CN1195055C - Method for establishing hematopoietic stem cells bank by extracting hematopoietic cells from placenta tissues - Google Patents
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Abstract
The present invention provides a new method for establishing a hemopoietic stem cell bank through extracting stem cells from a healthy puerpera's placenta so as to solves the technical problems. The present invention obtains the effect of changing waste into valuables because a large quantity of blood stem cells with favorable activity and high concentration are extracted from the placenta so as to be concentrated and purified by a mechanical method, an enzyme digestion method, and a method of monoclone antibodies and magnetic beads. Through cell culture, cell marking detection and animal experiments indicate that stem cells extracted from the placenta can strengthen the short term survival and the long term survival of transplanted hemopoietic stem cells; consequently, the stem cells can be used for establishing a hemopoietic stem cell bank of placentas and umbilical cord blood through freezing storage, applied when a placenta donor (self) or other people (variant) suffer from various hemopoietic stem cell transplanting adaptation diseases, and combined with various cell growth factors so as to clinically carry on cytothesis. The present invention clinically provides a new source of hemopoietic stem cells and stem cells and promotes the development of cell transplantation and tissue repair.
Description
Technical field
The present invention relates to belong to the method for setting up hemopoietic stem cell bank in International Classification of Patents C12N, A61K field, especially from placenta tissue, extract the method that hemopoietic stem cell is used to set up hemopoietic stem cell bank.
Background technology
The fertilized egg cell of human body can develop into embryonic cell in uterus, and this cell has and is divided into various histiocytic abilities, as neurocyte, muscle cell, liver cell, blood cell etc., so this cell is also referred to as multipotential stem cell.And the cell of directed a certain tissue differentiation also is called committed stem cell, as hemopoietic stem cell, neural stem cell etc.These cells can continue to be divided into specific cell or tissue, the blood hemopoietic stem cell can continue to be divided into red corpuscle, white corpuscle and thrombocyte.
Tumour cell is kind of the cell with unlimited multiplication capacity, and Chang Yong methods of treatment is strong dose thing and radiation clinically, and the hemopathy tumour as the leukemia has only the life that adopts medicine, radiation and hematopoietic stem cell transplantation could save patient.External hemopoietic stem cell can the patient's marrow behind chemotherapy and radiation in reconstitute hematopoiesis.This hematopoiesis has several characteristics.(1) and the quantity of external hemopoietic stem cell closely related, quantity is many more, easily more plants work.(2) relevant with the human leucocyte antigen (HLA) of donor and acceptor, the HLA site degree that conforms to is high more, easily more plants work, and rejection (GVHD) is also light more.(3) hemopoietic stem cell can be divided into two kinds according to the difference of its maturity: a kind ofly become the short-term hemopoietic stem cell for breaking up, be also referred to as 12 days spleen colonies and form cell (CFU-S), another kind is long-term hemopoietic stem cell, can patient's hematopoietic stem cell transplantation long-term surviving, closely related with this class cell.(4) relevant with stroma cell, stroma cell forms more early, and is many more, just can promote that hemopoietic stem cell adheres to stroma cell more, strengthen being in contact with one another and the secretion of correlation factor between the cell, hemopoietic stem cell presents form, amplification and the differentiation of " pebbles " sample on the stroma cell surface.
Comparatively sophisticated at present hematopoietic stem cell transplantation according to the source of cell, can be divided three classes: marrow hemopoietic stem cells is transplanted (BMT), and (Broxymeyer.PNAS 89 for hemocytoblast transplanting (MPST) and cord blood stem cell transplanting (UCBT) around mobilizing; 86:3828; 32).The former two's source of human stem cell is abundant, and general karyocyte number can reach 5-10 * 10
8/ Kg, CD34+ cell (a kind of surface markers of hematopoietic stem) can reach 1-5 * 10
6/ Kg owing to need the HLA Strict Compliance between donor and the acceptor, could guarantee the success of transplanting, otherwise that the donor hemopoietic stem cell is difficult in patient body's interplantation is alive, more serious graft also can be taken place arranges host disease (GVHD) even survive.In the children of one family, the probability that only has 1/4HLA to conform to, and in the unrelated donor of non-blood relationship, this probability has only one of percentage, has so just limited BMT or MPST greatly and has used widely clinically.Umbilical cord blood hematopoietic stem cell is implanted in successful clinically application (Gluckman e et al.New Engl J Med1989 321:1174-1178) has promoted the establishment of umbilical cord blood hematopoietic stem cell storehouse (UCBB) and the development of UCBT over surplus in the of nearly ten year.Cord blood comes from placenta, usually divide the puerperium discarded the women, find to contain in the Cord blood abundant hemopoietic stem cell now, the concentration and the marrow of its CD34+ cell are similar, account for the 0.1-0.5% of total cell count, and the more early stage hemopoietic stem cell of CD34-is high than marrow concentration also.Even find that clinically the UCBT superiority bigger than BMT is the difference that HLA has 1-2 site between donor and acceptor, serious GVHD reaction can not take place yet, also lower in Cord blood with the pollution rate of the various viruses of time image.Thereby people expect bleeding of the umbilicus collect, external concentrate and the liquid nitrogen profound hypothermia is preserved, set up umbilical cord blood bank (UCBB).When patient needs, can melt the cord blood cells of preservation at any time, clinical hematopoietic stem cell transplantation is provided.So far, nearly thousand routine UCBT have been carried out in the whole world, show that definitely UCBT is successful children or the lighter patient of body weight, and plant success ratio alive and can reach 80-90%, but the grownup big to body weight, effect is not ideal.Show that to plant live time long, 40 talentes that reach that have begin to have the cell growth, even plant work, the lymphsystem immunologic function of donor is also lower.Reason is that the content of each bleeding of the umbilicus is limited, on average is about about 100 milliliters, and the CD34+ cell is about 0.5-5.0 * 10
6Obviously, it is not enough being used for the big patient of body weight, and the somebody thinks going back to the nest (Homing) of umbilical hemopoietic stem cell and to be divided into the ability of stroma cell lower, and this also is one of long reason of implantation time.Simultaneously, the influence of thymus gland afterwards because the bleeding of the umbilicus lymphsystem does not stand to be born, this also causes in the grownup, even plant work, also also has one period that immunologic function weakens.In view of above reason, scientists is carried out 2 or the mixed transplantation of a plurality of bleeding of the umbilicus combined utilization in identical or similar HLA site, the content of this above umbilical hemopoietic stem cell number that can double, but clinical practice shows, can not shorten the aleukocytic time of patient, even plant work, also cord blood cells survival often.Another cord blood cells is repelled.Another solution route is exactly to cultivate at external hematopoietic stem cell expansion.Experimental results show that and adopt various cell stimulating factor and the suitable culture condition cord blood cells that can increase to reach tens thousand of times, but being this amplification, problem needs spended time, expense is also high, when the more important thing is owing to amplification, hemopoietic stem cell also breaks up, and the result of clinical application shows that cord blood cells amplification front and back are to transplanting no much difference.
