CN1397640A - Stem cell, and its preparing process and usage - Google Patents
Stem cell, and its preparing process and usage Download PDFInfo
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Abstract
A stem cell coming from human embryo, cord blood, or spina marrow, its preparing process and its application in treating or repairing the disfunctional organ or pathologic tissue are disclosed. The said stem cell is prepared through positive screening of immunomagnetic beads, MNC cell aquirement, CO34 antibody marking, sorting by flow cell sorter, separating by cell inoculation, testing stem cell reproduction by MTT method, and testing the transform of stem cell to liver cell by immunohisto chemical method.
Description
The present invention relates to a kind of stem cell, Its Preparation Method And Use, particularly from tissues such as people or other mammiferous embryos, Cord blood, spinal cord, cartilage the stem cell of preparation, its preparation method and in treatment, repair and lose the pathological tissues of function or the application in the organ.
Stem cell is the cell initiating cell that a class has self and differentiation potential.It comprises embryonic stem cell and adult stem cell.Embryonic stem cell is all-round, has all abilities of tissue and organ that is divided into almost, and for example in embryo's genesis and development, single zygote can be divided growth and is cellulous tissue or organ.Current research finds that adult stem cell can laterally be divided into the cell and the tissue of other types, and for example, in adult animals, normal physiological metabolism or pathology damage also can cause the reparative regeneration of tissue or organ.Because embryo's the regeneration that is differentiated to form with adult tissue is the further result of differentiation of stem cell, for the widespread use of stem cell provides the foundation.
Stem cell has the self ability, can produce well differentiated functioning cell.The tissue specificity stem cell has the potential that is divided into other cell or tissues equally, and this has started space widely for Application of stem cells.Therefore by directional induction, stem cell can be divided into the different tissues cell, comprises cornea, nerve and other organ cells, is called as " source cell ".Therefore cell, the tissue that cultivates by stem cells technology can be transplanted in the pathological tissues or organ that loses function, makes it recover normal function.
Present many research work all are to be the research object unfolded with mouse teratoma stem cell (EC cell), as: the neurogliocyte that dolantin medical group has been gone out by the ES cell cultures having transplanted in test mouse body of success last year.After this, the researchist of Missouri makes the cat of paralysis recover part limb activity ability by mouse blastocyte implantation technology.Along with the research of ES cell is deep day by day, life scientist has marched toward a new stage to the understanding of human ES cell.At the year ends 1998, two research groups of the U.S. successfully turn out human ES cell, and having kept the ES cytodifferentiation is various somatic totipotencies.So just make scientist utilize human ES cell therapy various diseases to become possibility.
But because mammal embryo is implanted postuterine inaccessiblility, the research of ES cell fluctuates always.Nineteen ninety-five Thomson separates and has set up ES clone from the blastocyst of rhesus monkey, and this is first embryonic stem cell of building the primate that is.This ES cell with normal X Y caryogram keep undifferentiated state to reach more than 1 year in continuing to go down to posterity, and it expresses the surface marker of some people's teratocarcinoma cell.In the cultivation of rhesus monkey ES cell, found the vital role of feeder cell and LIF (leukemia inhibitory factor), this lays a good foundation for the foundation of human ES clone, and provides reference relatively reliably for the external model of inquiring into people's fetal development, tissue differentiation.
In people ES cell research process, teratoma is main research material always.But the differentiation scope of embryo's tumor cell line of this people is very limited, and it is big to make a variation between the clone, and more crucial is the characteristics that it can not fully accurately represent normal differentiation; The scarcity in research object source and the constraint of ethics, the in vitro study that makes human embryo stem cell are blank always.This situation was broken in 1998, Thomson creates the beginning again, he is by Gardner, the G1.2 and the G2.2 substratum of DK invention, solved the dependency problem of body early embryo, thereby the outer zygote (IVF, in vitro fertilization) of fresh or freezing human body has been cultured to blastocyst stage by the 4-8 cell stage the uterine tube environment, going down to posterity gradually behind the separate inner cell mass (ICM, inner cell mass), to build be ES clone; The same period, John.D Gearheart has set up multipotential stem cell system---embryonic genital cell line (the EG cell similar to the ES cell function from people's archeocyte (primordial germ cell), embryonic germ cell), the result of these two experiment groups goes up once publishing at " Science " and " Proc.Natl.Acad.Sci.USA ", has caused investigators' very big interest immediately.This has not only started the embryonic stem cell research boom of a new round, also, make embryonic stem cell be applied to clinical studies and produced breakthrough progress because the hESC builds and is and inducing embryo stem cell is divided into neural stem cell and myocardium stem cell and succeeds.
The ES cell can be used as estimates new drug and the toxicity of chemical products and the detection system of usefulness.ES clone has the broad spectrum of tissue, cell, and it develops into the biosystem behind the idiosome, but complex interactions between the simulation cells in vivo, this is at medicine and agrochemicals are industrial has been widely used, can reduce the animal detection, reduce cost, have important commercial to be worth.The most noticeable part of ES cell is that it has the important clinical meaning, and promptly the ES cell might become the leading role of cell replacement therapy from now on.The ES cell is being followed the universal law of cytodifferentiation as the primordial stem cell at the beginning of the ontogeny, and this is that potentiality of development limits to gradually, the process of cell function Focus, and intension constantly increases, and extension is dwindled gradually, and last destiny is certain histocyte.Theoretically, the ES cell can ad infinitum provide can be transplants all specific cell types, hemopoietic system after displacement diseased tissue and the chemicotherapy damage, this solution for diseases such as inherited disease, tumour and agings provides new thinking, and many medical ramose researchs all can be benefited from this invention.
