CN102796699A

CN102796699A - Novel stem cells, method for screening same, kit and application thereof

Info

Publication number: CN102796699A
Application number: CN2011101496532A
Authority: CN
Inventors: 李福生; 卢磊磊; 卢淼淼; 卢晶晶
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-05-25
Filing date: 2011-05-25
Publication date: 2012-11-28

Abstract

The invention belongs to the technical field of cells and discloses novel adult stem cells, a method for screening the same, a kit and application thereof. Biopsy samples, in vitro tissue samples, and cell line samples of normal tissues and tumors, precancerous tissues and tumors, carcinogenic tissues and metastatic tumors or cancer tissues of rodent, human or other mammals are screened by using chemotherapeutics and cell markers which are combined so as to obtain the stem cell groups. Moreover, the invention also discloses a method for screening and identifying the stem cells from the mixed cell groups, and the kit; and the method and the kit have the advantages of strong specificity, simplicity, convenience, quickness, practicality and effectiveness. The novel stem cells, the method for screening the same and the kit can be applied to clinic treatment, basis and application research, treatment, tissue engineering, medicine screening, and repairing and regeneration of diseased tissues and organs which loose functions well, and have wide prospect.

Description

A kind of new dry cell, its screening method, test kit and uses thereof

Technical field

The invention belongs to the cell technology field; Particularly; Relate to a kind of novel adult stem cell, its screening method, test kit and uses thereof, particularly relate to method through the chemotherapy drugs in combination cell marking stem cell that screening obtains before mammiferous normal, the lesion/cancer of rodent, the mankind or other, the biopsy of lesion/cancer property, metastatic tumor/cancerous tissue, in vitro tissue sample, clone sample, more specifically; Relate to adopt chemotherapeutics in advance cell groups handle; Remove proliferative cell crowd wherein, and then screening, enrichment and activation population of stem cells wherein, afterwards; Employing can labeled stem cells mark to its with or the screening method discerned of filial generation; Especially, relate to a kind of being used for from the test kit of mixed cellularity group screening and the novel adult stem cell of mark, the novel adult stem cell of screening loses the pathological tissues of function or the application in the organ in clinical, basic and applied research, treatment, organizational project, drug screening, reparation and regeneration.

Background technology

As everyone knows, stem cell has following characteristics: 1) secular self ability; 2) can produce well differentiated other kind cell; 3) itself is in undifferentiated state; 4) generally be in slow cycle or quiescent condition; 5) be in the tissue in the corresponding microhabitat (niches).Stem cell comprise embryonic stem cell (Embryonic stem cells, ESCs) and adult stem cell (Adult Stem Cells, ASCs); Embryonic stem cell is totipotent; The tissue arbitrarily of in body, can regenerating in theory in the health almost, but because it has run into problem at aspects such as the ethics of obtaining, morals on the one hand, it has run into comparatively serious problem on using on the other hand; The embryonic stem cell poor controllability of for example transplanting; The risk that the tumour of being grown to is arranged, and it will receive immune repulsion of acceptor or the like behind transplant recipient, thus limited its application.

Seeing that the problems referred to above that embryonic stem cell research and application are run into, the mode that scientist's expectation is programmed through nuclear lets into the state that somatocyte can be got back to embryonic stem cell-like, through the research of decades; Finally, stretched more (YamanakaS, Cell in the Japanese scientist mountain in 2006; 2006126 (4): 663-676, Japanese Patent 2008-131577) adopt four inducible factor Oct4, nanog, sox2 etc. to import in the normal people inoblast, it is embryonic stem cell-like for successful reprogrammed; Be called as induce pluripotent stem cell (induced pluripotent stem cells, iPSCs), this cell has similar hESC's characteristic; Though it is similar with embryonic stem cell, has and to be divided into institute's potentiality in a organized way in the organism, Lowry and Plath discovery (Lowry etc.; Cell Stem Cell, 2009.5 (1): 111-123), when carrying out the molecular label comparison; Some expression of gene is obviously different with the iPS cell in the embryonic stem cell, compares with embryonic stem cell, and the iPS cell research still has problem to need to be resolved hurrily; IPSCs tool high gene distortion frequency (Loring etc. more for example; Cell Stem Cell, 2011.8 (1): 106-118), tumorigenicity risk, programming efficiency are low or the like need overcome, thereby; Although the researchist has obtained impressive progress, programme cell again is used to treat the also more more breakthroughs of needs of disease.

Adult stem cell is other one type of undifferentiated cell type that has reparation, regeneration, substitutes ability; Be usually located in the specific microhabitat; After receiving extraneous damage; It can mobilize or produce new stem cell, forms new functioning cell through the mode of breeding, break up, thereby regulates tissue and organ cell's quantity maintenance running balance.The adult stem cell that the versatility characteristics of adult stem cell at first obtain in marrow is able to find.Hemopoietic stem cell is one of the most detailed adult stem cell of research at present; Be the theme of stem cell area research in nearly in the past 40 years always; They carry out the cell fission of self in vivo; Be divided into all hematopoiesis compositions in unicellular level, and on function, make the myelosuppressive animal and human's of severe marrow be able to recover.

The basis of adult stem cell research is effective identification, the screening and separating to ancestral cells with one of its application prerequisite and most critical condition clinically.More and more evidences shows that adult stem cell is found in (the Moore KA of multiple mature tissue recently; Science; 2006.311 (5769): 1880-1885), and be likely by the stem cell coexistence (Li etc., the Science that are in tranquillization attitude (outer and low metabolism state of cell cycle) and activation (cell cycle is interior and can not keep dna marker) attitude; 2010.327 (5965): 542-545); But separate stem cells is still and has challenging task from adult, is its comparatively small amt on the one hand and more is difficult to location, the various tissue specificity stem cells of screening and separating, is to lack reliable specific molecular marker thing on the one hand in addition it is discerned.

Current, mainly contain following prior art and disclose the adult stem cell how to obtain being correlated with:

1) employing molecular marker screening candidate's stem cell, for example Thy-1 ^LowOr FLK2 ^-Lineage ^-Sca-1 ⁺C-Kit ⁺Sorting hemopoietic stem cell crowd (Christemsen JL etc., PNAS, 2001.98:14541-14546; Uchida N etc., Experimental hematology 1996.24:649-659), uses CD14 ⁺The method of immunomagnetic isolation is used α from the new population of stem cells (purification of PCT/055950 stem cell, identification and purposes) of peripheral blood screening ₆ ^Bri10G7 ^Dim(LiA etc., PNAS, 1998.95 (7): 3902-3907; Tani H etc., PNAS, 2000.97 (20): 10960-10965; Lavker RM etc., PNAS, 2000.97 (25): the epithelial stem cell that 13473-13475) identification is human, rodent is originated, but its validity is still waiting further confirmation;

2) carry out screening, the enrichment of stem cell with other technologies associating molecule marker: be the method for a kind of stem cell target-locating and enriching of CN108677A such as publication number; Adopt to the significant antigenic specific antibody of stem cell surface and combine with stem cell; Immunomagnetic beads on the mark again, transplant the back through two opposite poles with stem cell enrichment in target tissue.The persons of research group such as Azuma adopt machinery or enzymic digestion to separate the method that the back combines the known molecular mark; Hope direct separation from adult tissue goes out ancestral cells; Thereby it is differentiated and follow-up functional study (AzumaH etc., Hepatology, 2003.37 (6): 1385-1389; Wright N etc., Biochem Biophys Res Commun, 2008.366 (2): 367-372).Through experiments such as mouse genetics sign pedigree tests, in conjunction with the molecular marker of being found, for example Musashi-1, Lgr5 etc. provide possibility (Barker N etc., Nature, 2007.449 (7165): 1003-1007 of identifying stem cell; Haegebarth A etc., Am J Pathol, 2009.174 (3): 715-721), yet their validity is still waiting further confirmation.Weak point is, marked by magnetic bead may influence cell viability, and enzymic digestion is longer action time; Cell viability is not high after the enrichment; And these isolated cells crowd is not the homogeneity type, but is made up of the mixed cellularity group of many different differentiation states or multiplication capacity, and is very impure.

3) the side group sieve method (side population, SP), the ancestral cells that differentiation degree is lower is expressed some ATP binding transport albumen (ATP-binding cassette usually; ABC), cell often utilizes the ATP energy in cell, to discharge external toxic substance, to reach the purpose of protection self; (Goodell etc. such as early stage Goodell; J Exp Med, 1996.183 (4): discovering 1796-1806), after medullary cell being dyeed with Hoechst33342; Hemopoietic stem cell has the ability of very strong discharge dye liquor, is called as the side group cell.Recently; (Wulff etc. such as Wulff; Haematologica; 2003.88 (4): 368-378) in Mouse Liver, be separated to a group SP cell, CD45

or CD45-cell subsets, its shared per-cent is 1%; Vitro culture all can produce the clone of liver system and hematopoiesis system origin, and similarly result of study also is separated to similar SP cell in the mankind's adult hepatic tissue.This method has been used to screening potential ancestral cells crowd from other organs and tissue; Weak point is; After from solid tissue, filtering out the SP cell,,, this type of cell possibly influence their ability of reinventing after transplanting owing to obtain the limited in one's ability of viable cell.

4) adopt the experiment of damage combined transplantation to carry out the stem cell The Characteristic Study; For example: (Li WL etc., Stem cells, 2006.24 (2): such as Li 322-332) from retrorsine/partially hepatectomized (Retrorinine/Partial Hepatectomy; Rs/PH) be separated to a kind of cell mass in the liver of processing back adult mice; Between during cultivation, it can be divided into liver cell and bile duct epithelial cell, and Li etc. think that this group cell has ancestral cells potential; Be called as the liver epithelial progenitor cell (liver epithelial progenitor cells, LEPCs).It has the potential of differentiation the transplantation experiments prompting.Yet similarly the medicine that adds of damage test has carcinogenic nature usually, and the cell generation canceration during it causes organizing changes the precursor cell with stem cell characteristic into and it be unclear that.

