cell core B cell for re-starting can recognizing of having existed in the Autoantigen Immune identification treatment precursor CD4+CD8-that exotic antigen do not react with autoantigen or CD4 D8+ by the method for clear marrow, half clear marrow again, these antibody that can occur the cell of immune response to graft antigen or the autoantigen of variation and be generated by them are eliminated.There is document report, using a variety of B cell monoclonal antibodies（CD9, CD10, CD19, CD20) or T cell antibodies（CD2, CD3, CD4, CD5 etc.）Joint complement, and/or antibody binding cell toxicity medicament such as castor bean toxin, carry out the clear marrow treatment of these cells.
The removing for target cell specificity used at present is treated, and can use a variety of therapies, such as：
1. monoclonal antibody adds complement after being combined with corresponding target cell, target cell lysis is destroyed by activating complement classical pathway, reach purification purpose.
2. the cell toxicant method of immunotoxin mediation, this method is by specific antibody and cytotoxin（Ricin (WA)）With reference to immunotoxin is made, that is, there is powerful killing tumor effect, there is specificity again.
3. immune Physical, with monoclonal antibody coating microsphere or metallic particles, marking makes cell density increase or produce magnetic, again by precipitation or magnetic field adsorption method removing target cell after target cell.The above method cuts both ways.The anti-CD32 monoclonal antibodies of anti-BMDC can such as be used, the monoclonal antibodies such as anti-CD11 monoclonal antibody anti-CD79 monoclonal antibody anti-CD82 monoclonal antibodies combine complement, and/or antibody binding cell toxicity medicament, and/or antibody binding is coated with microsphere or metallic particles with monoclonal antibody, cell density is set to increase or produce magnetic after mark target cell, target cell is removed by precipitation or magnetic field adsorption method again, carry out the clear marrow treatment of anti-BMDC of cell, the monoclonal antibody joint complement of the cell for other preservation Autoantigen Immune identification storehouses can also be used, and/or antibody binding cell toxicity medicament carries out the clear marrow treatment of these cells.
Using a variety of B cell monoclonal antibodies (CD9, CD10, CD19, CD20) or T cell antibody (CD2, CD3, CD4, CD5 etc.）Joint complement, and/or antibody binding cell toxicity medicament such as castor bean toxin, and/or antibody binding is coated with microsphere or metallic particles with monoclonal antibody, cell density is increased or produce magnetic after mark target cell, again by precipitation or magnetic field adsorption method, carry out the clear marrow treatment of T cell, B cell.
The present invention is that do not have also by generation, and/or being recognized with Autoantigen Immune and/or the stem cell relevant with being immunized for Immune discrimination was carried out to the autoantigen of each histoorgan in body, progenitor cells etc. cell, for example from embryonated egg to the embryo of less than 7 months gestational ages each stage of development generation recognized with Autoantigen Immune and/or with immune relevant stem cell, such as candidate stem cell, and/or polytype cell with immune relevant HPC etc., by culture, amplification, by carrying out clear marrow, the clear marrow in part, or the bone-marrow transplantation of unclear marrow, human body is original, oneself kills through the ripe candidate stem cell with Immune discrimination function, score is killed or not killed, input again and being recognized with Autoantigen Immune and/or the stem cell relevant with being immunized for Immune discrimination also was not carried out to the autoantigen of each histoorgan in body by amplification, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell such as candidate stem cell and/or polytype HPC relevant with being immunized, recognize candidate stem cell and/or the cell of late period Immune discrimination hematopoiesis thousand to each histoorgan in body by these and early immune（Graft or graft antigen can be included）Autoantigen re-recognize.And/or by early immune recognize candidate stem cell and/or late period Immune discrimination candidate stem cell is carried out outside human body the progress that graft or graft antigen, variation histogenic immunity are recognized Immune discrimination stem cell, pass through bone-marrow transplantation or the method being directly inputted in receptor's hematological system, the treatment a variety of diseases related to disease of immune system, and can treat a variety of Different Organs, different types of disease using the method.
" tissue relevant with Autoantigen Immune identification, organ, cell, the factor " includes：Various cells relevant with Autoantigen Immune identification, hormone, various cell factors, polypeptide, protein, tissue, tissue fluid, cell included in thymus gland, spleen, lymph node and marrow etc. organ, and these organs etc..
Immune discrimination candidate stem cell needed for re-starting Autoantigen Immune identification is obtained from following approach：
1. clone cell or the source of embryonated egg：
1) from the somatic cells of graft receptor or immune system and Disease are transplanted to the people's ovum for taking out nucleus, the clone cell of formation；
2) from the somatic cells of receptor are transplanted to the animal ovum for taking out nucleus, the clone cell of formation；
3) sperm or ovum and donor or the sperm or ovum of other human or animals from receptor is combined, and forms fertilization Ovum；
4) combined from the sperm or ovum of volunteer donors and the sperm or ovum of receptor or other human or animals, form embryonated egg.
2. the source of human stem cell without Immune discrimination-
1) clone cell of these separate sources or embryonated egg are developed into the embryo of the stem cell stage of no Immune discrimination in tertium quid's body, the stem cell of no Immune discrimination is extracted from embryo；
2) culture is divided into the stem cell of no Immune discrimination in test tube；
3) embryo from human or animal and the fetal state, the early immune identification thin dried flank meat of Hematopoietic Stem carry out the early immune identification candidate stem cell extracted in Autoantigen Immune identification period, stillborn foetus of miscarrying；
4) after the embryo of human or animal and the fetal state and birth, the cell of Immune discrimination hematopoiesis thousand carries out the late period Immune discrimination candidate stem cell extracted in the period after Autoantigen Immune end of identification, stillborn foetus of miscarrying；
5) from the hematological system in receptor or donor or donor's body, late period Immune discrimination candidate stem cell is directly extracted.
The clone cell of these separate sources or embryonated egg are developed into early immune identification candidate stem cell and/or the embryo in Immune discrimination candidate stem cell stage in late period in tertium quid's body, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are extracted from embryo, or culture is divided into early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell in test tube.
The early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell extracted not only can be for treatment disease of immune system, and can treat other a variety of diseases, such as：The a variety of diseases of the disease of hematopoietic system that congenital heredity is caused etc..
Note：Current Westminster Parliament and multinational government, have agreed to carry out the therapeutic studies that scientific research personnel carries out human body cell clone.
Early immune recognizes that candidate stem cell and/or the approach of late period Immune discrimination candidate stem cell progress culture amplification have：
1. expanded in test tube.
Expanded 2. being implanted into tertium quid's body, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell after amplification are extracted later.
But in vitro expanded mostly at present.
Early immune recognizes that candidate stem cell and/or the approach of late period Immune discrimination candidate stem cell progress secondary immunity identification have：
1. carried out in vitro in test tube；
2. carried out in tertium quid's body；
3. carried out in recipient's body.
Early immune recognizes that candidate stem cell and/or late period Immune discrimination candidate stem cell are in vitro in test tube, or in tertium quid's body, the autoantigen of graft, and/or variation to donor carries out secondary immunity identification.
1. recognize candidate stem cell and/or late period Immune discrimination candidate stem cell in the external test tubes of a. the early immune obtained from several approach, b. in tertium quid's body, cultivated, expanded, expand to after the quantity for being adapted to transplant in vivo, carry out the graft antigen of donor and/or the Immune discrimination of receptor's intraindividual variation self-antigen.Amplification whether is needed to recognize that the cell of hematopoiesis thousand and/or late period Immune discrimination candidate stem cell quantity are determined according to required early immune, such as：Be transplanted to quantity required when secondary immunity identification is carried out in tertium quid's body oneself through meeting needs, perhaps avoid the need for progress and expand.
Early immune is recognized into candidate stem cell and/or late period Immune discrimination HSCT in tertium quid's body, used tertium quid can be：Immunodeficient animals or without immunodeficient animals.
, it is necessary to carry out clear marrow, removing and immune relevant cell, including " Autoantigen Immune recognizes storehouse " and the cell for storing Autoantigen Immune identification storehouse by the method for bone-marrow transplantation when using without immunodeficient animals.Then tertium quid's transplanting of the early immune identification cell of hematopoiesis thousand and/or late period Immune discrimination candidate stem cell is carried out.
2. recognize candidate stem cell and/or late period Immune discrimination HSCT to the identification of progress secondary immunity identification in tertium quid's body early immune. 1) graft or graft antigen and/or receptor's intraindividual variation self-antigen are in tertium quid's body.
2) transplanting early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are in tertium quid's body.
3) transplanting " tissue relevant with Autoantigen Immune identification, organ, cell, the factor ", start recognition factor including Autoantigen Immune identification, and/or Autoantigen Immune identification terminates recognition factor, activation secondary immunity identification terminates the factor of its identification after recognition.
4) cells such as the stem cell that carried out Immune discrimination after recognition, are extracted, are expanded（Can also be according to circumstances without amplification）, it is implanted into after amplification in recipient's body, then the graft transplantation of donor is entered in recipient's body.And secondary immunity identification is carried out in recipient's body, it is that the graft of first transplantation donor or graft antigen are implanted into recipient's body, then carry out receptor's secondary immunity identification.
3. early immune recognizes candidate stem cell and/or late period Immune discrimination HSCT to the identification of progress secondary immunity identification in test tube.
1) graft or graft antigen of receptor and/or donor and/or receptor's intraindividual variation self-antigen are implanted into external test tube（Its graft or graft antigen and/or receptor's intraindividual variation self-antigen can be tissue, cell or organ etc.）.
2) early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are transplanted again.
3) transplanting " tissue relevant with Autoantigen Immune identification, organ, cell, the factor ", start recognition factor including Autoantigen Immune identification, and/or Autoantigen Immune identification terminates recognition factor into test tube, this Immune discrimination organ can be organ can also organize, cell, the factor etc..
