CN101429495A - Cultivation method for human peripheral blood source hemopoietic stem cell - Google Patents
Cultivation method for human peripheral blood source hemopoietic stem cell Download PDFInfo
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Abstract
The invention relates to a method for culturing human peripheral blood hematopoietic stem cells and particularly provides a method for performing short-term culture on the human peripheral blood hematopoietic stem cells acquired in vitro to improve the antitumor activity of the human peripheral blood hematopoietic stem cells. The invention also relates to a culture medium for the short-term culture of hematopoietic stem cells. Finally, the invention also relates to a method for transplanting the hematopoietic stem cells, which can remarkably improve the survival ability of patients.
Description
[technical field]
The present invention relates to a kind of cultural method of human peripheral blood source hemopoietic stem cell, specifically, the invention provides a kind of human peripheral blood source hemopoietic stem cell and carry out Short-term Culture to improve the method for its anti-tumor activity to external collection.The invention still further relates to a kind of substratum that is used for hemopoietic stem cell is carried out Short-term Culture.The present invention also relates to a kind of implantation method of hemopoietic stem cell at last, and it can significantly improve patient's viability.
[background technology]
(hematopoietic stem cells HSC) is a small set of hemopoietic forebody cell with height the of self-replication capacity and multidirectional differentiation potential to hemopoietic stem cell.Its essential characteristic is: 1) Gao Du self or the of self-replication capacity; 2) can break up all types of hemocytes of generation.Under the normal circumstances, hemopoietic stem cell constantly produces a large amount of progenitor cells by the mitotic division of asymmetry, and hemopoietic progenitor cell is further bred and broken up, and replenishes and keep the human peripheral blood cell.Grow in the pedigree at hematopoietic cell, the top, position of hemopoietic stem cell has extremely important biological function.During the nearly last ten years, along with carrying out of autologous peripheral blood stemcell transplant treatment, deep day by day to the research of hemopoietic stem cell.The proliferation and differentiation of cell, reach maturity, move settle down, the many basic problems in the life science such as old and feeble apoptosis and canceration, become the main focus of fundamental research.
Hematopoietic stem cell transplantation (hematopoietic stem cell transplantation, HSCT) be a kind of methods of treatment that progressively grows up nearly decades, by heavy dose of chemicotherapy or other immunosuppression pre-treatment, remove acceptor interior tumor cell, unusual clone cell, the blocking-up pathogenesis, giving acceptor, make to be subjected to volume reconstruction normal hematopoiesis or immunologic function, thereby reach a kind of treatment means of therapeutic purpose then from body or allosome hematopoietic stem cell transplantation.The effective means that present hematopoietic stem cell transplantation is the pernicious or intractable hemopathy of treatment/radical cure, heredopathia, immunological disease and some solid tumor (referring to Paneesha S, Milligan DW.Stem celltransplantation for chronic lymphocytic leukaemia.Br J Haematol.2005Jan; 128 (2): 145-52.; Bredeson CN, Pavletic SZ.Considerations when designing aclinical trial of haematopoietic stem cell transplantation for autoimmune disease.BestPract Res Clin Haematol.2004 Jun; 17 (2): 327-43).Nineteen fifty-five Thomas etc. takes the lead in carrying out the work of people's bone marrow transplantation, and obtains Nobel Prize in medicine, and so far half a century, at present, hematopoietic stem cell transplantation is widely used in clinical at home and abroad.
Conventional hematopoietic stem cell transplantation method, source according to the transplantation donor cell is divided into autologous stem cell transplanting (Tomblyn M, Winter JN.Autologous hematopoietic stem cell transplant infirst remission in non-Hodgkin ' s lymphoma.Expert Rev Anticancer Ther.2003Jun; 3 (3): 281-94) with allosome hematopoietic stem cell transplantation (Bensiger WI, Clift R, Martin P, et al.Allogeneic peripheral blood stem cell transplantation in patients with advancedhematologic malignancies:aretrospective comparison with marrow transplantation.Blood, 1996,88:2794-2799.) two big classes.The allosome stem cell transplantation is an ideal mode comparatively in theory, but since exist donor source limited, join the type problem and transplant problems such as relevant case fatality rate height, its clinical application is subjected to certain limitation.The relevant case fatality rate of autologous stem cell transplantation is relatively low, so use more at present.Hematopoietic stem cell transplantation is divided into marrow, peripheral blood and umbilical cord blood hematopoietic stem cell according to the difference in blood source and is transplanted.Because having with respect to the stem cell transplantation in other sources, autologous peripheral blood stemcell transplant gathers safe and simple, hematopoiesis and immunity system is rebuild advantages such as fast, and present observed short term efficacy is satisfactory, is a kind of faster method of the more development of present clinical employing therefore.
