CN103087980A - Method for identifying and purifying stem cells by using lectin, kit and application method - Google Patents

Abstract

The invention belongs to the field of biotechnology, stem cells and oncology, and discloses a method for identifying and purifying the stem cells by using a lectin, a kit and an application method of the kit. More particularly, the invention relates to the method for identifying and purifying the stem cells in samples derived from rodent animals, humans or other mammalian by using new markers, the corresponding kit for identifying and purifying the stem cells and application of the kit. The method and the kit disclosed by the invention can effectively and accurately identify and purify the stem cells derived from cell populations of the humans or other mammalian, further, a purified sample containing the stem cells can contain no any external marks, in order to facilitate basic research and medical application; and simultaneously, a conventional buffer solution is added for adjusting the concentration and the volume of the sample. The kit disclosed by the invention has the following advantages that the kit is convenient to use, economical, capable of being industrially produced, practical and effective. The invention further relates to obtained stem cells and wide uses of the stem cells in researches and treatments.

Description

Lectin is used for method, test kit and the application method of identification, purifying stem cell

Technical field

the invention belongs to biotechnology, stem cell, oncology, relate to and adopt lectin to be used for identification, the method of purifying stem cell, test kit and application thereof, particularly, relating to the lectin binding substances that adopts specific recognition glycoprotein and/or glycolipid sugar-chain end is used for from rodent, identify in the cell mass that the mankind or other Mammalss are originated, the method of purifying stem cell, test kit, the method disclosed in the present, test kit can be effectively and is used for accurately identifying from described cell mass, the purifying adult stem cell, tumor stem cell, cancer stem cell, the described sample that contains stem cell is not with any external mark.The invention still further relates to the stem cell of acquisition and they such as the extensive use in research or treatment.

Background technology

" lectin " this term occurred as far back as 1888, it is found that it has the erythrocytic characteristic of aggegation.In simple terms, lectin is also a kind of protein, extensively separates from plant, animal, fungi, bacterium etc., has the characteristic (SharonN etc. in conjunction with glycolipid or glycopeptide end specificity carbohydrate, Science, 1972.177 (53): 949-59; Pusztai A, phytohemagglutinin, 1991. Cambridge: the Cambridge University Press), and its unique specificity is, they are distinguishing protein on one group of structure, but and the carbohydrate on the specific binding cytolemma, and further by aggegation (the Pusztai A of the crosslinked realization between the surface of cell membrane oligosaccharyl to cell, phytohemagglutinin, 1991. Cambridge: Cambridge University Press; Sharon N, Trends Biochemistry Sci, 1993.18 (6): 221-226).

As everyone knows, glycosylation is under the effect of enzyme, realizes the process to protein or lipid affix carbohydrate.This process is that corotation moves one of step that (co-translational) and rear transfer modify, and betides endoplasmic reticulum, and cell surface protein glycosylated probability occurs almost surpasses 50%.The eighties as far back as twentieth century, there is the investigator to find, lectin has in conjunction with or kills the ability of embryonal cell carcinoma and Genital carcinoma cells, found again afterwards, the lip-deep antigen of pluripotent stem cell usually is shown as glycoprotein or glycolipid (Andrews PW etc., Hybridoma, 1984.3 (4): 347-61; Pere MF etc., Differentiation, 1988.39 (2): 139-49), this prompting protein specific glycosylation may be the sign that cell has multipotency.

Above-mentioned result of study shows that lectin may have feature (Draber P etc., Somat Cell MolGenet, the 1984.10:435-443 with the cell interaction of versatility; Kosmehl H etc., Neoplasma 1989.36:29-39).And intracellular signal conduction (the Haltiwanger RS.Curr Opin Struct Biol2002.12 (5): 593-8 of the outer molecule of mediated cell or signal enabling is usually modified in the oligomeric saccharification of embrane-associated protein and corresponding part; Xia L etc., Blood, 2004.104 (10): 3091-6).It is found that, in the relevant event of many cells, such as cytodifferentiation (Moody AM etc., Cell, 2001.107 (4): 501-12) and tumour (Reis CA etc., J Clin Pathol, 2010.63 (4): 322-9) in the generation, all can be observed the dynamic change of protein glycosylation.

in embryo's level, for example, on the epithelial cell membrane of a plurality of histoorgans of mice embryonic, the investigator finds to have on it site (Carter WG etc., J.Biol.Chem, 1975.250 (7): 2756-62 of binding specificity lectin, Noguchi M etc., J.Embryol.Exp.Morphol, 1982.72:39-52), further observe and find, dissimilar lectin has otherness aspect identification embryo different tissues epithelium, what is interesting is, after with low dosage radiation part radiation exposure mice embryonic (0.25, 0.50 and 0.75Gy), on cytolemma in conjunction with SBA, the expression amount of PNA and three kinds of lectins of DBA increases (Nievergelt-Egido MC etc., Radiat Environ Biophys, 1993.32 (2): 119-28), and in embryo's different zones, the bonding strength of three kinds of lectins is different.Subsequently on the pancreas epithelium and tubulose cytolemma of growing embryonic stage, other investigators further find, but the site that the specific binding lectin is arranged on above-mentioned cytolemma, this shows the sign that the lectin of specificity sugar epi-position on the identification cytolemma may have versatility as an indicator cells, can be used for characterizing diabetes (Kobayashi etc., BBRC, 2002.293 (2): 691-7).Recent research is found, lewis oligosaccharide (Lewis X antigen) all has expression on the cytolemma of embryonic stem cell, multipotential cell and the embryonal carcinoma cell of mouse, and do not express (Muramatstu TA etc. on the corresponding embryonic stem cell of the mankind, inner cell mass or embryonal carcinoma cell, Glycoconjugate Joumal, 2004.21:41-45), also need further to study this discordance future.

