CN1353994B - Method for creating human/goat hemopoietic stem cell heteroplastic transplantation model - Google Patents
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- CN1353994B CN1353994B CN011322314A CN01132231A CN1353994B CN 1353994 B CN1353994 B CN 1353994B CN 011322314 A CN011322314 A CN 011322314A CN 01132231 A CN01132231 A CN 01132231A CN 1353994 B CN1353994 B CN 1353994B
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Abstract
The present invention belongs to the field of biological cytology, and relates to a method for creating hemopoietic stem cell heterograft model. It is characterized by separating human cord blood to obtain human HSC and grafting it into fetal goat body to create the human/goat hemopoietic steam cell heterograft model. The tests show that in the venous blood of grated goat the human HSC amplification to 1000-10000 times of displayed, and the human HSC phynotype can be stably retained for above 10 months, and can be in incomplete differentiation state. The graft model created by said invention can provide potential living body storage for storing and amplifying artificial blood stem cell/ancestral cell, can be made into the convenient source of artificial blood stem cell/ancestral cell, and can provide theoretical and technical basis for curing disease by means of intrauterine HSC graft.
Description
Technical field
The invention belongs to the biomass cells technical field, be specifically related to a kind of method of setting up hemopoietic stem cell heteroplastic transplantation model.
Background technology
Stem cell (stem cell) is a kind of undifferentiated, long-term existence and can be to the cell of all directions differentiation.It is characterized by not differentiation and do not have specificity, can self and the cell that produces one or more specificitys and have specific function.In recent years, characteristics of life science have been become for the research of stem cell.Particularly (hematopoietic stem cells HSC) transplants and to have become effective means (the Thomas ED.Arch.Immunol.Ther.Exp.1997 that treats numerous malignant tumours and heredopathia clinically hemopoietic stem cell; 45:1-5), yet in this methods of treatment of development, have at least be noticeable at 2, how the first obtains in a large number can be for the HSC that transplants, and it two is the HSC control of differentiation degree in vitro and in vivo.There is research once to attempt to use control (Williams DA.Blood, 1993 that external long-term cultivation HSC technology is come expanding stem cells and studied its differentiation; 81:3169-3172; Emerson SG.Blood, 1996; 87:3082-3088), but there is dispute in the result.
Human and mammiferous HSC transplantation experiments shows, only when the host was in immunological tolerance or immunization barrier and is not activated as yet, transplanting is (Thomas ED.World J.Surg., 2000 successfully; 24:815-818; Ball ED., Lister J., Law P.:Hematopoietic Stem Cell Therapy.Philadelphia, PA:Churchill Livingstone, 2000).Early stage in fetation, immune organ is undeveloped mature still, is in immune cubhood, so be the suitable acceptor of HSC transplanting.Zanjani etc. successfully will transplant in tire sheep from the isolating HSC of people's tire liver, obtain people HSC long-term surviving (Zanjani ED., Pallavicini MG., Ascensao JL.et al.J.Clin.Invest., 1992 in the sheep body; 89:1178-1188; Shimizu Y., Ogawa M., Kobayashi M., et al.Blood, 1998; 91:3688-3692).
Summary of the invention
The purpose of this invention is to provide a kind of method of setting up human/goat hemopoietic stem cell heteroplastic transplantation model, can obtain the human/goat hemopoietic stem cell follow board.Attainments are inquired into biological phenomena after HSC transplants in individual level, for HSC amplification and variation such as differentiation in vivo provides technology platform.And can open up the new way that the HSC intrauterine transplantation is studied, and not only can be the treatment disease theory and technology foundation is provided, also can improve the success ratio that HSC transplants.