Under given conditions, adult stem cell or produce new stem cell perhaps by the certain procedure differentiation, forms new functioning cell, thus the running balance that makes tissue and organ maintenance grow and fail.Think that in the past adult stem cell mainly comprises epithelial stem cell and hemopoietic stem cell.Studies show that recently, think that in the past unrenewable nervous tissue still comprises neural stem cell, the adult stem cell ubiquity is described, problem is how to seek and separate various tissue specificity stem cells.Adult stem cell often is arranged in specific microenvironment.Mesenchymal cell in the microenvironment can produce a series of somatomedins or part, interacts renewal and the differentiation of control stem cell with stem cell.
Hemopoietic stem cell is unique source of various hemocytes in the body, and it mainly is present in marrow, peripheral blood, the Cord blood.Scientists in 2000 have found to have the stem cell of hematopoietic potential again in muscle tissue.The transplanting of hemopoietic stem cell is treatment disease in the blood system, ancestor genetic diseases and a most effectual way multiple and the transitivity malignancy disease.
In clinical treatment, hemopoietic stem cell is used early, at 20th century the fifties, just begins to use bone marrow transplantation (BMT) method clinically and treats disease in the blood system.To late nineteen eighties, autologous peripheral blood stemcell transplant (PBSCT) technology is promoted gradually and is come, and overwhelming majority be autologous peripheral blood stem cell transplantation (APBSCT), improve treat efficient and shortening course of treatment aspect be better than conventional treatment, and effect is satisfactory.
Compare with both, the strong point that navel blood stem cell is transplanted is not have the restriction in source, and it is less demanding that HLA is joined type, the not pollution of susceptible viral or tumour.At 2000 beginning of the years, the first navel blood stem cell in the Northeast is transplanted successfully, is again that Made in China hemocytoblast implantation technique injects new vitality.Constantly perfect along with the navel blood stem cell implantation technique, it may replace the status of present APBSCT, for the patient of more hemopathy in the whole world and malignant tumour brings glad tidings.
Research is started late about neural stem cell, organizes difficulty to draw materials owing to separate the required fetal brain of neural stem cell, and the dispute of embryonic cell research is not calmed down as yet in addition, and the research of neural stem cell still is in the junior stage.Theoretically, any central nervous system disease all can be summed up as the disorder of neural stem cell function.Brain and spinal cord are owing to the existence of hemato encephalic barrier makes it can not produce immunological rejection in stem cell transplantation after central nervous system, as: contain the neural stem cell of Dopamine HCL founder cell for Parkinsonism patient's intracerebral transplantation, can cure part patient symptom.In addition, the function of neural stem cell may also extend into the drug testing aspect, has certain effect to judging drug effectiveness, toxicity.
Cell (or tissue) replacement therapy that grows up based on organizational project and genetic engineering technique and gene therapy are research focus and the forward positions in medical field and even the whole life science in recent years, provide new approach and hope for the mankind conquer multiple disease.One of them key issue is to select to meet the demands and the rake cell of easy handling, and stem cell is the first-selected target cell of cell therapy and gene therapy because of it has height self ability and multidirectional differentiation potential.
Mescenchymal stem cell is the early stage cell that the another kind of mesoderm except that hemopoietic stem cell is grown in the marrow, this class cell can be separated by external adherent culture, not only can be divided into hematopoiesis essence and stroma cell, also can be divided into multiple hematopoiesis tissue in addition, particularly mesoderm and neuroderm come the cell of source tissue, and be easy to the transfection and the expression of foreign gene, thereby may be cell therapy and gene therapy ideal target cell.In recent years, this class stem cell begins to be subjected to scholar's extensive concern.
1970's, the adherent BMNC that discoveries such as Friedenstech are cultivated in plastic culture dish can be divided into scleroblast, chondroblast, adipocyte and sarcoplast under certain condition, and these cells are through 20～30 culture cycle, still can keep its multidirectional differentiation potential, this class cell is called as mesenchymal stem cells MSCs (MSCs).These cells being implanted liver or the kidney coating of mouse finds to form bone sample and cartilage like cell.The multidirectional differentiation capability of mesenchymal stem cells MSCs has the evolution conservative of height.Confirm also that in recent years mesenchymal stem cells MSCs can participate in damaging the regeneration of muscular tissue in animal body and form star-shaped glial cell, thereby, cause widely and pay close attention to for the treatment of these cells, organ, system's relative disease provides new extremely valuable clue.
The cell in bone marrow interstital source, form becomes fiber-like, can be gathered into uniform colony, cell surface protein such as SH2, SH3, CD29, CD44, CD71, CD90, CD106, CD120a, CD124 etc. are positive, but CD14, CD34 and CD45 hematopoietic lineage mark then are negative.At present, the method that MSCs identifies all is by phenotypic differentiation occurring in culturing process, and backstepping learns whether be interstital stem cell then, does not still have direct method and can identify and obtain MSCs.