The autologous peripheral blood stemcell transplant step is divided into following several big basic step usually in the prior art at present:
1) mobilization of peripheral hematopoietic stem cells and collection;
2) preservation of peripheral hematopoietic stem cells, recovery;
3) peripheral hematopoietic stem cells is to the intravital feedback of acceptor patient;
4) clinical follow after the transplanting.And according to the difference of donor, the pre-treatment step that also can add prevention patient graft versus host disease (GVH disease) as required is (referring to autologous peripheral blood stemcell transplant [M], Beijing: People's Health Publisher, 2000; " allogeneic peripheral blood stem cell transplantation treatment malignant hematologic disease " Chinese Journal of Hematology the 23rd the 8th phase of volume of August in 2002).
But there is following shortcoming in the prior art in the above-mentioned autologous peripheral blood stemcell transplant:
1) in the prior art, also there is not a kind of extracorporeal culturing method of short-term raising hemopoietic stem cell anti-tumor activity at present, and the hemopoietic stem cell isolated culture base that is fit to;
2) in traditional hematopoietic stem cell transplantation method, after collecting hemopoietic stem cell in the body, hemopoietic stem cell places cell cryopreservation (frozen for autotransplantation usually, can be not frozen for heteroplastic transplantation, but the activation process that all lacks an external rapid lifting anti-tumor activity) preserve at-196 ℃ of liquid nitrogen in the liquid, thawing, the back is direct to be imported in the acceptor body, thereby the hemopoietic stem cell activity after preserving and all decline to some extent of quantity;
3) hemopoietic stem cell after transplanting is limited to the kill capability of tumour cell etc., so postoperative effect is general, and its inhibition ability to tumor growth remains further to be improved.
[summary of the invention]
The object of the invention is to remedy the defective that lacks hemopoietic stem cell isolated culture technology in the prior art, and a kind of effect cultural method of human peripheral blood source hemopoietic stem cell preferably is provided, and this cultural method can significantly improve the anti-tumor activity of hemopoietic stem cell.
The present invention also aims to provide a kind of special culture medium that is used for the hemopoietic stem cell in isolated culture human peripheral source, this substratum can effectively activate hemopoietic stem cell.
The present invention also aims to provide a kind of and cultivated the activatory hemopoietic stem cell that the back obtains by above-mentioned cell culture technology, and the application in oncotherapy.
The invention provides a kind of method of human peripheral blood source hemopoietic stem cell that improved to the kill capability of tumour, this method comprises the mobilization collection of peripheral blood source hemopoietic stem cell, place specific substratum to cultivate earlier the peripheral blood source hemopoietic stem cell that collects, and the cell after cultivating is carried out biologic activity detect.After the peripheral blood source hemopoietic stem cell collection after cultivating, washing, feed back and give tumour patient, can find that patient's appetite increases, sleep and physical situation are improved, cassette scoring index improves, and the patient of oedema, action obstacle is arranged individually, and symptom is clearly better after treatment.This show feed back to this cell in the body after, can improve immunizing power, play the treatment tumour purpose.
Used substratum called after IN-VITR03 in the above-mentioned cultural method of the present invention contains 1000 milliliters of the Dulbecco minimum mediums (IMDM) of Iscobe improvement, the lipid acid of 0.1-10 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 5-20 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
With 1000 milliliters be foundation, nutritional factor is: Regular Insulin, 2 mercapto ethanol, Sodium.alpha.-ketopropionate, NaH2CO3, glutamine.The usage quantity of nutritional factor is that convention amount gets final product, and the usage quantity of these components in substratum belongs to common practise.Inventive point of the present invention is the combination of said components, and the contriver has passed through multiple test, finds that above-mentioned combined effect is very good.Particularly, the present invention relates to following content:
The invention provides a kind of method that improves human peripheral blood source hemopoietic stem cell to the kill capability of tumour, this method comprises at first mobilizes and gathers the hemopoietic stem cell in peripheral blood source; Then the peripheral blood source hemopoietic stem cell that collects is carried out external short-term activation culture, and the cell after cultivating is carried out biologic activity detect; To feed back after the peripheral blood source hemopoietic stem cell collection after cultivating, the washing.