5) carry out the discriminating of stem cell according to nuclear morphology; For example; The PCT/021504 patent discloses a kind ofly identifies the method for stem cell according to karyomorphism, and through observing the karyomorphism that distributes in the tissue sample after treatment, the type of nuclear morphology type is selected from: bell, cigar shape, cohesion are spherical, spherical, avette, sausage shaped, kidney shape and bullet shaped; Thereby allowing microhistology/pathological technique personnel according to nuclear morphology, is stem cell/non-stem cell with cell differentiation.

6) growing animal pulse-chase method is carried out the identification of stem cell; Early stage discovers, when add can the mark of " mark " DNA after, and divide rapidly progenitor cell and compare; Stem cell has ability (the Bickenbach JR of long-term reservation dna marker owing to its characteristic with slow cycle or quiescent stage; J Dent Res, 1981.60 (Spec No C): 1611-1620), this method has been used to discern the stem cell in the tissues such as small intestine, uterine endometrium, pancreas, renal papilla.

7) based on the functional character of stem cell, for example tolerate the processing of medicine or other reagent, reported the method that is used to activate stem cell, (Gordon MY etc., Leukemia research, 1985.9:1017-1021; Berardi AC etc., Science, 1995.267:104-108; Sharkis SJ etc., Stem ceHs, 1997.15 (Suppl 1): 41-44; Juopperi TA etc., Experimental hematology, 2007.35:335-341); And separate these cells with some molecular surface affinity tags, yet, weak point; So still do not have very strong specificity at their employed molecule marker on the one hand, and wherein still unclear whether mixing other progenitor cell and former proliferative cell that pre-exists by stages arranged, wherein be still a foreign cell crowd (Berardi AC etc. probably; Science; 1995.267:104-108), the more important thing is, further do not adopt reliable and general effective means is used to discern these activated stem cells.

Other patents like publication number CN101768570 disclose a kind of enrichment and the method for extracting adult stem cell; It adopts the engineering materials heeling-in of a kind of porous three-dimensional tissue in human or animal body; Make and adult stem cell enrichment in material obtain stem cell thereby separate.

The method of above-mentioned disclosed description or technology had both had superiority also has inferior position, and for example fluidic cell separating method and immunomagnetic beads method sorting come the screening and separating stem cell according to the special mark of cell surface often, but special cell surface marker is difficult to reach common recognition at present; Lack specific molecular surface sign, in addition, need accomplish by professional person's operating instrument; Disengaging time is longer, expends higherly, and makes it with going up the mark that instrument can be discerned through certain processing; This chemical process pair cell state can affect; Misoperation in addition cause the differentiation or cause cell to move towards apoptosis, on the other hand, the instrument efficiency of separation is not high; Long assorting room; Cause cell viability to descend, cell state changes or the like problem, and these also are not suitable for stem cell enriched scientific research, the demand of using such as clinical of being used for of screening in enormous quantities.

Moreover, lack functional evidence and show that LRCs has the functional character of stem cell, and BrdU is not a cell surface marker (Blanpain etc., Cell, 2004.118 (5): 635-648); It is high to differentiate that through karyomorphism separate stem cells specialty and experience require, and is easy to occur the discriminating mistake, and operating process and time are long.

Thereby; The research of adult stem cell also provides the chance of a development new therapy gradually for clinical medicine; In cell therapy that organizational project and genetic engineering technique grow up and gene therapy is research focus and the forward position in the biomedical sector in recent years; Thereby directly using to the patient from the stem cell of body after modifying of for example use producing is used for gene therapy (Nathalie Cartier etc., Science, 2009.326 (5954): 818-823) and cell therapy; Adult stem cell is as the carrier and the first-selected target cell of gene or cell therapy, and one of them benefit is to reduce the immunological rejection that causes when transplanting.

Adult stem cell is at " seed bank " as clinical cell therapy, drug screening, artificial organ, rebuilds at tumour, trauma repair, tissue or organ dysfunction, the aspects such as treatment of various degenerative diseases will play an important role.

In view of the above; This area need overcome existing shortcoming in the prior art; In order to overcome the deficiency on the aforesaid method and further relevant advantage to be provided; The inventor screens a kind of novel population of adult stem cells, adopts a kind of simple, fast method to be used for from cell mass screening, the enrichment stem cell through the chemotherapeutics tolerance, and then the activated stem cell is carried out the systems approach of mark; And set up a kind of screening, identification said stem cell test kit; This test kit has general applicability, and further, stem cell and/or the filial generation that the present invention screens acquisition is exceedingly useful in the reparation of basis and clinical application research, treatment, drug screening, organizational project and impaired or illing tissue and regeneration.

Summary of the invention

The objective of the invention is to disclose a kind of novel adult stem cell that before mammiferous normal, the lesion/cancer of rodent, people or other, the biopsy of lesion/cancer property, metastatic tumor/cancerous tissue, in vitro tissue sample, clone etc., screens; Through the method for chemotherapy drugs in combination cell marking, said new dry cell mass can keep cell marking for a long time.

Another object of the present invention is to disclose a kind of before mammiferous normal, the lesion/cancer of rodent, people or other, the cell masses such as the biopsy of lesion/cancer property, metastatic tumor/cancerous tissue, in vitro tissue sample, clone the method for screening adult stem cell; This method is simple and efficient, removed the interference of proliferative cell in the cell mass; High specificity; Activated adult stem cell and to its with or filial generation carried out mark, practicability and effectiveness.

A time purpose of the present invention is to disclose a kind of test kit that before mammiferous normal, the lesion/cancer of said rodent, people or other, the biopsy of lesion/cancer property, metastatic tumor/cancerous tissue, in vitro tissue sample, clone etc., screens adult stem cell; That this test kit has is easy to use, high specificity, practicability and effectiveness, and application prospect is advantage widely.

To achieve these goals; The technical scheme that the present invention adopted is following: a kind of novel adult stem cell; Be with before mammiferous normal, the lesion/cancer of rodent, people or other, the biopsy of lesion/cancer property, metastatic tumor/cancerous tissue, in vitro tissue sample, clone etc. are raw material; Obtain through the screening of chemotherapy drugs in combination cell marking, wherein, said new dry cell mass can keep cell marking for a long time.

The present invention relates to the discovery of inventor's beyong contemplation, the chemotherapeutics that adds the finite concentration scope in advance is with after the cell mass that contains stem cell contacts, and the proliferative cell in the wherein said cell mass will be eliminated; And stem cell wherein can not be killed, and can tolerate the chemotherapeutics of adding, thereby has obtained negative sense selection and further enrichment; And it can further be activated; Afterwards, after adopting cell marking to merge, can follow up stem cell and or its filial generation of being screened.

The method and the test kit of a kind of novel adult stem cell of said screening have versatility; Briefly; Adopt chemotherapeutics to screen the adult stem cell in the cell mass, the population of stem cells after adopting cell marking to screening and activating is afterwards again discerned, and specifically may further comprise the steps:

At first, the said chemotherapeutics of finite concentration scope adds in the said cell mass in advance, directly removes proliferative cell crowd wherein, thus screening, enrichment population of stem cells wherein,

Further, obtain activate getting into the cell cycle after said population of stem cells is survivability-screened, wherein, add cell marking to screen the said population of stem cells of back activated with or its filial generation discern,

Wherein, said chemotherapeutics selects to have reagent or the material of removing proliferative cell and activating the population of stem cells effect, wherein, comprises one or more the combination of known chemotherapeutics,

Wherein, the mark of said labeled cell select BrdU, tritiated thymidine ( ³H-TdR), 5-ethynyl-2 ' deoxyuridine (EDU) but and the arbitrary molecule or the material of the said population of stem cells of other marks or its filial generation.

Wherein, the novel adult stem cell of above-mentioned screening also comprise adopt molecular marker to its with or filial generation carry out further somatotype, identification.

Further, can obtain its expressed specific molecular surface marker.

Further; Said from cell mass screening stem cell employed chemotherapeutics comprise that any known or potential can be used for removing the material of proliferative cell, comprise that microbiotic chemotherapeutics, antimetabolic chemotherapeutical medicine, alkylating agent chemotherapeutics, hormone and cryptorrheic chemotherapy medicine, plant chemotherapeutics, nitrosourea chemotherapeutics, antibody blocking thinner treat one or more combination of medicine, Asparaginase, procarbazine, RP-54780, platinum class, interior imines, hydroxyurea and decarbazine.

Wherein, the about 1mg/kg-100mg/kg cell of the concentration of the chemotherapeutics of said use weight, further, said chemotherapeutics function cells crowd's time is 0.1h-72h.

Further, the concentration of the chemotherapeutics of said use also can be selected about 10mg/kg-50mg/kg cell weight, wherein, said antitumor/chemotherapeutics function cells crowd's selection of time is 0.5h-36h.

Wherein, the about 0.1mg/kg-15mg/kg cell of the employed label concentration of said labeled cell weight, wherein, the time that said mark acts on said screening back population of stem cells is 0.01h-96h.

Further, the about 0.5mg/kg-10mg/kg cell of the employed label concentration of said labeled cell weight, wherein, the time that said mark acts on said screening back population of stem cells is 0.05h-72h.

Further, said mark can discern through the stem cell of tolerance screening with or the number of its filial generation be one or more,

Wherein, but also retention marker of the daughter cell of said cell mass, stem cell with or its filial generation retention marker for a long time.

Further, said filial generation is made up of one or more of the limited progenitor cell of potential, of short duration proliferative cell, noble cells, precursor cell or non-ancestral cells.

Further, preferred, the employed chemotherapeutics of said screening population of stem cells is any combination of the medication combined platinum-based chemotherapy medicine of antimetabolic chemotherapeutical medicine, plant chemotherapeutics, hormone and cryptorrheic chemotherapy.

Further, the population of stem cells of said screening and separating with or its filial generation be derived from one or more groups formed in liver organization, renal tissue, heart tissue, pancreatic tissue, intestinal tissue, face tissue, ovary tissue, oviduct tissue, testis tissue, nervous tissue, stomach-tissue, lung tissue, mammary tissue, fatty tissue, muscle tissue, Tiroidina, mesenchyme, spleen tissue, myeloid tissue, peripheral blood, Cord blood, menses, placenta, dental tissue, ocular tissue and the cerebral tissue.

Wherein, said tissue-derived in rodent, the mankind or other mammiferous biopsies or stripped healthy tissues.