At external MSCs to polytype cytodifferentiation.MSCs keeps the characteristic self of stem cell constantly to breed when vitro culture, can be to different pedigree differentiation under different inductive conditions.By density gradient centrifugation from dog or adult's marrow isolated M SCs, independent or combined induction can form aggregate or tubercle with dexamethasone, β-phospho-glycerol and xitix etc., and alkaline phosphatase activities increases, the obvious calcification of matrix can be divided into scleroblast.Some somatomedins such as BMP, TGF β, bFGF etc. also can induce the MSCs Osteoblast Differentiation.To the analysis revealed of matter precursor cell strain 2T3 differentiation between the immortalization in mouse skull source, part combines with bmp receptor IB, can cause Osteoblast Differentiation; Part combines with bmp receptor IA, can cause becoming the fat differentiation.May activate different signal transduction pathways because BMPR IA or BMPR IB and bmp receptor II form after the complex body, cause heterogeneic expression.Mechanism for the chondroblast differentiation it be not immediately clear, but MSCs can become the chondrocyte automatically or through inducing to break up in the vitro culture process.Fortier etc. (1998) are from the centrifugal MSCs that obtains of the bone marrow of sternum of horse, monolayer culture, its form gradually becomes circular from spindle shape inoblast sample profile, form the colony spot, and the transformation that experience is expressed to the II Collagen Type VI from the expression of type i collagen, the synthetic of proteoglycan also constantly increases, and shows from the MSCs of mature horses marrow to become the cartilage differentiation when the monolayer culture.Johnstone etc. (1998) separate the rabbit MSCs that obtains by monolayer culture, change over to and form aggregate in the test tube, are containing 10
-7Cultivate in the substratum of mol/L dexamethasone, detect, have II, X Collagen Type VI to express, show and induce into the cartilage differentiation through tissue staining and immunohistochemical methods; Handle with TGF β 1, have or do not have dexamethasone, all can induce all cells to break up, and follow the raising of aggregate cell alkaline phosphatase activities.Mackay etc. (1998) will be grown up the MSCs of derived from bone marrow under the inducing of dexamethasone and TGF β 3, and the 14th day, detect II Collagen Type VI, aggrecan in extracellular matrix; Handle with dexamethasone and thyroxine, MSCs can further break up to hypertrophic chondrocyte.Other have much experiment showed, MSCs formed aggregate after, have and be beneficial to into the cartilage differentiation, this situation with the interior cartilage development of body is similar.Lacking blood vessel at the inner cell of aggregate provides nutrient, relies on the interaction between cell and the cell, perhaps is into a favourable condition of cartilage differentiation.MSCs also orientablely is differentiated to form adipocyte and myocyte under the effect of external evoked factor.Pittenger etc. (1999) lure imported MSCs with 1-methyl-3 isobutyl-xanthine, dexamethasone, Regular Insulin and indomethacin, in cell, assemble gradually and contain the abundant vesicle of fat, these cell expressing peroxisome proliferation activated receptors 2 (PPAR2), lipoprotein lipase (LPL) and fatty acid binding protein α P2.Multiple induce to handle cause the cell directional 95% or more to be divided into adipocyte, and fat steeps and constantly grows up, merges, and finally is full of cell.But these adipocytes were vitro culture healthy growth at least 3 months.Wakitani etc. (1995) handle the s-generation MSCs in rat marrow source 24 hours through 5 U-18496s in culture dish, after 7～11 days, in some culture dish, can observe long multinuclear myotube, also can in the tenuigenin of cell, observe the positive droplet of sudan black.These observe the source that the MSCs that illustrates in the organism marrow of birth back can become the flesh progenitor cell, can be anathreptic clinical study the basis is provided.
In recent years, people constantly explore and utilize MSCs to carry out cell therapy and gene therapy.At first, can as the seed cell of organizational project, repair various tissues with the MSCs of cultured and amplified in vitro.Mentioned in the aforesaid experimentation on animals that the MSCs with cultured and amplified in vitro repairs various damages, and had good effect.Although people successfully utilize self scleroblast or chondrocyte's damage of repairing bone or cartilage, MSCs compares with these noble cellss, has bigger advantage.Because in the vitro culture process, MSCs can keep not phenotypic differentiation constantly to breed, and reaches required number, then through inducing the orientable desired phenotype that is divided into; The cell source of differentiation is limited, and when vitro culture, rate of propagation is slower, but also has the problem of dedifferenting.The second, can pass through transgenic method, foreign gene is imported MSCs.Can change the growth factor gene that is beneficial to directed differentiation over to MSCs, promote directed differentiation, accelerate the process that in-vivo tissue is repaired.Lou etc. pass through Adenovirus Transfection with people's BMP2 gene, and a mesenchymal progenitor cell can an orientation carry out the bone differentiation, and after being expelled to nude mice muscle, can develop into the bone with bone trabecula, marrow and cartilaginous tissue.Confirmations such as Guinn, transfection the MSCs of IX factor gene, after injecting the SDID mouse in an orderly manner, can reach for 8 weeks at least by secretory protein.So genetically engineered MSCs can be used as an effective carrier and treats such as hemophilia B and other genetic diseases that causes owing to the shortage circulating protein.The 3rd, implant MSCs under certain conditions in an orderly manner, substitute defective marrow, the offspring of MSCs is provided for other tissue.Express the sudden change glue protogene and weak young mouse, after with a large amount of implantation of the MSCs of normal mice, through growing, normal donorcells substitution rate can reach 30% in the bone, cartilage and the brain that become mouse.Also exist because the sudden change of type i collagen gene the mankind, child's bone forming imperfection, these results provide experimental basis for this type of disease of clinical treatment so, and the donor bone marrow that can transplant normal human leucocyte antigen (HLA) (HLA) coupling for young patient is alleviated severe bone defect symptom.In the future possible strategy is to separate MSCs on one's body from patient, when vitro culture, substitutes the type i collagen gene of sudden change by homologous recombination, and then Hui Zhi is on one's body the patient.The I clinical trial phase proves that it is safe implanting from body MSCs in an orderly manner.So, can utilize patient's self genetically engineered MSCs, treatment is such as diseases such as osteoporosis.
In a word, the interstital stem cell of derived from bone marrow, external can directed differentiation be dissimilar cells, can be as the source of different cells in the organizational project; Multiple tissue and organ can be distributed in the normal circulation in vivo, cell and gene therapy can be used for.
Once the someone reported and can isolate the comparatively cell of the one-tenth fiber shape of homogeneous of form from cartilage, but did not have the people that the cell of this class was identified, also whether this kind cell was not had multidirectional differentiation capability and inquired into.And the cartilaginous tissue composition is single, not resembling marrow, bleeding of the umbilicus, liver has multiple composition, so the application that utilizes the mescenchymal stem cell in cartilage source to study the molecular mechanism of the biological property of mescenchymal stem cell, directed differentiation condition, differentiation and cell therapy, gene therapy will have prospect widely.Because cartilaginous element is single, if can from cartilage, set up mescenchymal stem cell system, will than from the tissue in other sources, set up mescenchymal stem cell be more purifying, have more prospect.
