CN1502691A - Establishment identification and use for internal differentiation skin model of multipotential mesenchyme stem cell - Google Patents
Establishment identification and use for internal differentiation skin model of multipotential mesenchyme stem cell Download PDFInfo
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- CN1502691A CN1502691A CNA021454868A CN02145486A CN1502691A CN 1502691 A CN1502691 A CN 1502691A CN A021454868 A CNA021454868 A CN A021454868A CN 02145486 A CN02145486 A CN 02145486A CN 1502691 A CN1502691 A CN 1502691A
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Abstract
The present invention relates to a method for establishing model of internal differentiation of adult myeloid source plutipotential mesenchymal stem cell into dermal tissue. Its technical scheme includes the following steps: separating and obtaining the pluripotential messenchrymal stem cell from bone marrow of adult BALB/C mouse and making external culture and amplification, common-implating proper quantity of donor BALB/C mouse myeloid source pluripotential mesenchymal stem cell and a certain quantity of C57BH6 mouse bone marrow cell into the adult C57BL/6 mouse irradiated by lethal dose, after the receptor C57BL/6 mouse appears neonatal coat, using the dermal tissue of said region to make germ line genetic marker determination of protein and gene level so as to obtain said invented model.
Description
Technical field
The present invention relates to the multiple methods of organizing undifferentiated cell to separate and identify such as marrow, be specifically related to a kind of method that is divided into skin progenitor cell and skin histology model in the adult bone marrow multipotency mescenchymal stem cell body of setting up.
Background technology
In recent years, plastic research has become the focus of medical science and organizational engineering research to adult stem cell.Many research reports, the stem cell that comes from a kind of tissue can be divided into multiple histocyte under optimum conditions; Be implanted into intravital hemopoietic stem cell, can be divided into endotheliocyte, epidermic cell, muscle cell even liver cell and neurocyte, shown the application prospect that adult stem cell is wide.Especially adult bone marrow mescenchymal stem cell is more paid close attention to its stem cell than other sources because of its more easily separated acquisition relatively, vitro culture can increase in a large number, the inside and outside all has stronger multidirectional differentiation capability.
But the report of research previously focuses mostly in stem cell to bone, cartilage, fat, muscle, liver cell and neurocyte direction differentiation aspect, and is that the report of skin cells is less for differentiation of stem cells, and also has shortcoming aspect its evaluation.
For example, the method of human immunohistochemical methodss such as Korbling and FISH has detected Y chromosome and keratin positive cell (Korbling.M in the human female of having accepted male sex's donor hematopoietic stem cell transplantation, Katz RL, Khanna A, et al.Hepatocytes and epithelial cells of donor origin in recipients of peripheral-bloodstem cells.N Engl J Med.2002 Mar 7; 346 (10): 738-46.); Human FISH methods such as Krause, have in transplanting on the skin biopsy of female mice of male mice hemopoietic stem cell, confirmation has the (Krause that exists of Y chromosome, D.S.et al.Multi-organ, multi-lineage engraftment by a single bone marrow-derived stemcell.Cell.2001 May 4; 105 (3): 369-77).But people such as YING (Ying QL, Nichols J, Evans EP, Changing potency by spontaneous fusion.Nature.2002 Apr 4; 416 (6880): 545-8) and people (Terada N such as TERADA, Hamazaki T, Oka M et al.Bone marrow cells adopt the phenotype of othercells by spontaneous cell fusion.Nature.2002 Apr4; 416 (6880): 542-5) then think, because have some scavenger cells in the skin histology, it may engulf the skin cells of donor, perhaps the spontaneity because of two kinds of cells merges, cause its phenotype that obtains the donor's cells, thereby false positive results might appear in above-mentioned experiment.
Summary of the invention
The present invention is intended to fill up the blank of prior art, provides a kind of foundation in the adult bone marrow multipotency mescenchymal stem cell body to be divided into the animal model of skin progenitor cell and skin histology and the method for evaluation thereof.