Adopt cytobiology and biochemical method, from experimental studies have found that of a plurality of investigators, the glycosylation of hESC and its albumen closely related (Xia L etc., Blood, 2004.104:3091-6; Satomaa T etc., BMC Cell Biol, 2009.10:42; Venable A etc., BMCDev Biol 2005; 5:15).Specificity glycogen epi-position on cytolemma can be used as new a, specificity marker, the early differentiation state that is used for the reflection mice embryonic, it is expressed early than present embryo's specific antigens of finding, as SSEAl, CD9 and FA, and, development along with embryo's differentiation process, on its film, the expression of specificity glycogen epi-position reduces and disappear (Nash Rodney etc., 2006, stem cell.25 (4): 974-82) gradually.Embryonic stem cell or induced multi-potent stem cells that the employings such as Wang are cultivated are research object, find after with lectin chip analysis of cells extract, specific lectin can be used to identification and Isolation of Embryonic Stem Cell (Wang YC etc., Cell Research, 2011.1-13).。

In the adult level, for example, the ConA (PNA) of identification D-semi-lactosi can be used to rodentine hemopoietic stem cell (the Salner AL etc. that further hive off, 1982, J Natl Cancer Inst.68 (4): 639-41), even can be used to identify and isolate cell mass (Rietze RL etc., the Nature of the PNA positive from nervous tissue, 2001.412 (6848): 736-9), the cell of sorting may be stem cell.Nearest research finds, the N-glycosylation pattern of the human hematopoietic stem cell of CD133+ and progenitor cell and N-glycan structure and genetic expression closely related (Hemmoranta H etc., Exp Hematol, 2007.35 (8): 1279-92).

Lesion/cancer and lectin

Tumour is that body cell loses the true tumor that the normal regulation of its growth is formed, and is cell autogenous control and the interactional result of its residing microenvironment, and its treatment is still a world-famous puzzle.A study hotspot in tumor research field is cancer stem cell (Cancer Stem Cells, CSCs) at present.molecular biologist John Dick investigator by the University of Toronto proposes " cancer stem cell " theoretical (John Dick etc. in early days, Nature, 1994.17:645-648), this new theory shows, the generation of cancer and be difficult to effect a radical cure in the existence of cancer stem cell (CSCs) in the inner, result of study in a plurality of cancer researches is subsequently supported this theory (ReyaT etc., Nature, 2001.414:105-111), the cancer stem cell that further studies confirm that is derived from its hetero-organization exists, they are at for example blood, mammary gland, cerebral tissue, there are (Ai-Hajj M etc. in lung tissue and intestinal tissue, PNAS, 2003.100:3938-3988), He etc., Nat Genet, 2007.39:189-198, Kim CF etc., Cell, 2005.121:823-835, Lapidot T etc., Nature, 1994.367:645-648, Ricci-Vitiani L etc., Nature, 2006, Singh SK etc., Nature, 2004.432:396-401, Yilmaz OH etc., Nature, 2006.441:475-482).The proposition of " cancer stem cell " brings dawn to the treatment of tumour, thereby cancer stem cell will have and important clinical meaning if can identify, thereby such as curing tumour etc. by the target cancer stem cell, will produce great using value and economic benefit.Yet, regrettably, still do not have now effectively method to identify, isolate cancer stem cell from tumor tissues, this also means in treatment, can not accurately and up hill and dale dispose cancer stem cell.This has hindered the effective treatment for tumour greatly.The research discovery, in tumor cell surface and its residing microenvironment, a large amount of sugar chain modified changes and agglutinin receptor dysfunction are brought into play extremely important effect in tumor development and transfer process.Wherein, the interaction of the synthetic regulation and control of sugar chain and sugar chain and lectin has participated in all respects of oncobiology behavior.Lectin has been widely used in the research of tumor cell surface sugar connector, and it is playing a very important role aspect biological behaviour research, diagnosis, treatment and prognosis thereof of tumour.

Adult stem cell:

Adult stem cell is another special population of stem cells, increasing evidence shows that adult stem cell is found in the multiple (MooreKA of mature tissue, Science, 2006.311 (5769): 1880-1885), forming the basis of soma cell research and its application prerequisite and one of most critical condition clinically is effective and accurate screening and separating to stem cell.Current, the investigator has identified the relevant molecular surface sign of a plurality of adult stem cells, for example Thy-1low, FLK2-Lineage-Sca-l+c-Kit+ are used for identification hemopoietic stem cell (Hematopoietic stem cells, HSCs) (Christemsen JL etc., PNAS, 2001.98:14541-14546; Uchida N etc., Experimental hematology, 1996.24:649-659), ISL1+, Sca-l+ or c-kit+ (Laugwitz KL, etc., Nature.2004.433 (7026): 647-653; Okada S etc., Blood, 1991.78 (7): 1706-12; Matsuura K etc., J Biol Chem, 2004.279 (12): 11384-91), α 6bri10G7dim is for identification epidermal stem cells (LiA etc., PNAS, 1998.95 (7): 3902-7; Tani H etc., PNAS, 2000.97 (20): 10960-10965; Lavker RM etc., PNAS, 2000.97 (25): 13473-75), although obtaining certain progress aspect adult stem cell special molecular sign, but separate stem cells is still and has challenging task from adult, be its comparatively small amt on the one hand and more be difficult to location, the various tissue specificity stem cells of screening and separating, being to lack reliable specific molecular marker thing on the one hand in addition identifies it, special cell surface marker is difficult to reach common recognition at present, and its validity needs further to analyze.

Summary of the invention

The object of the invention is to disclose method, test kit and the application method thereof of a kind of identification, purifying stem cell, and further expand its application.

The inventor for achieving the above object, conduct in-depth research, the pleasantly surprised discovery of the inventor as a result, specific lectin not only can be used for identifying the healthy tissues in rodent, the mankind or other Mammalss sources or specificity adult stem cell, tumor stem cell, the cancer stem cell in organ, and can be used for being purified into described stem cell from the cell mass that derives from described species.

The invention discloses and a kind ofly adopt that lectin is used to identify, the method for purifying stem cell, it comprises:

(1) make described cell mass and cell pretreatment reagent in advance under contact conditions, make a kind of lectin of sample contact, the latter is contained: (a) at least a lectin, it can identify glycoprotein and/or glycolipid sugar-chain end specific site, and this lectin directly or indirectly is connected with (b) at least a binding substances; With

(2) will separate from described sample with the cell mass that described lectin binding substances is combined, obtain to be rich in the sample of stem cell, wherein, on described cell, existence shows that with the acceptor of described lectin pathoklisis they are stem cells.