Placenta is the place of fetus and the exchange of mother's blood, contains profuse method blood microcirculation.The people is in the interogestate stage of mother, and placenta is one of organ that at first forms.This shows in growth at embryonic stem cell, the atomization that being detained has a large amount of early stage stem cells in placenta, and probably these stem cells also continue the ability of in store differentiation hemopoietic stem cell in placenta.Although blood (Cord blood) contains abundant hemopoietic stem cell around fetus, the hemopoietic stem cell that is detained in this stem cell and the placenta may show at cytolemma and has difference on the acceptor, be that intraplacental hemopoietic stem cell has and organizes stronger avidity (function of going back to the nest), the stem cell and the cord blood cell one that can extract like this in the placenta tissue are used from transplanting, undoubtedly contain 1) placenta stem-cell can provide the microenvironment of making hematopoietic stem cell growth as the marrow blood cell, quickens growth, growth (the Hows JM.Lancet1992 of hemopoietic stem cell; 340:73-6).2) the placental blood stem cell is comparatively early stage stem cell, can be divided into various cells (as adipocyte, fibrocyte, chondrocyte etc.) in vivo, and this class cell has been formed the microenvironment of marrow just, promotes the generation and the differentiation of blood cell.3) hemopoietic stem cell in the placental blood has stronger going back to the nest (Homing) function, and this shows if extract such cell infusion gives patient, and these cells can be grown in patient's marrow sooner, more effective, grow.4) placental blood contains abundant early stage hemopoietic stem cell of various stages, if energy and cord blood cell are applied to patient together, has increased the content of hemopoietic stem cell undoubtedly greatly, makes this hilum-placental blood stem cell can be applicable to the adult patient.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method that hemopoietic stem cell is used to set up hemopoietic stem cell bank of extracting from placenta tissue.Extracting hemopoietic stem cell and set up hemopoietic stem cell bank from placenta tissue is main idea of the present invention, the technical scheme that realizes this purpose is to adopt mechanical mode separation, freezing long-term placenta tissue cell, and method concentrated, that purification of hematopoietic stem cells is used to set up hemopoietic stem cell bank, described method is divided into the process of collecting step, detecting step, extraction step, concentrated and purified step, wherein, the technical scheme of described concentrated and purified step is:
The placenta tissue cell and the hydroxyethylamyle that extract are pressed 1: 6 mixed;
In whizzer centrifugal 10 minutes, remove red corpuscle with 500g;
With the mixed of lymphocyte separation medium by 1: 4;
In whizzer centrifugal 30 minutes, remove remaining red corpuscle and mature granulocyte with 1000g;
Collect and wash the hemopoietic stem cell group of each different developmental phases;
Set up stem cell bank.
According to the above-mentioned method and the effect of having removed to improve UCBT by following several aspects: (1) placenta tissue stem cell; (2) placental blood and umbilical cord blood hematopoietic stem cell.Obviously, above-mentioned method is to be different from former marrow hemopoietic stem cells to transplant (BMT) method of blood hematopoietic stem cell transplantation and umbilical hemopoietic stem cell transplanting on every side fully.What its difference was at first to collect is the cord blood cell, remove various possible pollutions and infection then, be separated in the histocyte (stem cell that wherein contains the cytocerastic various stroma cells of hematopoiesis support and can be divided into hematopoietic cell) in the placenta at last.When need transplanting on the patients clinical, with above two kinds of ancestral cells that conform to patient HLA from the venoclysis patient.Another aspect of this invention is exactly the patient who when injects after this two classes cell is given high-dose chemotherapy and radiotherapy, the placental blood stem cell can be simultaneously and cord blood stem cell import by vein.Also can before it or thereafter, import.
The effect of hematopoietic stem cell transplantation treatment tumor disease has been strengthened in this invention greatly.Heavy dose of chemotherapy and radiation has also destroyed the microenvironment of marrow in the kill tumor cell, and the hematopoiesis that is implanted into only could begin differentiation and growth after rebuilding microenvironment.Just because of this reason, the most important aspect of this invention is exactly separation, purifying, profound hypothermia preservation, tissue and hemopoietic stem cell.When the HLA screening conforms to, flow to patient and be used to rebuild the marrow microcirculation, quicken the recovery of hemopoietic stem cell and the survival that guarantees graft.
Contribution of the present invention promptly is to adopt variety of way and easy equipment, from placenta, extract more hemopoietic stem cell and similar mesodermal stem cell (mesenchymal stem cellsCMSCs), under suitable culture condition and cell selection, these cells can be divided into various different types of cells, as the stroma cell of liver, skin, cartilage, marrow and the hematopoietic cell of various different stepss.Blood cell can directly be supported and instruct to marrow stromal cell, as increasing of hemopoietic stem cell etc. plant and break up (Herman P.LeuKemia 1998,12:735-45).Because before BMT, need carrying out heavy dose of chemotherapy (medicine) and radiotherapy (isotropic substance), patient goes the microenvironment eliminating the intravital tumour cell of patient and destroyed marrow inside.So how, accelerating and plant that the external cell of living survive in the patient body is exactly the individual very problem of key.A significant contribution of the present invention is exactly the hemopoietic stem cell of growing by from placenta extraction, these MSC cells of purifying and different steps, utilize the function of these cells to strengthen microenvironment after patient's marrow destroys, increase the content of the hemopoietic stem cell when transplanting, make this UCBT not only go for all crowds, and the zero cell stage after can shortening patient greatly and transplanting, thereby improved patient's survival rate.