The technical scheme and the concrete steps of the present invention's method are as follows:
(1) technical scheme of experimental model structure:
In the marrow of the BALB/C mice of growing up, separate obtaining and cultured and amplified in vitro multipotency mescenchymal stem cell, an amount of donor BALB/C mice bone marrow multipotency mescenchymal stem cell and a certain amount of C57BL/6 bone marrow cells in mice are implanted in the adult C57BL/6 mouse body of lethal quantity irradiation jointly.After treating that newborn fur appears in acceptor C57BL/6 mouse, it is that genetic marker is measured that the skin histology of getting this position carries out that immunohistochemical methods detects and plant, be divided into the model of skin histology in the multipotency mescenchymal stem cell body of mouse bone marrow cells source.
(2) concrete steps of experimental model structure:
1) preparation of donor bone marrow source multipotency mescenchymal stem cell:
The BALB/C mice (purchasing the Experimental Animal Center in the Department Of Medicine, Peking University) in age in 6-8 week is taken off ridge puts to death, with 75% alcohol-pickled sterilization, get the shin bone and the femur of its hind leg with aseptic operation scissors and tweezers, syringe needle with syringe inserts medullary space, HANKS liquid (D-Hanks liquid formula (g/L): 0.4g KCl, 0.06g KH with precooling
2P0
4, 8.00g NaCl, 0.35g NaHCO
3, 0.12g Na
2HP0
4.12H
20,0.01g is phenol red, adds ultrapure water to 1000ml; ) go out medullary cell, after blowing and beating cell dispersion repeatedly, after No. 4 syringe needles, make it into single cell suspension, once (centrifugal condition is 1000rpm to each washed cell for HANKS liquid and incomplete RPMI1640 nutrient solution (purchasing the company in LIFETECHNOLOGIES), 10min), obtain mononuclearcell, sub-elect the cell of CD45-Gly (-) again through magnetic bead (purchasing company) in MACS through Ficoll (purchasing) density gradient centrifugation in Inst. of Hematology, Chinese Academy of Medical Sciences.
(this substratum contains: DMEM, MCDB-201,2%FBS to add special substratum, 10ng/ml EGF, 10ng/mlPDGF-bb, 10ng/ml LIF, insulin transferring selenium, linoleic acid, bovine sermalbumin (bsa), 10-9M dexamethasone and 10-4M ascorbic acid 2-phosphate, all purchase company in sigma), by 8 * 10
5/ cm
2Be seeded in the culturing bottle, place 37 ℃ of 5%CO
2Cultivate in the incubator.After 24-72 hour, the reject suspension cell adds fresh culture and continues to cultivate, and changes liquid once in 2-3 days, after merging to cell 80-90%, and with 0.25% tryptic digestion that contains 0.02%EDTA 3 minutes, the cultivation of going down to posterity.
2) acceptor is with the preparation of strain bone marrow cells in mice:
The C57BL/6 mouse (being provided by animal portion of Inst. of Hematology, Chinese Academy of Medical Sciences) in age in 6-8 week is taken off ridge puts to death, aseptic shin bone and the femur of getting its hind leg, syringe needle with syringe inserts medullary space, goes out medullary cell with the HANKS liquid of precooling, after blowing and beating cell dispersion repeatedly, after No. 4 syringe needles, make it into single cell suspension, with EDTA-NH4Cl (prescription (g/L): 8.29g NH4Cl, 1.0012g KHCO3,29.225mg EDTA adds ultrapure water to 1000ml; ) remove red corpuscle, (centrifugal condition is 1000rpm, 10min) with HANKS washing secondary again.Calculate living cell rate according to a conventional method with the blue dyeing of 0.2% placenta, and it is standby to desired concn to adjust cell with HANKS.
3) preparation of acceptor mouse:
The C57BL/6 mouse (being provided by animal portion of Inst. of Hematology, Chinese Academy of Medical Sciences) in age in 10-12 week is carried out the lethal quantity irradiation, and (Gammacell 40, CES IUM 137, purchase company in Atomic Energy of Canada Limited Radiochemical), irradiation dose is 850Rad.
4) bone marrow transplantation:
With 1.5 * 10
7The medullary cell and 5 * 10 of C57BL/6 mouse
5BALB/C mice bone marrow mescenchymal stem cell by the common input of mouse tail vein in the C57BL/6 mouse body of lethal dose irradiation.