4) transplanting Autoantigen Immune identification startup factor, and/or Immune discrimination stops the factor, and the histoorgan of Autoantigen Immune identification is carried out, and/or the cell transplantation recognized with Autoantigen Immune of these histoorgans enters in external test tube.After Autoantigen Immune identification was carried out, the stem cell for carrying out Immune discrimination is extracted.
5) decided whether to be expanded according to cell quantity, be transplanted to after amplification in recipient's body.
6) graft transplantation of transplantation donor enters in recipient's body.
4. recognize candidate stem cell and/or late period Immune discrimination HSCT to the identification of progress secondary immunity identification in recipient's body early immune.
1) graft or graft antigen are transplanted in recipient's body, if need to transplant the state of an illness decision that can be treated as needed, such as treatment disease of immune system avoids the need for being transplanted.
2) early immune is recognized that candidate stem cell and/or the cell of late period Immune discrimination hematopoiesis thousand are transplanted in recipient's body, transplanting Autoantigen Immune identification startup factor can be needed to decide whether according to the state of an illness, and/or Autoantigen Immune identification stops the factor, and/or the cell transplantation of the histoorgan and/or histoorgan of progress Autoantigen Immune identification enters in vivo, secondary immunity identification is activated, its identification is terminated after recognition.
Note：Early immune recognizes that the purpose that candidate stem cell and/or late period Immune discrimination candidate stem cell carry out secondary immunity identification is exactly to recognize storehouse in order to which original autoantigen identification storehouse in recipient's body is replaced by into new autoantigen, and the method for replacing has two kinds of approach：
It is exactly the method using bone-marrow transplantation, such as 1. original autoantigen identification storehouse is thoroughly destroyed：Using radioactive ray, and/or cell toxicity medicament is used, and/or killed all cells for storing autoantigen identification storehouse in original recipient's body using monoclonal antibody combination cell toxicity medicament.
1) input early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell, and/or " tissue relevant with Autoantigen Immune identification, organ, cell, the factor ", the progress secondary immunity identification in recipient's body.
2) or input by carried out in vitro in receptor secondary immunity identification progress Immune discrimination stem cell and/or carried out new " Autoantigen Immune recognizes storehouse " of secondary immunity identification and/or store new " Autoantigen Immune recognizes storehouse " cell, use both approaches to set up new " Autoantigen Immune recognizes storehouse ".
2. on the basis of original " Autoantigen Immune recognizes storehouse ", the new storehouse content of addition, use the method for the clear marrow in part or non-clear marrow, do not remove or part is removed and stores the cell that Autoantigen Immune recognizes storehouse in vivo, candidate stem cell and/or late period Immune discrimination candidate stem cell are recognized by inputting early immune, and/or " tissue relevant with Autoantigen Immune identification, organ, cell, the factor ", secondary immunity identification is carried out in recipient's body.Or input is by receptor The external progress for having carried out secondary immunity identification the stem cell of Immune discrimination and/or store new " Autoantigen Immune recognizes storehouse " cell, increase the method for new storehouse content.Which kind of specifically used method, the result that can be tested is selected according to the specific needs of the state of an illness.
The current mankind do not separate startups also, promote and stop the factor of no Immune discrimination stem cell progress Autoantigen Immune identification, but from Nevada, USA university, Ai Si Mels-Zha Jiani（Esmail Zanjani) leader the experiment that carries out of group in prove that it is present, it is understood that there may be place be exactly " tissue relevant with Autoantigen Immune identification, organ, cell, the factor ", including：Each allows no Immune discrimination stem cell to carry out the organ of Autoantigen Immune identification, if T lymphocytes are in thymus gland progress Immune discrimination, and bone-marrow-derived lymphocyte is carried out in marrow in Immune discrimination etc. the internal organs and tissue relevant with autoimmunity identification, and the nude mice of thymus function missing is the immunologic function without T lymphocytes, such as BALB/cA-nude nude mices, the C. W. Friis of Denmark in 1973 have found the hairless mouse of spontaneous mutation in BALB/cA inbred mouses, the mutant mice thymic hypoplasia, immune t-cell is lacked, and cultivated into BALB/cA-nude, and there is no the B of maturation and T lymph corpuscles in C. B-17 SCID mice bodies, therefore thymus gland, spleen, the weight of lymph node is not as good as normal 30%, lymphocyte notable defect is shown as in histology.Thymus gland multidigit adipose tissue is surrounded, and without cortex structure, only remaining medullary substance, mainly there is epithelioid cell synthetic fibers cellularity, the accidental stove shape lymphocyte populations in edge.Splenic white pulp is not obvious, and red pulp is normal, and acini lienalis mainly has desmacyte composition without lymphocyte aggregation.Lymph node has desmacyte to occupy without obvious cortical area, cortical area missing.Assemble more rare with bronchial lymph under mucous membrane of small intestine, without lymphoid aggregates in structure.And both no T lymphocyte functions that thymus gland and spleen function are all lacked there is no bone-marrow-derived lymphocyte function yet.There is no the B of maturation and T lymph corpuscles in N0D-SCID Mice Bodies.
Therefore inference " the tissue relevant with Autoantigen Immune identification, organ, cell, multiple organs in the factor " are the vitals that early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell carry out Immune discrimination, in the blood of this multiple organ, tissue fluid, various cells, or in whole organ, should exist and start, promote, and/or stop the startup recognition factor of early immune identification candidate stem cell and/or the progress autoantigen identification of late period Immune discrimination candidate stem cell, promote recognition factor and terminate recognition factor, they can be：Polypeptide, protein, tissue, cell, organ etc..Before the mankind do not separate, the cell that will can be in embryo's Autoantigen Immune identification initial period and/or the organ relevant with Immune discrimination just in cognitive phase and/or termination phase, various organs, it is present in composition of blood and tissue fluid etc., it is added in culture medium, and/or be input in the body of people or tertium quid, carry out Autoantigen Immune identification to start early immune identification candidate stem cell and/or the cell of late period Immune discrimination hematopoiesis thousand in test tube or in people or tertium quid's body.The mankind can manufacture once finding to start and stopping to carry out the factor of Autoantigen Immune identification without Immune discrimination stem cell, now avoid the need for using these organ-tissues starting and/or terminating without Immune discrimination stem cell to carrying out Autoantigen Immune identification.
Miscarry, from the Human embryo corpse within pregnancy a couple of days to about four months, preferably in the corpse of the dead embryo of pregnancy less than three months, if embryo has early immune to recognize candidate stem cell and/or late period Immune discrimination candidate stem cell in the organ such as marrow or liver, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell can be just extracted in these organs, if do not had also, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell then can be directly extracted from the corpse of fetus.And " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " includes：Thymus gland, spleen, lymph node and marrow etc. organ.These fetuses without Immune discrimination stem cell, be that Immune discrimination was not carried out to the autoantigen of each histoorgan in fetal body,（Can also utilize and be obtained from other various originate, such as stem cell of other species, such as fat stem cell unicellular process conversion obtain without Immune discrimination stem cell, and embryonic stem cell by differentiation obtain without Immune discrimination stem cell）, will be without Immune discrimination stem cell, such as candidate stem cell and/or HPC are cultivated, expanded to certain quantity, reach about 1 (Γ6Quantity, preferably l (TVkBQuantity.Once the patient of organ transplant was carried out according to state of an illness needs, can be with immediate postoperative, it postoperative can also continue to take anti-immunity repulsion medicine, when physical recovery to a certain extent after, utilize the method for bone-marrow transplantation, removing and immune relevant cell, including " Autoantigen Immune recognizes storehouse " and the cell for storing Autoantigen Immune identification storehouse.Such as：Using medicine such as：Endoxan, and/or radioactive ray irradiation, such as：Cobalt6°, original candidate stem cell is killed in vivo by original in human body, Immune discrimination had been carried out to autoantigen candidate stem cell and/or HPC etc. for clinac, the method such as high-energy X-ray full-body exposure, will internal original T lymphocytes using medicine Killed Deng immunocyte, for example：The cell toxicity medicament formed after being combined using CD3 monoclonal antibodies with cytotoxin kills internal T lymphocytes etc. in vivo plus complement input, immunocyte in a variety of kill vivo immuning systems, and/or " Autoantigen Immune recognizes storehouse " and the medicine for the cell for storing Autoantigen Immune identification storehouse.Input again by cultivating, expanding without Immune discrimination stem cell, and/or input allows early immune to recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell carried out startup and/or the termination factor of Immune discrimination to the autoantigen of each histoorgan in fetal body, the identification of its own antigen immune starts and/or termination factor, can be one or more kinds of polypeptides, can also be one or more kinds of protein, can also be the cell or one or more histoorgan of one or more kinds of tissues.
Input simultaneously（Other various cells can also be included in the cell of input, can also be according to different diseases, different case needs, the stage will be killed without using medicine and/or radioactive ray irradiation by original candidate stem cell in vivo, directly will be internal without the input of Immune discrimination stem cell）.It is this after amplification is without Immune discrimination stem cell inputting, the hormone of " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " in the body（" Autoantigen Immune recognition factor ", and/or " promote（Make secretion）Various confactors required for Autoantigen Immune recognition factor ", and autoantigen progress Immune discrimination etc.）In the presence of, input by amplification can carry out once new Immune discrimination immediately without the cell of Immune discrimination thousand.The autologous tissue of the organ of transplanting and generation variation can be identified as the normal structure organ of itself by it, thus greatly reduced the dynamics of immune attack, will not even be produced new immune attack.So as to treat disease, it can substantially reduce the medical expense after various organ transplants.Profit can cure or mitigate various disease of immune system in this way, and because the organ, tissue or cell of transplanting can re-recognize, therefore can be by organ or the various diseases of tissue replacement therapies, such as：Diabetes carry out pancreatic islets transplantation treatment etc..Because input be health fetus by amplification without Immune discrimination stem cell, can thus produce normal blood cell, thus cure it is various because the disease of hematopoietic system that causes of congenital heredity such as：The diseases such as congenital chain globular anemia.It can also treat and treat other various types of diseases using the method, as long as this treat the rehabilitation for being conducive to patient.Input it is this with by amplification without the progress of the cell of Immune discrimination thousand once new Immune discrimination after, the external organization for entering back into human body or the autologous tissue for producing variation are considered as foreign matter, produce immune attack, therefore it will not produce because using immunodepressant in current treatment, and the immunologic function produced is the problem of reduce.