Wherein, method that can be by adding wash-out sugar is described lectin binding substances wash-out from the described cell mass, thereby obtains not contain any foreign labeled sample that is rich in stem cell.

Wherein, the cell pretreatment process choosing adopts the erythrocytic reagent in precipitation or cracking process precipitation or lysing cell group, and it is any of 0.1～20% hydroxyethylamyle, gelatin, dextran, polyvinylpyrrolidone, methylcellulose gum, carboxymethyl starch that described reagent may be selected to be mass concentration; Or may be selected to be any less than 0.9% normal saline solution of ammonium chloride erythrocyte cracked liquid, mass concentration; Maybe can select any cell pretreatment that carries out of Sodium Diatrizoate-dextran, HITOPAQUE, Ficoll, wherein, described reagent repeats to contact described cell mass once at least.

Wherein, lectin in described lectin binding substances comprises one or more combination of phytohemagglutinin natural or chemosynthesis, zoo-agglutinin or derivatives thereof, the concentration of described lectin is 0.001～40mg/ml, the time that contacts with described cell mass, incubation temperature was for being less than or equal to 38 ℃ in order to be less than or equal to 120 minutes.

Wherein, binding substances in described lectin binding substances can be selected in advance to carry out combination with any of biomacromolecule of fluorescence dye, magnetic microsphere or high molecular, or selects to have aggegation is have a kind of of affinity antibodies or antibody derivatives or antibody fragment.

Wherein, to between 800,000 dalton (Mw), the biomacromolecule of wherein said high molecular may be selected to be any of bovine serum albumin, hydroxyethylamyle, gelatin, methylcellulose gum, carboxymethyl starch, dextran, polyvinylpyrrolidone to the molecular weight of the biomacromolecule of described high molecular of being combined with lectin 10,000 dalton (Mw).

Wherein, the described cell mass that contains stem cell derives from tissue before rodent, the mankind or other mammiferous adult tissues, lesion/cancer, lesion/cancer tissue, metastatic tumor/cancerous tissue or other potential combinations that comprises one or more tissues of stem cell, or selection is from primary, the passage cell group's of described tissue one or more combination.

The present invention adopts openly also that lectin is used to identify, test kit and the application method thereof of purifying stem cell, wherein, described lectin can be used for also determining that in tissue, cell mass, stem cell exists and content, and it is in the purposes that is used for from described cell mass enrichment, purification stem cell.

Described test kit for identification, purifying stem cell, it comprises:

1) cell pretreatment reagent A;

2) identification, purified reagent B;

3) and optional container;

Wherein, to may be selected to be mass concentration be any of 01～20% hydroxyethylamyle, gelatin, dextran, polyvinylpyrrolidone, methylcellulose gum, carboxymethyl starch to described cell pretreatment reagent A; Or may be selected to be any less than 0.9% normal saline solution of ammonium chloride erythrocyte cracked liquid, mass concentration; Described cell pretreatment reagent maybe can be selected any of Sodium Diatrizoate-dextran, HITOPAQUE, Ficoll, and wherein, described reagent repeats to contact described cell mass once at least.

wherein, identification, purified reagent B is the lectin binding substances, include but not limited to phytohemagglutinin natural or synthetic, the combination of one or more of zoo-agglutinin or derivatives thereof, binding substances in wherein said lectin binding substances can be selected in advance and fluorescence dye, the biomacromolecule of magnetic microsphere or high molecular any carries out combination, or select to have aggegation is have a kind of of affinity antibodies or antibody derivatives or antibody fragment, the concentration of described lectin is 0.001～40mg/ml, the time that contacts with described cell mass is for being less than or equal to 120 minutes, incubation temperature is for being less than or equal to 38 ℃.

Wherein, the described cell mass that contains stem cell be rodent, the mankind or other Mammalss sources normal adult and or pathology, tumour, cancer before, carcinous, metastasis of cancer tissue, or from primary, the passage cell group's of described tissue one or more combination.

Wherein, can select subsequently to adopt fluorescent activation cell sorting method for the identification of with the cell mass of sorting binding lectin binding compounds or the described cell mass of selective precipitation method separation and combination lectin, further, the described stem cell that separates, it comprises the acceptor that can be combined with lectin on cytolemma.

Wherein, the stem cell of the purifying that obtains according to the inventive method, test kit, can be further method by adding wash-out sugar described lectin binding substances wash-out from the described cell, thereby make the stem cell of acquisition not be with any external marker.

Wherein, the stem cell of described identification, purifying comprises myeloid-lymphoid stem cell, multipotential stem cell, special energy stem cell, adult stem cell, tumor stem cell, cancer stem cell, and further, it contacts with known stem cell markers.

Wherein, test kit of the present invention also comprises wash-out sugar, be used for described lectin binding substances wash-out from the described cell, thereby the sample that makes described acquisition contain stem cell is not with any external mark.

Wherein, the stem cell of described acquisition also can add physiological saline, PBS damping fluid, HBSS damping fluid to adjust concentration and the volume of stem cell.

Wherein, according to test kit application method of the present invention, its application method comprises the following steps:

(1) described cell sample contacts with the cell pretreatment reagent A: as add the erythroprecipitin agent, select to get supernatant; As add erythrocyte cracked liquid, select to abandon supernatant after centrifugal and get cell precipitation;

(2) the described cell mass through step (1) is contacted with described identification, purified reagent B, the concentration of described lectin is 0.001～40mg/ml, the time that contacts with described cell mass, incubation temperature was for being less than or equal to 38 ℃ in order to be less than or equal to 120 minutes;

(3) separate the cell mass that obtains the lectin positive: can select to adopt fluorescent activation cell sorting, magnetic bead sorting method or staticly settle method;

(4) add wash-out sugar described lectin binding substances from wash-out on the described cell of step (3) acquisition, thereby the stem cell that makes acquisition is not with any external marker, further, the stem cell of acquisition also can add physiological saline, PBS damping fluid, HBSS damping fluid to adjust concentration and the volume of stem cell.

The stem cell of described separation is used for, the further analysis of biological example chemistry, molecular biology or marker levels, further, described cell can be same phenotype or not isophenic set, and the stem cell of wherein said identification comprises at least 1, and the stem cell of described purifying comprises and is at least 1%, preferably comprise at least 50%, more preferably comprise at least 70%, more preferably comprise at least 80%, most preferably comprise at least 90%.