The present invention separates acquisition people HSC from people's bleeding of the umbilicus, is expelled in the tire goat body, has successfully set up human/goat hemopoietic stem cell heteroplastic transplantation model.Transplant in the goat for 50 in experimental group, 35 goats are successfully transplanted, and show that people HSC is expanded to 1000-10000 doubly, more meaningfully, transplant in stable its phenotype that keeps of the intravital human hematopoietic stem cell of goat and reach more than 10 months, and be in incomplete differentiation state.And the peculiar CD34 of hemopoietic stem cell that has no talent in the blood of 40 Nonimplantation goats of control group
+Cell, difference between the two be [P＜0.01] extremely significantly.
The inventive method technical scheme and step are as follows:
1, the basic line of transplantation model structure:
At first collect the Freshman bleeding of the umbilicus, isolate people HSC then, isolating HSC is implanted in the tire goat abdominal cavity.Whole process was finished in 8-16 hour.Treat the tire goat divide 3 months puerperiums, 6 months, 10 months respectively venous blood samples detect whether embedding tenant in common HSC and content and differentiation degree in the checking goat body.
2, the concrete steps of transplantation model structure:
(1) obtains fresh people's bleeding of the umbilicus.
(2) the fresh bleeding of the umbilicus of income earner is at first used NycoPrepTM (1.077g/mL, Nycomed Pharma AS, Oslo, Norway) mononuclearcell in the gradient centrifugation separating umbilical blood, use " the former progenitor cell enrichment of people cocktail " (HumanPrimitive Progenitor Enrichment Cocktail then, Stemcell Technologies, Vancouver, Canada) test kit further separates acquisition people HSC (CD34+CD38-Lin-cell).
(3) will separate the people HSC that obtains and transplant in the tire goat, gestational age is 55-65 days.She-goat needs fasting 24 hours before the art, intramuscular anesthesia agent then " 846 " (0.01mL/kg, Chinese military supplies university product), cut open the belly expose the uterus after, directly to tire goat intraperitoneal injection people HSC, injected dose is every HSC 1-5 * 10 by Uterus wall
5After closing the parent abdominal cavity, intravenous injection " dose of sobriety " (Chinese military supplies university product) impels goat to regain consciousness.
3, detect the structure model:
Life birth is 39 in the tire goat of 50 transplanting.Treat the tire goat divide 3 months puerperiums, 6 months, 10 months respectively venous blood samples detect, measure whether successfully to be implanted into the i.e. embedding tenant in common HSC whether of people HSC in the goat body, and detect the amount of people HSC in the goat body and the differentiation degree of HSC thereof.
(1) carry out facs analysis:
In the body of identifying the human/goat hemopoietic stem cell transplantation model before the whether chimeric HSC that goes into the people, comparative analysis normal goats and people's cell is to the difference of mouse-anti people monoclonal antibody binding ability, to find out the FACS comparative analysis that people's cell-specific is used for the human/goat model to the not specified mouse-anti people of goat cell monoclonal antibody.
The present invention adopts material source: the mouse-anti people T cell differentiation antigen of PE (phycoerythrin) and FITC covalent labeling, the antigenic monoclonal antibody of GPA and HLA are available from U.S. B.D. company, and flow cytometer is a U.S. B.D. company product.The analytical procedure of FACS is undertaken by the operational guidance of U.S. B.D. company among the present invention, by the comparative analysis of mouse-anti people monoclonal antibody to normal goats and people's cell binding ability between the two.
The FACS measuring method is as follows: every group of monoclonal antibody 10 μ L add 50 μ L goat whole blood samples, thoroughly at room temperature hatch 30 minutes behind the mixing, add 1mL 1 * FAGS then
TMLysate (B.D. company, San Jose, CA, USA), and mixing, room temperature is washed secondary with the PBS that contains 0.5% bovine serum albumin (BSA) and 0.1% sodium azide after following 15 minutes, and is centrifugal again 1, and 500rpm was hatched 15 minutes, abandoned supernatant liquor, got 1-2 * 10
5Cell carries out at room temperature carrying out flow cytometry analysis after the dyeing of monoclonal antibody.Analytical results of the present invention demonstrates and is specific to CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD34, CD38, CD42a, CD45, CD56, the mouse-anti people monoclonal antibody of CD62 and CD71 is insensitive to the goat cell, on this basis, and the conventional CD20/CD7 that selects; GPA/CD45; CD34/CD15; CD14/38 (PE/FITC mark) and CD3, CD4, CD8, monoclonal antibodies such as CD56 carry out FACS and measure.