Because family planning is a fundamental state policy of Chinese Government, therefore abortion operation is universal, legal progress all over China, miscarriage simultaneously must can be just carried out below three months in gestational age, it is the stem cell relevant with being immunized in the fetus of embryo, such as candidate stem cell and/or polytype HPC, the last stage of primary immune response identification is carried out to the autoantigen of each histoorgan in body.Fetus after is a bitty stillborn foetus.Therefore stem cell is extracted from a bitty stillborn foetus, such as candidate stem cell does not have moral problem, while fetus and immune relevant stem cell, such as diversity in the HLA sources of candidate stem cell can be protected.Therefore the candidate stem cell autoantigen that is highly suitable as being grown up is extracted with the obtained stillborn foetus of the miscarriage dilatation and curettage of the uterus and re-recognizes treatment with disease of immune system.
It is used in the present invention without Immune discrimination stem cell, this stem cell：Can use being recognized with Autoantigen Immune of being extracted from the stillborn foetus below the gestational age three months of miscarriage and/or with immune relevant stem cell, such as candidate stem cell and/or polytype HPC relevant with being immunized, the primary embryonic stem cell obtained from internal or external various sources can also be used, and by natural breed or by artificial culture in embryoid body or in blake bottle, manual intervention, each stage that the differentiation of a variety of stem cells or conversion that primary embryonic stem cell or various sources are obtained are trained recognized with Autoantigen Immune and/or with immune relevant stem cell, this stem cell can include：Myeloid-lymphoid stem cell（Embryonic stem cell）, multipotential stem cell（Such as candidate stem cell）With the stem cell in unipotent stem cell stage（Such as a variety of HPCs）Each stage changed into recognized with Autoantigen Immune and/or with immune relevant stem cell, such as candidate stem cell and/or polytype HPC relevant with being immunized, certainly the stem cell in each stage of the above can also be used according to treatment requirement, unipotent stem cell is also named specially can stem cell.Embryonic stem cell is " original " cell for not being divided into various tissues in embryo also（That is myeloid-lymphoid stem cell）Whole cells that adult body has can be broken up.The various kinds of cell of human body 200, can be produced by replicating.Last cell can produce tissue, tissue and synthetic organ, just turn into human body.Embryonic stem cell is primarily present among body early embryo.Embryonic development has individual process, sperm and ovum two basic change, produce embryonated egg, embryonated egg is further split into mulberry body, and it is exactly blastaea that mulberry body enters Walk differentiation again, and blastaea produces internal layer cell, in this former stage, as long as extracting a cell, adult body can be developed, so being all-round stem cell.It is how competent thin Born of the same parents oneself through be development late period.In marrow there is also much recognized with Autoantigen Immune and/or with immune relevant stem cell, such as cell of hematopoiesis thousand, these are all multipotential stem cell.Candidate stem cell is a kind of tissue specifc stem cells, it is derived by the mesoblastema of embryonic period, embryonic phase yolk bag, intraembryonic blood forming organ, liver, spleen are travelled in succession down to marrow, on the one hand self invariable number is maintained by asymmetry mitosis, on the other hand each system's progenitor cells are constantly be generated, the normal hematopoiesis function of body is maintained.The cell of hematopoiesis thousand（HSC) be carry out hematopoietic tissue transplanting after rebuild long term hematopoietic key factor.
A variety of progenitor cells can be divided into.Leukocytoblast, red blood cell, blood platelet etc. can be broken up in marrow, be multipotency.Another is single energy or special energy, can only be divided into a kind of cell, or is divided into two kinds of close cells of Relationship Comparison, and this is unipotent stem cell（Such as a variety of HPCs）.As long as each stage, various types of stem cells can be broken up by various stem cell lines to candidate stem cell induced orientation is converted into no Immune discrimination stem cell, it is possible to re-recognize the treatment with disease of immune system for autoantigen of being grown up.
Be irradiated with medicine kill original thousand cell of hematopoiesis when because the stem cell of active stage can only be killed, can not be killed without the stem cell in active stage, it is therefore desirable to kill the stem cell of resting stage, have a variety of methods-
1. the medicine for allowing the stem cell in resting stage to enter active stage is used, such as：5- FU etc. suppress candidate stem cell division, so stimulate body to allow the candidate stem cell in resting stage to enter active stage by suppressing the medicine of DNA fragmentation, and radiating irradiation is then carried out again or medicine kills the candidate stem cell for being in active stage；Various hematopoiesis stimulating factors can also be used, the internal candidate stem cell in resting stage is irritated and enters active stage, radiating irradiation is then carried out again or medicine kills the candidate stem cell in active stage.
2. use the antibody targeted chemotherapy of the stem cell for carrying out Immune discrimination（Antibody- mediated chemotherapy), combined using the monoclonal antibody or monoclonal antibody fragment of anti-candidate stem cell, and/or various immune progenitor cells, and/or the immunocyte of each stage of development, such as：Monoclonal antibody 3A5 Fab' fragments, bispecific antibody, three specific antibodies, antibody cytokine fusion protein etc..Novel gene engineered antibody continuously emerges, such as humanized antibody, monovalent small molecular antibody（Fab, single-chain antibody, single domain antibody, hypervariable region polypeptide etc.）, multivalence small molecular antibody（Double-chain antibody, three chain antibodies, miniantibody etc.）, some type-specifics（Bispecific antibody, three specific antibodies, antibody cytokine fusion protein, antigenized antibody, intrabody, catalytic antibody, immunoliposome etc.）And antibody fusion protein（Immunotoxin, immune adherence element）Various cytotoxins（Such as ricin (WA)）Deng combination.Monoclonal antibody is formed by being chemically crosslinked the immune conjugate constituted, the medicine of killing candidate stem cell, and/or various immune progenitor cells, and/or various immunocytes, 1 can be made) antibody targeted chemotherapy（Antibody- mediated chemotherapy), there will be killing activity cell drug to be connected with monoclonal antibody, the immunologic cytotoxicity chemicals of preparation has specific strong killing activity cytotoxic chemotherapy medicine to candidate stem cell：Such as Ah's toxin, cis-platinum, fluorouracil, vincristine, adriamycin；2) Immunotoxin Therapies（Immunotoxintherapy), toxin is connected with monoclonal antibody, the immunotoxin of preparation has specific strong killing activity to original stem cell for carrying out Immune discrimination in recipient's body, or a certain, certain several cell therein.Conventional toxin has two classes：One class is phytotoxin, including numb seed toxin of combing（RT), abrin（Abrin), momordin（MD) etc..Another kind of is cytotoxin, including diphtheria toxin（DT), Pseudomonas Exotoxin（PE) etc..The specific antibody of candidate stem cell and/or various immune progenitor cells can be used, such as using monoclonal antibodies such as CD34+ or A133 candidate stem cell specific antibodies, combined with cell toxicity medicament, such as combined with ricin (WA), diphtheria toxin cell toxicity medicament, the guided missile of specific kill candidate stem cell is made, the candidate stem cell, and/or various immune progenitor cells, and/or various immunocytes of remaining in vivo is killed.Description of drawings 1. is the flow chart of a kind of carry out early immune identification candidate stem cell and/or the extraction of late period Immune discrimination candidate stem cell, culture and amplification method of the present invention；
Fig. 2 are that adult's autoantigen for carrying out of patient and disease of immune system patient of the organ transplant of the present invention re-recognizes treatment method flow chart with disease of immune system；
Fig. 3 are that a kind of of the present invention carries out acquisition, culture, amplification and progress secondary immunity identification that early immune recognizes candidate stem cell and/or late period Immune discrimination candidate stem cell, treatment graft rejection reaction outside human body, and/or are immunized The flow chart of systemic disease method；
Fig. 4 are one kind progress early immune identification candidate stem cell and/or the extraction of late period Immune discrimination candidate stem cell, culture, amplification and progress secondary immunity identification in recipient's body outside human body of the present invention, treat graft rejection reaction, and/or disease of immune system method flow chart；
Fig. 5 are that a kind of early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell of the present invention carry out the experimental method flow chart of secondary immunity identification in tertium quid's body；
Another early immune to carry out bone-marrow transplantation that Fig. 6 are the present invention recognizes the flow chart that candidate stem cell and/or late period Immune discrimination candidate stem cell are extracted, cultivate and expanded；
Fig. 7 are the patients of another organ transplant of the present invention and adult's autoantigen of disease of immune system patient re-recognizes treatment method flow chart with disease of immune system；
Fig. 8 are the extraction flows of the candidate stem cell and thymocyte of one embodiment of the present of invention, bone marrow cell；Fig. 9 are that the autoantigen for carrying out organ transplant in animals of the present invention re-recognizes the embodiment with disease of immune system.Embodiment below in conjunction with the accompanying drawings, further illustrates each embodiment.Embodiment 1
A kind of carry out early immune identification candidate stem cell of Fig. 1 present invention and/or the flow chart of the extraction of late period Immune discrimination candidate stem cell, culture and amplification method.Stem cell without Immune discrimination obtains, below by taking the acquisition of candidate stem cell as an example.
1. the stillborn foetus obtained from miscarriage：The liver and marrow of stillborn foetus are stripped out under the microscope, because marrow is easier to obtain, so preferably obtaining the stem cell of no Immune discrimination from marrow, but because fetus is liver hematopoiesis, therefore the stem cell of no Immune discrimination can also be obtained from liver, or obtain from other organs or tissue the stem cell of no Immune discrimination.