As seen, obtain stem cell, and further detect its activity and lose the pathological tissues of function or the application in the organ, can provide new solution thinking for diseases such as inherited disease, tumour and agings in treatment, reparation.
The objective of the invention is to disclose a kind of stem cell of preparation from tissues such as people or other mammiferous embryos, Cord blood, spinal cord, cartilage, described stem cell has biologic activity, can be divided into cell or tissue.
Another object of the present invention is to disclose a kind of method of obtaining stem cell from tissues such as people or other mammiferous embryos, Cord blood, spinal cord, cartilage, this method is simple and efficient, high specificity, practicability and effectiveness.
An of the present invention purpose is that open stem cell is in the purposes that is used for preparing treatment various diseases medicine.
To achieve these goals, the technical solution used in the present invention is: a kind of stem cell is to be organized as raw material with people or other mammiferous embryos, Cord blood, spinal cord etc., by the preparation of immunomagnetic beads positive-selecting.
Specifically, the preparation method of stem cell of the present invention comprises the steps:
4) the raw material dilution is obtained diluent;
5) monocytic acquisition;
6) immunomagnetic beads positive-selecting.
Wherein the dilution of the raw material in the step 1) is to adopt: the PBS buffered soln with 20ml Citric Acid-Sodium Citrate (being called for short ACD) and glucose (being called for short CB) dilutes Cord blood or spinal cord piece etc., obtains Cord blood or spinal cord diluent; ACD in the buffered soln: CB=1: 6, CB: PBS=1: 4.
Or with people or other mammiferous embryo's homogenate.
Step 2) obtain monocyte by following method in: according to Ficoll: CB=3: the Ficoll layering of 7 amount,, centrifugal 30 minutes of 4 ℃ of 800g, after collecting interfacial layer, centrifugal 20 minutes of 4 ℃ of 500g, abandon supernatant, obtain the spinal cord monocyte and collect interfacial layer.
Also comprise among the above-mentioned preparation method and adopt the selected by flow cytometry apoptosis stem cell.
When adopting Cord blood to be raw material, also comprise and to abandon throw out behind the supernatant with 4 ℃ of following cracking of erythrocyte cracked liquid 10 minutes.With PBS damping fluid washed cell, centrifugal 20 minutes of 300g adopts the selected by flow cytometry apoptosis stem cell, obtains the monocyte (MNC) of Cord blood.
Immunomagnetic beads positive-selecting in the step 3) is: adopt MNC: Dynabeads M-450CD
34: Detachabead CD
34=4 * 10
8: 4 * 10
7: 4 * 10
7=1ml: 100 μ l: 100 μ l.Dynabeads M-450 CD
34Washing placed magnetic sheet (MPC) 1 minute, abandoned clear liquid, PBS damping fluid washing 2 times.The monocytic CD that mixes magnetic bead (MACS, Miltenyi company product) and Cord blood
34Solution, 4 ℃ of little revolving are hatched 30 minutes, positive-selecting CD
34+ cell.
Rose ring-type cell-magnetic bead hangs again, places magnetic sheet (MPC) 2 minutes, abandons clear liquid, repeats 3 times, gets Rose ring-type cell-magnetic bead, is suspended from 100 μ l PBS damping fluids.Detachabead CD
34Dissociated cell, little revolving hatched 45 minutes, adds 2ml PBS damping fluid, and mixing placed magnetic sheet (MPC) 2 minutes, and the collecting cell clear liquid repeats 3 times, compiles cell supernatant, places MPC 2 minutes, removes residual magnetic bead, and cell supernatant is moved into the taper test tube.Centrifugal, washing 2 times, cell counting, flow cytometer FACS detects.
For avoiding intracellular ice crystal damage and solution damage, adopt the method for glass frozen preservation of human embryonic cells more application.Ice bath precooling liquid storage at first, cell centrifugation pipe place the ice-water bath operation, along the slow frozen storing liquid (which kind of frozen storing liquid) that adds of centrifugal tube wall, to final concentration of cells be 1 * 10
61.5 * 10
6, pressure-vaccum is even.Be sub-packed in the frozen pipe, flame sealing directly drops into liquid nitrogen container with frozen pipe and preserves, and cooling rate is about 600 ℃/min, carries out cell cryopreservation.During cell cultures, cell concn transfers to 1 * 10
6Cultivate.
Adopt mtt assay to measure stem cells hyperplasia among the present invention and identify the activity of gained stem cell.Be specially: prepare single cell suspension with serum free medium, be inoculated in the culture plate, add MTT (Chinese name) solution, hatch, centrifugal, abandon and cultivate supernatant in the plate hole, add dimethyl sulfoxide (DMSO).Enzyme joins the detector mensuration OD570 detection stem cell of exempting from service and had significant proliferation whether occurs, has activity if any the prepared stem cell of propagation explanation.
Adopt the immunohistochemical methods method to check that whether stem cell transforms to liver cell, is specially among the present invention: get cell smear, acetone fixed adds dilution one anti-(albumin 1: 50; CK81: 10), 37 ℃ 40 minutes, PBS washes 3 times, each 3 minutes.Add two anti-(dilutions in 1: 50) of FITC mark, preserved 40 minutes under 37 ℃ of conditions.PBS wash 3 times each 3 minutes.Buffering glycerine mounting, whether fluorescent microscope is checked to transforming to liver cell down.