The placenta tissue stem cell of the present invention preparation generally is input as the master with vein, but also can directly enter in the myeloid tissue part, also can be by any other approach, as method infusions such as artery injections to patient.
Pharmacy composition that the present invention mainly uses or content have physiological saline, low molecular dextran, umbilical cord blood plasma, DMEM (Dulbecco ' s Modified Essential Medium) nutrient solution, water for injection etc.All above compositions all are patients aseptic, that pyrogen-free prepares and is suitable for clinical transplantation.
Can take number of ways about the method for from placenta, extracting tissue stem cell, particularly adopt the method for mechanical disintegration placenta as (1); (2) method of application collagenase or protease digestion tissue; (3) mixing of above two kinds of methods; (4) adopt other to pulverize the method for placenta.
The present invention can be intravenous injection, arterial infusion about using the approach of placenta tissue stem cell, also can be local injection, also adopts the application of alternate manner certainly except not.
The present invention can be that (1) and cord blood stem cell inject together about the placenta tissue Application of stem cells time; (2) injecting the day before yesterday at navel blood stem cell uses; (3) repeatedly in different time, import the placenta tissue stem cell respectively.
The content of use placenta tissue stem cell in hematopoietic stem cell transplantation is crucial.Say that generally each placenta tissue is separable to go out 1-4 * 10
9Mononuclearcell, like this, even, also can have 1 * 10 at least to the patient of 100 kg body weight
7The mononuclearcell of/Kg.This numeral is the patient who is suitable for bone marrow transplantation fully.The patient of BMT, roughly have only 1 * 10
4The cell that/Kg is relevant with stroma cell is no more than 7 * 10 usually
5/ Kg, thereby the common application of placenta tissue stem cell and navel blood stem cell will inevitably strengthen the effect of hematopoietic stem cell transplantation.
No matter on the quality and quantity of the present invention in hematopoietic stem cell transplantation all be a huge improvement, it has not only quickened the umbilical cord blood transplantation recovery time in umbilical cord blood transplantation, the quantity of hemopoietic stem cell is increased greatly, not only can make bleeding of the umbilicus be widely used in various ill crowds, and can to shorten patient's cell count in the umbilical cord blood transplantation greatly be time of zero, reduce the incidence of polluting, can save more patients' life.
After placenta and umbilical cord are overflowed, can collect placenta when collecting bleeding of the umbilicus, separate the hematopoietic stem in more each period from placenta, in addition profound hypothermia is preserved, when patient uses, can be simultaneously and bleeding of the umbilicus together infusion to patient.
The main range of application of the present invention is: various malignant hematologic diseases, as leukemia, lymphoma, multiple myeloma; Heredopathia is as congenital hemolytic anemia; Blood hypoplasia disease is as aplastic anemia; Medicine or chemical element are as benzene, mercury etc.; Radiation disease is as radioactive rays accident etc.Methods of treatment is to adopt various means such as chemotherapy and radiotherapy to destroy ill medullary cell and the immunity system of patient self, the hemopoietic stem cell of input health then, owing to destroyed the interior environment of patient's self marrow simultaneously, the hemopoietic stem cell of input only could begin the reconstruction of hematopoietic cell after the environment in rebuilding marrow later on.So just delayed the time that hemocyte is rebuild, and if just import the cell that is equivalent to environment in the marrow that HLA conforms in advance, this has just quickened the time that hemopoietic stem cell is rebuild undoubtedly, accelerates patient's recovery.
The present invention also is applicable to suffer from congenital or Acquired immunity defective disease (AIDS), and these patients are diseases of suffering from the lymphocytic immunity system, import normal immune system cell, obtains function of immune system and rebuilds, and also can cure these patients undoubtedly.
The present invention also is applicable to the treatment of gene transfection.As everyone knows, the ectosome gene transfection be subjected to the cell of gene transfection the more the better, and placenta stem-cell has very strong hyperplasia and differentiation capability, is the desirable cell of gene transfection.
The present invention also is applicable to the treatment of multipotential stem cell to various histocyte differentiation, confirmed that now embryonic stem cell, medullary cell, adipocyte all contain multipotential stem cell, these cells can be divided into various histocytes under certain environment, as fat, marrow, nerve, liver etc.The present invention finds also to contain such multipotential stem cell in placenta.
In all operations step of the present invention, it all is the routine operation method that pharmacopeia allows, this comprises employing aseptic technique, stroke-physiological saline solution washing, sterilization, standard contents such as aseptic cell concentration and purifying, applied medicine, the picture methyl-sulphoxide, stroke-physiological saline solution, low molecular dextran, sterile distilled waters etc. all are the requirements that meet China health organ, do conventional virus behind the end of operation, cytoscopy and bacterium, mould are cultivated, and the placental blood cell that only meets the requirements just can be applied to clinical.
The placental blood cell that adopts present method extraction is in 2-4 * 10
9The mononuclearcell number, it is the CD34 positive cell that 0.3-0.7% is wherein arranged approximately, and external granulocyte colony is cultivated (CFU-GM), and the visible huge cell mass of colony is generally in 5-10 * 10
6Between.This colony group and marrow, mobilization peripheral blood and cord blood stem cell have evident difference, show that colony is big, cell sample growth in an overlapping, and the time that the colony group occurs is also different, demonstrates the placental blood stem cell and has different steps.And in long-term cell cultures, the amplification ability of placental blood cell manys 1-3 doubly than cord blood cells approximately.The collection capacity of general cord blood cells is about 0.8-1.5 * 10
9, and the CD34 positive cell is between 0.1-0.5%, like this if placental blood cell and cord blood cells are used for patient simultaneously, then total cell count can increase 3-5 doubly, complete needs applicable to all patients.