2. extract karyocyte.
3. the step separating method of immunomagnetic beads-flow cytometer two can be utilized, the stem cell of no Immune discrimination is sub-elected from marrow, for example, utilizes the stem cell antigen of stem cell surface specifically expressing（Stemcellantigen, Sea) monoclonal antibody and be coated in the secondary antibody of magnetic particle surface, using magnetic suck cell sorting method（MACS the candidate stem cell without Immune discrimination in marrow) is isolated.The stem cell of no Immune discrimination can also be sub-elected using other methods.
Restricted dilution test shows：The cell that can be persistently implanted into for a long time is the CD34+ KDR in KDR+, marrow+It is candidate stem cell that cell, which has 20%,；The candidate stem cell for having the ability of being chronically implanted is CD34+KDR+ subgroups, therefore can use CD34+, KDR+ monoclonal antibody sorted.The mark of other selection candidate stem cells can also be used to be sorted, such as： AC133、 CD50+Etc. mark.
Can be serum-free medium 4. the stem cell without Immune discrimination sorted out is incubated in the nutrient solution combined containing different hemopoieticgrowth factors.That identifies at present can act on candidate stem cell culture amplification（Propagation）Stimulation and inhibiting factor it is quite a lot of, but only several cell factors can individually play a role.A variety of Porcine HGFs of hematopoiesis thousand can be added in serum-free medium, such as add under SCF, IL- 3, IL- 6, a variety of hemopoieticgrowth factor combination conditions of TP0, Flt3- L, G-CSF and Epo, carry out amplification in vitro culture, wherein SCF, IL- 3 and GM- CSF are mainly the survival for maintaining primitive hematopoietic cell, but it can not be induced to breed, proliferative induction needs the synergy between the factor, and can be co-cultured using mesenchymal stem cells MSCs and candidate stem cell plays the role of to strengthen amplification to candidate stem cell.Its number of nucleated cells (NC), surface antigen and Colony forming ability are monitored.The general time-of-week of amplification in vitro culture about 2.It is of course possible to be increased according to actual conditions or reduce number of days.
5. when reaching time of amplification in vitro culture, and/or reach to carry out after the stem cell requirement without Immune discrimination of bone-marrow transplantation, bone-marrow transplantation can be immediately available for, cryogenic freezing preservation can also be carried out. Embodiment 2
Fig. 2 are the patients of organ transplant of the present invention and adult's autoantigen of disease of immune system patient re-recognizes treatment method flow chart with disease of immune system.
It may be referred to " organ transplant clinical guidelines "（Chief editor：Leaf is inspired, Beijing Science Press in July, 1999 first edition）One book
1. the organ transplant stage：
1) donor organs, histocyte are carried out first transplant the various preparations of early stage.
2) donor organs, histiocytic donor are carried out and by the HLA distribution type between patient receptor being transplanted, prevents rejection so that the organ after transplanting can be survived.
3) organ transfer operation is carried out.
4) rejection is prevented using anti-rejection medication.
No Immune discrimination stem cell bone-marrow transplantation is ready for after patient host transplants or in the case where the patient such as disease of immune system state of an illness allows.
2. bone-marrow transplantation, the secondary Autoantigen Immune cognitive phase of progress：
1) using medicine such as：Endoxan, and radioactive ray irradiation is such as：Cobalt6°, that human body is original, the ripe candidate stem cell for having carried out Immune discrimination of clinac high-energy X-ray full-body exposure (TBI) kills.Into bio-clean treatment unit.
2) input by culture amplification without thousand cells for carrying out Immune discrimination, such as candidate stem cell.According to different diseases, different case needs the stage can also will be killed without using medicine and/or radioactive ray irradiation by original candidate stem cell in vivo, directly will be internal without the input of Immune discrimination stem cell.
3) input by culture amplification without the medicine that promotion candidate stem cell after the stem cell for carrying out Immune discrimination, can be needed to use to go back to the nest according to the state of an illness（Candidate stem cell（H S C) go back to the nest and refer to that H S C enter receptor by vein transplantation through Peripheral Circulation after, its identification and positioning in marrow mediated through complicated intermolecular interaction.）.And carry out internal expansion of stem cells treatment (exviV0).It can use stem cell factor SCF and G-CSF that the thousand cells/progenitor cells (PBPC) for stimulating amplification internal are used in combination.
4) prevention and treatment of complication are wanted after bone-marrow transplantation：Such as prevent early stage rejection, use anti-rejection medication etc., the various treatments of prevention treatment of infection, etc..Prevention implantation syndrome (engraftment syndrome), uses the drug therapies such as corticosteroid hormone.Because to carry out the Immune discrimination of autoantigen within a period of time after HSCT, and immune system is rebuild, therefore within this period is without anti-infection ability, it is necessary to which strict gnotobasis, and antibacterium viral infection resisting are treated.
5) Immune discrimination of autoantigen, is that those factors promote candidate stem cell to carry out autoimmunity identification, and immune system rebuilds required time.Now to track every immunology assay index.
6) bio-clean treatment unit is left after transplanting successfully, immunologic function is carried out later and is resumed treatment, various vaccines are such as injected.
Organ transplant stage and bone-marrow transplantation, the secondary Autoantigen Immune cognitive phase of progress, it can carry out simultaneously, graft antigen can also be first transplanted to carry out bone-marrow transplantation, carry out secondary Autoantigen Immune identification, when body immune system by graft antigen recognizing be autoantigen after, organ transplant is being carried out, can thus improve graft survives probability.Embodiment 3
Fig. 3 are a kind of acquisition, culture, amplification and progress secondary immunity identification that early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are carried out outside human body of the present invention, treat graft rejection reaction, and/or disease of immune system method flow chart.
1. embryonated egg（It can also be the egg cell of clone）Can be obtained from several lower several respects： 1) after normal pregnancy; miscarry, no early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are extracted from the stillborn foetus miscarried, and/or need the graft of transplanting; this graft can be organ, tissue, cell, such as：Liver, kidney, pancreas islet e cells etc..
2) somatic cells of receptor or donor are cloned into the egg cell for the kind identical with receptor or donor for taking out nucleus, clone's egg cell of formation, no early immune identification candidate stem cell and/or Immune discrimination candidate stem cell stage in late period are developed into test tube or in human body or in tertium quid's body, extract no Immune discrimination thousand are thin, and/or need the graft born of the same parents of transplanting.
3) somatic cells of receptor or donor are cloned into the different genera tertium quid's egg cell for taking out nucleus, form clone's egg cell, early immune identification candidate stem cell and/or Immune discrimination candidate stem cell stage in late period are developed into test tube or in tertium quid's body or in human body, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are extracted, and/or needs the graft of transplanting.
4) embryonated egg that the sperm or ovum of receptor and different in nature sperm or ovum fertilization with kind are obtained, the embryonated egg combined including receptor and/or donor father sperm and female ovum, early immune identification candidate stem cell and/or Immune discrimination candidate stem cell stage in late period are developed into test tube or in tertium quid's body or in human body, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are extracted, and/or needs the graft of transplanting.
5) embryonated egg can also be obtained using different methods from a variety of different approach.
2. embryonated egg is subjected in vitro culture and/or development in vivo obtains early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell and there are a variety of methods, wherein can use：
1) method of in vitro culture is that have the embryonated egg side of being put into the blake bottle of culture medium to cultivate, and adds the candidate stem cell directed differentiation factor, is divided into early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell.
2) it will be fertilized in implantation of ovum tertium quid or human body, such as：Developed in the uterus of people or in the uterus of animal such as mouse or rabbit, the time of development can be determined according to the specific needs of medical condition, for example, waits until that embryonic development recognizes that candidate stem cell and/or late period Immune discrimination candidate stem cell carry out any suitable time in the stage before autoimmunity identification to early immune therein.Early immune is extracted by set method and recognizes candidate stem cell and/or the cell of late period Immune discrimination hematopoiesis thousand, for example, is extracted using flow cytometer.It can also be expanded as needed after early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell is extracted in blake bottle.
3. allow early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell progress autoantigen to re-recognize has number of ways with graft culture development.For example:
1) carried out in tertium quid's body, early immune is recognized that candidate stem cell and/or late period Immune discrimination HSCT are carried out in immune deficiency tertium quid's body including 1., such as carried out in the small BALB/cA-nude nude mices, SCID mouse, NOD- SCID mouse bodies of immune deficiency.Candidate stem cell and/or late period Immune discrimination candidate stem cell and/or graft transplantation can also be recognized to without immune deficiency tertium quid's body early immune by the method for bone-marrow transplantation.
2) carried out in blake bottle.
3) carried out in recipient's body.
4) candidate stem cell separated out of recipient's body, extracted and/or immune stem cell.
5) what is extracted out of aborted fetus body needs the organ of transplanting without Immune discrimination stem cell, and/or separation.
4. allow early immune to recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell carry out autoantigen in tertium quid's body and re-recognized（Secondary immunity is recognized）Specific method：
1) secondary immunity identification-a. transplanting is carried out in tertium quid's body and prepares what is transplanted in human body or animal body（Donor）Graft or graft antigen or variation（Receptor's）Autoantigen is to without immunologic function（Or immune deficiency）Tertium quid's body in, and/or kill by the method using bone-marrow transplantation the stem cell for carrying out Immune discrimination and with immune relevant cell, and/or relevant with Immune discrimination organ it is illuminated after lose in tertium quid's body of due function.Can also first transplantation donor candidate stem cell and/or immune stem cell and the Immune discrimination factor and/or thymocyte, bone marrow cell.Autoantigen Immune identification is carried out, graft transplantation is then carried out again.