Further, the population of stem cells of said screening and separating and or its filial generation be derived from before the lesion/cancer in rodent between healthy tissues and cancer/tumprigenicity tissue, the mankind and other Mammalss source pathological tissues arbitrarily.

Further, the population of stem cells of said screening and separating with or its filial generation be derived from liver cancer, kidney, carcinoma of the pancreas, intestinal cancer, ovarian cancer, mammary cancer, the cancer of the brain, lung cancer, liver's metastatic tumor, pulmonary metastases, tumor cell line and or one or more groups of forming of materials such as lesion/cancer biopsy, in vitro tissue in.

Wherein, tumor cell line comprises the tumor cell line that is derived from rodent, the mankind or other mammiferous tumor tissues foundation.

Another object of the present invention is to provide a kind of method of before mammiferous normal, the lesion/cancer of rodent, people or other, the biopsy of lesion/cancer property, metastatic tumor/cancerous tissue, in vitro tissue sample, clone etc., screening adult stem cell; The interference that this method is simple and efficient, removed proliferative cell; High specificity; Activated adult stem cell and to its with or filial generation carried out mark, practicability and effectiveness.

Further, thus the present invention obtains normal stem cell, tumor stem cell and or its filial generation according to above-mentioned screening method.

Further, the population of stem cells of screening according to the invention identification or generation is of value to the cell therapy as organ disease, cell source, drug screening, gene therapy, reparation and the regeneration of damaged of organizational project or the application in illing tissue, basis and clinical and the applied research thereafter.

According to another aspect of the present invention, of the present invention time a purpose is, the test kit of a kind of screening, the novel adult stem cell of identification is provided, and wherein, said test kit comprises:

1) chemotherapeutics;

2) cell washing liquid;

3) cell marking;

4) combination of cytokines;

5) specification sheets of method of use is described, and choose wantonly;

6) mixing vessel and device.

Wherein, said chemotherapeutics comprises that microbiotic chemotherapeutics, antimetabolic chemotherapeutical medicine, hormone and cryptorrheic chemotherapy medicine, alkylating agent chemotherapeutics, plant chemotherapeutics, nitrosourea chemotherapeutics, antibody blocking thinner treat one or more combination of medicine, Asparaginase, procarbazine, RP-54780, platinum class, tetrahydroform, hydroxyurea and decarbazine;

Wherein, said chemotherapeutics is more preferably selected with antimetabolic chemotherapeutical medicine, alkylating agent chemotherapeutics, microbiotic chemotherapeutics, any combination of plant chemotherapy drugs in combination platinum-based chemotherapy medicine;

Wherein, said cell washing liquid can be saline water, PBS or other damping fluids;

Wherein, the mark of said labeled cell select BrdU, tritiated thymidine ( ³H-TdR), 5-ethynyl-2 ' deoxyuridine (EDU) but and the arbitrary molecule or the material of the said population of stem cells of other marks or clone or its filial generation;

Wherein, said combination of cytokines comprises that EGF, B27, bEGF, N2, Heparin, LIF are basic combination of cytokines;

Wherein, the specification sheets of said description method of use comprises the method for screening, identification survivability-screened novel adult stem cell of the present invention.

Further, the mark that carries of the labeled stem cells of test kit according to the invention also may be selected to be any stem cell that can let said screening of martial ethiops mark, quantum dot-labeled, GFP mark, RFP mark or other and or filial generation carry discernible mark.

The population of stem cells of further, test kit according to the invention screening, identification from rodent, the mankind and other mammiferous healthy tissuess, clone, cancer before one or more combination of tissue, cancer/tumprigenicity tissue, metastatic tumor/cancerous tissue.

Further, adopt monoclonal antibody or polyclonal antibody to identify the mark that mixes with specificity, said antibody is immunoglobulin molecules; Can combine target, institute's digit synbol among the present invention, its combination is to discern an antigen site of the variable region of Tegeline at least; Antibody according to the invention not only comprises the whole antibody molecule, also comprises their fragment, for example Fab, Fab ', Fv, strand (ScFv) etc.; Perhaps their two mutants; Or comprise the fusion rotein of antibody moiety, change fully or the humanized antibody of partization and any other adorned immunoglobulin molecules, said immunoglobulin molecules comprises an antigen binding site.

Further, said antibody carries resorcinolphthalein, vitamin H, radio-labeling, enzyme labelling, fluorogenic substrate mark, chromogenic substrate mark, chemiluminescent labeling etc.

Further, the novel adult stem cell according to test kit screening acquisition according to the present invention loses the pathological tissues of function or the application in the organ as basic, clinical and applied research, organizational project, treatment, drug screening, reparation.

Description of drawings

Screening, the identification of Fig. 1 New Liver stem cell

This figure shows chemotherapy drugs in combination BrdU label screening, the identification New Liver stem cell of different concns; X-coordinate is represented the time (h) that medicine and mark add; Ordinate zou is represented cell survival rate and mark rate (%), representes that respectively medicine capecitabine and concentration thereof are respectively 2mg/mL, 6mg/mL, 10mg/mL, 14mg/mL, 20mg/mL, the curvilinear motion of cell survival and mark rate; Each gradient repeats 3 holes, and the corresponding label concentration that adds is 0.5mg/kg cell weight.Drug treating time is 16 hours, whenever carries out survival rate at a distance from sampling in 4 hours and detects, and wherein, mark whenever added once at a distance from 12 hours, and effect total time is 24 hours, and wherein, marker detection is carried out in sampling in per 6 hours.

The screening of the novel cardiac stem cells of Fig. 2, identification

This figure show different concns chemotherapy drugs in combination IdU label screening, discern novel cardiac stem cells; X-coordinate is represented the time (h) that medicine and mark add; Ordinate zou is represented cell survival rate and mark rate (%), representes that respectively Mitomycin C and concentration thereof are respectively 4mg/mL, 10mg/mL, 18mg/mL, 24mg/mL, 28mg/mL, the curvilinear motion of cell survival and mark rate; Each gradient repeats 3 holes, and the corresponding label concentration that adds is 0.8mg/kg cell weight.Drug treating time is 24 hours, whenever carries out survival rate at a distance from sampling in 6 hours and detects, and wherein, mark whenever added once at a distance from 6 hours, and effect total time is 24 hours, and wherein, marker detection is carried out in sampling in per 6 hours.

Fig. 3 screens novel cancer stem cell and it is discerned from liver cancer

This figure shows that different chemotherapy drugs in combination BrdU marks screen, discern the novel liver cancer stem cell from liver cancer; X-coordinate is represented the time (h) that medicine and mark add; Ordinate zou is represented cell survival rate and mark rate (%), representes that respectively medicine Fluracil, endoxan, carboplatin and adding concentration thereof correspond to 10mg/mL, 4mg/mL, 4mg/mL, the curvilinear motion of cell survival and mark rate; Each gradient repeats 3 holes, and the corresponding label concentration that adds is 2mg/kg cell weight.Drug treating time is 16 hours, whenever carries out survival rate at a distance from sampling in 4 hours and detects, and wherein, mark whenever added once at a distance from 12 hours, and effect total time is 36 hours, and wherein, marker detection is carried out in sampling in per 12 hours.

Fig. 4 screens novel breast carcinoma stem cell from isolating stripped breast cancer tissue

This figure shows that different chemotherapy drugs in combination BrdU marks screen, discern novel breast carcinoma stem cell from mammary cancer; X-coordinate is represented the time (h) that medicine and mark add; Ordinate zou is represented cell survival rate and mark rate (%); Represent that respectively medicine Fluracil, taxol, capecitabine, endoxan and adding concentration thereof correspond to 2mg/mL, 10mg/mL, 10mg/mL, 2mg/mL; The curvilinear motion of cell survival and mark rate, each gradient repeats 3 holes, and the corresponding label concentration that adds is 5mg/kg cell weight.Drug treating time is 8 hours, whenever carries out survival rate at a distance from sampling in 2 hours and detects, and wherein, mark whenever added once at a distance from 12 hours, and effect total time is 48 hours, and wherein, the detection of mark is carried out in sampling in per 12 hours.

Fig. 5 screens the lung cancer stem cell from lung cancer cell line

This figure shows that different chemotherapy drugs in combination BrdU marks screen, the novel lung cancer stem cell of mark from lung cancer cell line; X-coordinate is represented the time (h) that medicine and mark add; Ordinate zou is represented cell survival rate and mark rate (%), representes that respectively medicine cis-platinum, taxol, etoposide and adding concentration thereof correspond to 20mg/mL, 5mg/mL, 15mg/mL, the curvilinear motion of cell survival and mark rate; Each gradient repeats 3 holes, and the corresponding label concentration that adds is 4mg/kg cell weight.Drug treating time is 10 hours, whenever carries out survival rate at a distance from sampling in 2.5 hours and detects, and wherein, mark whenever added once at a distance from 12 hours, and effect total time is 32 hours, and wherein, the detection of mark is carried out in sampling in per 8 hours.

The test kit of Fig. 6 screening, identification new dry cell is formed

The composition of the test kit of the clear screening of this chart, identification new dry cell; Comprise and be provided with the corresponding screens groove of placing reagent bottle in box body, the box body; Be placed with 6 reagent bottles that closing cap is set, and working instructions in the box body, test kit (1) comprising: screening, identification agent bottle combination (2), cell washing reagent bottle (5); Cytokine reagent bottle (6); Mixing vessel or device, wherein (2) comprise screening reagent bottle (3) and corresponding mixing vessel (7), identification agent bottle (4) and corresponding mixing vessel (8), the reagent in said reagent bottle (3), (4), (5), (6) is respectively chemotherapeutics, cell marking, cell washing liquid, the various kinds of cell factor; Said (7) are exclusively used in and mix the chemotherapy pharmaceutical agent, and said (8) are exclusively used in the cell mixing mark.The shape of said 6 reagent bottles is not limit, its inclusion is aseptic, and its bottle stopper can be pressure, screw or other.

Embodiment

Below in conjunction with specific embodiment to specify advantage of the present invention; Those skilled in the art is to be understood that; The embodiments of the invention purpose is for advantage of the present invention clearly is described; Be not intended to limit the invention, anyly all drop within spiritual category of the present invention and the protection domain based on modification of the present invention, replacement, change.