Experimentize CD according to experiment grouping situation
34+ cell is marked the tail vein injection stem cell drugs with Hoechest33342, PKH26 are two.Get the observation internal organs and make frozen section, paraffin section.Morphocytology identifies and adopts the Ultraluminescence micro-imaging that the laser co-focusing cell instrument is observed.The cell humanized detects the employing round pcr, extracts the DNA in the tissue, with the specific amplification of the method people Alu sequence of PCR, is used to distinguish the cell of people and mouse.If pcr amplification has positive findings, can be further nucleic acid probe by the personnel selection Alu sequence carry out in situ hybridization and identify.Cell hemopoietic stem cell character detects and adopts CD
34+ CD
38+ two marks, Confolcal detects.CD
34+ cell, the two marks of Hoechest33342, PHK26, tail vein injection is gone into nude mice, adds stem cell inducible factor SIF, gets liver and makes frozen section, and the laser co-focusing cell instrument detects.CD
34+ cell, the two marks of Hoechest33342, PHK26, tail vein injection is gone into nude mice, adds the PBS damping fluid of SIF, gets liver and makes frozen section, and the laser co-focusing cell instrument detects.CD
34+ cell, the two marks of Hoechest33342, PHK26, tail vein injection is gone into nude mice, gets liver and makes frozen section, and the laser co-focusing cell instrument detects.
The detection of liver property, adopt the immunohistochemical methods method: available CK14, CK19, white protein, AFP identify respectively.The also available HepParl of liver cell mark, OC can adopt OV-6 and CD-117 mark.The contrast of immunohistochemical methods setter liver-yang, the contrast of mouse liver-yin.
In another embodiment of the invention, also relate to preparation from people or other mammiferous cartilaginous precursors to be the mescenchymal stem cell in feedstock production cartilaginous precursor source.
The mescenchymal stem cell in cartilaginous precursor source is by following method preparation: get people or other mammiferous cartilages, by trypsinase, collagen protein enzymic digestion, cultivate in substratum, go down to posterity, obtain mescenchymal stem cell.
Owing to cartilaginous precursor is exactly to grow from mesenchyme in fetal development period, estimate in the fetus cartilage, may have a part of mescenchymal stem cell, to remedy because the cartilage defect that accident causes.Cartilage to the embryo among the present invention passes through pancreatin, collagenase digesting, separating mesenchymal stem cell from cartilaginous precursor, obtained the relatively cell mass of homogeneous, the surface marker of the surface marker of this cell mass and the mescenchymal stem cell of bibliographical information is quite similar, and cell proliferation is vigorous, form is homogeneous comparatively, external it is carried out skeletonization, becomes fat, becomes neural multidirectional differentiation capability to detect, and finds that this group cell has skeletonization, becomes fat, becomes neural multidirectional differentiation capability.
Stem cell of the present invention and preparation method thereof has following advantage:
1. simple and efficient: through the MNC cell obtain, CD34 antibody labeling, selected by flow cytometry apoptosis and cell inoculation separate stem cells.
2. high specificity: measure stem cells hyperplasia by mtt assay, the immunohistochemical methods method checks whether stem cell transforms to liver cell, and the result proves that stem cell of the present invention can directedly transform.
3. practicability and effectiveness:, be used for the stem-cell therapy various diseases and carry out the screening of cell drug through inducing orientable conversion.
Describe the present invention in detail below in conjunction with the drawings and specific embodiments, described embodiment is used to describe the present invention, rather than restriction the present invention.
Description of drawings:
Fig. 1 .CD
34+ cell, the two marks of Hoechest33342, PHK26, stem cell inducible factor SIF acts on the laser co-focusing cell instrument detected result figure of liver;
Fig. 2 .CD
34+ cell, Hoechest33342, PHK26 are two to be marked, and adds the PBS damping fluid of SIF, gets liver and makes frozen section, laser co-focusing cell instrument detected result figure;
Fig. 3 .CD
34+ cell, the two marks of Hoechest33342, PHK26 are got liver and are made frozen section, laser co-focusing cell instrument detected result figure;
Fig. 4. mescenchymal stem cell flow cytometer qualification result figure of the present invention;
Fig. 5-mescenchymal stem cell flow cytometer qualification result figure 8. of the present invention;
Get the about 5g of people's 14 all embryonic spinal cords and shred, the PBS buffered soln dilution with 20ml Citric Acid-Sodium Citrate (being called for short ACD) and glucose (being called for short CB) obtains the spinal cord diluent; ACD in the buffered soln: CB=1: 6, CB: PBS=1: 4.
The spinal cord diluent is with Ficoll (Sigma company product) layering, Ficoll: CB=3: centrifugal 30 minutes of 7,4 ℃ of 800g.After collecting interfacial layer,, abandon supernatant, obtain spinal cord monocyte (MNC) centrifugal 20 minutes of 4 ℃ of 500g.
With magnetic bead (MACS, Miltenyi company product) positive-selecting CD
34(antibody) and spinal cord monocyte (MNC), used antibody are Dynabeads M-450 CD
34With Detachabead CD
34, with ratio and the consumption of MNC be: MNC: Dynabeads M-450 CD
34Antibody (Dynal company product): Detachabead CD
34Antibody (Dynal company product)=4 * 10
8: 4 * 10
7: 4 * 10
7(concentration)=1ml: 100 μ l: 100 μ l (consumption).
Positive-selecting CD
34+ cell (MNC)---mix magnetic bead and cell, 4 ℃ of following little revolving are hatched 30 minutes.
At first use Dynabeads M-450 CD
34Washing spinal cord monocyte placed magnetic sheet (MPC) 1 minute, abandon clear liquid after, with PBS damping fluid washing 4 times.
Positive-selecting CD
34+ cell (MNC), magnetic bead hangs again---with Rose ring-type cell---, places magnetic sheet (MPC) 2 minutes, abandons clear liquid, repeats 3 times, gets Rose ring-type cell-magnetic bead, is suspended from the 100mlPBS damping fluid.Use Detachabead CD
34Dissociated cell, little revolving hatched 45 minutes, adds 2ml PBS damping fluid mixing, places magnetic sheet (MPC) 2 minutes, and the collecting cell clear liquid repeats 3 times, compiles cell supernatant, places magnetic sheet (MPC) 2 minutes, removes residual magnetic bead, and cell supernatant is moved into the taper test tube.Centrifugal, the washing 2 times, cell counting, CD
34, CD
3Two marks, flow cytometer (FACS) detect MACS positive-selecting CD
34+ cell.Obtain cord stem cell.