B. transplanting people or tertium quid start or carry out Autoantigen Immune cognitive phase embryo it is immune Recognize organ, and/or according in the contents such as variety classes, the different types of tissue fluid inside the variety classes in the organ of design requirement transplanting embryo Jing Guo selection, the different types of tissue relevant with Autoantigen Immune identification, cell, and/or these organs relevant with Immune discrimination to tertium quid's body.Recover immune deficiency tertium quid, and/or by radiation and/or the defective immune organ function of the tertium quid of drug therapy.
C. in tertium quid's body that transplanting early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are arrived.D. startup factor can be inputted according to design requirement makes early immune recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell carry out Autoantigen Immune identification.Starting or carrying out the Immune discrimination organ in the embryo of Autoantigen Immune cognitive phase for transplanting people or tertium quid can also be used, and/or according to variety classes, the different types of cell in the organ of design requirement transplanting embryo Jing Guo selection, and/or the content such as variety classes, the different types of tissue fluid inside organ in tertium quid's body when the startup factor that has existed, and/or stop the factor.
E. decide whether that input stops the factor and early immune is recognized that candidate stem cell and/or late period Immune discrimination candidate stem cell stop the termination factor that Autoantigen Immune is recognized according to design requirement.
F. flow cytometer or miscellaneous equipment are used, the stem cell for carrying out Immune discrimination is extracted out of tertium quid body.G. early immune is recognized into candidate stem cell and/or late period Immune discrimination candidate stem cell or carries out the stem cell progress cell amplification of Immune discrimination.
H. thousand cells of Immune discrimination will be carried out by bone-marrow transplantation, and/or be grafted directly in recipient's body.
I. can will carry out the stem cell transplantation of Immune discrimination to before in recipient's body, simultaneously or after, carry out the organ, tissue, cell transplantation of graft.And/or treatment disease of immune system.
2) secondary immunity identification-a. is carried out in test tube, blake bottle and transplants the graft for preparing to be transplanted in human body or graft antigen or the autoantigen of variation into test tube.
B. transplanting people or tertium quid start or carry out the Immune discrimination organ in the embryo of Autoantigen Immune cognitive phase, and/or according to contents such as variety classes, the different types of tissue fluid inside the variety classes in the organ of design requirement transplanting embryo Jing Guo selection, the different types of tissue relevant with Autoantigen Immune identification, cell, and/or these organs relevant with Immune discrimination into test tube.
C. transplanting people early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are into test tube.
D. input startup factor makes early immune recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell carry out Autoantigen Immune identification.And/or transplanting people or tertium quid start or carry out the Immune discrimination organ in the embryo of Autoantigen Immune cognitive phase, and/or according to variety classes, the different types of cell in the organ of design requirement transplanting embryo Jing Guo selection, and/or the content such as variety classes, different types of tissue fluid inside organ is into test tube.
E. the termination factor can be inputted as needed makes early immune recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell stop Autoantigen Immune identification.
F. extract into the stem cell for carrying out Immune discrimination.
G. the stem cell for carrying out Immune discrimination is expanded.
H. by the method for foregoing bone-marrow transplantation, and/or it is grafted directly in recipient's body.
I. the stem cell transplantation of Immune discrimination can will be being carried out to after in recipient's body, carry out the organ, tissue, cell transplantation of graft.And/or treatment disease of immune system.Because now internal immune system oneself through graft is identified as into autoantigen, therefore HLA and receptor even if graft is mismatched will not also occur immunological rejection.Tertium quid can be used to play a part of the organ of transplanting required for culture simultaneously.The organ origin of transplanting required for further expanding.Embodiment 4
Fig. 4 are one kind progress early immune identification candidate stem cell and/or the extraction of late period Immune discrimination candidate stem cell, culture, amplification and progress secondary immunity identification in recipient's body outside human body of the present invention, treat graft rejection reaction, and/or disease of immune system method flow chart.
1. embryonated egg（It can also be the egg cell of clone）Can obtain -1 from several lower several respects) after normal pregnancy; miscarry; no early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are extracted from the stillborn foetus miscarried, and/or needs the graft transplanted, this graft can be organ, Tissue, cell, such as：Liver, kidney, beta Cell of islet etc..
2) somatic cells of receptor or donor are cloned into the egg cell for the kind identical with receptor or donor for taking out nucleus, clone's egg cell of formation, no early immune identification candidate stem cell and/or Immune discrimination candidate stem cell stage in late period are developed into test tube or in human body or in tertium quid's body, the dry thin of no Immune discrimination is extracted, and/or needs the graft born of the same parents of transplanting.
3) somatic cells of receptor or donor are cloned into the different genera tertium quid's egg cell for taking out nucleus, form clone's egg cell, early immune identification candidate stem cell and/or Immune discrimination candidate stem cell stage in late period are developed into test tube or in tertium quid's body or in human body, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are extracted, and/or needs the graft of transplanting.
4) embryonated egg that the sperm or ovum of receptor and different in nature sperm or ovum fertilization with kind are obtained, the embryonated egg combined including receptor and/or donor father sperm and female ovum, early immune identification candidate stem cell and/or Immune discrimination candidate stem cell stage in late period are developed into test tube or in tertium quid's body or in human body, early immune identification candidate stem cell and/or the cell of late period Immune discrimination hematopoiesis thousand are extracted, and/or needs the graft of transplanting.
5) embryonated egg can also be obtained using different methods from a variety of different approach.
2. embryonated egg is subjected in vitro culture and/or development in vivo obtains early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell and there are a variety of methods, wherein can use：
1) receptor's in vitro culture is carried out, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell is divided into.
2) it is implanted into tertium quid or human body, according to design requirement in the appropriate period before embryonic development to early immune recognizes candidate stem cell and/or late period Immune discrimination candidate stem cell carries out autoimmunity identification.Take out embryo and extract early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell, and/or the various progenitor cells relevant with being immunized（Multipotential stem cell）, and/or the various unipotent stem cells relevant with being immunized.
3) candidate stem cell separated out of recipient's body, extracted and/or immune stem cell.
4) what is extracted out of aborted fetus body needs the organ of transplanting without Immune discrimination stem cell, and/or separation.
3. early immune is recognized that candidate stem cell and/or late period Immune discrimination candidate stem cell carry out cell amplification in test tube, blake bottle or culture vessel.
4. transplanting prepares the graft transplanted in human body or graft antigen into the human body.To carry out anti-rejection medication treatment simultaneously.If a variety of diseases such as treatment autoimmune disease avoid the need for carrying out this step.
5. early immune is recognized into the method that candidate stem cell and/or late period Immune discrimination candidate stem cell pass through bone-marrow transplantation（Can be with the concrete condition of the state of an illness, using clear marrow bone-marrow transplantation, the clear marrow bone-marrow transplantation in part or nonmyeloablative bone marrow transplantation）It is transplanted in recipient's body.
6. transplanting embryo is starting or carried out Immune discrimination organ in Autoantigen Immune cognitive phase in receptor's human body.And/or input startup factor makes early immune recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell carry out Autoantigen Immune identification.This step can decide whether to carry out according to actual conditions during treatment.
Early immune is set to recognize that candidate stem cell and/or the cell of late period Immune discrimination hematopoiesis thousand stop Autoantigen Immune identification 7. the termination factor can be inputted as needed.
Treat rejection and the disease of immune system of graft.This step can decide whether to carry out according to actual conditions during treatment.Embodiment 5
Fig. 5 are that a kind of early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell of the present invention carry out the experimental method flow chart of secondary immunity identification in tertium quid's body.
The tertium quid's experiment for carry out early immune identification candidate stem cell and/or the extraction of late period Immune discrimination candidate stem cell, culture, expanding and secondary immunity identification is carried out in tertium quid's body can be adopted with the following method：Tertium quid is divided into two parts while carrying out culture experiment.
First group：Be transplantation donor graft or graft antigen to group in tertium quid's body. 1. the tertium quid used can use a. immunodeficient animals, such as：Nude mice, SCID mice, N0D-SCID mouse etc., the nonimmune deficient animals of b. carry out clear marrow, half clear marrow or the processing of unclear marrow, can carry out the animal of early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell bone-marrow transplantation.
2. the graft or graft antigen of transplantation donor and/or receptor are in tertium quid's body.When autoantigen identification is carried out, donor and/or the autoantigen of receptor can be identified as into autoantigen without Immune discrimination stem cell.Because receptor without the Immune discrimination stem cell autoantigen with receptor in itself, and have a strong expression, thus can be decided whether by experiment in addition transplant recipient graft antigen in tertium quid's body.
3. trnasplantion immunity organ is to the cell relevant with autoimmunity identification in tertium quid's body, such as transplanted in people's thymus gland, spleen, people's myeloid tissue, organ organ, or these tissue, organs etc..This step trnasplantion immunity organ can decide whether to use according to requirement of experiment.
4. input startup factor and/or termination factor（" tissue relevant with Autoantigen Immune identification, organ, cell, the factor "）." tissue relevant with Autoantigen Immune identification, organ, cell, the factor " with kind is preferably transplanted or inputs, can also using " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " of different genera ,/this step is transplanted that use can be decided whether according to requirement of experiment.
5. transplanting is with kind or different genera without the progress autoantigen identification of Immune discrimination stem cell.Now also by graft antigen recognizing be autoantigen.
6. now tertium quid turns into：If that graft is people, for example, transplant the candidate stem cell of people, then this animal now have people or by progress graft or graft antigen recognizing immune system animal.This have the progress of people the immune system of graft or graft antigen recognizing animal, not exclusively can be used for this experiment, can be used for other all kinds, the experiment of various purposes.
7. extracted using methods such as immunomagnetic beads, and/or flow cytometers and carried out Immune discrimination human stem cell.
8. extraction is transplanted in second group of tertium quid's body for carrying out Autoantigen Immune identification without Immune discrimination human stem cell.
Second group：Be not the graft of transplantation donor or graft antigen to group in tertium quid's body.