The invention provides a kind of novel adult stem cell; Wherein said stem cell is before mammiferous normal, the lesion/cancer of rodent, people or other, the biopsy of lesion/cancer property, metastatic tumor/cancerous tissue, in vitro tissue sample, clone etc., to obtain through the screening of chemotherapy drugs in combination cell marking, and wherein said new dry cell mass keeps cell marking for a long time.

Wherein, novel adult stem cell of the present invention is derived from one or more groups formed in the tissues such as normal liver organization, renal tissue, heart tissue, pancreatic tissue, small intestine, face tissue, ovary tissue, testis tissue, nervous tissue, stomach-tissue, lung tissue, mammary tissue, fatty tissue, myeloid tissue, cerebral tissue, ocular tissue, muscle tissue, Tiroidina.

Wherein, said cell source is derived from rodent, the mankind or other mammiferous biopsy or in vitro tissue.

Wherein, novel adult stem cell of the present invention is organized before being derived from lesion/cancer, comprises rodent, people or other mammiferous pathological tissues between normal and pathological tissues.

Wherein, Novel adult stem cell of the present invention is derived from lesion/cancer sex organization; The biopsy of metastatic tumor/cancerous tissue, in vitro tissue sample, clone comprise in one or more the group such as liver tumor/cancer, tumor of kidney/cancer, pancreas tumor/cancer, intestinal tumor/cancer, ovarian tumor/cancer, breast tumor/cancer, cerebral tumor/cancer, lung tumor/cancer, tumor of testis/cancer, dermatoma/cancer, blood cancer, liver's metastatic tumor, pulmonary metastases, metastatic encephaloma, lesion/cancer in vitro tissue, SMMC-7721, lung cancer cell line, ovarian cancer cell line.

The present invention relates to the discovery of inventor's beyong contemplation; The chemotherapeutics that adds certain limit in advance is with after the cell mass that contains stem cell contacts, and proliferative cell wherein will be eliminated, and can not be killed and stem cell can tolerate chemotherapeutics; Thereby obtained negative sense and selected and further enrichment; And it can be activated, and after adding the cell marking fusion, can follow up stem cell and or its filial generation of being screened.

Therefore, the invention provides a kind of method of screening novel adult stem cell, this method has versatility; Briefly, comprise and adopt chemotherapeutics to add in advance in the said cell mass, directly remove proliferative cell crowd wherein; Stem cell has been carried out the screening of negative sense resistance, high specificity, simple and efficient; Thereby screening, enrichment and activation adult stem cell wherein, afterwards through cell marking to the stem cell after activating with or its filial generation carry out mark, specifically may further comprise the steps:

At first, the chemotherapeutics of finite concentration scope adds in the said cell mass in advance, directly removes proliferative cell crowd wherein, thus screening, enrichment population of stem cells wherein,

Further, the survivability-screened back of said population of stem cells is activated and is got into the cell cycle, wherein, add cell marking to screen the said population of stem cells of back activated with or its filial generation discern,

Wherein, the mark of said labeled cell select BrdU, tritiated thymidine ( ³H-TdR), 5-ethynyl-2 ' deoxyuridine (EDU) but and the arbitrary molecule or the material of the said population of stem cells of other marks or clone or its filial generation.

Wherein, the novel adult stem cell of above-mentioned screening comprises that also the employing molecular marker carries out further somatotype, identification to it.

Further, obtain the specific molecular surface marker that said stem cell is expressed.

Further; Said from cell mass screening stem cell employed chemotherapeutics comprise that any known or potential can be used for removing the material of proliferative cell, comprise that microbiotic chemotherapeutics, antimetabolic chemotherapeutical medicine, alkylating agent chemotherapeutics, hormone and cryptorrheic chemotherapy medicine, plant chemotherapeutics, nitrosourea chemotherapeutics, antibody blocking thinner treat one or more combination of medicine, Asparaginase, procarbazine, RP-54780, platinum class, tetrahydroform, hydroxyurea and decarbazine.

Wherein, the about 1mg/kg-100mg/kg cell of the concentration of the chemotherapeutics of said use weight, further; Said chemotherapeutics function cells crowd's time range is 0.1h-72h, and preferred, the concentration of the chemotherapeutics of said use also can be selected about 10mg/kg-50mg/kg cell weight; Wherein, Said antitumor/chemotherapeutics function cells crowd's selection of time is 0.5h-36h, preferred, the said chemotherapeutics function cells crowd's who uses selection of time is 2h-24h.

Wherein, said antitumor/number of times of chemotherapeutics and cell mass effect can also may be selected to be repeatedly effect for once, for example divides 1-10 time, and is preferred, branch 2-6 secondary action.

Wherein, the number of times of said cell marking and cell mass effect can also may be selected to be repeatedly effect for once, for example divides 1-20 time, and is preferred, divides the 4-10 secondary action.

Wherein, The about 0.1mg/kg-15mg/kg cell of the employed label concentration of said labeled cell weight, further, the time that said mark acts on said screening back population of stem cells is 0.01h-96h; Preferably; The about 0.5mg/kg-10mg/kg cell of the employed label concentration of said labeled cell weight, wherein, the time that said mark acts on said screening back population of stem cells is 0.05h-72h.

Wherein, the daughter cell of said cell mass also can be mixed mark, stem cell with or its filial generation retention marker for a long time.

Further, said chemotherapeutics is more preferably selected with antimetabolic chemotherapeutical medicine, alkylating agent chemotherapeutics, microbiotic chemotherapeutics, any combination of plant chemotherapy drugs in combination platinum-based chemotherapy medicine.

Further, be of value to known or unknown molecular marker to said ancestral cells crowd or its filial generation is further screened, somatotype.

Further, said chemotherapeutics acts in the biopsy or one or more groups formed in stripped normal liver tissue, renal tissue, heart tissue, pancreatic tissue, intestinal tissue, face tissue, ovary tissue, oviduct tissue, testis tissue, nervous tissue, stomach-tissue, lung tissue, mammary tissue, fatty tissue, muscle tissue, Tiroidina, mesenchyme, spleen tissue, myeloid tissue, peripheral blood, Cord blood, menses, placenta, dental tissue, ocular tissue and the cerebral tissue in rodent, the mankind and other Mammalss source.

Further, said chemotherapeutics acts on pathological tissues before the lesion/cancer in rodent between healthy tissues and cancer/tumprigenicity tissue, the mankind and other Mammalss source.

Further, said chemotherapeutics act on rodent, the mankind and other Mammalss source biopsy or stripped liver cancer, kidney, carcinoma of the pancreas, intestinal cancer, ovarian cancer, mammary cancer, the cancer of the brain, lung cancer, liver's metastatic tumor, pulmonary metastases, tumor cell line and or one or more groups of forming of lesion/cancer in vitro tissue etc. in.

Further, the population of stem cells of screening according to the invention or clone or thereafter the generation as the cell therapy of organ disease, cell source, drug screening, gene therapy, reparation and the regeneration of damaged of organizational project or the application in illing tissue, basis and clinical and the applied research.

In preferred implementation of the present invention, preferred cell marking is chosen as BrdU, further, the material of cell marking also may be selected to be tritiated thymidine ( ³H-TdR), 5-ethynyl-2 ' deoxyuridine (EDU) and other for a long time mark this state cell mass or clone or the molecule in generation thereafter.

In a preferred implementation of the present invention; Wherein, But the preferred said stem cell of mark of embodiment be labeled as BrdU; The mark that cell kept is the BrdU molecule, and wherein said cell mass or its filial generation are and continue to be BrdU positive (BrdU ).The liver organization of drawing materials is handled the back and is obtained cell mass, and said cell mass derives from the stripped or biopsy of rodent, mankind or other mammalian livers; Preferred medicine is a capecitabine, and the final concentration in substratum is 2mg/mL, and function cells crowd's time is 16 hours (h); Whenever detected cell survival rate at a distance from 4 hours; Said mark gives dosage 0.5mg/kg cell weight, at 37 ℃, and 5%CO ₂Under the condition, preferred every separated 12h adds mark once, acts on 24h altogether.

Further; Adopt the capecitabine (6mg/mL, 10mg/mL, 14mg/mL, 20mg/mL) of different concns gradient to handle cell mass; 3 holes of repetition under each concentration; Add respective markers (0.5mg/kg cell weight) under each concentration the activated population of stem cells discerned, said afterwards cell mass in the substratum of factor-containing combination, 37 ℃, 5%CO ₂Cultivate under the condition, wherein, said substratum is preferably DMEM/F12K; And interpolation penicillium mould and Streptomycin sulphate; Concentration is respectively 100U/mL and 100 μ g/mL, and wherein, the ancestral cells quantity of BrdU institute mark is one or more ancestral cells; Further comprise the filial generation of the said cell mass of mark, wherein adopt known molecular marker with progressive identification BrdU ⁺The cell mass of mark or its daughter cell, further, said filial generation is made up of one or more of the limited progenitor cell of potential, of short duration proliferative cell, noble cells, precursor cell or non-ancestral cells.

Wherein, Comprise the molecular marker that the said cell mass of further screening is expressed; Should be appreciated that any known or unknown molecule of employing or screening substances separate said cell mass or its filial generation is very useful to research and application, all belong to spiritual category of the present invention and protection domain.

In another preferred embodiment of the present invention, wherein, but the preferred said stem cell of mark of embodiment be labeled as IdU, the mark that cell kept is the IdU molecule, wherein said cell mass or its filial generation are and continue to be the positive (IdU of IdU ⁺).The heart tissue of drawing materials is handled the back and is obtained cell mass, and said cell mass derives from rodent, people or other mammalian heart tissues, biopsy or the fresh heart tissue that exsomatizes; Preferred medicine is a MTC, and the final concentration in substratum is 4mg/mL, and function cells crowd's time is 24 hours; Whenever carrying out survival rate at a distance from sampling in 6 hours detects; Said mark gives dosage 0.8mg/kg cell weight, at 37 ℃, and 5%CO ₂Under the condition, preferred every separated 6h adds mark once, is divided into 4 addings, acts on 24h altogether.Further; Adopt the MTC (10mg/mL, 18mg/mL, 24mg/mL, 28mg/mL) of different concns gradient to handle cell mass; Cell mass is 3 holes of repetition under each concentration; Add respective markers (0.8mg/kg cell weight) afterwards under each concentration the activated population of stem cells discerned, said afterwards cell mass in the substratum of factor-containing combination, 37 ℃, 5%CO ₂Cultivate under the condition, wherein, said substratum is preferably DMEM/F12K; And interpolation penicillium mould and Streptomycin sulphate; Concentration is respectively 100U/mL and 100 μ g/mL, and wherein, the ancestral cells quantity of IdU institute mark is one or more ancestral cells; Further comprise the filial generation of the said cell mass of mark, wherein adopt known molecular marker with further identification IdU ⁺The cell mass of mark or its daughter cell, further, said filial generation is made up of one or more of the limited progenitor cell of potential, of short duration proliferative cell, noble cells, precursor cell or non-ancestral cells.