For avoiding intracellular ice crystal damage and solution damage, adopt the method for glass frozen preservation of human embryonic cells more application.Ice bath precooling liquid storage at first, cell centrifugation pipe place the ice-water bath operation, add frozen storing liquid along centrifugal tube wall is slow, and final concentration of cells is 1 * 10
61.5 * 10
6, pressure-vaccum is even.Be sub-packed in the frozen pipe, flame sealing directly drops into liquid nitrogen container with frozen pipe and preserves, and cooling rate is about 600 ℃/min, carries out cell cryopreservation.
Embodiment 2
The method of reference example 1, concrete operations are as follows:
Get the about 100ml of human cord blood,, obtain the diluent of Cord blood, ACD in the buffered soln: CB=1 with the PBS buffered soln dilution of Citric Acid-Sodium Citrate (being called for short ACD) and glucose (being called for short CB): 6, CB: PBS=1: 4.
The human cord blood diluent is with Ficoll (Sigma company product) layering, Ficoll: CB=3: centrifugal 30 minutes of 7,4 ℃ of 800g.After collecting interfacial layer,, abandon supernatant centrifugal 20 minutes of 4 ℃ of 500g.Throw out is used 4 ℃ of following cracking of erythrocyte cracked liquid 10 minutes.With PBS damping fluid washed cell, centrifugal 20 minutes of 300g adopts the selected by flow cytometry apoptosis stem cell, cell counting, and FASC detects, and adjusts cell to 4 * 10
7, obtain the monocyte (MNC) of Cord blood.
Adopt MNC: Dynabeads M-450 CD
34: Detachabead CD
34=4 * 10
8: 4 * 10
7: 4 * 10
7=1ml: 100 μ l: 100 μ l.Dynabeads M-450 CD
34Washing placed magnetic sheet (MPC) 1 minute, abandoned clear liquid, PBS damping fluid washing 2 times.The monocytic CD that mixes magnetic bead (MACS, Miltenyi company product) and Cord blood
34Solution, 4 ℃ of little revolving are hatched 30 minutes, positive-selecting CD
34+ cell.
Rose ring-type cell-magnetic bead hangs again, places magnetic sheet (MPC) 2 minutes, abandons clear liquid, repeats 3 times, gets Rose ring-type cell-magnetic bead, is suspended from 100 μ l PBS damping fluids.Detachabead CD
34Dissociated cell, little revolving hatched 45 minutes, adds 2ml PBS damping fluid, and mixing placed magnetic sheet (MPC) 2 minutes, and the collecting cell clear liquid repeats 3 times, compiles cell supernatant, places MPC 2 minutes, removes residual magnetic bead, and cell supernatant is moved into the taper test tube.Centrifugal, washing 2 times, cell counting, flow cytometer FACS detects.
With embodiment 1, it is raw material that difference is to adopt 10 all pig embryonic spinal cords, obtains the Medulla Sus domestica stem cell.
Embodiment 4
With embodiment 2, it is raw material that difference is to adopt the ox Cord blood, obtains the ox cord blood stem cell.
Embodiment 5
Present embodiment relates to the preparation of the mescenchymal stem cell in cartilage source
15 all embryos carry out conventional aseptic sterilization, fully after the joint cartilage of exposure four limbs it is cut, remove the compositions such as perichondrium of articular cartilage surface, rinse well repeatedly with PBS, with the scissor cut cartilage to being not more than 1 cubic centimetre of size, in 37 ℃ of water-baths according to cartilage and 1: 5 ratio of trypsinase, with 0.25% tryptic digestion 30 minutes, after removing trypsinase, add the 0.2%II collagen type enzyme enzyme that contains serum according to cartilage and 1: 5 ratio of trypsinase, 37 ℃ of stirred in water bath digestion are after 90 minutes, sieve, with cold hanks buffered soln thorough washing 5 times, obvious to cell precipitation, plant in 75cm
2In the culturing bottle, substratum is 10ml IMDM (Hyclone company product, the abbreviation of ISCOVE ' S MODIFIED DUIBECLO ' S MEDIUM)+1ml 10% foetal calf serum+2.5umol/L amphotericin B+two anti-(100 μ g/ml penicillin+100 μ/ml Streptomycin sulphates).When cell grows to 80%～90% fusion, add the conventional digestion of 0.25% pancreatin-1mmol/L EDTA according to 1: 3 ratio, go down to posterity liquid nitrogen cryopreservation with 1: 3 (1 bottle passed for 3 generations) ratio.
Find through flow cytometer identification of cell surface antigen, mescenchymal stem cell CD45 of the present invention (-), CD44 (+) (-) is referring to Fig. 4; CD117 (-), CD147 (+) is referring to Fig. 5; CD34 (-), CD147 (+) is referring to Fig. 6; (-) is the surface antigen feminine gender, and (+) is surface antigen positive.
The present invention extracts the cell mass of mescenchymal stem cell surface marker basically identical of the derived from bone marrow of surface marker and bibliographical information first from cartilage, and prove that the isolated cells group not only has into the ability to the mesoderm differentiation of fat, skeletonization from cartilage, and has the neural ability that transforms to the ectoderm differentiation.Compare with the mescenchymal stem cell in other sources of present bibliographical information, the isolated cells group has that cellular constituent is single, the characteristics of purifying from cartilage, is convenient to research and sets up clone.
Embodiment 6
With embodiment 5, difference is to adopt rabbit embryo's second trimester of pregnancy four limbs cartilage to prepare mescenchymal stem cell.