1. the tertium quid used can use a. immunodeficient animals, such as：Nude mice, SCID mice or N0D-SCID mouse etc., the nonimmune deficient animals of b., but clear marrow processing was carried out, the animal of early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell bone-marrow transplantation can be carried out.It can be tested by taking SCID mice or N0D-SCID mouse as an example.
2. the graft of transplant recipient or graft antigen are in tertium quid's body.Because receptor without the Immune discrimination stem cell autoantigen with receptor in itself, and have a strong expression, thus can be decided whether by experiment in addition transplant recipient graft antigen in tertium quid's body.And now tertium quid is because without immunologic function, therefore graft is easy to transplant successfully, can using this tertium quid as graft culture, smaller graft is allowed to be grown in tertium quid's body, reach after the size that can be transplanted, graft is taken out out of tertium quid body, is transplanted in recipient's body.
3. immune organ is transplanted in tertium quid's body, such as transplant allogenic animal or heterogenous animal or people's thymus gland, spleen, people's myeloid tissue.
4. input startup factor and/or termination factor.
5. transplanting carries out autoantigen identification without the cell of Immune discrimination people thousand.
6. now tertium quid turns into：If that graft is all people, this animal now has the animal of the immune system of people（The graft Immune discrimination of donor was not carried out now）.This has the animal of the immune system of people, not exclusively can be used for this experiment, can be used for other all kinds, the experiment of various purposes.
There is the tertium quid of the immune system of people to test-A. groups being divided into two groups of progress for this:
1. the method for using the clear marrow of clear marrow, part or non-clear marrow to carry out bone-marrow transplantation, what transplanting was extracted out of first group tertium quid's body transplants without Immune discrimination human stem cell.
2. it is to tertium quid's body body or internal to transplant graft or graft antigen.
3. observation whether there is rejection. 4. experiment terminates.
This is observed after thousand cell transplantations for carrying out Immune discrimination, if can be treated graft rejection reaction, or can be treated disease of immune system.
B. group：
Because now the immune system in animal body has the immune system of people, no longer it is immunodeficient animals, therefore now carry out that the transplanting of graft will carry out that the related distribution type of tissue, the graft that only HLA is matched entirely, or HLA half match again can just transplant success.The graft that use HLA half to match in this experiment is transplanted, and anti-rejection medication is taken after transplanting.
1. carrying out the method for bone-marrow transplantation using clear marrow, bone-marrow transplantation will be carried out without Immune discrimination human stem cell,
2. input startup factor and/or termination factor.
3. disable anti-rejection medication.
4. observation whether there is graft rejection reaction.
5. experiment terminates.
This is the method that observation carries out early immune identification candidate stem cell and/or late period Immune discrimination HSCT using the clear marrow in part or all clear marrow, see whether that autoantigen can be re-recognized, graft is identified as autoantigen, and graft rejection reaction is treated, or disease of immune system can be treated.
Note：The method that wherein early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell can use Fig. 3 to be mentioned in illustrating is obtained.Immune organ transplanting immune organ used can also use animal with user.
Recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell have with the stem cell methods for carrying out Immune discrimination using early immune：
1. by graft transplantation to recipient's body, then early immune identification candidate stem cell and/or late period Immune discrimination HSCT are carried out, early immune is set to recognize that candidate stem cell and/or late period Immune discrimination candidate stem cell, by secondary immunity identification is carried out in body, treat rejection.
2. first will in vitro be carried out in receptor the progress of graft secondary immunity identification thousand cells of Immune discrimination be transplanted in recipient's body, then again by graft transplantation to recipient's body, graft is just without causing rejection.Embodiment 6
Another early immune to carry out bone-marrow transplantation that Fig. 6 are the present invention recognizes the flow chart that candidate stem cell and/or late period Immune discrimination candidate stem cell are extracted, cultivate and expanded.
Early immune recognizes that candidate stem cell and/or late period Immune discrimination candidate stem cell are obtained, below by taking the acquisition of candidate stem cell as an example.
1. the stillborn foetus obtained from miscarriage：The organ carried out required for secondary immunity identification can be extracted by peeling off：Such as thymus gland, bone, liver.The liver, thymus gland and skeleton of stillborn foetus are stripped out, because thymus gland, bone stock are easier to obtain,
" tissue relevant with Autoantigen Immune identification, organ, cell, the factor ", is obtained preferably from thymus gland, bone.Because fetus is liver hematopoiesis, therefore the early immune identification cell of hematopoiesis thousand and/or late period Immune discrimination candidate stem cell, or acquirement early immune identification candidate stem cell and/or the cell of late period Immune discrimination hematopoiesis thousand from other organs or tissue can be obtained from liver.
2. tire liver is made into single cell suspension, the karyocyte of the early immune identification candidate stem cell containing higher concentration and/or late period Immune discrimination candidate stem cell is extracted with density gradient centrifugation method.Thymus gland and bone can be grafted directly in body, thymus cell suspension can also be made, and bone marrow cell suspension, the method that can also be separated by using special cells such as immunomagnetic beadses, extract in " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " with Autoantigen Immune recognize it is relevant required for cell, further cultivated, or directly used.
3. the karyocyte of the early immune identification candidate stem cell containing higher concentration and/or late period Immune discrimination candidate stem cell can be extracted with density gradient centrifugation method, the step point of immunomagnetic beads-flow cytometer two can also be further utilized Method is selected, early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell are sub-elected from fetal liver cell suspension, for example, utilizes the stem cell antigen of stem cell surface specifically expressing（Stemcellantigen, Sea) monoclonal antibody and be coated in the secondary antibody of magnetic particle surface, using magnetic suck cell sorting method（MACS ：) isolate the candidate stem cell without Immune discrimination in marrow.Early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell can also be sub-elected using other methods.Thymocyte, bone marrow cell from thymus gland, bone marrow suspension required for extraction Immune discrimination.
Restricted dilution test shows：The cell that can be persistently implanted into for a long time is the CD34+ KDR in KDR+, marrow+It is candidate stem cell that cell, which has 20%,；The candidate stem cell for having the ability of being chronically implanted is CD34+KDR+Subgroup, therefore CD34 can be used+, KDR+ monoclonal antibody sorted.The mark of other selection candidate stem cells can also be used to be sorted, such as： AC133、 CD50 .1Etc. mark.
Can be serum-free medium 1. the thymocyte sorted out, bone marrow cell, early immune are recognized into candidate stem cell and/or late period Immune discrimination candidate stem cell are incubated in the nutrient solution containing different combinations of. growth factors and expanded.That identifies at present can act on candidate stem cell culture amplification（Propagation）Stimulation and inhibiting factor it is quite a lot of, but only several cell factors can individually play a role.A variety of hematopoietic stem cell growth factors can be added in serum-free medium, such as add under a variety of hemopoieticgrowth factor combination conditions of SCF, IL- 3, IL- 6, TP0, Flt3-L, G- CSF and Epo, carry out amplification in vitro culture, wherein SCF, IL- 3 and GM- CSF are mainly the survival for maintaining primitive hematopoietic cell, but it can not be induced to breed, proliferative induction needs the synergy between the factor, and can be co-cultured using mesenchymal stem cells MSCs and candidate stem cell plays the role of to strengthen amplification to candidate stem cell.Its number of nucleated cells (NC), surface antigen and Colony forming ability are monitored.The general time-of-week of amplification in vitro culture about 2.It is of course possible to be increased according to actual conditions or reduce number of days.
2. when the time for reaching amplification in vitro culture, and/or reach to carry out after the early immune identification candidate stem cell of bone-marrow transplantation and/or late period Immune discrimination candidate stem cell requirement, bone-marrow transplantation can be immediately available for, cryogenic freezing preservation can also be carried out.Embodiment 7
Fig. 7 are the patients of another organ transplant of the present invention and adult's autoantigen of disease of immune system patient re-recognizes treatment method flow chart with disease of immune system.
It may be referred to " organ transplant clinical guidelines "（Chief editor：Leaf is inspired, Beijing Science Press in July, 1999 first edition）One book
1. the organ transplant stage：
1) donor organs, histocyte are carried out first transplant the various preparations of early stage.
2) the tissue associated antigen distribution type between donor organs, patient host of histocyte and trophy always is carried out, rejection is prevented so that the organ after transplanting can be survived.
3) organ transfer operation is carried out.
4) rejection is prevented using anti-rejection medication.
This organ transfer operation can be carried out before autoantigen secondary immunity identification is carried out, graft antigen can also first be transplanted in vivo, first carry out autoantigen secondary immunity identification, the graft antigen recognizing that will be transplanted is autoantigen, carrying out organ transfer operation, the distribution type of tissue associated antigen can need not be thus carried out, postoperative rejection is also prevent.
No Immune discrimination stem cell bone-marrow transplantation is ready for after patient host transplants or in the case where the patient such as disease of immune system state of an illness allows.
2. the bone-marrow transplantation stage-
1) removing and immune relevant cell, including " Autoantigen Immune recognizes storehouse " and the cell for storing Autoantigen Immune identification storehouse.It can be removed using non-specific removing, and/or specific immunity blood method, the relevant cell with being immunized, including：" Autoantigen Immune recognizes storehouse " and the cell that Autoantigen Immune recognizes storehouse is stored, and original immunocyte in peripheral blood and immuning tissue, including：The T cell and B cell of Solid phase and positive selection were carried out in thymus gland and marrow. 2) early immune that input is expanded by " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " and/or by culture recognizes candidate stem cell and/or the cell of late period Immune discrimination hematopoiesis thousand.
3) can also be according to different diseases, different case needs, the stage will be killed without using medicine and/or radioactive ray irradiation by original candidate stem cell in vivo, early immune is directly recognized into candidate stem cell and/or late period Immune discrimination candidate stem cell, and/or " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " is inputted in vivo.
4) Immune discrimination of autoantigen, is to promote early immune to recognize candidate stem cell and/or late period Immune discrimination candidate stem cell in the presence of the material required for the factors such as " Autoantigen Immune recognition factors " in " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " and/or the identification of various carry out autoimmunities.Carry out Autoantigen Immune identification.Immune system is observed in the middle of this and rebuilds required time.Now to track every immunology assay index.