Further, its cell surface of the stem cell of described separation has the acceptor of can the lectin in the lectin binding substances being combined, and further described cell contacts with known stem cell markers, for example Sca-l, Alb, c-Kit, CD90, Nkx2.5 molecule.

Further, according to method of the present invention, can be used for using for the preparation of the diagnostic reagent of stem cell existence and/or content in the diagnosis sample.

Wherein, the stem cell of test kit identification of the present invention, purifying is adult stem cell, tumor stem cell, cancer stem cell, and the stem cell of described identification comprises at least 1, and the purity of the stem cell of purifying is at least 1%.Adult stem cell of the present invention, the stem cell that preferred postnatal tissue obtains.In the Mammals such as the mankind, preferred, the adult stem cell that obtained large individuality from least 1 year old, preferred postdevelopmental individuality is for example grown up.In a preferred embodiment of the invention, described stem cell is adult stem cell, tumor stem cell and/or cancer stem cell.

Wherein, detect or the method for separate stem cells comprise adopt fluorescent activation cell sorting method (FACS) for the identification of with the cell of purifying binding lectin binding substances.

Wherein, the stem cell of the separation that obtains according to the method for the invention, test kit, further, and the stem cell of separation, it comprises the acceptor that can be combined with lectin on cytolemma.

Accordingly, the present invention realizes identification from the sample that described rodent, the mankind or other Mammalss are originated, purifying stem cell by aforesaid method, test kit, and further acquisition not with any foreign labeled sample that contains described stem cell, the population of stem cells of separation of the present invention basis and applied research, organizational project, treatment, reparation is impaired or pathological tissues, drug screening etc. in application.

Test kit of the present invention has general applicability, can be used for stem cell accurate and that identification fast, purifying rodent, the mankind or other Mammalss are originated.Further, but described test kit has the simple and direct suitability for industrialized production of use, high specificity, practicability and effectiveness, and application prospect is advantage widely.

Description of drawings

The figure as a result of Fig. 1 identification, purifying Lung stem cells, concrete as embodiment 1.The streaming figure (n=2) of A figure expression homotype control group; B figure expression separates, the streaming figure of purifying Lung stem cells, and the positive viable cell quantity of lectin purified from cell mass is about 4.5% (n=2); The purity of the described Lung stem cells of C figure expression.

Identification in Fig. 2 marrow, Cord blood, purifying stem cell figure as a result, concrete as embodiment 2.

The HPL (PE-HA) of A figure expression employing PE mark is identified from the medullary cell group, the result of purifying stem cell, and positive cell quantity is 2.6% (n=2), and B figure expression cell purity is 95.7%; The Ulex europaeus lectin (PE-UEA) of C figure expression employing PE mark is identified from the cord blood cell group, the result of purifying stem cell, and positive cell quantity is 3.5% (n=2), and D figure expression cell purity is 96.8%.

The figure as a result of Fig. 3 identification, purifying liver-cancer stem cell, concrete as embodiment 3.A figure expression homotype control group, cellular control unit quantity is 0.13% (n=2); B figure expression adopts the lectin SJ (T-SJ) of TRITC mark as the result of molecule marker purification stem cell from liver cancer tissue, and the lectin positive cell quantity is 5.6% (n=2); The new T-SJ masculine liver cancer population of stem cells that separates of C figure expression, asterisk refers to CD90+ (green) cell, and wherein nucleus dyes with DAPI, and the magnification of picture is 100 times.

Fig. 4 adopts that test kit of the present invention is identified from cell mass, the figure as a result of purifying liver stem cells and Lung stem cells, and is concrete as embodiment 4.Purifying and the microscopic examination result of A-C figure expression liver stem cells, wherein, A figure expression homotype control group, cellular control unit quantity is 0.05% (n=2); The lectin PT that the gelatin combination is adopted in B figure expression purifying stem cell and carry out the result of streaming identification and analysis with FITC-PT from the liver cell group, the lectin positive cell quantity is 2.6% (n=2); The new cell mass that separates of C figure expression, short arrow refers to the F-PT+/Alb+ liver stem cells, asterisk refers to the F-PT+/Alb-liver stem cells.Purifying and the microscopic examination result of A '-C ' figure expression Lung stem cells, wherein, A ' figure expression homotype control group, cellular control unit quantity is 0.19% (n=2); The lectin RC that the gelatin combination is adopted in B ' figure expression purifying stem cell and carry out the result of streaming identification and analysis with TRITC-RC from the lungs cell mass, the lectin positive cell quantity is 4.3% (n=2); The new cell mass that separates of C ' figure expression, arrow refers to T-RC+ (redness)/c-Kit+ (green) Lung stem cells.Nucleus dyes with DAPI, and the magnification of picture is 100 times.

Fig. 5 adopts that test kit of the present invention is identified, the figure as a result of purifying heart stem cell from cell mass, concrete as embodiment 5.A figure expression homotype control group, cellular control unit quantity is 0.1% (n=2); B figure expression adopts the lectin SJ (F-SJ) of FITC mark as the result of molecule marker purifying stem cell from the heart cell group, and the lectin positive cell quantity is 4.9% (n=2); The cell mass of the new F-SJ positive of separating of C figure expression, wherein nucleus dyes with DAPI, and short arrow refers to the positive cardiac stem cells of F-SJ, and long arrow refers to the F-SJ+/Nkx2.5+ cardiac stem cells, and the magnification of picture is 100 times.

Embodiment

Below in conjunction with specific embodiment to describe advantage of the present invention in detail; 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, any all drop on spiritual category of the present invention and protection domain based on modification of the present invention, replacement, change within.

As described herein, unless context separately has clearly indication, otherwise do not limit the implication of object.

As skilled in the art to understand, in literary composition of the present invention, " a kind of " refers to " at least a ".Term " comprises ", " comprising ", " containing ", " selection " are synonyms, is that have a pardon or open, and does not get rid of the extra member, key element or the method steps that do not describe in detail.As described herein, term " cell mass " refers to the combination of one or more cell, typically refer to one group of cell, except as otherwise noted, otherwise this term refers to be formed or comprised by the cell of separation of the present invention the cell colony of the cell that separates herein.