(2) DNA analysis
1. extracting DNA;
2. design the PCR primer: by Genbank respectively inquirer CD34, glycophorin A (GlucophorinA GPA) and the sequence of sry gene, carries out the homology comparison with the sequence of goat, chooses nonhomologous partial design primer;
Table 1 is concrete primer table.
3. detect primer specificity: with synthetic each to the primer people's gene group that increases respectively, detect the specificity of DNA and control group sheep DNA;
4. nest-type PRC detects the human/goat hemopoietic stem cell follow board:
The pcr amplification of CD34 gene: extractive DNA as template, is done amplification for the first time with HC1/HC4, and amplification condition is: 94 ℃ * 5 ' → (94 ℃ * 1 ' → 61 ℃ * 45 " → 72 ℃ * 1 ') * 15cycles → 72 ℃ * 10 ', the amplification volume is 15 μ L.Get 5 μ L amplified productions and do amplification for the second time with HC2/HC3 again, amplification condition is: 94 ℃ * 5 ' → (94 ℃ * 1 ' → 61 ℃ * 45 " → 72 ℃ * 1 ') * 28cycles → 72 ℃ * 10 ', the amplification volume is 25 μ L.Wherein male can amplify the specific band of 437bp.
The pcr amplification of GPA gene: extractive DNA as template, is done amplification for the first time with GA2/GA3, and amplification condition is: 94 ℃ * 5 ' → (94 ℃ * 1 ' → 61 ℃ * 45 " → 72 ℃ * 1 ') * 15cycles → 72 ℃ * 10 ', the amplification volume is 15 μ L.Get 5 μ L amplified productions and do amplification for the second time with GA1/GA4 again, amplification condition is: 94 ℃ * 5 ' → (94 ℃ * 1 ' → 61 ℃ * 45 " → 72 ℃ * 1 ') * 28cycles → 72 ℃ * 10 ', the amplification volume is 25 μ L.Wherein male can amplify the specific band of 479bp.
The pcr amplification of sry gene: extractive DNA as template, is done amplification for the first time with HS1/HS4, and amplification condition is: 94 ℃ * 5 ' → (94 ℃ * 1 ' → 56 ℃ * 45 " → 72 ℃ * 1 ') * 15cycles → 72 ℃ * 10 ', the amplification volume is 5 μ L.Get 15 μ L amplified productions and do amplification for the second time with HS2/HS3 again, amplification condition is: 94 ℃ * 5 ' → (94 ℃ * 1 ' → 58 ℃ * 45 " → 72 ℃ * 1 ') * 28cycles → 72 ℃ * 10 ', the amplification volume is 25 μ L.Male can amplify the specific band of 302bp.
5. PCR-Southern blot detects: the PCR product that the CD34 gene that will increase obtains is transferred on the nylon membrane, uses the 437bp fragment that increases from people DNA to hybridize as probe.
6. RT-PCR detects the human/goat hemopoietic stem cell follow board
Extracted total RNA: use QIAGEN company
RNA Blood Kit extracted total RNA from the sheep peripheral blood.With the RNA reverse transcription is cDNA: use the reverse transcription test kit of BRL company to carry out reverse transcription reaction.
Pcr amplification CD34 gene, GPA gene: the cDNA that generates with reverse transcription is a template, and concrete amplification procedure is the same.
7. the tire sheep of transplanting the back miscarriage is carried out histology and immunohistochemical detection.
8. the maternal blood to transplanted tire sheep carries out hematological examination.