5) needed that the prevention and treatment of complication can be carried out according to the state of an illness after bone-marrow transplantation：Such as prevent early stage rejection, use anti-rejection medication etc., the various treatments of prevention treatment of infection, etc..Prevention implantation syndrome (engraftment syndrome), uses the drug therapies such as corticosteroid hormone.Because to carry out the Immune discrimination of autoantigen within a period of time after HSCT, and immune system is rebuild, therefore within this period is without anti-infection ability, it is necessary to which strict gnotobasis, and antibacterium viral infection resisting are treated.
6) after transplanting or treatment of autoimmune diseases are successful, immunologic function can be carried out as needed later and resumed treatment, such as inject various vaccines.Embodiment 8
Fig. 8 are the extraction flows of the candidate stem cell and thymocyte of one embodiment of the present of invention, bone marrow cell：Fetus：34 monthly ages
Tissue/cell is obtained：
1st, 75% ethanol immersion fetus 25 minutes, sterilization
2nd, thymus gland, liver, femur, upper arm stock, shin bone are taken out, is placed in nutrient solution（Containing 0. 02%EDTA RPMI1640 nutrient solutions）
3rd, extract thymus gland and extract appendicular skeleton
Thymus gland is ground to form into unicellular solution in serum-free cell culture medium, tissue grinder is lightly ground thymus gland, and thymus cell suspension is made, and bone marrow cell is gone out with nutrient solution
4th, splitting erythrocyte, sieving filters off impurity, and specific cells extracting method secretes the cell of " Autoantigen Immune recognition factor ", and cell culture amplification is extracted：Autoantigen Immune recognition factor, transgenic cell clone, is prepared：Autoantigen Immune recognition factor
5th, liver is taken out
1) it is cut into small pieces in culture dish（5 Hidden or so）, cell suspension is made with tissue grinder in batches
2) supernatant is abandoned in centrifugation, and precipitation adds erythrocyte cracked liquid, and lucifuge is cracked 10 minutes
3) supernatant is abandoned in centrifugation, is resuspended and precipitated with 1640 culture medium
4) 100 200 aim cells are sieved through filter hepatocyte suspension
5) density gradient centrifugation is separated
Percoll each 3ml of cell separation liquid that proportion is 1. 110 are added in centrifuge tube bottom, each 3ml of Percoll cell separation liquid that proportion is 1. 077 and 1. 035 is then successively respectively added slowly on its upper strata.The hepatocyte suspension filtered through cell sieve is added to separating liquid upper strata, 1500rpm is centrifuged 20 minutes.
6) remove supernatant liquid, take precipitation to be cleaned with 11640 nutrient solutions 2 times.
7) people CD34+ antibody immune magnetic beads separation is purified
Three of the above cell（Thymus gland, marrow, liver）It is 5X10 with the nutrient solution adjustment concentration containing 10%DMS06Left and right, freezes in liquid nitrogen.
If adding the cell separation liquid 3ml that proportion is 1. 077 in centrifuge tube bottom, the cell separation liquid 3ml that proportion is 1. 035 is then slowly added on its upper strata.The hepatocyte suspension filtered through cell sieve is added to separating liquid upper strata, 1500rpm, Centrifugation 20 minutes.Remove supernatant liquid, take precipitation to be cleaned with 11640 nutrient solutions 2 times.Then the hepatocyte suspension after separation, employment CD34 antibody labelings, flow cytomery candidate stem cell ratio are taken.The purity that human hematopoietic stem cell can be obtained is 13 30%.The suitable concentration of method choice that gradient of continuous density can also be used to centrifuge is centrifuged.
Embodiment 9
Fig. 9 are that the autoantigen for carrying out organ transplant in animals of the present invention re-recognizes the embodiment with disease of immune system.
The present embodiment is the experiment in order to verify the theory of the present invention and make, but this experiment is also demonstrated simultaneously：It can be included in the animal bodies for having certain immunologic function by transplanting " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " of people：Thymus gland, thymocyte, marrow, bone marrow cell and Immune discrimination candidate stem cell and the experimental animal for obtaining the function of immune system with people, the animal of this immunologic function with people, because the immune system with people, therefore human body can be substituted and carry out various medical experiments, so so that many pairs because there is the scientific experiment that necessarily damages and can not carry out to human body, it can be tested with this animal, and have and human experimentation identical effect, because sage's Judas's child study hospital of the U.S.（St. Jude Children's Research Hospital) it is published on May 15th, 2005《Immunology》Magazine（Thejournal of Immunology P6540-6545) on article, describe they
NOD/LtSz-scid IL2R Y nu11The candidate stem cell that people is transplanted with mouse completes the experimental animal model of human immune system.Simply NOD/LtSz-scid IL2R Y "u" mouse is that NK cells are non-functional, it is impossible to kill the human hematopoietic stem cell of transplanting, therefore this mouse is rejection for graft, received completely without any immunologic function.The NOD/LtSz-scid IL2R y completely without immunologic function have been used in their experimentnu" NOD-scid mouse of the mouse as experimental group and with certain immunologic function as a control group, in NOD/LtSz- scid IL2R ynu11The cell transplantation of hematopoiesis thousand success of people in the experimental group of mouse so that NOD/LtSz- scid IL2R ynu11Mouse obtains the immunocyte of people, immunologic function with people, and the HSCT failure of the NOD-scid mouse people with certain immunologic function, the HSCT failure result of this NOD-scid mouse people can be used as experimental comparison group of the invention, it was demonstrated that the animal of the immunologic function with people can be made in method used in the present invention with the animal with normal immunological function using method used in the present invention.
Used in the present invention is to have NK cell functions（With certain immunologic function）NOD-scid mouse, generally in human body carry out HSCT when need carry out human leucocyte antigen (HLA)（HLA) distribution type, needs to take anti-rejection medication, otherwise can not carry out transplanting successfully after the transplanting of half matching.And in the experiment of the present embodiment, the cell with the people of the entirely different kind of experimental animal is used, major histocompatibility antigen complex can not be carried out at all（MHC) antigen distribution type, because that people is HLA, mouse is that H-2 is two kinds of different MHC systems, therefore the successful of transplanting does not almost have, and " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " injected in the experiment of the present embodiment in Mice Body includes：Thymus gland, thymocyte, marrow, bone marrow cell and Immune discrimination candidate stem cell, it is a kind of there is very strong antigenic graft, and do not take any anti-rejection medication to mouse in migration process, and this transplanting mode be equal to graft antigen transplanting be with the cell transplantation of hematopoiesis thousand be simultaneously progress, the transplanting success of the present embodiment, it can be really between different types of animal of the same race to demonstrate the present invention, and progress immune system and organ are mutually moved between humans and animals, and overcome rejection.Demonstrate the correctness of the theory and practice of the present invention.Also demonstrating the present invention simultaneously can be included with many animals with immunologic function by " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " of transplanting people：Thymus gland, thymocyte, marrow, bone marrow cell and Immune discrimination candidate stem cell, and the immunocyte of people is obtained, the experiment basis of this method of the experimental animal of the immunologic function with people." tissue relevant with Autoantigen Immune identification, organ, cell, the factor " of animal can certainly be carried out between different types of animal and animal to be included：Thymus gland, thymocyte, marrow, bone marrow cell and Immune discrimination HSCT, make receptor have the animal of donor immune systems' function.Embodiments of the invention prove that the immune system of people can be set up with the animal with immunologic function, can substitute people and do the immune system experiment of various people, and tissue, cell and organ of people etc. can be cultivated in the body of animal. " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " of same kind should be transplanted in the zoopery of the present embodiment, at that time because of mouse from by precise and penetrating birth only 29 days or so, simultaneously as the Autoantigen Immune identification period of mouse is not clear in which day, " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " of people have to be transplanted, this result to experiment has a great impact, but the result of formula this experiment can also prove the principle correctness of the present invention.
Zoopery process：
1. the first stage tests：
1) 9 N0D/SCID mouse, the cGy of TBI 1. 0 are irradiated.
2) thymus gland and marrow in transplanting " tissue relevant with Autoantigen Immune identification, organ, cell, the factor ", the human foetus liver cells of 6. 24 preservations（People's CD34+ antibody labelings, flow cytomery candidate stem cell ratio.For 30%).
), recovery, nutrient solution is washed 2 times.
Count results-fetal liver cell： 2. 3 xlO8
9 N0D/SCID mouse,
Tail vein injection, every 0. 2ml cell suspension of inoculation, the xlO of liver cell 4. 67
Continue to raise in Laminar Flow Room, observe 2- 3 months.
Vena ophthalmica blood was taken after 12 weeks, anti-CD45 SABC pathological sections are done, the lymphocyte growth in thymus gland, spleen, the marrow of bone, positive visible anti-CD45 in the organ such as liver.Prove the immunocyte of acquisition people in HSCT success, animal bodies.
The present invention is to utilize the early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell obtained from a variety of methods, and the autoantigen that be grown up re-recognizes what is design with the method for the treatment of disease of immune system for embodiment, but early immune identification candidate stem cell and/or late period Immune discrimination candidate stem cell of the stem cell of each stage of development, various species by conversion acquisition, the disease for the various species of stem-cell therapy for carrying out Immune discrimination can also be utilized.
As described above, with reference to each accompanying drawing, highly preferred embodiment of the present invention is described in detail, still, it is not considered that the design of the present invention is only limited to each above-mentioned embodiment.Those skilled in the art, the inspiration conceived by the various embodiments described above, it is not difficult to re-recognize the adult autoantigen of the present invention to make with the method for the treatment of disease of immune system and various improve, changes or replacement, and in the treatment applied to various diseases, therefore, these improve, change or replaced, it is not considered that the design of the present invention has been had disengaged from, or appended claims limited range.