As described herein, term " tissue " comprises tissue before the healthy tissues that obtains from rodent, the mankind or other Mammalss or organ sample, lesion/cancer, lesion/cancer tissue, metastatic tumor/cancerous tissue.described cell mass is derived from rodent, the mankind or other Mammals healthy tissuess or organ sample, tissue before lesion/cancer, the lesion/cancer tissue, metastatic tumor/cancerous tissue, be derived from primary cell group prepared in above-mentioned species, obtain in the passage cell of described cell mass or the clone of setting up, it can form or comprise and at least part ofly have altogether that isophenic cell forms by having altogether isophenic cell, when cell substantially similar or when consistent on one or more notable features, can think that cell has common phenotype, its feature includes but not limited to the form outward appearance, certain cellular constituent or product (RNA, protein or other material) the having or not or level of expression, the vigor of certain biochemical route, multiplication capacity and or dynamic behavior, differentiation potential and or to response or the vitro culture behavior of differentiation signal.Therefore this notable feature can be decided to be a cell mass or its part.

As described herein, term " stem cell " refers to the progenitor cell of self (namely do not break up and can breed) for a long time, be in tranquillization or propagation, wherein the offspring of stem cell or at least its part substantially kept parental generation stem cell specialization or phenotype, differentiation potential and multiplication capacity relatively less specialization.This term comprises the stem cell of unlimited self basically, that is: compare with parental generation, offspring or its part further ability of propagation significantly do not reduce, and the stem cell that shows limited self, that is: compare with parent cell, offspring or its part further ability of propagation significantly reduce.Based on its celliferous type, described stem cell or progenitor cell can be multipotency, all-round, specially can or one or more combination of monoenergetic.Term " cell mass that contains stem cell " refers to contain at least a stem cell or progenitor cell in the present invention, perhaps comprises the cell mass of part progenitor cell or stem cell.Usually, the stem cell of described part or progenitor cell can have common phenotype, also can have different phenotypes.

As used herein, in literary composition of the present invention, term " rodent " refers to rat, mouse etc.; " Mammals " refers to the mankind, ox, horse, dog, rabbit, monkey etc.As used herein, term " exsomatizes " and comprises the tissue that leaves animals or humans or cell and in external preservation or propagation, for example be kept in culture vessel.Term " biopsy " comprises that the method that adopts this area generally to understand obtains tissue from animal or human tissue or organ.That " healthy tissues or organ sample " herein refers to is healthy, unconverted, non-malignant, non-carcinous or nononcogenic tissue or organ sample.Whether any pathologist, those skilled in the art can determine to organize is healthy, unconverted, non-malignant, non-carcinous or nononcogenic.

The present invention comes from the inventor's further investigation, the pleasantly surprised discovery specific agglutination element of the inventor can be used for identification tissue, lesion/cancer tissue, metastatic tumor/cancerous tissue before rodent, the mankind or other Mammals healthy tissuess or organ sample, lesion/cancer, is purified into adult stem cell, tumor stem cell or cancer stem cell, further can be used for identification from the clone of the primary cell group that derives from above-mentioned tissue, passage cell group, foundation, is purified into described stem cell.

The object of the invention is to disclose a kind of method that adopts specific agglutination element binding substances to be used for identification, purifying stem cell, comprising, adopt specific lectin binding substances for the method for the sample identification of originating from rodent, the mankind or other Mammalss, purifying adult stem cell, tumor stem cell, cancer stem cell, wherein, the described sample source primary cell group, passage cell group, the clone that mammiferously exsomatize, prepare in biopsy or any one or the two or more tissue that obtain by conventional means from rodent, the mankind or other.

Another object of the present invention is to disclose a kind of from the cell mass that contains stem cell test kit and the application method thereof of identification and purifying stem cell, wherein, described cell mass is derived from the clone of the primary cell group, passage cell group or the foundation that prepare in rodent, the mankind or other mammiferous any one or two or more tissue.

Further, but described test kit have and use simple and direct, high specificity, practicability and effectiveness suitability for industrialized production, application prospect advantage widely.

The invention discloses and a kind ofly adopt that lectin is used to identify, the method for purifying stem cell, it comprises:

(1) make described cell mass and cell pretreatment reagent in advance under contact conditions, make a kind of lectin of sample contact, the latter is contained: (a) at least a lectin, it can identify glycoprotein and/or glycolipid sugar-chain end specific site, and this lectin directly or indirectly is connected with (b) at least a binding substances; With

(2) will separate from described sample with the cell mass that described lectin binding substances is combined, obtain to be rich in the sample of stem cell, wherein, on described cell, existence shows that with the acceptor of described lectin pathoklisis they are stem cells.

Wherein, method that can be by adding wash-out sugar is described lectin binding substances wash-out from the described cell mass, thereby obtains not contain any foreign labeled sample that is rich in stem cell.

Wherein, the cell pretreatment process choosing adopts the erythrocytic reagent in precipitation or cracking process precipitation or lysing cell group, and it is any of 0.1～20% hydroxyethylamyle, gelatin, dextran, polyvinylpyrrolidone, methylcellulose gum, carboxymethyl starch that described reagent may be selected to be mass concentration; Or may be selected to be any less than 0.9% normal saline solution of ammonium chloride erythrocyte cracked liquid, mass concentration; Maybe can select any cell pretreatment process of carrying out of Sodium Diatrizoate-dextran, HITOPAQUE, Ficoll, wherein, described reagent repeats to contact described cell mass once at least.

Wherein, the lectin in described lectin binding substances comprises one or more combination of phytohemagglutinin natural or chemosynthesis, zoo-agglutinin or derivatives thereof, and the concentration of described lectin is 0.001～40mg/ml, further, the concentration of described lectin is at least 0.001mg/ml, is at least 0.5mg/ml, is at least 3mg/ml, be at least 6mg/ml, be at least 12mg/ml, be at least 20mg/ml, be at least 25mg/ml, be at least 30mg/ml, be no more than 40mg/ml.