By following detailed description, will be to working method of the present invention, characteristics and superiority are further illustrated.Certainly, although should it is worthy of note that this paper has described following several cases, people also can be according to basis of the present invention, and the form that proposes various variations and individual character also is possible.
Description of drawings
Fig. 1: be placenta tissue cell and cord blood cell co-cultivation, visible placental blood hemopoietic stem cell is the growth of " pebbles " sample on the placenta tissue attached cell.
Fig. 2: the placenta tissue cell forms adherent fibroblast-like cell through reaching the vitro culture in January, and this cell begins to irregular, and cell is huge, monokaryon or double-core, and when being paved with whole culture dish, cell becomes fusiformis gradually.Repeatedly go down to posterity and profound hypothermia is preserved, melt in vitro culture, cytoactive is still fine.
Fig. 3: the colony of placenta tissue cell is cultivated (CFU-GM).The time that the placenta tissue cell forms colony be evening than cord blood cell, but Once you begin forms, and cell is grown very fast, the overlapped layering of iuntercellular, and colony again more than 3 times can be arranged.
Fig. 4: the placenta tissue cell is carried out the analysis of two colour developing-fluorescence activated cells (FACS), find that the CD34+ cell is a height than cord blood cells in placenta tissue, mean value high approximately 50%.
Among the figure: Fig. 1 is meant: for from mazolytic histocyte and cord blood cell co-cultivation, visible placental blood hemopoietic stem cell is the growth of " pebbles " sample on the placenta tissue attached cell.
Fig. 2 is meant: placenta tissue forms the fibroblast-like cell of adherent shape, its cell begin for irregular, cell is huge, after adherent, cell is the shuttle profound hypothermia gradually preserve, melt after, cytoactive is still fine.
Fig. 3 is meant: the colony of placenta tissue cell is cultivated (CFU-GM): incubation time is the 9th day, and the time that forms colony be evening (7 days) than cord blood cells, but Once you begin forms, and the cell growth is very fast, presents overlappedly, and the formation of colony again more than 3 times can be arranged.
Fig. 4 is meant: the histocyte after same cord blood is separated carries out the flow cytometer laser analysis, about CD34 positive cell (being red point symbol on the figure) doubles than bleeding of the umbilicus in placenta tissue.
Embodiment
The technical scheme that the present invention relates to is a kind of method that hemopoietic stem cell is used to set up hemopoietic stem cell bank of extracting from placenta tissue, described method is to adopt mechanical mode separation, freezing long-term placenta tissue cell, and method concentrated, that purification of hematopoietic stem cells is used to set up hemopoietic stem cell bank, described method is divided into the implementation procedure of collecting step, detecting step, extraction step, concentrated and purified step, wherein, described concentrated and purified step is:
The placenta tissue cell and the hydroxyethylamyle that extract are pressed 1: 6 mixed;
In whizzer centrifugal 10 minutes, remove red corpuscle with 500g;
With the mixed of lymphocyte separation medium by 1: 4;
In whizzer centrifugal 30 minutes, remove remaining red corpuscle and mature granulocyte with 1000g;
Collect and wash the hemopoietic stem cell group of each different developmental phases;
Set up stem cell bank.
Say that further described collection step is meant: collection Cord blood and placenta also adheres to bar coded sticker and deposits in 4 ℃ of refrigerators, is no more than 24 hours.
And described detection step is meant: institute's refrigerated Cord blood and placenta are extracted sample and carry out following detection:
Hepatitis virus and corresponding antibodies,
The venereal disease detection,
The malicious antibody test of AIDS (AIDs),
The heredity hemolytic disease,
Have nuclear leukocyte and mononuclearcell the counting,
Hematopoietic stem detection by quantitative (CD34),
Tissue matching (HLA),
The detection of hematopoietic stem qualitative detection (CFU-GM).
Say that further the detailed process of described extraction step is meant: a pair of placenta tissue is under sterile environment and condition:
Clean placenta with stroke-physiological saline solution, wash all blood clots and hematocele, adopt sterilizing agent to kill any possible bacterial contamination;
Use phosphoric acid buffer (PH7.4) once more, low molecular dextran and cell culture fluid (DMEM) clean in order;
To mix jointly through umbilical cord blood plasma, DMEM nutrient solution and placenta cells after selecting and spend the night; Wipe out the placental membrane on umbilical cord and top layer, application is torn, and grinds and the DMEM process of washing is collected placenta cells in 50 milliliters the centrifuge tube, and with 1000rpm, 5 minutes, 4 ℃ centrifugal, abandoning supernatant in whizzer;
Adopt the syringe needle of 16G, 18G and 20G to filter, placenta cells is made individual cells; Placenta cells after the preparation is done following detection:
Cell and mould are cultivated,
Nuclear leukocyte and mononuclearcell counting is arranged,
Hematopoietic stem detection by quantitative (CD34),
Hematopoietic stem qualitative detection (CFU-GM),
The activity of hematopoietic stem (platform is expected blue dyeing),
The mononuclearcell that the placenta cells of the placenta tissue after detecting is produced is with 1 * 10
8/ ml and DMSO mix, and cool to subzero 80 ℃ with 10% DMSO and low molecular dextran concentration in the liquid nitrogen minuent, transfer to profound hypothermia preservation in liquid nitrogen (186 ℃) liquid phase then.
For realizing the object of the invention, the step of described concentrated and purified hemopoietic stem cell is absolutely necessary herein, and the step of concentrating hematopoietic stem cells wherein can also be:
Accurately measure isolating histocyte volume from placenta;
Histocyte is washed secondary with the DMEM liquid that does not contain serum;
With the ratio adding hydroxyethylamyle (HES) of the histocyte after the washing, on omnipotent shaking table, mixed 5 minutes in 1: 6;
Get the tissue that shakes after mixing with the speed of 200g under 12 ℃ of temperature, the condition of 5 minutes time, go in the whizzer centrifugally, afterwards, oppress with the blood plasma squeezer and to be rich in histiocytic liquid and to discard to another centrifuge tube and be rich in erythrocytic throw out, obtain supernatant liquor;
Once more with 12 ℃ of temperature, the condition of 15 minutes time is centrifugal with the speed of 800g, afterwards abandoning supernatant with the supernatant liquor that obtains;
Collection is sunken to the pipe cell at the end, adds DMEM liquid sedimentation cell is made suspension, is added to the upper strata of lymphocyte separation medium with 1: 4 ratio, then with 800g, and 12 ℃ of temperature, 30 minutes time is centrifugal;
The cell of absorption between lymphocyte separation medium and supernatant liquid adds the DMEM nutrient solution, washs 2 times, does cell counting and active the detection at last;
Set up stem cell bank on the basis of the above.