Experimental example 1
The active authentication method of stem cell is as follows:
Mtt assay is measured stem cells hyperplasia: the stem cell cell concn of embodiment 1-4 is transferred to 1 * 10 respectively
6, prepare the single cell suspension of the foregoing description respectively with serum free medium, by every hole 1 * 10
4Individual cell inoculation is in cultivate in 96 holes, and every pore volume is 200 μ l, cultivates inoculating cell 3 days.Every hole adds MTT solution 20 μ l (5mg/ml), hatches 4 hours.1000 rev/mins, centrifugal 5 minutes.Abandon and cultivate supernatant in the plate hole.Every hole adds 150 μ l dimethyl sulfoxide (DMSO), shakes 15 minutes.Enzyme joins the detector of exempting from service and measures 0D570, and the experimental result of embodiment 1-4 all shows: had significant proliferation appears in stem cell, illustrates that prepared stem cell has activity.
Experimental example 2
This experimental example relates to the evaluation of stem cell into hepatocyte activity of conversion:
Adopt the immunohistochemical methods method to check whether stem cell transforms to liver cell: to get cell smear, acetone fixed 10 minutes.Add dilution one anti-(albumin 1: 50; CK81: 10), placed 40 minutes down for 37 ℃, the PBS damping fluid is washed 3 times, each 3 minutes.Add two anti-(dilutions in 1: 50) of FITC mark, preserved 40 minutes under 37 ℃ of conditions.PBS wash 3 times each 3 minutes.Buffering glycerine mounting, fluorescent microscope is checked down.Experimental result shows: human stem cell is bred and is transformed to liver cell.
Experimental example 3
This experimental example relates to the reparation of the stem cell in Cord blood source for hepar damnification.
The nude mice liver injury model is made film with 40%CCl
4Sweet oil, 0.3ml/100g, 2 times/week, subcutaneous injection, 6 weeks were made Liver Fibrosis Model.The immunomagnetic beads positive-selecting obtains the required Cord blood CD of experiment
34+ cell, and set up cell drug screening platform.
Experimentize CD according to experiment grouping situation
34+ cell is marked with Hoechest33342, PKH26 are two, tail vein injection hepatocyte growth promoting factors (commercially available prod), and factor dosage doubles first, and hepatocyte growth promoting factors is appended in later subcutaneous injection every day.Regularly put to death animal, get liver, spleen, make frozen section, paraffin section.With Ultraluminescence micro-imaging, laser co-focusing cell instrument, carry out morphocytology and identify.Referring to Fig. 1-3.
Carrying out the cell humanized with round pcr detects.Extract the DNA in the tissue,, be used to distinguish the cell of people and mouse with the specific amplification of the method people Alu sequence of PCR.The contrast of setter's liver-yang, the contrast of mouse liver-yin.PCR step: 1mm
3The tissue block of size places the EP pipe, add 25 μ l, 1 * TE damping fluid, smash tissue block to pieces, get 1 μ l tissue milk and place the PCR reaction tubes, gene mentation releasing agent 20 μ l vibrate 10-30 second, add one deck paraffin oil, put in the microwave oven and heated 5-7 minute, put on the amplification instrument that transfers to the 80-90 degree in advance, add primer, Mg respectively on request
2+, dNTP, ddH
2O, TaqDNA polysaccharase react.Reaction is carried out 2% agarose gel electrophoresis after finishing, and observes amplified band.If pcr amplification has positive findings, can be further nucleic acid probe by the personnel selection Alu sequence carry out in situ hybridization and identify.CD
34+ CD
38+ two marks, Confolcal detect and carry out the detection of cell hemopoietic stem cell character.
Adopt the immunohistochemical methods method to carry out the detection of liver property, the contrast of setter's liver-yang, the contrast of mouse liver-yin.The immunohistochemical methods step: slide glass pickling, pre-treatment, paraffin section are handled, frozen section is handled.Staining procedure: 3%H
2O
2Incubated at room 5～10 minutes is to eliminate endogenous peroxidase activity, distilled water flushing, PBS immersion 5 minutes.Saturated processing endogenous vitamin H will be cut into slices and be immersed in the 25 μ g/ml avidin solution 15 minutes, and PBS cleans after 15 minutes and can dye.Protease digestion or antigen retrieval are specifically carried out on request.The animal serum sealing (PBS dilution) in 5-10% normal two anti-sources, incubated at room 10 minutes, the serum deprivation that inclines is not washed, and drips an anti-working fluid of suitable proportion dilution, and 37 degree are hatched and were spent night in 1-2 hour or 4.(putting in the wet box) PBS flushing, 5 minutes 3 times.Drip biotin labeled two anti-(1%BSA-PBS dilutions) of suitable proportion dilution, 37 degree were hatched 10-30 minute.The PBS flushing, 5 minutes 3 times.Drip the strepto-avidin (PBS dilution) of the LRP mark of suitable proportion dilution, 37 degree were hatched 10-30 minute, PBS flushing, 5 minutes 3 times.Chromogenic reagent (DAB or AEC), tap water fully washes, and redyes (Hematorylin), and the normal sheep serum mounting is observed.
The above results shows, prepared stem cell can be used for the treatment of, repair pathology or lose the tissue or the organ of function.
Claims (9)
1. stem cell is to be raw material with people or other mammiferous embryos, Cord blood, myeloid tissue, by the preparation of immunomagnetic beads positive-selecting.
2. a kind of stem cell according to claim 1, its preparation method comprises the steps:
1) the raw material dilution is obtained diluent;
2) monocytic acquisition;
3) immunomagnetic beads positive-selecting.
3. a kind of stem cell according to claim 2, wherein the dilution of the raw material in the step 1) is: the PBS buffered soln with 20ml Citric Acid-Sodium Citrate (being called for short ACD) and glucose (being called for short CB) dilutes Cord blood or spinal cord piece etc., obtains Cord blood or spinal cord diluent; ACD in the buffered soln: CB=1: 6, CB: PBS=1: 4;
Or will dilute after people or other the mammiferous embryo's homogenate.