4, experimental result of the present invention is as follows:
(1) people's bleeding of the umbilicus HSC transplants behind 50 the first-born goats, wherein 11 former thereby miscarriages because of operation wound or acute immune response etc., other 39 the first-born goat gestation is to the childbirth of mature back, through FACS and DNA analysis, confirm 35 and have inlaying of human hematopoietic stem cell, and stably continue to birth after back 10 months and do not have an immunological rejection.Table 2 is the result of people HSC intrauterine transplantation behind the tire goat.
The facs analysis result shows in the goat blood of 35 tribal chief's ancestral cells transplanting survivals people CD34 is arranged all
+Cell (2.58 ± 0.54%), and the CD34 that has no talent in the blood of 40 Nonimplantation contrast goats
+Cell exists extremely significant difference (P＜0.01), not people CD34 between homologous transplantation sheep individuality between the two
+The quantity of cell has difference, but has nothing to do with sex of acceptor goat and the age of ewe.In addition, also exist in the blood of transplanting survival sheep and express CD14, the positive cell of CD20 and GPA etc. is not expressed CD3, CD4, the cell of CD8 and CD56 but detect.
(2) frequency (%) of each subgroup of human hematopoietic cell is represented with mean value ± SE in the goat blood.Has significant difference { P＜0.01} between transplanting survival group and the control group.Table 3 is the result of transplantation group and control group goat facs analysis.
After the tire goat birth, different times by its body of FACS dynamic monitoring in human hematopoietic cell have a situation, the result shows that human hematopoietic cell can more stably be expressed kinds of surface antigen in the goat body, it is back more than 10 months that its quantity also remains to birth.
(3) detected result of primer specificity:
All can amplify specific band in CD34, GPA and three kinds of genes of SRY among the human gene group DNA, not amplify corresponding band in the sheep genomic dna, the present invention proves that the synthetic primer specificity is better, can be used for Molecular Detection subsequently.
(4) the nest-type PRC detected result of human/goat hemopoietic stem cell follow board:
The result of the pcr amplification of CD34 gene: Fig. 2 confirms that the specific fragment that can amplify 437bp from the lamb peripheral blood among 8 DNA that extract is arranged, and proves to have people HSC in its peripheral blood.
The result of the pcr amplification of GPA gene: Fig. 3 1-8 as can be seen is positive.Have in 8 lamb peripheral blood DNAs and can amplify people GPA gene.
The result of the pcr amplification of sry gene: Fig. 4 is 2,3,5,8,10 positive as can be seen, transplants in these several the intravital HSC of lamb all to come from the male sex, and the result conforms to practical situation.
(5) the RT-PCR detected result of human/goat hemopoietic stem cell follow board:
The amplification of CD34 Gene RT-PCR: 1,2,4,5,6,7,8 is positive as can be seen from Figure 5.Have people CD34 expression of gene is arranged in 7 sheep peripheral bloods.
The amplification of GPA Gene RT-PCR: 1-8 is all positive as can be seen from Figure 6.Have people GPA expression of gene is arranged in 8 lamb peripheral bloods.
(6) PCR-Southern blot hybridization confirms above PCR result.
(7) the tire goat of transplanting miscarriage has carried out histology and immunohistochemical inspection: show that not only blood vessel contains in human hematopoietic stem cell but also the part organs and tissues, relatively large people's hepatocellular gathering is arranged in liver organization especially.
(8) finder's hemopoietic stem cell during the maternal blood of transplanting tire goat is checked.
Result of the present invention shows, separates the HSC that obtains from people's bleeding of the umbilicus and also can successfully transplant in the goat body, and its technical scheme, detection integrity and good result do not appear in the newspapers both at home and abroad.As accounting for goat peripheral blood mononuclear cell sum (10 according to human hematopoietic stem cell in the goat body of every transplanting survival
11-10
12) 1-3% calculate, people HSC has been expanded to 10 in the goat body so
3-10
4Doubly, in other words, as with 1 * 10
5People HSC transplant in goat, will have 10
8-10
9Human hematopoietic stem cell be circulated in the goat body, so the human/goat HSC transplantation model that the present invention set up can be and store and amplification artificial blood ancestral cells provides potential live body warehouse, can become a kind of source easily of artificial blood ancestral cells.