The present invention also provides a kind of method of external short-term activation culture hemopoietic stem cell, and this method is: after the physiological saline washing, with aseptic PBS two-fold dilution, promptly ratio is the dilution of 1:1 with the peripheral blood source hemopoietic stem cell of fresh collection; Remove red corpuscle with the Ficoll parting liquid, centrifugation purpose hemopoietic stem cell, centrifugal condition is: 1800rpm, 20min; With PBS washing hemopoietic stem cell twice, 1400rpm, 15min is centrifugal; Trypan blue exclusion method counting sample, adding contains the serum free medium of rhIL-2 100-5000U/mL and IFN-γ 100-5000U/mL and adjusts cell concn is 5 * 106/mL, places incubator to cultivate, culture condition is 37 ℃, 5%CO2; Collecting cell behind cultivation activation 3～4h, and carry out biological activity assay.
Cultural method as indicated above, the concrete composition of wherein said substratum is: contain 1000 milliliters of the Dulbecco minimum mediums (IMDM) of Iscobe improvement, the lipid acid of 0.1-10 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 5-20 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
With 1000 milliliters be foundation, the nutritional factor that above-mentioned substratum can also add is: Regular Insulin, 2 mercapto ethanol, Sodium.alpha.-ketopropionate, NaH
2CO
3, glutamine.Can add according to conventional usage quantity.
A kind of hemopoietic stem cell that adopts method short-term activation culture mentioned above to obtain.
Hemopoietic stem cell as indicated above application in preparation medicine for treating tumor thing.
Application as indicated above, wherein said tumour are lymphoma, lung cancer, colorectal carcinoma or mammary cancer.
[description of drawings]
Fig. 1: the phenotype characteristics before and after peripheral blood source hemopoietic stem cell collection, the cultivation;
Fig. 2: the unicellular colony form of grain that forms before and after peripheral blood source hemopoietic stem cell is cultivated is (before the A. activation; B. after the activation);
Fig. 3: the different targets of imitating are cultivated the killing activity of front and back to multiple noumenal tumour than following peripheral blood source hemopoietic stem cell;
Fig. 4: 4-1 before the cell colony that adopts the cultivation of substratum of the present invention and cultural method to obtain is cultivated, cultivate back 4-2.
[embodiment]
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Following embodiment is not to be restriction protection scope of the present invention only for the activation culture method of the hemopoietic stem cell that inventor's peripheral blood is originated is described.The tumor cell line of used chemical reagent, cytokine and experiment usefulness is all purchased if no special instructions in GIBICO company among the following embodiment; Involved detection among the following embodiment, and evaluation method is and well known to a person skilled in the art detection, evaluation method.
Embodiment 1
The collection of peripheral blood source hemopoietic stem cell
Following embodiment is not to be restriction protection scope of the present invention only for the activation culture method of the hemopoietic stem cell that inventor's peripheral blood is originated is described.The tumor cell line of used chemical reagent, cytokine and experiment usefulness is all purchased if no special instructions in GIBICO company among the following embodiment; Involved detection among the following embodiment, and evaluation method is and well known to a person skilled in the art detection, evaluation method.
Embodiment 2
The external Short-term Culture activation of peripheral blood source hemopoietic stem cell.
Embodiment 2-1
With the peripheral blood source hemopoietic stem cell sample of fresh collection after the physiological saline washing, with aseptic PBS two-fold dilution.Remove red corpuscle with the Ficoll parting liquid, centrifugation purpose hemopoietic stem cell, centrifugal condition is: 1800rpm, 20min.With PBS washing hemopoietic stem cell twice, 1400rpm, 15min is centrifugal.Trypan blue exclusion method counting sample, adding contains the serum free medium of rhIL-2 5000U/mL and IFN-γ 1000U/mL and adjusts cell concn is 5 * 106/mL, places incubator to cultivate, culture condition is 37 ℃, 5%CO2; Cultivate activation 3 back collecting cells, carry out biological activity assay.