(3) identify the experimental model that makes up
1. detect newborn skin cells with the immunohistochemical methods test
1) negative control C57BL/6 mouse (MHC-I class antigen is H-2Kb), positive control BALB/C mice (MHC-I class antigen is H-2Kd) and the skin of experimental mice are made frozen section, anti-with biotinylated anti-H-2Kd monoclonal antibody (purchasing company) as one in BD, be that Color Appearance System carries out the test of direct immunization group with DAB (purchasing company) in DAKO A/S.As a result, do not see brown xanchromatic H-2Kd positive cell in the negative control group section, and visible significantly black hair fragment in the hair follicle; As seen stronger pale brown look painted H-2Kd positive cell in the positive controls section, not seeing in the hair follicle has black hair fragment; In experimental group section, not seeing in the hair follicle has black hair fragment, and around the hair follicle of epidermis and skin corium visible brown xanchromatic H-2Kd positive cell, point out these cells really to originate for BALB/C mice.
2) use at two kinds of monoclonal antibodies of P63 (a kind of transcription factor of promoting epidermization propagation can be used as the comparatively special sign of cutin stem cell) and H-2Kd anti-ly, the frozen section of above-mentioned mouse skin tissue has been carried out two mark immunohistochemical methodss tested as one.At first carry out indirect immunohistochemical methods test, as Color Appearance System, the positive result of mazarine occurs with BCIP/NBT with anti-P63 monoclonal antibody (purchasing company) in SANTA CRUZ BIOTECHNOLOGY; After adding two mark blocking-up liquid, carry out the test of direct immunization group with anti-H-2Kd monoclonal antibody, with AEC (purchasing company) in DAKO A/S as Color Appearance System, the positive result of bright red.As seen the result contains the visible obviously two positive cells in skin progenitor cell place at the hair follicle lower position in the positive controls section, is the single positive cell of red H-2Kd around it, prompts for ripe skin cells; In the negative control group section, the visible mazarine P63 positive cell at the hair follicle place, and redfree H-2Kd positive cell; Show then in the experimental group section that the hair follicle place is the positive skin progenitor cell of mazarine P63, is the red H-2Kd positive cell that dyes on every side.
3) under aseptic condition, obtain skin and the negative control C57BL/6 mouse back skin that experimental mice contains adularescent hair place, and by collagenase, neutral protease, trypsin all available from SIGMA company) step such as digestion, isolating skin cells puts in the culturing bottle and cultivates, after 7-10 days, after cell reaches 90% fusion, harvested cell, the centrifugal cell smear of making, and then carry out the test of above-mentioned two mark immunohistochemical methods, and redye with the Maxwell Hematorylin.Positive and the red H-2Kd positive of both visible mazarine P63 in the experimental group section, promptly two positive cells, pointing out this type of cell is the skin progenitor cell in donor source, the red color visible H-2Kd positive again, the P63 feminine gender, promptly single positive cell, pointing out this type of cell is the ripe skin cells in donor source; From the also visible two class cells of negative control group section, a class is H-2Kd feminine gender and P63 positive cell, and pointing out this type of cell is receptor's skin progenitor cell, and another kind of is H-2Kd and P63 jack to jack adapter sexual cell, prompts for receptor's skin mature cell.Thereby the possibility of having got rid of the systematic error of bringing because of the test of tissue slice immunohistochemical methods.From protein level explanation tested tissue is the positive mouse of H-2Kd source really.
2. identify skin cells with the RT-PCR means from transcriptional level.
Under aseptic condition, obtain the skin that experimental mice contains adularescent hair place, and pass through collagenase, neutral protease, steps such as tryptic digestion, isolating skin cells puts in the culturing bottle and cultivates, after 7-10 days, after cell reaches 90% fusion, harvested cell, with TRIZOL (purchasing company) lysing cell in GIBCO, obtain total RNA, utilize RT-PCR test kit (purchasing company) and specificity H-2Kd cDNA primer (purchasing company) in TAKARA in TAKARA, having amplified the H-2Kd cDNA fragment of length-specific, is the positive mouse of H-2Kd source from gene level explanation tested tissue really.
3. with simple 1.5 * 10
7The medullary cell of C57BL/6 mouse inject in the C57BL/6 mouse body of same lethal dose irradiation through the tail vein, then do not see the appearance of white hair, illustrate that the transformation of this kind hair and irradiation are irrelevant.