Result of the present invention further supports following viewpoint: the early stage fetus of gestation is because of being in the immune response cubhood, thereby is the desirable acceptor of HSC transplanting.Its distinctive feature is that normal HSC can transplant successfully and long-term surviving, and need not carry out the destruction and the immunosuppressant therapy of hemopoietic tissue.Human/goat HSC transplantation model of the present invention provides the theory and technology foundation for treating disease by HSC transplanting approach in utero.
Result of study clearlys show, from the intraperitoneal injection of tire goat behind the human hematopoietic stem cell, human hematopoietic stem cell does not exist only in the blood of tire goat, and shows that people's histocyte is present in the tire goat respective organization, as there being people's liver cell in the tire Hepar Naemorhedi.
Table 1: used PCR primer table look-up in the experiment
| The primer title | Primer sequence |
| HC1? | 5’-CATGGAGCTCAGTGGAACTTAG-3’ |
| HC2? | 5’-CTGGTGACCAAGTCCACAGT-3’ |
| HC3? | 5’-CACCTCAGAGGCTGTTCTTG-3’ |
| HC4? | 5’-ATGCTGGAGGTGACATCTCT-3’ |
| GA1? | 5’-GGACACATATGCAGCCACTC-3’ |
| GA2? | 5’-TATGTCCACGCAGTCACCTC-3’ |
| GA3? | 5’-CCACAGCCACTGTCTGAATC-3’ |
| GA4? | 5’-CTGTTCCACCTGTGCTAACC-3’ |
| HS1? | 5’-AGCGAAGTGCAACTGGACAA-3’ |
| HS2? | 5’-GCAGGCTCACTTCTGGATGT-3’ |
| HS3? | 5’-AAGAATGGAGCACCAGCTAGG-3’ |
| HS4? | 5’-GGTAGTCGGCGTTCTCAACA-3’ |
Table 2, people's bleeding of the umbilicus HSC intrauterine transplantation are in the result of tire goat
Table 3, facs analysis artificial blood ancestral cells are transplanted the result behind the goat
Description of drawings
Fig. 1 transplants in the goat blood for facs analysis and contains people CD34 and the antigenic hemopoietic stem cell of GPA.N wherein: Nonimplantation goat; T: transplant goat
Fig. 2 is the pcr amplification result of people CD34 gene in the sheep peripheral blood DNA
M:100bp DNA ladder wherein; 1-9: the pcr amplification result of experiment sheep peripheral blood DNA; 10: blank; 11: the pcr amplification result (negative control) of contrast sheep peripheral blood DNA; 12: the pcr amplification result (positive control) of human peripheral DNA.
Fig. 3 detects people GPADNA fragment in the transplanting Sanguis Naemorhedi, 2% agarose gel electrophoresis, wherein M:100bp gradient molecular mass mark for using PCR; The 1-11:11 head is transplanted goat, has the 479bp amplified fragments that is specific to people GPA gene; 12: negative control; 13: blank; 14: positive control (people DNA).
Fig. 4 is the pcr amplification result of people's sry gene in the sheep peripheral blood DNA
M:100bp DNA ladder wherein; 1-11: the pcr amplification result of experiment sheep peripheral blood DNA; 12: the pcr amplification result (positive control) of man's peripheral blood DNA; 13: the pcr amplification result (negative control) of contrast sheep periphery DNA.