Wherein, Comprise the molecular marker that the said cell mass of further screening is expressed; Should be appreciated that any known or unknown molecule of employing or screening substances separate said cell mass or its filial generation is very useful to research and application, all drop in spiritual category of the present invention and the protection domain.

In another preferred embodiment of the present invention; Wherein, But the preferred said stem cell of mark of embodiment be labeled as BrdU; The mark that cell kept is the BrdU molecule, and wherein said cell mass or its filial generation are and continue to be BrdU positive (BrdU

).The liver cancer tissue of drawing materials is handled the back and is obtained the liver cancer cell crowd, and said cell mass derives from rodent, people or other mammiferous biopsies or stripped fresh HCC tissue; Preferred drug regimen is Fluracil, endoxan, carboplatin; Final concentration in substratum is respectively 10mg/mL, 4mg/mL, 4mg/mL, and function cells crowd's time is 16 hours, whenever carries out survival rate at a distance from sampling in 4 hours and detects; 3 holes of repetition under each concentration; Said mark gives dosage 2mg/kg cell weight, at 37 ℃, and 5%CO ₂Under the condition, preferred every add mark once, be divided into 3 adding at a distance from 12h, mark effect 36h altogether, said afterwards cell mass in the substratum that factor-containing makes up, 37 ℃, 5%CO ₂Cultivate under the condition, wherein, said substratum is preferably DMEM/F12K, and adds penicillium mould and Streptomycin sulphate, and concentration is respectively 100U/mL and 100 μ g/mL.Wherein, the quantity of the liver-cancer stem cell of BrdU institute mark is one or more, further comprises the filial generation of the said cell mass of mark, wherein adopts known molecular marker with further identification BrdU ⁺The cell mass of mark or its daughter cell; Wherein, Comprise the molecular marker that the said cell mass of further screening is expressed, further, said filial generation is made up of one or more of the limited progenitor cell of potential, of short duration proliferative cell, noble cells, precursor cell or non-ancestral cells.Should be appreciated that any known or unknown molecule of employing or screening substances separate said cell mass or its filial generation is very useful to research and application, all belong to spiritual category of the present invention and protection domain.

In another preferred embodiment of the present invention, wherein, but the preferred said stem cell of mark of embodiment be labeled as BrdU, the mark that cell kept is the BrdU molecule, wherein said cell mass or its filial generation are and continue to be the positive (BrdU of BrdU ⁺).The breast cancer tissue that exsomatizes draws materials; Handle the back and obtain cell mass, said cell mass derives from rodent, people or other mammiferous mammary cancer excision or biopsy, and preferred drug regimen is Fluracil, taxol, capecitabine, endoxan; Final concentration in substratum is respectively 2mg/mL, 10mg/mL, 10mg/mL, 2mg/mL; Function cells crowd's time is 8 hours, whenever carries out survival rate at a distance from sampling in 2 hours and detects, and said mark gives dosage 5mg/kg cell weight; At 37 ℃, 5%CO ₂Under the condition, preferred every add mark once, be divided into 3 adding at a distance from 12h, mark effect 36h altogether, said afterwards cell mass in the substratum that factor-containing makes up, 37 ℃, 5%CO ₂Cultivate under the condition, wherein, said substratum is preferably DMEM/F12K, adds penicillium mould and Streptomycin sulphate, and concentration is respectively 100U/mL and 100 μ g/mL.Wherein, The quantity of the breast carcinoma stem cell of BrdU institute mark is one or more; Further comprise the filial generation of the said cell mass of mark; Wherein adopt cell mass or its daughter cell of known molecular marker with further identification BrdU+ mark, further, said filial generation is made up of one or more of the limited progenitor cell of potential, of short duration proliferative cell, noble cells, precursor cell or non-ancestral cells.

In another preferred embodiment of the present invention, but wherein the preferred said stem cell of mark of embodiment be labeled as BrdU, the mark that cell kept is the BrdU molecule, wherein said cell mass or its filial generation are and continue to be the positive (BrdU of BrdU ⁺).Lung cancer cell line; Said clone derives from rodent, people or other mammiferous lung cancer excision or biopsy, and preferred drug regimen is cis-platinum, taxol, etoposide, and the final concentration in substratum is respectively 20mg/mL, 5mg/mL, 15mg/mL; 3 holes of repetition under each concentration; Said mark gives dosage 4mg/kg cell weight, at 37 ℃, and 5%CO ₂Under the condition, preferred every add mark once, be divided into 2 adding, be total to mark effect 10h at a distance from 5h, said afterwards cell mass in the substratum of factor-containing, 37 ℃, 5%CO ₂Cultivate under the condition.Wherein, the quantity of the lung cancer stem cell of BrdU institute mark is one or more, further comprises the filial generation of the said cell mass of mark, wherein adopts known molecular marker with further identification BrdU ⁺The cell mass of mark or its daughter cell, further, one or more compositions of progenitor cell, of short duration proliferative cell, noble cells, precursor cell or non-ancestral cells that said filial generation mountain potential is limited.

In another embodiment of the invention, also relate to the test kit of a kind of screening, the novel adult stem cell of identification, wherein, said test kit comprises:

1) chemotherapeutics;

2) cell washing liquid;

3) cell marking;

4) combination of cytokines;

5) specification sheets of method of use is described, and choose wantonly;

6) mixing vessel and device.

Wherein, said chemotherapeutics comprises that microbiotic chemotherapeutics, antimetabolic chemotherapeutical medicine, alkylating agent chemotherapeutics, hormone and cryptorrheic chemotherapy medicine, plant chemotherapeutics, nitrosourea chemotherapeutics, antibody blocking thinner treat one or more combination of medicine, Asparaginase, procarbazine, RP-54780, platinum class, tetrahydroform, hydroxyurea and decarbazine;

Wherein, the mark of said labeled cell select BrdU, tritiated thymidine ( ³H-TdR), 5-second piece base-2 ' deoxyuridine (EDU) but and the arbitrary molecule or the material of the said population of stem cells of other marks or clone or its filial generation;

Wherein, said combination of cytokines comprises that EGF, B27, bEGF, Heparin, N2, LIF are basic combination of cytokines, further; Said EGF adds when stem cell is cultivated, to final concentration be 5ng/mL-100ng/mL, preferred 10ng/mL-50ng/mL; Said bFGF adds when stem cell is cultivated, to final concentration be 5ng/mL-100ng/mL, preferred 10ng/mL-50ng/mL; Said Heparin adds when stem cell is cultivated, to final concentration be 2 μ g/mL-40 μ g/mL, said B27, N2 add when stem cell is cultivated; To final concentration be 2ng/mL-50ng/mL, preferred 10ng/mL-30ng/mL, said LIF (LIF) adds when stem cell is cultivated; To final concentration be 100U/mL-1500U/mL, preferred 500U/mL-1000U/mL.

Wherein, the specification sheets of said description method of use comprises the method for screening survivability-screened novel adult stem cell of the present invention.

Wherein, Said screening, identification new dry cell reagent box (1) comprising: screening, identification agent bottle combination (2); Cell washing reagent bottle (5), cytokine reagent bottle (6), mixing vessel or device; Wherein (2) comprise screening reagent bottle (3) and corresponding mixing vessel (7), identification agent bottle (4) and corresponding mixing vessel (8); Reagent in said reagent bottle (3), (4), (5), (6) is respectively chemotherapeutics, cell marking, cell washing liquid, the various kinds of cell factor, and said (7) are exclusively used in and mix the chemotherapy pharmaceutical agent, and said (8) are exclusively used in the cell mixing mark.

Wherein, the shape of various said reagent bottles is not limit, and its bottle stopper can be pressure, screw or other.

Further, its inclusion of said reagent bottle is aseptic, wherein, and endotoxin content≤0.5EU/mL.

Wherein, the said material that holds reagent is that the material that is usually used in reagent storage is processed, and further, said material does not influence the activity of reagent.

Further, the mark that carries of the labeled stem cells of test kit according to the invention also may be selected to be any stem cell that can let said screening of martial ethiops mark, quantum dot-labeled, GFP mark, RFP mark or other and or the mark that carries of filial generation.

Further, adopt monoclonal antibody or polyclonal antibody to identify the mark that mixes with specificity, said antibody is immunoglobulin molecules; Can combine target, digit synbol among the present invention, its combination is to discern an antigen site of the variable region of Tegeline at least; Antibody according to the invention not only comprises the whole antibody molecule, also comprises their fragment, for example Fab, Fab ', Fv, strand (ScFv) etc.; Perhaps their two mutants; Or comprise the fusion rotein of antibody moiety, change fully or the humanized antibody of partization and any other adorned immunoglobulin molecules, said immunoglobulin molecules comprises an antigen binding site.

Screening, the identification of embodiment 1 New Liver stem cell

The liver organization of drawing materials, liver tissue source be in rodent, people or other Mammals normal liver tissues, and biopsy or stripped hepatic tissue with PBS buffer solution for cleaning tissue 2～3 times, shred written treaty 1mm with eye scissors with liver organization afterwards again ³Fritter, add pancreatin (concentration is 0.1%～0.3%) back at 37 ℃ of digestion 10mim～60min, add the DMEM/F12K that contains 10% foetal calf serum (U.S. Hyclone company) termination reaction with pancreatin liquor capacity equivalent; Low temperature is with the centrifugal 3mim～5min of 800～1000g; Abandon supernatant, the PBS washing precipitation that contains 2% foetal calf serum of adding low temperature precooling 2～3 times is filtered with aseptic 100 orders～400 eye mesh screens; Thereby leach cell suspension, trypan blue dyeing viable cell>=96.7%.