Same, the step of purification of hematopoietic stem cells wherein can also be:
Use the selected by flow cytometry apoptosis hemopoietic stem cell, and:
Sorting has fluorescently-labeled anti-CD34 monoclonal antibody;
To and have the antigenic monoclonal antibody of the anti-CD34 of fluorescence through the histocyte after concentrating and mix mutually, in room temperature or 4 ℃, hatch 30 minutes, wash 3 times with the DMEM nutrient solution that does not contain serum then;
With the histocyte after the washing with have fluorescently-labeled anti-CD34 monoclonal antibody and combine and put into flow cytometer through laser radiation;
According to the power that fluorescence takes place, collect the hemopoietic stem cell of 90% above purity;
Set up stem cell bank on the basis of the above.
In the method that describedly concentrates, can also adopt in the step of purification of hematopoietic stem cells antibody-magnetic bead mode purification of hematopoietic stem cells or with the method for vitamin H purification of hematopoietic stem cells, they are respectively:
The method implementation step of antibody-paramagnetic particle method purification of hematopoietic stem cells is: the carrier that sorting can combine with anti-CD34 monoclonal antibody, antibody magnetic bead; The spissated placenta tissue population of stem cells of sorting; The placenta tissue population of stem cells is contacted with the antibody magnetic bead, the hematopoietic stem in the cell mass is combined with antibody, and adhere on the magnetic bead; Remove the cell that do not combine with inhaling iron device absorption magnetic bead and washing with magnetic bead; Extract hematopoietic stem, its purity is more than at least 80%.And the method for vitamin H purification of hematopoietic stem cells the steps include: the monoclonal antibody that sorting can combine with vitamin H, contains the biological post of antibiotic antibody; The spissated placenta tissue population of stem cells of sorting; The placenta tissue population of stem cells is combined with the biological post that contains antibiotic antibody; Wash the not cell of binding antibody; Change the pH value of washings, extract hematopoietic stem, its purity is more than at least 80%.
Following examples are analysed it so, again:
Case one
Adopt the method for machinery to separate the placenta tissue stem cell
Placenta and Cord blood all come from local obstetrics and gynecology hospital or section office.At first, after obtaining pregnant woman or its direct relatives and agreeing to contribute placenta, consult all survey reports of pregnant woman, confirm the virus infectiones relevant such as no any virus, syphilis with blood, medical histories such as perquisition pregnant woman's fertility, disease, heredity, infection then, after all were normally, Cord blood (patented technology: " using the device of soft pocket vertical collection bleeding of the umbilicus ", the patent No.: ZL99244936.7) was collected in beginning according to a conventional method.Cord blood after having collected and placenta are deposited in 4 ℃ of refrigerators all with bar coded sticker.Shelf-time generally is no more than 24 hours.
According to the numbering of barcode, storing in the pertinent data input computer.In gnotobasis, the numbering of pressing barcode is extracted sample from relative bleeding of the umbilicus, is provided with down detecting:
---hepatitis virus and corresponding antibodies: as hepatitis B, hepatitis C etc.
---venereal disease detects; Picture syphilis etc.
---the malicious antibody test of AIDS (AIDs)
---the heredity hemolytic disease
---nuclear leukocyte and mononuclearcell counting is arranged
---hematopoietic stem detection by quantitative (CD34)
---tissue matching (HLA)
---hematopoietic stem qualitative detection (CFU-GM)
(Robinstein P.Blood1993 80:1679-90) carries out the mononuclearcell concentration to cord blood cell, and adds stored frozen liquid (DMSO), preserves in the liquid nitrogen profound hypothermia midium or long term by external conventional technological method.
Corresponding placenta tissue is by handling under follow procedure gnotobasis and the condition: clean placenta with stroke-physiological saline solution, wash all blood clots and hematocele, adopt sterilizing agent to kill any possible bacterial contamination, and then application phosphoric acid buffer (PH7.4), low molecular dextran and cell culture fluid (Dulbecco ' s Modified Eagles Mediam) also are called for short DMEM and clean in order, adopt the umbilical cord blood plasma (AB blood group) after selecting at last, DMEM nutrient solution and placenta are mixed jointly to spend the night.Wipe out the placental membrane on umbilical cord and top layer, application is torn, and grinds and DMEM nutrient solution process of washing, placenta cells is collected in 50 milliliters the centrifuge tube, and with 1000rpm, 5 minutes, 4 ℃ centrifugal in whizzer, and abandoning supernatant is once more with nutrient solution cleaning 3 times.Adopt the syringe needle of 16G, 18G and 20G to filter at last, placenta cells is made individual cells, the placenta cells after the preparation is done following detection:
---cell and mould are cultivated
---nuclear leukocyte and mononuclearcell counting is arranged
---hematopoietic stem detection by quantitative (CD34)
---hematopoietic stem qualitative detection (CFU-GM)
---the activity (platform is expected blue dyeing) of hematopoietic stem
The mononuclearcell of producing from placenta is with 1 * 10
8/ ml and DMSO mix.DMSO with 10% and low molecular dextran concentration cool to subzero 80 ℃ in the liquid nitrogen minuent, transfer to profound hypothermia preservation in liquid nitrogen (186 ℃) liquid phase then.
Case two
Adopt the method for enzymic digestion to extract the placental blood stem cell
Placenta adopts aseptic method to collect from obstetrics and gynecology hospital, and and corresponding Cord blood send into the navel blood stem cell center together, before the collection placenta, should obtain pregnant woman and corresponding lineal relative's agreement, and relevant heredity and infect medical history in investigation pregnant woman and the family, and all relevant virus checkings of hospital could be collected after negative.