The present invention has filled up the blank of prior art, has set up the animal model that is divided into skin progenitor cell and skin histology in a kind of adult bone marrow mescenchymal stem cell body, also can do multifaceted detection and evaluation to this model at albumen and gene level simultaneously.
The structure of this model provides adult bone marrow mescenchymal stem cell can be divided into skin progenitor cell and skin histology first, for the plastic theory of adult stem cell provides new direct evidence; The clinical treatment of the two skin injury that also causes for various factors (as burn, RADI etc.) provides a kind of new approach, for solid basis has been established in the widespread use of adult bone marrow mesenchymal stem cell transplantation in skin regeneration medical science.
Description of drawings:
Fig. 1 acceptor C57BL/6 mouse is transplanted the 50th day the photo in back
The single mark of Fig. 2 skin histology section immunohistochemical methods result
A experimental group (40 *), b experimental group (100 *), c positive control (40 *), d negative control (40 *)
The two mark of Fig. 3 skin histology section immunohistochemical methods result
A positive controls (20 *), b experimental group (20 *), c negative control (20 *)
Fig. 4 skin cells two mark immunohistochemical methods result (100 *) and RT-PCR result
A, b are two mark immunohistochemical methods results of the experimental mice skin cells in the different visuals field.Empty arrow indication C57BL/6 source skin cells; The skin progenitor cell that tail filled arrows indication BALB/C source is arranged; The ripe skin cells in anury filled arrows indication BALB/C source;
Two mark immunohistochemical methods results of the negative control group mice skin cells of c.The skin progenitor cell that tail filled arrows indication C57BL/6 source is arranged; The ripe skin cells in anury filled arrows indication C57BL/6 source.
Embodiment
The invention will be further described below in conjunction with specific embodiment.
Embodiment 1 makes up experimental model:
1) preparation of donor bone marrow source multipotency mescenchymal stem cell:
The BALB/C mice (purchasing the Experimental Animal Center in the Department Of Medicine, Peking University) in age in 6-8 week is taken off ridge puts to death, with 75% alcohol-pickled sterilization, get the shin bone and the femur of its hind leg with aseptic operation scissors and tweezers, syringe needle with syringe inserts medullary space, HANKS liquid (D-Hanks liquid formula (g/L): 0.4g KCl, 0.06g KH with precooling
2P0
4, 8.00g NaCl, 0.35g NaHCO
3, 0.12g Na
2HPO
4.12H
2O, 0.01g is phenol red, adds ultrapure water to 1000ml; ) go out medullary cell, after blowing and beating cell dispersion repeatedly, after No. 4 syringe needles, make it into single cell suspension, once (centrifugal condition is 1000rpm to each washed cell for HANKS liquid and incomplete RPMI1640 nutrient solution (purchasing the company in LIFETECHNOLOGIES), 10min), obtain mononuclearcell, sub-elect the cell of CD45-Gly (-) again through magnetic bead (purchasing company) in MACS through Ficoll (purchasing) density gradient centrifugation in Inst. of Hematology, Chinese Academy of Medical Sciences.
(this substratum contains: DMEM to add special substratum, MCDB-201,2%FBS, 10ng/ml EGF, 10ng/mlPDGF-bb, 10ng/ml LIF, insulin transferring selenium, linoleic acid, bovine sermalbumin (bsa) .10-9M dexamethasone, with 10-4M ascorbic acid 2-phosphate, all purchase company in sigma), by 8 * 10
5/ cm
2Be seeded in the culturing bottle, place 37 ℃ of 5% CO
2Cultivate in the incubator.After 24-72 hour, the reject suspension cell adds fresh culture and continues to cultivate, and changes liquid once in 2-3 days, after merging to cell 80-90%, and with 0.25% tryptic digestion that contains 0.02%EDTA 3 minutes, the cultivation of going down to posterity.