4. a kind of stem cell according to claim 2, step 2 wherein) obtain monocyte by following method in: according to Ficoll: CB=3: 7 amount with Ficoll with spinal cord or the layering of embryo's diluent, centrifugal 30 minutes of 4 ℃ of 800g, after collecting interfacial layer, centrifugal 20 minutes of 4 ℃ of 500g, abandon supernatant, obtain the spinal cord monocyte and collect interfacial layer.
5. a kind of stem cell according to claim 4, on also comprise and adopt the selected by flow cytometry apoptosis stem cell.
6。A kind of stem cell according to claim 2, step 2 wherein) obtain monocyte by following method in:: according to Ficoll: CB=3: 7 amount with Ficoll with the layering of Cord blood diluent, centrifugal 30 minutes of 4 ℃ of 800g, after collecting interfacial layer, centrifugal 20 minutes of 4 ℃ of 500g, abandon supernatant, to abandon throw out behind the supernatant with 4 ℃ of following cracking of erythrocyte cracked liquid 10 minutes, with PBS damping fluid washed cell, centrifugal 20 minutes of 300g, adopt the selected by flow cytometry apoptosis stem cell, obtain the monocyte (MNC) of Cord blood.
7. a kind of stem cell according to claim 2, the immunomagnetic beads positive-selecting in the step 3) is: Dynabeads M-450 CD
34Washing placed magnetic sheet (MPC) 1 minute, abandoned clear liquid, PBS damping fluid washing 2 times, the monocytic CD of mixing magnetic bead (MACS, Miltenyi company product) and Cord blood
34Solution, 4 ℃ of little revolving are hatched 30 minutes, positive-selecting CD
34+ cell;
Rose ring-type cell one magnetic bead hangs again, places magnetic sheet (MPC) 2 minutes, abandons clear liquid, repeats 3 times, gets Rose ring-type cell-magnetic bead, is suspended from 100 μ l PBS damping fluids; Detachabead CD
34Dissociated cell, little revolving hatched 45 minutes, adds 2ml PBS damping fluid, and mixing placed magnetic sheet (MPC) 2 minutes, and the collecting cell clear liquid repeats 3 times, compiles cell supernatant, places MPC 2 minutes, removes residual magnetic bead, and cell supernatant is moved into the taper test tube; Centrifugal, washing 2 times, cell counting, flow cytometer FACS detects.
MNC wherein: Dynabeads M-450 CD
34: Detachabead CD
34=4 * 10
8: 4 * 10
7: 4 * 10
7=1ml: 100 μ l: 100 μ l;
8. stem cell is by following method preparation: get people or other mammiferous cartilages, by trypsinase, collagen protein enzymic digestion, cultivate, go down to posterity the mescenchymal stem cell that obtains in substratum.
9. a kind of stem cell according to claim 8 prepares by following method:
The embryo carries out conventional aseptic sterilization, fully after the joint cartilage of exposure four limbs it is cut, remove the compositions such as perichondrium of articular cartilage surface, rinse well repeatedly with PBS, with the scissor cut cartilage to being not more than 1 cubic centimetre of size, in 37 ℃ of water-baths according to cartilage and 1: 5 ratio of trypsinase, with 0.25% tryptic digestion 30 minutes, after removing trypsinase, add the 0.2%II collagen type enzyme enzyme that contains serum according to cartilage and 1: 5 ratio of trypsinase, 37 ℃ of stirred in water bath digestion are after 90 minutes, sieve, with cold hanks buffered soln thorough washing 5 times, obvious to cell precipitation, plant in 75cm
2In the culturing bottle, substratum is 10ml IMDM (Hyclone company product, the abbreviation of ISCOVE ' S MODIFIED DUIBECLO ' S MEDIUM)+1ml 10% foetal calf serum+2.5umol/L amphotericin B+two anti-(100 μ g/ml penicillin+100 μ/ml Streptomycin sulphates); When cell grows to 80%～90% fusion, add the conventional digestion of 0.25% pancreatin-1mmol/L EDTA according to 1: 3 ratio, go down to posterity liquid nitrogen cryopreservation with 1: 3 (1 bottle passed for 3 generations) ratio.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1898380A (en) * | 2003-12-23 | 2007-01-17 | 卡拉格股份公司 | Method for separating cell from mammal gland secretion |
| CN101085983A (en) * | 2006-06-06 | 2007-12-12 | 刘永详 | Autologous stem cell, target cell transformed therefrom and its uses |
| CN104725511A (en) * | 2015-02-16 | 2015-06-24 | 华南农业大学 | Separation culture method for pig intestinal stem cells |
| CN105497069A (en) * | 2014-10-14 | 2016-04-20 | 讯联生物科技股份有限公司 | Composition for skin care, pharmaceutical composition and preparation method thereof |
| CN109045282A (en) * | 2018-08-14 | 2018-12-21 | 东营凤起生物科技发展有限公司 | A method of delaying premature ovarian failure and treatment osteoporosis using navel blood stem cell infusion joint autologous peripheral blood stem cells |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1898380A (en) * | 2003-12-23 | 2007-01-17 | 卡拉格股份公司 | Method for separating cell from mammal gland secretion |
| CN101085983A (en) * | 2006-06-06 | 2007-12-12 | 刘永详 | Autologous stem cell, target cell transformed therefrom and its uses |
| CN105497069A (en) * | 2014-10-14 | 2016-04-20 | 讯联生物科技股份有限公司 | Composition for skin care, pharmaceutical composition and preparation method thereof |
| CN104725511A (en) * | 2015-02-16 | 2015-06-24 | 华南农业大学 | Separation culture method for pig intestinal stem cells |
| CN104725511B (en) * | 2015-02-16 | 2018-01-12 | 华南农业大学 | A kind of isolated culture method of pig intestinal stem cell |
| CN109045282A (en) * | 2018-08-14 | 2018-12-21 | 东营凤起生物科技发展有限公司 | A method of delaying premature ovarian failure and treatment osteoporosis using navel blood stem cell infusion joint autologous peripheral blood stem cells |
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