Wherein cultivating used medium component is: 1000 milliliters contain the Dulbecco minimum medium (IMDM) of Iscobe improvement, the lipid acid of 10 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 5 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
Embodiment 2-2
Method is as described in the embodiment 2-1, wherein cultivates used medium component to be: 1000 milliliters contain the Dulbecco minimum medium (IMDM) of Iscobe improvement, the lipid acid of 0.1 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 20 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
Embodiment 2-3
With the peripheral blood source hemopoietic stem cell sample of fresh collection after the physiological saline washing, with aseptic PBS two-fold dilution.Remove red corpuscle with the Ficoll parting liquid, centrifugation purpose hemopoietic stem cell, centrifugal condition is: 1800rpm, 20min.With PBS washing hemopoietic stem cell twice, 1400rpm, 15min is centrifugal.Trypan blue exclusion method counting sample, adding contains the serum free medium of rhIL-2 100U/mL and IFN-γ 5000U/mL and adjusts cell concn is 5 * 106/mL, places incubator to cultivate, culture condition is 37 ℃, 5%CO2; Collecting cell behind the cultivation activation 4h carries out biological activity assay.
Wherein cultivating used medium component is: 1000 milliliters contain the Dulbecco minimum medium (IMDM) of Iscobe improvement, the lipid acid of 5 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 10 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
Embodiment 2-4
Method is as described in the embodiment 2-1, wherein cultivates used medium component to be: 1000 milliliters of the Dulbecco minimum mediums (IMDM), the lipid acid of 0.1-10 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 5-20 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml, Regular Insulin, 2 mercapto ethanol, Sodium.alpha.-ketopropionate, NaH2CO3, the glutamine that contain the Iscobe improvement.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
Embodiment 2-5
Method is as described in the embodiment 2-1, wherein cultivates used medium component to be: 1000 milliliters of the Dulbecco minimum mediums (IMDM), the lipid acid of 0.1-10 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 5-20 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml, Regular Insulin, 2 mercapto ethanol, Sodium.alpha.-ketopropionate, NaH2CO3, the glutamine that contain the Iscobe improvement.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
Embodiment 2-6
Method is as described in the embodiment 2-1, wherein cultivates used medium component to be: 1000 milliliters of the Dulbecco minimum mediums (IMDM), the lipid acid of 0.1-10 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 5-20 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml, Regular Insulin, 2 mercapto ethanol, Sodium.alpha.-ketopropionate, NaH2CO3, the glutamine that contain the Iscobe improvement.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
Embodiment 2-7
Method is as described in the embodiment 2-1, wherein cultivates used medium component to be: 1000 milliliters of the Dulbecco minimum mediums (IMDM), the lipid acid of 0.1-10 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 5-20 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml, Regular Insulin, 2 mercapto ethanol, Sodium.alpha.-ketopropionate, NaH2CO3, the glutamine that contain the Iscobe improvement.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
Embodiment 2-8
Method is as described in the embodiment 2-1, wherein cultivates used medium component to be: 1000 milliliters of the Dulbecco minimum mediums (IMDM), the lipid acid of 0.1-10 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 5-20 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml, Regular Insulin, 2 mercapto ethanol, Sodium.alpha.-ketopropionate, NaH2CO3, the glutamine that contain the Iscobe improvement.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
The bioactive detection of the peripheral blood source hemopoietic stem cell after cultivating
3.1 phenotype detects
Adopt conventional direct immunofluorescence to measure the expression of cultivating CD3+CD 4+, CD3+CD 8+, CD3-CD 56+CD 16+, CD8+CD 28+, CD45RO+CD 8+, CD4+CD 25+, CD25+, CD 69+ in the peripheral blood source hemopoietic stem cell of front and back respectively in conjunction with flow cytometer.Get 3 * 106 cells, 1400 rev/mins, centrifugal 5 minutes.Abandon supernatant, with the PBS washed twice, the same centrifugal cell is suspended among the 0.5mlPBS, counting moving into cell in U type 96 orifice plates, and every porocyte number adds fluorescence antibody mixing gently respectively for being no less than 5 * 105, and 4 ℃, lucifuge were hatched 30-60 minute.1000 rev/mins, centrifugal 5 minutes, abandon supernatant.Each adds twice, 1000 rev/min of 200 μ l PBS washing, centrifugal 5 minutes.Every hole adds 100 μ l, 1% Paraformaldehyde 96 re-suspended cell and fixing.30min before last machine is analyzed, every pipe accurately add 100 μ l Flow-Count fluorescent microspheres, mixing gently, lucifuge.Draw cell to the flow cytometer detector tube, and add an amount of PBS (0.5-1ml), go up machine testing behind the re-suspended cell.