Fig. 5 uses PCR and detects people CD34DNA fragment in the transplanting Sanguis Naemorhedi, 2% agarose gel electrophoresis, wherein M:100bp gradient molecular mass mark
The 1-6:6 head is transplanted goat, has the 437bp amplified fragments that is specific to people CD34 gene
7: negative control (Nonimplantation goat)
8: blank
9: positive control (people DNA)
Fig. 6 is a GPA Gene RT-PCR amplification among the total RNA of sheep peripheral blood
M:100bp DNA ladder wherein; 1-8: the RT-PCR amplification of the total RNA of experiment sheep peripheral blood; 9: the RT-PCR amplification (negative control) of the total RNA of contrast sheep peripheral blood; 10: the RT-PCR amplification (positive control) of the total RNA of human peripheral; 11: blank.
Fig. 7 detects the people CD34 gene of transplanting goat for PCR-Southern, and the CD34DNA fragment of amplification is transferred to S﹠amp; On the S nylon membrane, then with (α-
32P) the parallel radioautograph of the people CD34 gene probe of dCTP mark hybridization, 1,2,4,5,7,8,9: transplant goat, show positive hybrid belt
3,6: ewe, amixia band 10: negative control (Nonimplantation goat)
11: blank 12: positive control (people DNA)
Fig. 8 transplants the expression of goat (#3) birth back different times human hematopoietic cell surface marker for using the FACS performance analysis.
Relatively be born back 3 months to 10 months, CD34, CD20, CD14, CD7, CD38, CD15, the frequency of CD45 and GPA is almost similar.
Claims (5)
1. method of setting up human/goat hemopoietic stem cell heteroplastic transplantation model is characterized in that may further comprise the steps:
A) collect Freshman bleeding of the umbilicus, separation of human hemopoietic stem cell;
B) to be injected into gestational age be in 55-65 days the abdominal cavity of tire goat to the human hematopoietic stem cell that step a) is obtained;
C) treat that the tire goat divides the puerperium, extract its venous blood and detect as chimeric human hematopoietic stem cell arranged, its content is expanded to 1000-10000 doubly, and stable its phenotype that keeps is in incomplete differentiation state more than 10 months, then shows the modelling success.
2. the method for claim 1 is characterized in that: finished in 8-16 hour to the whole process that transplanting people HSC goes into the tire goat abdominal cavity from collector's bleeding of the umbilicus.
3. the method for claim 1 is characterized in that: it is to divide 3 months puerperiums, 6 months, 10 months to extract respectively the tire goat that described venous blood samples detects.
4. the method for claim 1, it is characterized in that: described detection to tire goat venous blood comprises facs analysis and DNA analysis.
5. method as claimed in claim 4, it is characterized in that described DNA analysis comprises extracting DNA, design PCR primer, detects the nido and the RT-PCR detection human/goat hemopoietic stem cell follow board of primer specificity and PCR detection human/goat hemopoietic stem cell follow board.
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| CN011322314A CN1353994B (en) | 2001-11-19 | 2001-11-19 | Method for creating human/goat hemopoietic stem cell heteroplastic transplantation model |
| US10/178,036 US20030096410A1 (en) | 2001-11-19 | 2002-06-24 | Methodology for constructing human / goat chimeras for the production of human cells |
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| CN011322314A CN1353994B (en) | 2001-11-19 | 2001-11-19 | Method for creating human/goat hemopoietic stem cell heteroplastic transplantation model |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064081C (en) * | 1994-11-04 | 2001-04-04 | 上海贝特生物技术有限公司 | Cloning method of hemotopoietic stem cell, ancestor cell and megacaryocyte |
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2001
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064081C (en) * | 1994-11-04 | 2001-04-04 | 上海贝特生物技术有限公司 | Cloning method of hemotopoietic stem cell, ancestor cell and megacaryocyte |
Non-Patent Citations (3)
| Title |
|---|
| 李续建 楼方定.造血干细胞移植术发展史.《中华医史杂志》.2001, * |
| 杜亚利.脐血造血干细胞研究进展.《医师进修杂志》.1998,第21卷(第5期), * |
| 裴雪涛 等.脐带血造血干细胞的"质""量"研究.《中华放射医学与防护杂志》.1997, |
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