Wherein, the time that described lectin binding substances contacts with described cell mass is for being less than or equal to 120 minutes, further, be at least duration of contact 1min, be at least 10min, be at least 20min, be at least 30min, be at least 40min, be at least 1hrs, be at least 2hrs.

Wherein, the incubation temperature of described lectin binding substances and cell mass is for being less than or equal to 38 ℃.Further, be at least 2 ℃, be at least 8 ℃, be at least 12 ℃, be at least 16 ℃, be at least 20 ℃, be at least 24 ℃, be at least 28 ℃, be at least 32 ℃, be no more than 38 ℃.

Wherein, binding substances in described lectin binding substances can be selected in advance to carry out combination with any of biomacromolecule of fluorescence dye, magnetic microsphere or high molecular, or selects to have aggegation is have a kind of of affinity antibodies or antibody derivatives or antibody fragment.

Wherein, to between 800,000 dalton (Mw), the biomacromolecule of wherein said high molecular may be selected to be any of bovine serum albumin, hydroxyethylamyle, gelatin, methylcellulose gum, carboxymethyl starch, dextran, polyvinylpyrrolidone to the molecular weight of the biomacromolecule of described high molecular of being combined with lectin 10,000 dalton (Mw).

Wherein, the described cell mass that contains stem cell derives from tissue before rodent, the mankind or other mammiferous adult tissues, lesion/cancer, lesion/cancer tissue, metastatic tumor/cancerous tissue or other potential combinations that comprises one or more tissues of stem cell, or selection is from primary, the passage cell group's of described tissue one or more combination.

The present invention adopts openly also that lectin is used to identify, test kit and the application method thereof of purifying stem cell, wherein, described lectin can be used for also determining that in tissue, cell mass, stem cell exists and content, and it is in the purposes that is used for from described cell mass enrichment, purification stem cell.

Described test kit for identification, purifying stem cell, it comprises:

1) cell pretreatment reagent A;

2) identification, purified reagent B;

3) and optional container;

Wherein, to may be selected to be mass concentration be any of 0.1～20% hydroxyethylamyle, gelatin, dextran, polyvinylpyrrolidone, methylcellulose gum, carboxymethyl starch to described cell pretreatment reagent A; Or may be selected to be any less than 0.9% normal saline solution of ammonium chloride erythrocyte cracked liquid, mass concentration; Described cell pretreatment reagent or may be selected to be any of Sodium Diatrizoate-dextran, HITOPAQUE, Ficoll, wherein, described reagent repeats to contact described cell mass once at least.

wherein, identification, purified reagent B is the lectin binding substances, include but not limited to phytohemagglutinin natural or synthetic, the combination of one or more of zoo-agglutinin or derivatives thereof, binding substances in wherein said lectin binding substances can be selected in advance and fluorescence dye, the biomacromolecule of magnetic microsphere or high molecular any carries out combination, or select to have aggegation is have a kind of of affinity antibodies or antibody derivatives or antibody fragment, the concentration of described lectin is 0.001～40mg/ml, the time that contacts with described cell mass is for being less than or equal to 120 minutes, incubation temperature is for being less than or equal to 38 ℃.

Wherein, the described cell mass that contains stem cell be rodent, the mankind or other Mammalss sources normal adult and or pathology, tumour, cancer before, carcinous, metastasis of cancer tissue, or from primary, the passage cell group's of described tissue one or more combination.

Wherein, can select subsequently to adopt fluorescent activation cell sorting method for the identification of with the cell mass of sorting binding lectin binding compounds or the described cell mass of selective precipitation method separation and combination lectin, further, the described stem cell that separates, it comprises the acceptor that can be combined with lectin on cytolemma.

Wherein, the stem cell of the purifying that obtains according to the inventive method, can be further method by adding wash-out sugar described lectin binding substances wash-out from the described cell, thereby make the stem cell of acquisition not be with any external marker.

Wherein, test kit of the present invention also comprises wash-out sugar, be used for described lectin binding substances wash-out from the described cell, thereby the sample that makes described acquisition contain stem cell is not with any external mark.

Wherein, the stem cell of described identification, purifying comprises myeloid-lymphoid stem cell, multipotential stem cell, special energy stem cell, adult stem cell, tumor stem cell, cancer stem cell, and further, it contacts with known stem cell markers.

Wherein, the stem cell of described acquisition also can add physiological saline, PBS damping fluid, HBSS damping fluid to adjust concentration and the volume of stem cell.

Wherein, according to test kit application method of the present invention, its application method comprises the following steps:

(1) described cell sample contacts with the cell pretreatment reagent A: as add the erythroprecipitin agent, select to get supernatant; As add erythrocyte cracked liquid, select to abandon supernatant after centrifugal and get cell precipitation;

(2) the described cell mass through step (1) is contacted with described identification, purified reagent B, the concentration of described lectin is 0.001～40mg/ml, the time that contacts with described cell mass, incubation temperature was for being less than or equal to 38 ℃ in order to be less than or equal to 120 minutes;

(3) separate the cell mass that obtains the lectin positive: can select to adopt fluorescent activation cell sorting, magnetic bead sorting method or staticly settle method;

(4) add wash-out sugar described lectin binding substances from wash-out on the described cell of step (3) acquisition, thereby the stem cell that makes acquisition is not with any external marker, further, the stem cell of acquisition also can add physiological saline, PBS damping fluid, HBSS damping fluid to adjust concentration and the volume of stem cell.

Wherein, the stem cell of identification of the present invention, purifying is adult stem cell, tumor stem cell, cancer stem cell, further, its cell surface of described separate stem cells has the acceptor of can the lectin in the lectin binding substances being combined, further contact with known stem cell markers, for example Sca-1, Alb, c-Kit, CD90, Nkx2.5 molecule.

Wherein, the present invention comprises that also adopting described lectin to be used for definite sample stem cell exists and content.

Wherein, the binding substances of binding lectin of the present invention also can be selected to be combined with the material that allows radiological imaging, positron emission computerized tomography, Magnetic resonance imaging or X ray or computed tomography.

Wherein, the purification of lectin positive cell group (purifying) can adopt flow cytometer (lectin of being combined with fluorescein), magnetic bead screening (being coated with the magnetic bead of lectin), adsorption column or other known purification means to obtain, for example, the affine purification of immunity, binding compounds is combined with solid support, for another example, elutriation, binding compounds is combined with the tissue culture ware.