Cord blood after the collection and corresponding placenta need (4-20 ℃) at a certain temperature, at 24 hours with interior my center of delivering to.Need do corresponding virus once more at the center and detect, have only virus negative bleeding of the umbilicus and placenta just can enter my computer registration procedure, after the corresponding bar code number of record, transport to the sterile workshop at my center.
The sample of getting in sterile workshop in the Cord blood is done the HLA detection, and record is input to computer system, and bleeding of the umbilicus separates, adds freezing protective material, program control cooling routinely, preserves in the liquid nitrogen profound hypothermia.
Corresponding placenta, wash with stroke-physiological saline solution earlier, remove the cell that all contain female blood, aseptic sterilization in addition in special container then, remove the source of all possibility cell contaminations, after this adopt cell culture fluid, as DMEM (GIBCO, Grand Island.NY.USA) washed, remove possible female blood stains once more and dye.Shred placenta then, remove after birth and umbilical cord, use collagenase (0.2g/ml) digestion 15 minutes, 20 minutes and 30 minutes.We find to digest 15 minutes obtained cells for well.Wherein cell activity is good, the rate of recovery is high; collect postdigestive placenta cells at last; centrifugal in special centrifuge tube, rotating speed is 1000rpm, 10 minutes; with low molecular dextran washing three times; collected cell is made individual cells suspension, white corpuscle and mononuclearcell counting, measure cytoactive, calculate the white corpuscle colony (CFU-GM) of CD34, CD4, cd8 cell content and vitro culture; adding before the freezing protective material (10%DMSO), getting a sample and do cell and cultivate.
Case three
Adopt physics and biological method to concentrate and purification of hematopoietic stem cells
Use isolating hemopoietic stem cell in above-mentioned two kinds of method people placentas, in fact also be a group cell, it contains red corpuscle, thrombocyte, has the adult stem that is divided into the different tissues cell, the hemopoietic stem cell of different times and mature granulocyte and lymphsystem cell.The present invention adopts following method to concentrate and purification of hematopoietic stem cells.
1. adopt precipitation, centrifugation method concentrating hematopoietic stem cells
Accurately measure by above-mentioned two kinds of methods isolating histocyte volume from placenta, use the DMEM liquid that does not contain serum and wash secondary, add hydroxyethylamyle (HES) in 1: 6 ratio, on omnipotent shaking table, mixed 5 minutes, then in whizzer with 200g, 12 ℃ of temperature, 5 minutes time, be rich in histiocytic liquid to another centrifuge tube with the compressing of blood plasma squeezer, discard and be rich in erythrocytic throw out, supernatant liquor recentrifuge 800g, 12 ℃ of temperature, 15 minutes time, abandoning supernatant.Collection is sunken to the pipe cell at the end, adds DMEM liquid sedimentation cell is made suspension, is added to the upper strata of lymphocyte separation medium with 1: 4 ratio, then with 800g, and 12 ℃ of temperature, 30 minutes time is centrifugal.The cell of absorption between lymphocyte separation medium and supernatant liquid adds the DMEM nutrient solution, washs 2 times, does cell counting and active the detection at last.
2. adopt biological method purification of hematopoietic stem cells.
2.1: use the selected by flow cytometry apoptosis hemopoietic stem cell
Progenitor cell film surface after hemopoietic stem cell and early stage differentiation is all contained a kind of protein and is called CD34 antigen.Anti-CD34 monoclonal antibody can be discerned this class human hematopoietic cell (Krause, Blood 1996 87:1-13).Therefore can carry out fluorescence-activated cell sorting and sorting cells.At first, the histocyte after above-mentioned process concentrated and have the antigenic monoclonal antibody of the anti-CD34 of fluorescence and mix was mutually hatched 30 minutes in room temperature or 4 ℃, washed 3 times with the DMEM nutrient solution that does not contain serum then.Hematopoietic stem in the placenta tissue just can and have fluorescently-labeled anti-CD34 monoclonal antibody to combine like this.After this group cell was put into flow cytometer, each cell was all by laser radiation, and according to the power that fluorescence takes place, instrument can be collected the cell that these have fluorescence automatically, and this method can provide the hemopoietic stem cell of 90% above purity.
2.2: adopt antibody-paramagnetic particle method purification of hematopoietic stem cells
Another scheme of the present invention is to adopt anti-CD34 monoclonal antibody and a kind of carrier to combine, and this supporting carrier is magnetic bead often.Above-mentioned spissated placenta tissue population of stem cells and this antibody are contacted, and meeting of the hematopoietic stem in the cell mass and antibody combine like this, and adhere to supporting carrier.At this moment, if magnetic bead, available suction iron device absorption magnetic bead.Also kept simultaneously hematopoietic stem, the cell that combines with magnetic bead is removed not in washing.If the monoclonal antibody that employing and vitamin H combine then can be used the biological post that contains antibiotic antibody, when the cell of binding antibody does not pass through this biology pearl, all be washed away.At last, change the pH value of washings, washing is the CD34+ cell down.Adopt above two kinds of methods, the purity of the hematopoietic stem of acquisition can reach more than 80%.
3. the hemopoietic stem cell biological nature after concentrated and purified.
The present invention relatively comes from the biological nature of histocyte, medullary cell and the cord blood cell of placenta respectively.Cultivate the histocyte colony come from placenta as table 1 for cell colony and form laterly, but the colony growth is fast, and granulocyte colony is big, can form repeatedly colony.When this three classes cell long-period is cultivated, from the histocyte of placenta can be very fast the formation attached cell, and these attached cells are very easy repeatedly goes down to posterity.This three classes cell with contain the hemopoietic stem cell stimulating factor, as SCF (10 μ g/ml), IL-3 (100 μ g/ml) and FLT-3 (50ng/ml), during long-term cultivation, the placenta tissue cell length of holding time, hematopoietic stem hyperplasia amount is many.The present invention adopts placenta tissue cell, cord blood cell respectively, the placenta tissue cell of balanced mix and cord blood cell are injected in the mouse of the immunodeficiency of isotropic substance and lethal quantity, find that this three classes cell can both grow in mouse bone marrow cells, the placenta tissue cell injects no any untoward reaction behind the mouse.