Claims (24)

  1. Claim
    Raw material needed for 1. one kind re-starts Autoantigen Immune identification obtains and methods for using them, it is characterised in that raw material needed for re-starting Autoantigen Immune identification includes：The tissue relevant with Autoantigen Immune identification, organ, cell, the factor, and/or Autoantigen Immune identification candidate stem cell.
    Raw material needed for 2. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, the tissue relevant with Autoantigen Immune identification, organ, cell, the factor are to participate in related organization's organ that Immune discrimination candidate stem cell carries out Immune discrimination to autoantigen in body, and/or tissue, and/or cell, and/or the hormone relevant with Autoantigen Immune identification, cell factor, polypeptide, protein.
    Raw material needed for 3. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterised in that the tissue relevant with Autoantigen Immune identification, organ, cell, the factor include：The embryo of human or animal and the fetal state, participate in Immune discrimination candidate stem cell and carry out Immune discrimination period to autoantigen in body.Immune discrimination candidate stem cell is provided to the microenvironment required for autoantigen progress Immune discrimination in body, the cell for promoting Immune discrimination candidate stem cell to the secretion " Autoantigen Immune recognition factor " required for autoantigen progress Immune discrimination in body is provided, and/or secretion " promotees（Make secretion）Related organization's organ of the cell of various confactors required for the cell of Autoantigen Immune recognition factor ", and/or secretion autoantigen progress Immune discrimination, using complete histoorgan, or uses portion of tissue organ.
    Raw material needed for 4. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterised in that the tissue relevant with Autoantigen Immune identification, organ, cell, the factor include：The embryo of human or animal and the fetal state, participate in Immune discrimination candidate stem cell and carry out Immune discrimination period to autoantigen in body.Immune discrimination candidate stem cell is provided to the microenvironment required for autoantigen progress Immune discrimination in body, the cell for promoting Immune discrimination candidate stem cell to the secretion " Autoantigen Immune recognition factor " required for autoantigen progress Immune discrimination in body is provided, and/or secretion " promotees（Make secretion）Whole cells of related organization's organ of the cell of various confactors required for the cell of Autoantigen Immune recognition factor ", and secretion autoantigen progress Immune discrimination, and/or part cell, and/or one or more of which special cells.
    Raw material needed for 5. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterised in that the tissue relevant with Autoantigen Immune identification, organ, cell, the factor include：" Autoantigen Immune recognition factor ", and/or " promote（Make secretion）Autoantigen Immune recognition factor ", and/or autoantigen carry out the various confactors required for Immune discrimination.
    Raw material needed for 6. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterised in that the tissue relevant with Autoantigen Immune identification, organ, cell, the factor include：Thymic tissue, thymocyte, and/or myeloid tissue, bone marrow cell.
    Raw material needed for 7. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, characterized in that, the Immune discrimination candidate stem cell for re-starting the required people of Autoantigen Immune identification and/or animal is obtained from following approach：
    A. the source of clone cell or embryonated egg：
    A. from the somatic cells of graft receptor or immune system and Disease are transplanted to the people's ovum for taking out nucleus, the clone cell of formation；
    B. from the somatic cells of receptor are transplanted to the animal ovum for taking out nucleus, the clone cell of formation；C is combined from the sperm or ovum and donor of receptor or the sperm or ovum of other human or animals, forms fertilization Ovum；
    D. combined from the sperm or ovum of volunteer donors and the sperm or ovum of receptor or other human or animals, form embryonated egg.
    B. Immune discrimination derived from hematopoietic precursor cells：
    A., the clone cell of these separate sources or embryonated egg are developed into the embryo in Immune discrimination candidate stem cell stage in tertium quid's body, Immune discrimination candidate stem cell is extracted from embryo；
    B. culture is divided into the cell of Immune discrimination hematopoiesis thousand in test tube；
    C. the embryo from human or animal and the fetal state, the early immune that early immune identification candidate stem cell extracted in Autoantigen Immune identification period, stillborn foetus of miscarry recognize candidate stem cell；
    D. after the embryo of human or animal and the fetal state and birth, Immune discrimination candidate stem cell carries out the late period Immune discrimination candidate stem cell extracted in the period after Autoantigen Immune end of identification, stillborn foetus of miscarrying；
    E. from the hematological system in receptor or donor or donor's body, late period Immune discrimination candidate stem cell is directly extracted.
    Raw material needed for 8. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, Immune discrimination candidate stem cell is that by density gradient centrifugation, and/or immuno magnetic cell separation method is separated from the hepatocyte suspension of the liver of fetus.
    9. raw material needed for one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterised in that Immune discrimination candidate stem cell is in vitro expanded, or it is implanted into tertium quid's body and is expanded.
    Raw material needed for 10. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, input in vivo " tissue relevant with Autoantigen Immune identification, organ, cell, the factor ", and/or inputs vivo immunization and recognizes the cell of hematopoiesis thousand when re-starting Autoantigen Immune identification.
    Raw material needed for 11. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterised in that Immune discrimination candidate stem cell has in the approach for carrying out secondary immunity identification：Carry out, or carried out in tertium quid's body in test tube in vitro, or carried out in recipient's body.
    Raw material needed for 12. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, the donor and receptor of graft or graft antigen are to come from same kind of people, and/or animal, or from different types of people, and/or animal.
    Raw material needed for 13. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, Immune discrimination candidate stem cell carries out Autoantigen Immune identification, it is in vitro in test tube, or in tertium quid's body, or in recipient's body, the autoantigen of graft, and/or variation to donor carries out secondary immunity identification.
    Raw material needed for 14. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, before people and/or the identification of animal autoantigen secondary immunity, using non-specific therapy, and/or specific therapy part is removed, part is removed or is not removed and immune relevant cell.
    Raw material needed for 15. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, before the identification of autoantigen secondary immunity, use non-specific therapy, and/or specific therapy is removed, part is removed or does not remove the cell relevant with being immunized, including remove " Autoantigen Immune recognizes storehouse " and store the cell that Autoantigen Immune recognizes storehouse.And/or it is original in immune system in peripheral blood and immuning tissue, including：The immunocyte of Solid phase and positive selection was carried out in thymus gland and marrow, including：T cell and B cell.
    Raw material needed for 16. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, graft or graft antigen are transplanted in recipient's body, using the marrow transplant techniques of clear marrow, the clear marrow in part or non-clear marrow, Immune discrimination candidate stem cell, and/or the tissue relevant with Autoantigen Immune identification, organ, cell, the factor are transplanted in recipient's body, make Immune discrimination candidate stem cell by progress secondary immunity identification in body.
    Raw material needed for 17. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, transplanting " tissue relevant with Autoantigen Immune identification, organ, cell, the factor ", and/or Immune discrimination candidate stem cell is in tertium quid's body.
    Raw material needed for 18. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, use immunodeficient animals, or nonimmune deficient animals, carry out Immune discrimination candidate stem cell bone-marrow transplantation so that animal turn into people or by immune system animal.
    Raw material needed for 19. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, tertium quid uses immunodeficient animals, or nonimmune deficient animals, in the graft or graft antigen of transplantation donor and/or receptor to tertium quid's body：Trnasplantion immunity recognizes that the cell of hematopoiesis thousand, and/or the tissue relevant with Autoantigen Immune identification, organ, cell, the factor carry out secondary Autoantigen Immune identification：So that animal turn into donor, and/or by progress graft or graft antigen recognizing immune system animal.
    Raw material needed for 20. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, first using the marrow transplant techniques of clear marrow, the clear marrow in part or non-clear marrow, the Immune discrimination HSCT of graft or the identification of graft antigen secondary Autoantigen Immune will be carried out in vitro in receptor in recipient's body, then again by graft transplantation to recipient's body.
    Raw material needed for 21. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, the organ transfer operation of receptor is carried out prior to or while autoantigen secondary immunity identification is carried out, or graft antigen is first transplanted in vivo, carry out autoantigen secondary immunity identification, it is autoantigen by the graft antigen recognizing of transplanting, then carries out organ transfer operation.
    Raw material needed for 22. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, first carry out clear marrow, the clear marrow in part or non-clear marrow treatment, then it is implanted into " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " in vivo, and/or input vivo immunization identification hematopoiesis thousand cell progress re-starts Autoantigen Immune and recognized.
    Removing and immune relevant cell, and/or " Autoantigen Immune recognizes storehouse " and the method for the clear marrow of clear marrow or part for the cell for storing Autoantigen Immune identification storehouse are removed, including：
    1) it is non-specific to remove and immune relevant cell, including：
    A. medicine is utilized；
    B. radioactive ray irradiate.
    2) specificity removes the cell relevant with being immunized, and/or removes " Autoantigen Immune recognizes storehouse " and store the cell that Autoantigen Immune recognizes storehouse, including：
    A. original one or more cells relevant with being immunized in vivo is killed using medicine, or will be inactivated using medicine with immune relevant cell factor；
    B. specific cells, and/or and/or the killed cells factor are removed using immunological method.
    23. raw material needed for one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterised in that receptor or supply be between one species, and/or different types of animal and animal, and people and dynamic Immune system and organ are carried out between thing mutually to move, and are included by transplant recipient or " tissue relevant with Autoantigen Immune identification, organ, cell, the factor " that supplies：Thymus gland, thymocyte, marrow, bone marrow cell and Immune discrimination candidate stem cell, and receptor is obtained the animal of function of immune system supplied.
    Raw material needed for 24. one kind according to claim 1 re-starts Autoantigen Immune identification obtains and methods for using them, it is characterized in that, the immune system of people is set up with the animal with immunologic function, substitute people and do the immune system experiment of various people, and tissue, cell and the organ of people can be cultivated in the body of animal.
CNA2005800370506A 2004-11-04 2005-11-04 Method for acquiring and applying needed raw material for reccogniting self antigen immunity Pending CN101048497A (en)

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