Further, if use fluorescein-labelled lectin, the method purification of target cell from cell mass that utilizes flow cytometer is preferred, and more preferably fluorescence activated cell sorter (FACS) separates described cell, by using this device, can automatically separate, reclaim target cell.

Further, the stem cell that the method according to this invention, test kit are identified comprises at least 1, further, the purity at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5% or at least 1% of the method according to this invention, stem cell that test kit is purified.

Should be appreciated that many lectins are all information known in the art.Term " lectin " refers to total protein group in conjunction with glycolipid or the specific carbohydrate group characteristic of glycoprotein.Described lectin can be from natural origin purifying lectin, for example from plant, animal, fungi, algea and bacteria, the lectin or derivatives thereof of perhaps selecting to modify (natural or synthetic), perhaps by chemosynthesis it.One or more subunits in the lectin derivative comprises many-subunit's lectin.In a preferred embodiment, lectin can be selected phytohemagglutinin, and lectin can commercially obtain from many commercial supplier there, for example Sigma company.

Further, the binding substances of described binding lectin also can be selected to be combined with the material that allows radiological imaging, positron emission computerized tomography, Magnetic resonance imaging or X ray or computed tomography.

The invention still further relates to the lectin binding substances is being used for from the purposes of described cell mass identification, purifying stem cell.

Lectin compositions in the bottle of test kit of the present invention can be the form of pharmaceutically acceptable solution, for example with Sterile Saline, PBS damping fluid or other pharmaceutically acceptable sterile liquid combination.Perhaps, can freeze-drying or dry lectin compositions, in this type of situation, test kit is the optional pharmaceutically acceptable solution that is contained in container that comprises also, such as physiological saline, PBS damping fluid etc., preferred aseptic, to dissolve described freeze-drying or dry lectin compositions.

Sample of the present invention comprises, thereby for example can be by organizing or organ sample mechanical shearing or be broken into less tissue block obtain single cell suspension from solid organ.Choose wantonly, can by such as chemicals for example EDTA and or by using, the fritter of organizing that the digestion characteristics of collagenase, Dispase, trypsinase or zymine will obtain further is separated into single cell.Marrow and or (umbilical cord) blood, urine, cerebrospinal fluid can be regarded as the n cell suspension.

Wherein, stem cell according to the binding lectin binding substances of method of the present invention, the obtainable separation of test kit, when needs during from lectin binding substances separate stem cells, can separate by the several different methods known to those skilled in the art, for example, by adding wash-out sugar, or carry out separating of described stem cell and lectin binding substances by changing change pH values or salt concn.The stem cell and the filial generation thereof that it will be understood by those of skill in the art that described separation also belong to spiritual category of the present invention and protection domain.The stem cell that separates, it comprises the acceptor that can be combined with lectin on cytolemma.Based on the stem cell of separation of the present invention, can carry out genetic modification to it, for example, described stem cell is contacted with modifying factor group sequence with nucleic acid, then separate the wherein adorned stem cell of genome sequence, be applied to afterwards the application such as treatment.Further; any modification, application to the stem cell that obtains based on the present invention; the stem cell that for example obtains by any method of the present invention or the purposes of composition in the medicine of or illing tissue impaired for the preparation for the treatment of of stem cell, this all belongs to spiritual category of the present invention and protection domain.

Further, adopt population of stem cells that identification purification process of the present invention, test kit obtain basis and applied research, organizational project, treatment, reparation is impaired or pathological tissues, drug screening etc. in application.

Embodiment

The identification of embodiment 1, Lung stem cells and purifying thereof

Material: DAPI, DBL DBA be available from Sigma, collagenase (Sigma), foetal calf serum, DMEM/F-12K substratum, blood counting chamber, ammonium chloride (1 *).Lavation buffer solution: PBS (the pH value is 7.4 ± 0.1); Sealing damping fluid: add 3%BSA in lavation buffer solution (PBS); Antigen retrieval liquid: citrate buffer solution; Permeable membrane damping fluid: TBS (0.1%Triton+PBS).Wherein, the binding substances of lectin is chosen as the FITC mark.

a) fresh biopsy lung tissue is put into sterile petri dish, with PBS buffer solution for cleaning 3 times, each 3～5min, subsequently, shear with aseptic eye scissors and be organized as fine grained chippings, change in the 50ml centrifuge tube that fills collagenase/tryptic digestive juice, at 37 ℃, 100rpm digests 45min～60min, what add subsequently 4 ℃ of precoolings contains 10% foetal calf serum (Fetal bovine serum, FBS) DMEM/F12K substratum termination reaction, to blow and beat into single cell suspension through the tissue block of digestion with disposable syringe again, afterwards, at 4 ℃, with the centrifugal 3～5min of 200g, abandon the PBS that contains 2%FBS that adds 4 ℃ of precoolings after supernatant, mixing gently, be filtered in another 50ml centrifuge tube through 180 eye mesh screens, add mixing after the NH4Cl lysate, room temperature is placed 3～5min, at 4 ℃, with the centrifugal 3～5min of 200g, add 4 ℃ of precoolings of 1ml to contain the PBS mixing of 2%FBS after abandoning supernatant, with blood counting instrument counting cells quantity, cell is divided into two parts, portion adds F-DBA, wherein the concentration of lectin is 5mg/ml, portion adds the excessive homotype contrast (Fig. 1 .A) of equal-volume, two parts of cells are all hatched 30～60min 37 ℃ of lucifuges, afterwards, at 4 ℃, after the centrifugal 3～5min of 200g, abandon supernatant, the PBS re-suspended cell that contains 2%FBS that adds 4 ℃ of precoolings, selected by flow cytometry apoptosis F-DBA+ cell mass (Fig. 1 .B), the stem cell purity of purifying is 94% (Fig. 1 .C).Before analysis, interpolation propidium iodide (PI) comes the dead cell in Exclusion analysis.

Result: adopt method of the present invention can identify the stem cell in lung tissue and further can carry out purifying (Fig. 1) to it, can further add N-Acetygalactose amine described lectin binding substances wash-out from described cell mass, thereby obtain not to be with any foreign labeled cell sample, and further, add physiological saline to regulate concentration and the volume of stem cell in described sample.