Hemopoietic stem cell result after cultivating activation as shown in Figure 1.The activation of this presentation of results short-term can not influence the distribution and the ratio of various main cell subsets in the procedure for peripheral blood stem cells, but has increased the ratio of T and NK cell greatly, for the rising of antitumor killing activity provides strong cytology basis.
3.2 clonality detects
Adopt conventional semi-solid agarose method to measure the quantity of cultivating GM-CFU in the peripheral blood source hemopoietic stem cell of front and back.It is GM-CSF50ng, rIL-35ng that every milliliter of culture system of CFU-GM contains G CFS.More than every milliliter of soft agar that contains 20%FCS, 10% people AB serum (ABS), 1%FVBSA, 2 mercapto ethanol 5 * 10-5mol/L, 0.9% methylcellulose gum or 0.3% respectively, every milliliter of system adds 2 * 105 mononuclearcells, put the 35mm culture dish, cultivated 14 days at 37 ℃ of saturated humidity carbonic acid gas incubators, count colony down with inverted microscope, CFU-GM 〉=40 cell is 1 colony unit, with the formed colony of semi-solid soft agar assay, the substantive dyeing of available quick blood cell staining fluid, the form and the kind of observing its cell colony.
Detected result as shown in Figure 2.This presentation of results short-term activation can not influence quantity and the function of stem cell in the procedure for peripheral blood stem cells.
Embodiment 4
The killing activity of the hemopoietic stem cell before and after the activation is measured and is compared
Adopt conventional LDH method to measure cultivation front and back peripheral blood source hemopoietic stem cell and than under lymphoma raji cell, lung cancer 95D cell, colorectal carcinoma HCT-8 and mammary cancer MCF-7 (are available commercially from ATCC at difference effect target, U.S. typical case thing preservation center claims US mode typical case thing to collect the center again) killing activity.Method is summarized as follows: peripheral blood source hemopoietic stem cell is as the different effect cell before and after collecting activation respectively, and with RPMI-1640 washing 2 times, counting is adjusted cell concn to 4 * 106/ml.Collect target cell raji, 95D, HCT-8 and MCF-7, the washing counting is adjusted cell concn to 1 * 105/ml.Every kind of effector cell does 3 multiple holes, every hole 100 μ l, add in the 96 hole U templates, and compare 40:1 according to imitating target, 20:1,10:1 adds target cell, establishes the maximum release of target cell simultaneously, naturally discharge, nutrient solution blank and volume are corrected contrast, 200 μ l/ holes, 3 every group multiple holes, centrifugal 250g 4 minutes, 37 ℃ of 5%CO2 incubators were cultivated 6 hours, cultivating preceding 45 minutes of end, took out 96 orifice plates, correct the every hole adding of control group lysis buffer 20 μ l in maximum release group of target cell and volume, centrifugal 250g 4 minutes continues to cultivate after 45 minutes and takes out, and sucking-off 50 μ l supernatants in every hole are transferred to another 96 orifice plate, every hole adds substrate solution 50 μ l, lucifuge incubated at room 15 minutes adds 50 μ l stop buffers, with vibrator pigment granule is broken up, survey the absorbancy at wavelength 490nm place, press following formula:
CTL activity (%)=(experimental group-effector cell's nature release group-target cell nature release group)/(the maximum release group of target cell-volume is corrected control group-target cell nature release group) * 100%
Proofread and correct each hole absorbancy with the nutrient solution control group as blank.