Said isolating primary cell crowd is inoculated in Tissue Culture Flask or Tissue Culture Plate or the petridish, 37 ℃, 5%CO ₂Cultivate under the condition, add the chemotherapeutics capecitabine in advance, the final concentration in substratum is 2mg/mL, at 37 ℃, and 5%CO ₂Culture condition is effect 16h down, afterwards, and collecting cell.

1) whenever detect at a distance from 4 hours collecting cells, low temperature abandons supernatant with the centrifugal 3mim～5min of 830g, and cell survival rate is calculated in 1 * PBS washing.

2) cell after the collection and treatment is inoculated once more, wherein contains the B27 of 5ng/mL, the N2 of 10ng/mL, the EGF of 8ng/mL, the bFGF of 10ng/mL, the Heparin of 5 μ g/mL, the LIF of 800U/ml.

3) add the BrdU mark subsequently the stem cell after screening, activating is discerned, wherein, the final concentration that said BrdU is marked in the substratum is a 0.5mg/kg cell weight, at 37 ℃, and 5%CO ₂Under the condition, every add mark once at a distance from 12h, act on 24h altogether after, collecting cell.

4) whenever carried out the detection of cell marking at a distance from 6 hours, low temperature abandons supernatant with the centrifugal 3mim～5min of 830g, and 1 * PBS washing is got 50 μ L and carried out the per-cent that immunohistochemical methods detects the BrdU mark.

5) cell cultures to cell 60% merges, repeating step 1)-3) operation four times, the final concentration of capecitabine is respectively 6mg/mL, 10mg/mL, 14mg/mL and 20mg/mL, cell mass is 3 holes of repetition under each concentration.

6) add the chemotherapeutics capecitabine, the final concentration in substratum is 20mg/mL, at 37 ℃, and 5%CO ₂Act on 16h under the condition, collecting cell, low temperature is with the centrifugal 3mim～5min of 830g; Abandon supernatant, 1 * PBS washing, cell counting; Cell after the collection and treatment is inoculated in Tissue Culture Flask or the Tissue Culture Plate once more, wherein, contains the B27 of 5ng/mL, the N2 of 10ng/mL, the EGF of 8ng/mL, the bFGF of 10ng/mL, the Heparin of 5 μ g/mL; The LIF of 800U/ml, at 37 ℃, 5%CO ₂Being cultured to cell 60% under the condition merges.

7) repeat above-mentioned steps 6) cultivate 1 time.

8) add the chemotherapeutics capecitabine, the final concentration in substratum is 20mg/mL, at 37 ℃, and 5%CO ₂Cultivate under the condition, obtain the liver stem cells of stabilizing amount at last, said stem cell can be used for using in basis and applied research, treatment, directional induction differentiation, organizational project, reparation or regeneration of damaged or the pathological tissues.

Screening, the identification of embodiment 2 novel cardiac stem cells

The heart tissue of drawing materials, heart tissue derives from rodent, people or the normal heart tissue of other Mammalss, and the biopsy or the fresh heart tissue that exsomatizes with PBS buffer solution for cleaning tissue 2～3 times, shred written treaty 0.5～1mm with eye scissors with heart tissue afterwards again ³Fritter, add collagenase (concentration be 2～10mg/mL) backs at 37 ℃ of digestion 10mim～30min, add the DMEM/F12K that contains 10% foetal calf serum (U.S. Hyclone company) termination reaction with collagenase solution volume equivalent; Low temperature is with the centrifugal 3mim～5min of 900g; Abandon supernatant, the PBS washing precipitation that contains 2% foetal calf serum of adding low temperature precooling 2～3 times is filtered with aseptic 200～400 eye mesh screens; Thereby leach cell suspension, trypan blue dyeing viable cell>=95%.

Said cell mass is inoculated in the 6 porocyte culture plates, 37 ℃, 5%CO ₂Cultivate, add the chemotherapeutics MTC in advance, the final concentration in substratum is 4mg/mL, at 37 ℃, and 5%CO ₂Culture condition is effect 24h down, afterwards, and collecting cell.

1) whenever detect at a distance from 6 hours collecting cells, low temperature abandons supernatant with the centrifugal 3mim～5min of 900g, and cell survival rate is calculated in 1 * PBS washing.

2) cell after the collection and treatment is inoculated in the 6 porocyte culture plate corresponding apertures once more, wherein contains the B27 of 10ng/mL, the N2 of 10ng/mL, the EGF of 30ng/mL, the bFGF of 30ng/mL, the Heparin of 10 μ g/mL, the LIF of 600U/ml, and at 37 ℃, 5%CO ₂Cultivate under the condition.

3) add the IdU mark subsequently the cardiac stem cells after screening, activating is carried out mark, wherein, the final concentration that said IdU is marked in the substratum is a 0.8mg/kg cell weight, at 37 ℃, and 5%CO ₂Act on 24h under the condition, be divided into 4 addings, promptly whenever added once, afterwards collecting cell at a distance from 6 hours.

4) whenever carried out the detection of cell marking at a distance from 6 hours, low temperature abandons supernatant with the centrifugal 3mim-5min of 900g, and 1 * PBS washing is got 50 μ L and carried out the per-cent that immunohistochemical methods detects the IdU mark.

5) be cultured to cell 60% and merge repeating step 1)-3) operation four times, the final concentration of MTC is respectively 10mg/mL, 18mg/mL, 24mg/mL and 28mg/mL cell mass 3 holes of repetition under each concentration.

6) add the chemotherapeutics MTC, the final concentration in substratum is 28mg/mL, at 37 ℃, and 5%CO ₂Culture condition is effect 24h down, collecting cell, and low temperature is with the centrifugal 3mim～5min of 900g; Abandon supernatant, 1 * PBS washing 2 times, cell counting; Cell after the collection and treatment is inoculated in the 6 porocyte culture plate corresponding apertures once more, wherein contains the B27 of 10ng/mL, the N2 of 10ng/mL, the EGF of 30ng/mL, the bFGF of 30ng/mL, the Heparin of 10 μ g/mL, the LIF of 600U/ml; At 37 ℃, 5%CO ₂Being cultured to cell 60% under the condition merges.

7) repeat above-mentioned steps 7) cultivate 1 time.

8) add the chemotherapeutics MTC, the final concentration in substratum is 28mg/mL, at 37 ℃, and 5%CO ₂Cultivate under the condition, obtain the cardiac stem cells of stabilizing amount at last, said stem cell can be used for that the molecular surface sign is analyzed, basis and applied research, treatment, directional induction differentiation, organizational project, reparation is impaired or diseased heart etc. in use.

Embodiment 3 screens novel cancer stem cell from liver cancer

The liver cancer tissue of drawing materials, liver cancer tissue derives from rodent, people or other mammiferous liver cancer tissues, biopsy or stripped fresh HCC tissue, with PBS buffer solution for cleaning tissue 2～3 times, with eye scissors liver cancer tissue is shredded written treaty 1mm more afterwards ³Fritter; (concentration is that concentration is that 1～10mg/mL) back digests 5mim～60min at 37 ℃, and the DMEM/F12K that contains 10% foetal calf serum (the U.S. Hyclone company) termination reaction of adding and enzyme solution volume equivalent is blown and beaten cell suspension 2～3 times gently to add pancreatin (concentration is 0.1%～0.3%) and collagenase; Low temperature is with the centrifugal 3mim～5min of 800～1000g; Abandon supernatant, the PBS washing precipitation that contains 2% foetal calf serum of adding low temperature precooling two to three times is filtered with aseptic 200 orders-400 eye mesh screen; Thereby leach cell suspension, trypan blue dyeing viable cell>=96%.

Said isolating primary cell crowd is inoculated in Tissue Culture Flask or the Tissue Culture Plate, 37 ℃, 5%CO ₂Cultivate, add chemotherapeutics Fluracil, endoxan, carboplatin in advance, the final concentration in substratum is respectively 10mg/mL, 4mg/mL, 4mg/mL, at 37 ℃, and 5%CO ₂Culture condition is effect 16h down, afterwards, and collecting cell.

1) whenever detect at a distance from 4 hours collecting cells, low temperature abandons supernatant with the centrifugal 3mim～5min of 900g, and cell survival rate is calculated in 1 * PBS washing.

2) cell after the collection and treatment is inoculated once more, wherein contains the B27 of 10ng/mL, the N2 of 5ng/mL, the EGF of 5ng/mL, the bFGF of 5ng/mL, the Heparin of 10 μ g/mL, the LIF of 900U/ml.

3) add the BrdU mark subsequently the liver-cancer stem cell after screening, activating is carried out mark, wherein, the final concentration that said BrdU is marked in the substratum is a 2mg/kg cell weight, at 37 ℃, and 5%CO ₂Under the condition, every add mark once at a distance from 12h, act on 36h altogether after, collecting cell.

4) whenever carried out the detection of cell marking at a distance from 12 hours, low temperature abandons supernatant with the centrifugal 3mim～5min of 900g, and 1 * PBS washing is got 50 μ L and carried out the per-cent that immunohistochemical methods detects the BrdU mark.

5) cell after the collection and treatment is inoculated in Tissue Culture Flask or the Tissue Culture Plate once more, wherein, the Heparin of the EGF of the B27 of 10ng/mL, the N2 of 5ng/mL, 5ng/mL, the bFGF of 5ng/mL, 10 μ g/mL, the L1F of 900U/ml, at 37 ℃, 5%CO ₂Be cultured to cell 60% under the condition and merge, add chemotherapeutics Fluracil, endoxan, carboplatin once more, the final concentration in substratum is respectively 10mg/mL, 4mg/mL, 4mg/mL, at 37 ℃, and 5%CO ₂Culture condition is effect 16h down, afterwards, and collecting cell.

6) repeat above-mentioned steps 5) cultivate 2 times, obtain stable liver-cancer stem cell thus, said stem cell can be used for basis and applied research, drug screening, cell analysis etc.