The present invention find medullary cell, cord blood cells and placenta tissue cell with the co-cultivation that contains the hematopoietic stimulation factor in (CFU-GM), formed hematopoietic cell colony has evident difference, and is as shown in table 1: _
Table 1: the comparison of medullary cell, cord blood cells and placenta tissue cell CFU-GM
| Colony begins formation time | The colony size | The granulocyte colony form | Repeatedly colony is cultivated | Attached cell | Attached cell is gone down to posterity | |
| Medullary cell | 5 days | Colony includes the about 50-200 of cell | Cell is little, circle, and growth disperses | Only can generate colony one time | The small amount of fibers like cell | Difficulty |
| The placenta tissue cell | 9 days | Colony includes cell greater than 1,000, and form superpose cell | Cell is big, circle, periphery is irregular, growth is concentrated, and is overlapped | Can generate colony four times | A large amount of or sheet, fibroblast-like cell cover with culture dish | 10-20 generation |
| Cord blood cells | 7 days | Colony includes the about 200-1,000 of cell | Cell is big, circle, and it is comparatively concentrated to grow | Can generate the secondary colony | The more fibrocyte of shape looses | 2-4 generation |
Case four
Set up the allosome placenta---umbilical cord blood bank
From Robinstein (Rubinstein P, et al; Proc.Natl.Acad.Sci.USA92,10 119-10122,1995) set up since the umbilical cord blood bank, worldwide carried out 2,000 many cases umbilical hemopoietic stem cell is transplanted, saved many patients' life, since karyocyte in (1) Cord blood and ancestral cells limited very be not suitable for the big patient of body weight and transplant; (2) may since the Cord blood inner cell contain stroma cell maybe can change into stroma cell than in the marrow for few, thereby the bleeding of the umbilicus poor growth in the patient after transplanting.The placenta tissue cell can remedy the characteristics of Cord blood just, thereby can set up a large-scale placenta---umbilical cord blood bank, supply the patient of any age and body weight to carry out hematopoietic stem cell transplantation, its step is by the following (Zhou Shengli that forms, Chinese experimental hematology 2001,2:153-160).
1. set up computer networking and bar code system: be convenient to data storage, guarantee all data accurately, and can be rapidly join the type result queries to placenta---the cord blood stem cell that conforms to patient HLA, in order to clinical transplantation according to patient's HLA.
2. coact with hospital and collect placenta and Cord blood: solicit under pregnant woman's situation about agreeing, collect the Cord blood and the placenta of no correlated inheritance medical history in every detection and the pregnant woman family.
3. detect: the every detection relevant that all need try again of all Cord bloods of collecting from hospital with virocyte, mould and inherited disease, have only the bleeding of the umbilicus of every detection index feminine gender just can offer clinical transplantation.
4. to join the accuracy of type be to be related to whether to transplant one of chief factors of success to tissue matching (HLA): HLA, and the HLA antigen that is usually used in umbilical cord blood transplantation has I class: A, B and II class DR.The type of joining this two class HLA need adopt molecular biology (DNA) to detect, and detects and more need use high resolution to II class DR.
5. placenta and the preservation of bleeding of the umbilicus ancestral cells, extraction, purifying: spissated Cord blood ancestral cells and placenta tissue ancestral cells and stored frozen agent (DMSO) mix, profound hypothermia cools to-80 ℃ in liquid nitrogen, transfers to (196 ℃) prolonged preservation in the liquid nitrogen then.
6. freeze thawing and hematopoietic stem cell transplantation: when clinical affirmation need be used certain routine bleeding of the umbilicus in the hemopoietic stem cell bank and placenta tissue stem/progenitor cells, before patient's bed, in liquid nitrogen, take out bleeding of the umbilicus and placenta tissue ancestral cells, in 37 ℃ of electronic water baths, melt, when hemocyte almost is about to freeze thawing and finishes, add the low molecular dextran balanced mix, give patient's infusion in order.
Case five
Foundation is from the body placenta---umbilical cord blood bank
From the body placenta---umbilical cord blood bank is that the pregnant woman is after newborn infant's birth, store placenta tissue cell and Cord blood ancestral cells, its objective is (1) because placenta and Cord blood contain abundant hematopoietic stem, storage has just been avoided after the child grows after the birth, if suffer from tumour, especially during hematologic disease, the danger when lacking donor, this is particularly suitable for having in the family tumour or hemopathic newborn infant; (2) present, a large amount of scientific experiments prove, contain can be divided into various histocytes in early days in tissue, as the stem cell of liver, skin, nerve etc.Life is early stage more, and the content of this stem cell is just high more.If can preserve the histocyte in the placenta, set up a human bank to the newborn infant beyond doubt.In neonatal growth growth course,, all can be divided into needed cell, for clinical application by the placenta tissue cell in vitro culture because a variety of causes as wound, organ failure etc., and needs cells of organs to transplant.Because these cells are to come from neonatal autogenous cell, do not exist and repel and problem such as can not grow, say in some sense, can provide the chance of life for the second time for the newborn infant.Some, remaining and allosome placenta---umbilical cord blood bank is the same except that following for specific procedure.
1. when collecting placenta and bleeding of the umbilicus, pregnant woman or its lineal relative will with our book of signing an agreement, agreement has valency to preserve neonatal placenta and navel blood stem cell by us, and we need guarantee that also not getting the pregnant woman family members agrees, must not be used for anyone to this part cord blood cells.
2. bleeding of the umbilicus ancestral cells and placenta tissue cell will divide many bags of storages, so that use to the newborn infant in the different time gradation.