2) acceptor is with the preparation of strain bone marrow cells in mice:
The C57BL/6 mouse (being provided by animal portion of Inst. of Hematology, Chinese Academy of Medical Sciences) in age in 6-8 week is taken off ridge puts to death, aseptic shin bone and the femur of getting its hind leg, syringe needle with syringe inserts medullary space, goes out medullary cell with the HANKS liquid of precooling, after blowing and beating cell dispersion repeatedly, after No. 4 syringe needles, make it into single cell suspension, with EDTA-NH4Cl (prescription (g/L): 8.29g NH4Cl, 1.0012g KHC03,29.225mg EDTA adds ultrapure water to 1000ml; ) remove red corpuscle, (centrifugal condition is 1000rpm, 10min) with HANKS washing secondary again.Calculate living cell rate according to a conventional method with the blue dyeing of 0.2% placenta, and it is standby to desired concn to adjust cell with HANKS.
3) preparation of acceptor mouse:
The C57BL/6 mouse (being provided by animal portion of Inst. of Hematology, Chinese Academy of Medical Sciences) in age in 10-12 week is carried out the lethal quantity irradiation, and (Gammacell 40, CES IUM 137, purchase company in Atomic Energy of Canada Limited Radiochemical), irradiation dose is 850Rad.
4) bone marrow transplantation:
With 1.5 * 10
7The medullary cell and 5 * 10 of C57BL/6 mouse
5BALB/C mice bone marrow mescenchymal stem cell by the common input of mouse tail vein in the C57BL/6 mouse body of lethal dose irradiation.
Embodiment 2 identification experiment models
1) detects newborn skin cells with the immunohistochemical methods test
A) negative control C57BL/6 mouse (MHC-I class antigen is H-2Kb), positive control BALB/C mice (MHC-I class antigen is H-2Kd) and the skin of experimental mice are made frozen section, anti-with biotinylated anti-H-2Kd monoclonal antibody (purchasing company) as one in BD, be that Color Appearance System carries out the test of direct immunization group with DAB (purchasing company) in DAKO A/S.As a result, do not see brown xanchromatic H-2Kd positive cell in the negative control group section, and visible significantly black hair fragment in the hair follicle; As seen stronger pale brown look painted H-2Kd positive cell in the positive controls section, not seeing in the hair follicle has black hair fragment; In experimental group section, not seeing in the hair follicle has black hair fragment, and around the hair follicle of epidermis and skin corium visible brown xanchromatic H-2Kd positive cell, point out these cells really to originate for BALB/C mice.
B) use at two kinds of monoclonal antibodies of P63 (a kind of transcription factor of promoting epidermization propagation can be used as the comparatively special sign of cutin stem cell) and H-2Kd anti-ly, the frozen section of above-mentioned mouse skin tissue has been carried out two mark immunohistochemical methodss tested as one.At first carry out indirect immunohistochemical methods test, as Color Appearance System, the positive result of mazarine occurs with BCIP/NBT with anti-P63 monoclonal antibody (purchasing company) in SANTA CRUZ BIOTECHNOLOGY; After adding two mark blocking-up liquid, carry out the test of direct immunization group with anti-H-2Kd monoclonal antibody, with AEC (purchasing company) in DAKO A/S as Color Appearance System, the positive result of bright red.As seen the result contains the visible obviously two positive cells in skin progenitor cell place at the hair follicle lower position in the positive controls section, is the single positive cell of red H-2Kd around it, prompts for ripe skin cells; In the negative control group section, the visible mazarine P63 positive cell at the hair follicle place, and redfree H-2Kd positive cell; Show then in the experimental group section that the hair follicle place is the positive skin progenitor cell of mazarine P63, is the red H-2Kd positive cell that dyes on every side.
C) under aseptic condition, obtain skin and the negative control C57BL/6 mouse back skin that experimental mice contains adularescent hair place, and by collagenase, neutral protease, trypsin all available from SIGMA company) step such as digestion, isolating skin cells puts in the culturing bottle and cultivates, after 7-10 days, after cell reaches 90% fusion, harvested cell, the centrifugal cell smear of making, and then carry out the test of above-mentioned two mark immunohistochemical methods, and redye with the Maxwell Hematorylin.Positive and the red H-2Kd positive of both visible mazarine P63 in the experimental group section, promptly two positive cells, pointing out this type of cell is the skin progenitor cell in donor source, the red color visible H-2Kd positive again, the P63 feminine gender, promptly single positive cell, pointing out this type of cell is the ripe skin cells in donor source; From the also visible two class cells of negative control group section, a class is H-2Kd feminine gender and P63 positive cell, and pointing out this type of cell is receptor's skin progenitor cell, and another kind of is H-2Kd and P63 jack to jack adapter sexual cell, prompts for receptor's skin mature cell.Thereby the possibility of having got rid of the systematic error of bringing because of the test of tissue slice immunohistochemical methods.From protein level explanation tested tissue is the positive mouse of H-2Kd source really.