Embodiment 4 screens novel breast carcinoma stem cell from isolating stripped breast cancer tissue

The biopsy or the fresh breast cancer tissue that exsomatizes with PBS buffer solution for cleaning tissue 2～3 times, shred written treaty 1mm with eye scissors with breast cancer tissue again ³Fritter; Add pancreatin (concentration is 0.1%～0.3%) low temperature digested overnight, (concentration 4～8mg/ml) adds the DMEM/F12K that contains 10% foetal calf serum (the U.S. Hyclone company) termination reaction with enzyme solution volume equivalent at 37 ℃ of digestion 5mim～60min to collagenase afterwards; Low temperature is with the centrifugal 3mim～5min of 800～1000g; Abandon supernatant, the PBS washing precipitation that contains 2% foetal calf serum of adding low temperature precooling 2～3 times is filtered with aseptic 200 orders～400 eye mesh screens; Thereby leach cell suspension, trypan blue dyeing viable cell>=%%.

Said isolating primary cell crowd is inoculated in Tissue Culture Flask or the Tissue Culture Plate, 37 ℃, 5%CO ₂Cultivate, add chemotherapeutics Fluracil, taxol, capecitabine, endoxan in advance, the final concentration in substratum is respectively 2mg/mL, 10mg/mL, 10mg/mL, 2mg/mL, at 37 ℃, and 5%CO ₂Culture condition is effect 8h down, afterwards, and collecting cell.

1) whenever detect at a distance from 2 hours collecting cells, low temperature abandons supernatant with the centrifugal 3mim～5min of 900g, and cell survival rate is calculated in 1 * PBS washing.

2) cell after the collection and treatment is inoculated once more, wherein contains the B27 of 5ng/mL, the N2 of 2ng/mL, the EGF of 5ng/mL, the bFGF of 5ng/mL, the Heparin of 15 μ g/mL, the LIF of 900U/ml.

3) add the BrdU mark subsequently the breast carcinoma stem cell after screening, activating is carried out mark, wherein, the final concentration that said BrdU is marked in the substratum is a 5mg/kg cell weight, at 37 ℃, and 5%CO ₂Under the condition, every add mark once at a distance from 12h, act on 48h altogether after, collecting cell.

5) cell after the collection and treatment is inoculated in Tissue Culture Flask or the Tissue Culture Plate once more, wherein, the Heparin of the EGF of the B27 of 5ng/mL, the N2 of 2ng/mL, 5ng/mL, the bFGF of 5ng/mL, 15 μ g/mL, the LIF of 900U/ml, at 37 ℃, 5%CO ₂Be cultured to cell 60% under the condition and merge, add chemotherapeutics Fluracil, taxol, capecitabine, endoxan once more, the final concentration in substratum is respectively 2mg/mL, 10mg/mL, 10mg/mL, 2mg/mL, at 37 ℃, and 5%CO ₂Culture condition is effect 6h down, afterwards, and collecting cell.

6) repeat above-mentioned steps 5) cultivate 3 times, obtain stable breast carcinoma stem cell thus, said stem cell can be used for basis and applied research, drug screening, cell analysis etc.

Embodiment 5 screens the lung cancer stem cell from lung cancer cell line

Cancer clone, cell survival rate>=96% adds chemotherapeutics cis-platinum, taxol, etoposide in advance, and the final concentration in substratum is respectively 20mg/mL, 5mg/mL, 15mg/mL, at 37 ℃, 5%CO ₂Culture condition is effect 10h down, afterwards, and collecting cell.

1) whenever carry out survival rate at a distance from 2.5 hours collecting cells and detect, low temperature abandons supernatant with the centrifugal 3mim-5min of 800g, and cell survival rate is calculated in 1 * PBS washing.

2) cell after the collection and treatment is inoculated once more, wherein contains the B27 of 8ng/mL, the N2 of 5ng/mL, the EGF of 10ng/mL, the bFGF of 10ng/mL, the Heparin of 10 μ g/mL, the LIF of 900U/ml.

3) add the BrdU mark subsequently the lung cancer stem cell after screening, activating is carried out mark, wherein, the final concentration that said BrdU is marked in the substratum is a 4mg/kg cell weight, at 37 ℃, and 5%CO ₂Under the condition, every add mark once at a distance from 8h, act on 32h altogether after, collecting cell.

4) detection of cell marking, low temperature abandons supernatant with the centrifugal 3mim～5min of 800g, and 1 * PBS washing is got 50 μ L and is carried out the per-cent that immunohistochemical methods detects the BrdU mark.

5) cell after the collection and treatment is inoculated in Tissue Culture Flask or the Tissue Culture Plate once more; Wherein, 5 contain the B27 of 8ng/mL, the N2 of 5ng/mL, the EGF of 10ng/mL, the bFGF of 10ng/mL, the Heparin of 10 μ g/mL, the LIF of 900U/ml; At 37 ℃, 5%CO ₂Be cultured to cell 60% under the condition and merge, add chemotherapeutics cis-platinum, taxol, etoposide once more, the final concentration in substratum is respectively 20mg/mL, 5mg/mL, 15mg/mL, at 37 ℃, and 5%CO ₂Culture condition is effect 10h down, afterwards, and collecting cell.

6) repeat above-mentioned steps 5) cultivate 2 times, obtain stable lung cancer stem cell thus, said stem cell can be used for basis and applied research, drug screening, cell analysis etc.

The test kit of embodiment 6 screenings, identification new dry cell is formed

The test kit (1) of screening, identification new dry cell is formed; Comprise: screening, identification agent bottle combination (2); Cell washing reagent bottle (5), cytokine reagent bottle (6), mixing vessel or device; Wherein (2) comprise screening reagent bottle (3) and corresponding mixing vessel (7), identification agent bottle (4) and corresponding mixing vessel (8); Reagent in said reagent bottle (3), (4), (5), (6) is respectively chemotherapeutics, cell marking, cell washing liquid, the various kinds of cell factor, and said (7) are exclusively used in and mix the chemotherapy pharmaceutical agent, and said (8) are exclusively used in the cell mixing mark.

Wherein, the shape of said reagent bottle is not limit, and its bottle stopper can be pressure, screw or other.

Further, its inclusion of said reagent bottle is aseptic.Wherein, endotoxin content≤0.5EU/mL.

Wherein, the chemotherapeutics placed of said screening reagent bottle (3) selects to comprise that microbiotic chemotherapeutics, antimetabolic chemotherapeutical medicine, alkylating agent chemotherapeutics, hormone and cryptorrheic chemotherapy medicine, plant chemotherapeutics, nitrosourea chemotherapeutics, antibody blocking thinner treat one or more combination of medicine, Asparaginase, procarbazine, RP-54780, platinum class, tetrahydroform, hydroxyurea and decarbazine;

Wherein, said identification agent bottle (4) cell marking of placing select to comprise BrdU, tritiated thymidine ( ³H-TdR), 5-ethynyl-2 ' deoxyuridine (EDU) but and the arbitrary molecule or the material of the said population of stem cells of other marks or clone or its filial generation;

Wherein, the cytokine that said cytokine reagent bottle (6) is placed selects to comprise that EGF, B27, bEGF, Heparin, N2, LIF are basic combination of cytokines, further; Said EGF adds when stem cell is cultivated, to final concentration be 5ng/mL-100ng/mL, preferred 10ng/mL-50ng/mL; Said bFGF adds when stem cell is cultivated, to final concentration be 5ng/mL-100ng/mL, preferred 10ng/mL-50ng/mL; Said Heparin adds when stem cell is cultivated, to final concentration be 2 μ g/mL-40 μ g/mL, said B27, N2 add when stem cell is cultivated; To final concentration be 2ng/mL-50ng/mL, preferred 10ng/mL-30ng/mL, said LIF (LIF) adds when stem cell is cultivated; To final concentration be 100U/mL-1500U/mL, preferred 500U/mL-1000U/mL.

Wherein, the cell washing agent of said cell washing reagent bottle (5) may be selected to be saline water, PBS or other damping fluids.

Embodiment 7 uses test kit from small intestine, to screen, discern stem cell

Of the present invention being used for is made up of following from the test kit of small intestine screening stem cell:

1) chemotherapeutics is Fluracil, FUDR;

2) cell washing liquid is saline water;

3) cell marking;

4) combination of cytokines;

5) specification sheets of description method of use, and;

6) mixing vessel.

Mentioned reagent liquid is prepared under aseptic condition, test endotoxin content≤0.5EU/mL.

Wherein, said cell marking preferentially is chosen as BrdU;

Wherein, said cytokine is preferentially selected the combination of B27, EGF, bFGF, N2, Heparin, LIF;

Wherein, the specification sheets of said description method of use comprises the method for the stem cell for preparing mark of the present invention.

Adopt mentioned reagent box method of operating may further comprise the steps:

1) cell pre-treatment: after small intestine's pre-treatment of acquisition was single cell suspension, cell survival rate was calculated in trypan blue dyeing, and subsequently, low temperature abandons supernatant with the centrifugal 3mim～5min of 800g, the cell weight of weighing;

2) mix screening reagent: mix the reagent chemotherapeutics Fluracil that is comprised in the screening reagent bottle (3), FUDR with mix reagent bottle (7), final concentration is respectively 15mg/kg, 2mg/kg;

3) screening stem cell: add mixed screening reagent, at 37 ℃, 5%CO ₂Cultivate after 6 hours collecting cell under the condition of factor-containing;

4) calculate cell survival rate: low temperature abandons supernatant with the centrifugal 3min～5min of 800g, and the weight of the cell of weighing adds the cell washing liquid saline water re-suspended cell deposition in the reagent bottle (5) again, calculates cell survival rate;

5) cell mixing mark: the cell marking reagent BrdU that is comprised in the identification agent bottle (4), mix with mix reagent bottle (7), final concentration is a 2.5mg/kg cell weight;

6) identification stem cell: add the cell marking in the cell marking reagent bottle (4), at 37 ℃, 5%CO ₂Cultivate under the condition of factor-containing, mark is every to add once at a distance from 6h, adds altogether 4 times; Amount to 24h, collecting cell, wherein; Said cytokine comprises the combination that EGF, B27, bFGF, Heparin, N2 form, and further, said EGF adds when stem cell is cultivated; To final concentration be 10ng/mL, said bFGF adds when stem cell is cultivated, to final concentration be 10ng/mL.Said Heparin adds when stem cell is cultivated, to final concentration be 4 μ g/mL, said B27, N2 are the cell cultures additive, when stem cell is cultivated, add, to final concentration be 5ng/mL, said LIF adds when stem cell is cultivated, to final concentration be 800U/mL;

7) low temperature abandons supernatant with the centrifugal 3min～5min of 800g, adds cell washing reagent saline water re-suspended cell deposition again;

8) repeat above-mentioned steps each 2 times, obtain the intestines population of stem cells of stabilizing amount.