3. when the purposes that needs except that hematopoietic stem cell transplantation, for example need to be used for hepar damnification, dermatoplasty etc., press clinical requirement freeze thawing placenta tissue cell, in cell culture fluid (as 1640, DMEM), cultivate, treat that the placenta tissue cell covers with culturing bottle after, use the nutrient solution that contains stimulating factor accordingly instead, as liver cell stimulating factor, neurocyte stimulating factor, impel the placenta tissue cell to corresponding cytodifferentiation, treat that cytodifferentiation fully after, use to patient.
4. work as the newborn infant in growth, growth course, any reason causes that bleeding of the umbilicus and the tissue stem cell that can use its storage at any time substitute its ill cell when needing hematopoietic stem cell transplantation to save its life.
Gene therapy is the effective means of current treatment various diseases.Placenta tissue cell and bleeding of the umbilicus ancestral cells are the fine carriers of gene transfection.The plasmid that has normal or the gene of curing the disease can be effectively chimeric be gone in the DNA chain of placenta tissue cell, and constantly duplicates along with the division of cell, be transcribed into can marking protein RNA, thereby play the effect for the treatment of disease.
Claims (3)
1. one kind is extracted the method that hemopoietic stem cell is used to set up hemopoietic stem cell bank from placenta tissue, and described method is divided into the process of collecting step, detecting step, extraction step, concentrated and purified step, and it is respectively:
(1) collect step: collection Cord blood and placenta also adheres to bar coded sticker and deposits in 4 ℃ of refrigerators, is no more than 24 hours;
(2) detect step: institute's refrigerated Cord blood and placenta are extracted sample and carry out following detection,
Hepatitis virus and corresponding antibodies,
The venereal disease detection,
The malicious antibody test of AIDS (AIDs),
The heredity hemolytic disease,
Have nuclear leukocyte and mononuclearcell the counting,
Hematopoietic stem detection by quantitative (CD34),
Tissue matching (HLA),
The detection of hematopoietic stem qualitative detection (CFU-GM);
(3) extraction step: placenta tissue under sterile environment and condition, is cleaned placenta with stroke-physiological saline solution, wash all blood clots and hematocele, adopt sterilizing agent to kill any possible bacterial contamination;
Use phosphoric acid buffer (PH7.4) once more, low molecular dextran and cell culture fluid (DMEM) clean in order;
To mix jointly through umbilical cord blood plasma, DMEM nutrient solution and placenta cells after selecting and spend the night; Wipe out the placental membrane on umbilical cord and top layer, application is torn, and grinds and the DMEM process of washing is collected placenta cells in 50 milliliters the centrifuge tube, and with 1000rpm, 5 minutes, 4 ℃ centrifugal, abandoning supernatant in whizzer;
Adopt the syringe needle of 16G, 18G and 20G to filter, placenta cells is made individual cells;
Placenta cells after the preparation is done following detection:
Cell and mould are cultivated,
Nuclear leukocyte and mononuclearcell counting is arranged,
Hematopoietic stem detection by quantitative (CD34),
Hematopoietic stem qualitative detection (CFU-GM),
The activity of hematopoietic stem (platform is expected blue dyeing);
The mononuclearcell that the placenta cells of the placenta tissue after detecting is produced is with 1 * 10
8/ ml and DMSO mix, and with 10% DMSO and low molecular dextran concentration, cool to subzero 80 ℃ in the liquid nitrogen minuent, transfer in liquid nitrogen (186 ℃) liquid phase profound hypothermia then and preserve;
(4) concentrated and purified step: the placenta tissue cell and the hydroxyethylamyle that extract are pressed 1: 6 mixed;
In whizzer centrifugal 10 minutes, remove red corpuscle with 500g;
With the mixed of lymphocyte separation medium by 1: 4;
In whizzer centrifugal 30 minutes, remove remaining red corpuscle and mature granulocyte with 1000g;
Collect and wash the hemopoietic stem cell group of each different developmental phases;
Set up stem cell bank.
2. according to claim 1ly from placenta tissue, extract the method that hemopoietic stem cell is used to set up hemopoietic stem cell bank, it is characterized in that the step of described concentrated and purified hemopoietic stem cell can also be:
Accurately measure isolating histocyte volume from placenta;
Histocyte is washed secondary with the DMEM liquid that does not contain serum;
With the ratio adding hydroxyethylamyle (HES) of the histocyte after the washing, on omnipotent shaking table, mixed 5 minutes in 1: 6;
Get the tissue that shakes after mixing with the speed of 200g under 12 ℃ of temperature, the condition of 5 minutes time, go in the whizzer centrifugally, afterwards, oppress with the blood plasma squeezer and to be rich in histiocytic liquid and to discard to another centrifuge tube and be rich in erythrocytic throw out, obtain supernatant liquor;
Once more with 12 ℃ of temperature, the condition of 15 minutes time is centrifugal with the speed of 800g, afterwards abandoning supernatant with the supernatant liquor that obtains;
Collection is sunken to the pipe cell at the end, adds DMEM liquid sedimentation cell is made suspension, is added to the upper strata of lymphocyte separation medium with 1: 4 ratio, then with 800g, and 12 ℃ of temperature, 30 minutes time is centrifugal;
The cell of absorption between lymphocyte separation medium and supernatant liquid adds the DMEM nutrient solution, washs 2 times, does cell counting and active the detection at last;
Set up stem cell bank on the basis of the above.
3. according to claim 1ly from placenta tissue, extract the method that hemopoietic stem cell is used to set up hemopoietic stem cell bank, it is characterized in that the step of described concentrated and purified hemopoietic stem cell can also be:
Use the selected by flow cytometry apoptosis hemopoietic stem cell, and:
Sorting has fluorescently-labeled anti-CD34 monoclonal antibody;
To and have the antigenic monoclonal antibody of the anti-CD34 of fluorescence through the histocyte after concentrating and mix mutually, in room temperature or 4 ℃, hatch 30 minutes, wash 3 times with the DMEM nutrient solution that does not contain serum then;
With the histocyte after the washing with have fluorescently-labeled anti-CD34 monoclonal antibody and combine and put into flow cytometer through laser radiation;
According to the power that fluorescence takes place, collect the hemopoietic stem cell of 90% above purity;
Set up stem cell bank on the basis of the above.
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