2) identify skin cells with the RT-PCR means from transcriptional level.
Under aseptic condition, obtain the skin that experimental mice contains adularescent hair place, and pass through collagenase, neutral protease, steps such as tryptic digestion, isolating skin cells puts in the culturing bottle and cultivates, after 7-10 days, after cell reaches 90% fusion, harvested cell, with TRIZOL (purchasing company) lysing cell in GIBCO, obtain total RNA, utilize RT-PCR test kit (purchasing company) and specificity H-2Kd cDNA primer (purchasing company) in TAKARA in TAKARA, having amplified the H-2Kd cDNA fragment of length-specific, is the positive mouse of H-2Kd source from gene level explanation tested tissue really.
3) with simple 1.5 * 10
7The medullary cell of C57BL/6 mouse inject in the C57BL/6 mouse body of same lethal dose irradiation through the tail vein, then do not see the appearance of white hair, illustrate that the transformation of this kind hair and irradiation are irrelevant.
Embodiment 4
Finish two groups in totally 12 C57BL/6 mouse (6 the every group) bodies behind the structure of skin differentiation model, newborn fur to all mouse has carried out immunohistochemistry detection and DNA detection, found that: after 40 days, in 12 acceptor mouse, have to have occurred white hair in the back black wools of 11 mouse, and expand to 3-4cm gradually
2, at its belly and neck white hair has also appearred simultaneously.The repetition rate that the model that makes up with the present invention's method is described can reach more than 90%.
Claims (6)
1. set up the method that is divided into the model of skin histology in the adult bone marrow multipotency mescenchymal stem cell body, it is characterized in that, in the marrow of the BALB/C mice of growing up, separate and obtain and cultured and amplified in vitro multipotency mescenchymal stem cell, an amount of donor BALB/C mice bone marrow multipotency mescenchymal stem cell and a certain amount of C57BL/6 bone marrow cells in mice are together implanted in the adult C57BL/6 mouse body of lethal quantity irradiation, after treating that newborn fur appears in acceptor C57BL/6 mouse, the kind that the skin histology of getting this position carries out albumen and gene level is that genetic marker is measured, be divided into the model of skin progenitor cell and skin histology in the adult bone marrow multipotency mescenchymal stem cell body.
2. the method for claim 1 is characterized in that, described acceptor C57BL/6 mouse is 10-12 age in week.
3. the method for claim 1 is characterized in that, this method comprises the steps:
A) prepare donor bone marrow source multipotency mescenchymal stem cell, acceptor respectively with strain bone marrow cells in mice and acceptor mouse;
B) donor bone marrow source multipotency mescenchymal stem cell and acceptor are together implanted in the acceptor mouse body with the strain bone marrow cells in mice, the newborn fur of acceptor mouse is carried out albumen and the gene level kind is that genetic marker is measured.
4. method as claimed in claim 3, it is characterized in that, described protein level kind is that genetic marker is determined as to detect with single mark immunohistochemical methods and two mark immunohistochemical methods methods and is subjected to the newborn skin of mouse, particularly identifies with two mark immunohistochemical methods methods to be subjected to the newborn skin histology stem cell of mouse.
5. method as claimed in claim 3 is characterized in that, described gene level kind is that genetic marker is determined as to detect with the RT-PCR method and is subjected to the newborn skin of mouse.
6. with the application in experimental study and/or the clinical and experimental study before the skin regeneration clinical medicine of the animal model of the method for claim 1 foundation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531615A (en) * | 2015-01-05 | 2015-04-22 | 山东中医药大学 | Method for collecting mesenchymal stem cells through rat stress fracture model |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104531615A (en) * | 2015-01-05 | 2015-04-22 | 山东中医药大学 | Method for collecting mesenchymal stem cells through rat stress fracture model |
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