According to the intestines stem cell of test kit of the present invention screening, can be used as the application tool in fields such as clinical, basis and applied research, organizational project, treatment, drug screening, reparation damaged tissue.

Embodiment 8 uses test kit from face tissue, to screen stem cell

The present invention is used for being made up of following from the test kit of face tissue screening stem cell:

1) chemotherapeutics is bleomycin, Fluracil;

2) cell washing liquid is PBS;

3) cell marking;

4) combination of cytokines;

5) specification sheets of description method of use, and;

6) mixing vessel.

Mentioned reagent liquid is prepared under aseptic condition, the test endotoxin content≤0.5EU/mL, afterwards, be stored in aseptic container with or the device in.

Wherein, said cell marking preferentially is chosen as BrdU;

Adopt mentioned reagent box method of operating may further comprise the steps:

1) cell pre-treatment: ordinary method obtains face tissue, and after pre-treatment was single cell suspension, cell survival rate was calculated in trypan blue dyeing, and subsequently, low temperature abandons supernatant with the centrifugal 3mim～5min of 800g, the cell weight of weighing;

2) mix screening reagent: the reagent chemotherapeutics bleomycin, the Fluracil that are comprised in the screening reagent bottle (3), concentration is respectively 3mg/kg, 10mg/kg;

3) screening stem cell: add mixed screening reagent, at 37 ℃, 5%CO ₂Cultivate after 8 hours collecting cell under the condition of factor-containing;

4) calculate cell survival rate: low temperature abandons supernatant with the centrifugal 3min～5min of 900g, and the weight of the cell of weighing adds the cell washing liquid PBS damping fluid re-suspended cell deposition in the reagent bottle (5) again, calculates cell survival rate;

5) cell mixing mark: the cell marking reagent BrdU that is comprised in the identification agent bottle (4), mix with mix reagent bottle (7), final concentration is a 4.5mg/kg cell weight;

6) identification stem cell: add the cell marking in the cell marking reagent bottle (4), at 37 ℃, 5%CO ₂Contain under the condition of reagent 5 cytokines and cultivate, mark is every to add once at a distance from 6h, adds altogether 8 times; Amount to 48h, collecting cell, wherein; Said cytokine comprises the combination that EGF, B27, bFGF, Heparin, N2 form, and further, said EGF adds when stem cell media; To final concentration be 12ng/mL, said bFGF adds when stem cell is cultivated, to final concentration be 12ng/mL.Said Heparin adds when stem cell is cultivated, to final concentration be 8 μ g/mL, said B27, N2 are the cell cultures additive, when stem cell is cultivated, add, to final concentration be 10ng/mL, said LIF adds when stem cell is cultivated, to final concentration be 900U/mL;

7) low temperature abandons supernatant with the centrifugal 3min～5min of 900g, adds reagent 6 cell washing liquid PBS damping fluid re-suspended cells deposition again;

According to the epidermal stem cells of test kit of the present invention screening, can be used as the application tool in fields such as clinical, basis and applied research, organizational project, treatment, drug screening, reparation damaged tissue.

Obviously do not violating under the field of the invention and the category; Can to the above-mentioned test kit that is used to screen the new dry cell carry out composition modification and or increase; The present invention is the explanation of carrying out test kit with the foregoing description; Be not meant to limit the use of test kit, what this was carried out anyly is equal to replacement, modification, increases and all belong in the protection category of test kit of the present invention.

Claims

1. new dry cell; It is characterized in that: before population of stem cells derives from mammiferous normal, the lesion/cancer of rodent, people and other, lesion/cancer property, the biopsy of metastatic tumor/cancerous tissue, vitro samples tissue, clone; Obtain through the screening of chemotherapy drugs in combination cell marking, wherein this new dry cell mass can keep cell marking for a long time.

2. a kind of method of screening novel adult stem cell according to claim 1 is characterized in that said screening, recognition methods have versatility, comprising:

Adopt the adult stem cell in the chemotherapeutics cell groups to screen, the step that adopts cell marking that the population of stem cells after screening is discerned afterwards again,

Said chemotherapeutics adds in the said cell mass in advance, directly removes proliferative cell crowd wherein, thus screening, enrichment population of stem cells wherein,

The survivability-screened back of said population of stem cells is activated and is got into the cell cycle, afterwards, add cell marking to screen the said population of stem cells of back activated with or its filial generation discern,

Wherein, Said chemotherapeutics selects to have reagent or the material of removing proliferative cell and activating the population of stem cells effect; Wherein, Comprise that known microbiotic chemotherapeutics, antimetabolic chemotherapeutical medicine, alkylating agent chemotherapeutics, hormone and cryptorrheic chemotherapy medicine, plant chemotherapeutics, nitrosourea chemotherapeutics, antibody blocking thinner treat one or more one or more combination of combination of medicine, Asparaginase, procarbazine, RP-54780, platinum class, tetrahydroform, hydroxyurea and decarbazine; Or select with said antimetabolic chemotherapeutical medicine, alkylating agent chemotherapeutics, microbiotic chemotherapeutics, any combination of plant chemotherapy drugs in combination platinum-based chemotherapy medicine; Wherein, The about 1mg/kg-100mg/kg cell of the concentration of the chemotherapeutics of said use weight; And said chemotherapeutics function cells crowd's time range is 0.1h-72h, or selects to use the about 10mg/kg-50mg/kg cell of the concentration weight of described chemotherapeutics, and said chemotherapeutics function cells crowd's time is 0.5h-36h.

Wherein, the mark of said labeled cell select BrdU, tritiated thymidine ( ³H-TdR), 5-ethynyl-2 ' deoxyuridine (EDU) but and the arbitrary molecule or the material of the said population of stem cells of other marks or clone or its filial generation; Wherein, The time that the about 0.1mg/kg-15mg/kg cell of the label concentration of said use weight, said mark act on said screening back population of stem cells is 0.01h-96h, perhaps; The time that the about 0.5mg/kg-10mg/kg cell of the label concentration of said use weight, said mark act on said screening back population of stem cells is 0.05h-72h.

3. method according to claim 2 is characterized in that, said mark can discern through the stem cell of tolerance screening with or the number of its filial generation be one or more, wherein, stem cell with or its filial generation retention marker for a long time.

4. method according to claim 1 and 2; It is characterized in that, said population of stem cells with or its filial generation be derived from one or more groups formed in normal liver tissue, renal tissue, heart tissue, pancreatic tissue, intestinal tissue, face tissue, ovary tissue, testis tissue, nervous tissue, stomach-tissue, lung tissue, mammary tissue, fatty tissue, muscle tissue, Tiroidina, mesenchyme, spleen tissue, myeloid tissue, peripheral blood, Cord blood, menses, placenta, dental tissue, ocular tissue and the cerebral tissue of rodent, the mankind or other Mammals biopsies/stripped.

5. method according to claim 1 and 2; It is characterized in that, the population of stem cells of said screening and separating and or its filial generation be derived from pathological tissues before the lesion/cancer between normal and cancer/tumprigenicity, liver cancer, kidney, carcinoma of the pancreas, intestinal cancer, ovarian cancer, mammary cancer, the cancer of the brain, lung cancer, liver's metastatic tumor, pulmonary metastases, tumor cell line and or one or more groups of forming of lesion/cancer in vitro tissue etc. in.

6. according to any normal stem cell, tumor stem cell and or its filial generation that said method obtains in the claim 1 to 5.

7. one kind prepares the described test kit of claim 6, it is characterized in that said test kit comprises:

1) chemotherapeutics;

2) cell washing liquid;

3) cell marking;

4) combination of cytokines;

5) specification sheets of method of use is described, and choose wantonly;

6) mixing vessel and device.

Wherein, said chemotherapeutics is selected with antimetabolic chemotherapeutical medicine, alkylating agent chemotherapeutics, microbiotic chemotherapeutics, any combination of plant chemotherapy drugs in combination platinum-based chemotherapy medicine;

Wherein, said cell washing test solution can be saline water, PBS or other damping fluids;

Wherein, said combination of cytokines comprises that EGF, B27, bEGF, N2, Heparin, LIF are basic combination of cytokines, and said EGF adds when stem cell is cultivated; To final concentration be 5ng/mL-100ng/mL, preferred 10ng/mL-50ng/mL, said bFGF adds when stem cell is cultivated; To final concentration be 5ng/mL-100ng/mL, preferred 10ng/mL-50ng/mL, said Heparin adds when stem cell is cultivated; To final concentration be 2 μ g/mL-40 μ g/mL, said B27, N2 add when stem cell is cultivated, to final concentration be 2ng/mL-50ng/mL; Preferred 10ng/mL-30ng/mL; Said LIF (LIF) adds when stem cell is cultivated, to final concentration be 100U/mL-1500U/mL, preferred 500U/mL-1000U/mL;

Wherein, the specification sheets of said description method of use comprises any described method in the claim 1 to 7.

8. test kit according to claim 7; It is characterized in that the mark that the labeled stem cells of said test kit is carried also may be selected to be any stem cell that can let said screening of martial ethiops mark, quantum dot-labeled, GFP mark, RFP mark or other and or the mark that carries of filial generation.

9. test kit according to claim 7; It is characterized in that, the population of stem cells of the screening of said test kit, identification from the mammiferous normal stripped or biopsy of rodent, mankind and other, clone, cancer before one or more combination of tissue, cancer/tumprigenicity tissue, metastatic tumor/cancerous tissue.

10. according to claim 1 to 9; It is characterized in that, said new dry cell, thereafter generation and or method, the test kit of screening new dry cell be of value to the application in basic, clinical and applied research, organizational project, treatment, drug screening, reparation and fields such as regeneration of damaged or illing tissue.