Experimental result as shown in Figure 3, this result show no matter be imitate target than 40:1 or the situation of 20:1 under, the hemopoietic stem cell of collection is after the method for the invention is cultivated activation, its tumor killing effect all has remarkable increase.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, not departing from the these modifications or improvements basically of spirit of the present invention, all belong to the scope of protection of present invention.
Claims (6)
1, a kind of method that improves human peripheral blood source hemopoietic stem cell to the kill capability of tumour, this method comprise at first mobilizes and gathers the hemopoietic stem cell in peripheral blood source; Then the peripheral blood source hemopoietic stem cell that collects is carried out external short-term activation culture, and the cell after cultivating is carried out biologic activity detect; To feed back after the peripheral blood source hemopoietic stem cell collection after cultivating, the washing.
2, a kind of method of external short-term activation culture hemopoietic stem cell, this method is: with the peripheral blood source hemopoietic stem cell of fresh collection after the physiological saline washing, with aseptic PBS two-fold dilution; Remove red corpuscle with the Ficoll parting liquid, centrifugation purpose hemopoietic stem cell, centrifugal condition is: 1800rpm, 20min; With PBS washing hemopoietic stem cell twice, 1400rpm, 15min is centrifugal; Trypan blue exclusion method counting sample, adding contains the serum free medium of rhIL-2 100-5000U/mL and IFN-γ 100-5000U/mL and adjusts cell concn is 5 * 106/mL, places incubator to cultivate, culture condition is 37 ℃, 5%CO2; Cultivate activation 3
-Collecting cell behind the 4h, and carry out biological activity assay.
3, cultural method as claimed in claim 2, the concrete composition of wherein said substratum is: contain 1000 milliliters of the Dulbecco minimum mediums (IMDM) of Iscobe improvement, the lipid acid of 0.1-10 μ g/ml, the cholesterol of 1.5 μ g/ml, the glyceryl ester of 5-20 μ g/ml, the Transferrins,iron complexes of 300 μ g/ml.Buffered soln is sodium bicarbonate or HEPES, and the pH value of substratum is 7.0.
4, a kind of hemopoietic stem cell that obtains as method short-term activation culture as described in claim 2 or 3.
5, the application of hemopoietic stem cell as claimed in claim 4 in preparation medicine for treating tumor thing.
6, application as claimed in claim 6, wherein said tumour are lymphoma, lung cancer, colorectal carcinoma or mammary cancer.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102988415A (en) * | 2012-08-15 | 2013-03-27 | 王沁怡 | Natural killer cells (NK) prepared by industrializing human allogeneic nucleated cells and injection |
| CN104371973A (en) * | 2013-08-15 | 2015-02-25 | 协和华东干细胞基因工程有限公司 | Serum-free medium of immune cells |
| CN104388385A (en) * | 2014-11-25 | 2015-03-04 | 广州赛莱拉干细胞科技股份有限公司 | Culture method and application of human peripheral blood mesenchymal stem cell |
| WO2020147272A1 (en) * | 2019-01-16 | 2020-07-23 | 浙江大学 | Method for preparing heterogeneous hematopoietic stem/progenitor cells by non-mobilized peripheral blood |
-
2007
- 2007-11-09 CN CNA2007101662818A patent/CN101429495A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102988415A (en) * | 2012-08-15 | 2013-03-27 | 王沁怡 | Natural killer cells (NK) prepared by industrializing human allogeneic nucleated cells and injection |
| CN104371973A (en) * | 2013-08-15 | 2015-02-25 | 协和华东干细胞基因工程有限公司 | Serum-free medium of immune cells |
| CN104371973B (en) * | 2013-08-15 | 2017-12-05 | 协和华东干细胞基因工程有限公司 | A kind of serum free medium of immunocyte |
| CN104388385A (en) * | 2014-11-25 | 2015-03-04 | 广州赛莱拉干细胞科技股份有限公司 | Culture method and application of human peripheral blood mesenchymal stem cell |
| CN104388385B (en) * | 2014-11-25 | 2018-06-08 | 广州赛莱拉干细胞科技股份有限公司 | A kind of cultural method of human peripheral mescenchymal stem cell and application |
| WO2020147272A1 (en) * | 2019-01-16 | 2020-07-23 | 浙江大学 | Method for preparing heterogeneous hematopoietic stem/progenitor cells by non-mobilized peripheral blood |
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Open date: 20090513 |