The identification of embodiment 2, marrow, cord blood stem cell and purifying thereof

Prepare respectively marrow, Cord blood.

Material: with embodiment 1, wherein dextran (Sigma), HP, UEA are available from Vector, and the binding substances of lectin is bovine serum albumin (BSA).

Method:

a): add the dextran of 6～15% mass concentrations in bone marrow cell suspension, thereby the time of exposing cell is 30min precipitation red corpuscle, draw subsequently supernatant at 4 ℃, with the centrifugal 3～5min of 230g, abandon the PBS that contains 2%FBS that adds 4 ℃ of precoolings after supernatant, mixing gently, be filtered in another centrifuge tube with blood counting instrument counting cells quantity through 200 eye mesh screens, the blue staining cell survival rate 98% of placenta, with the lectin HP that adds the BSA combination, wherein, the concentration of lectin is 1.2mg/ml, after contact 60min under 35 ℃ of conditions, draw lower floor's cell precipitation, the cell mass of sorting can further elute the lectin binding substances with wash-out sugar N-acetylgalactosamine from cytolemma, at 4 ℃, with the centrifugal 2～3min of 230g, add 4 ℃ of precoolings to contain the PBS mixing of 1%FBS after abandoning supernatant.Subsequently, at 4 ℃, after the centrifugal 3～5min of 230g, abandon supernatant, add the PBS re-suspended cell that contains 1%FBS of 4 ℃ of precoolings, thereby obtain not contain any foreign labeled cell mass, further can add the PBS damping fluid to regulate concentration and the volume of stem cell.Adopt quantity and the purity of the described cell mass of flow cytometer identification and analysis, result shows that the per-cent of positive cell is 2.6% (Fig. 2 .A), and the stem cell purity of identification, purifying is 95.7% (Fig. 2 .B).Before analysis, interpolation propidium iodide (PI) comes the dead cell in Exclusion analysis.

b) add the dextran of 6～15% mass concentrations in the cord blood cell suspension, thereby the time of exposing cell is 40min precipitation red corpuscle, draw subsequently supernatant at 4 ℃, with the centrifugal 3～5min of 220g, abandon the PBS that contains 2%FBS that adds 4 ℃ of precoolings after supernatant, mixing gently, be filtered in another centrifuge tube with blood counting instrument counting cells quantity through 160 eye mesh screens, the blue staining cell survival rate 98% of placenta, the centrifugal lectin UEA that adds the BSA combination after supernatant that abandons, wherein, the concentration of lectin is 2.1mg/ml, after contact 45min under 37 ℃ of conditions, draw lower floor's cell precipitation, identification, the cell mass of purifying can further elute the lectin binding substances with wash-out sugar trehalose from cytolemma, at 4 ℃, with the centrifugal 2～3min of 220g, add 4 ℃ of precoolings to contain the PBS mixing of 1%FBS after abandoning supernatant.Subsequently, at 4 ℃, after the centrifugal 3～5min of 220g, abandon supernatant, add the PBS re-suspended cell that contains 1%FBS of 4 ℃ of precoolings, thereby obtain not contain any foreign labeled cell mass, further can add physiological saline to regulate concentration and the volume of stem cell.Adopt the quantity of flow cytometer identification and analysis positive cell group, result shows that positive cell quantity is 3.5% (Fig. 2 .C), and the stem cell purity of identification, purifying is 96.8% (Fig. 2 .D).Before analysis, interpolation propidium iodide (PI) comes the dead cell in Exclusion analysis.

Result: adopt the method for the invention can identify, be purified into stem cell (Fig. 2) from marrow, Cord blood.

The identification of liver-cancer stem cell and purifying thereof in embodiment 3, liver cancer tissue

Tissue preparation: prepared the liver cancer tissue from the adult animal, tissue cleans each 3～5min 3 times through the PBS buffer solution for cleaning.

Material: with embodiment 1, wherein the SJ lectin (T-SJ) of TRITC mark is available from Sigma.

Method:

a) after under the aseptic condition, acquisition fresh HCC tissue changes in sterile petri dish, shear with aseptic eye scissors and be organized as fine grained chippings, change over to again in the 50ml centrifuge tube that fills the loose enzymic digestion liquid of collagenase/trypsinase part, at 37 ℃, after 100rpm digestion 45min～60min, what add 4 ℃ of precoolings contains 10% foetal calf serum (Fetal bovine serum, FBS) DMEM/F12K substratum termination reaction, to blow and beat into single cell suspension through the tissue block of digestion with disposable syringe again, afterwards, at 4 ℃, with the centrifugal 3～5mm of 180g, abandon the PBS that contains 2%FBS that adds 4 ℃ of precoolings after supernatant, mixing gently, be filtered in another 50ml centrifuge tube through 200 eye mesh screens, add 0.2% sodium-chlor splitting erythrocyte, be 30second～1mm action time, adding 1.6% sodium-chlor to recover solution oozes to waiting again, afterwards at 4 ℃, with the centrifugal 3～5min of 180g, add 4 ℃ of precoolings to contain the PBS mixing of 2%FBS after abandoning supernatant, with blood counting instrument counting cells quantity, cell is divided into two parts, portion adds T-SJ, lectin concentration is 0.03mg/ml, portion adds the excessive homotype contrast of equal-volume, two parts of cells all under 4 ℃ of conditions lucifuge hatch 100mm, afterwards, at 4 ℃, after the centrifugal 3～5min of 180g, abandon supernatant, the PBS re-suspended cell that contains 2%FBS that adds 4 ℃ of precoolings, selected by flow cytometry apoptosis T-SJ+ cell mass, the stem cell purity of purifying is 85%.Before analysis, interpolation propidium iodide (PI) comes the dead cell in Exclusion analysis.

b) 4% paraformaldehyde solution fixed cell 30～40mim, after room temperature is placed 40min～60min, again successively through (55 ℃～75 ℃ of antigen retrieval, 30min～80min), room temperature is cooling (adds permeable membrane damping fluid and fixed cell to hatch after 30min～60min), incubation time is 20～40min, add afterwards BSA sealing (room temperature, 30min～60min), PBS washes away residue not in conjunction with after BSA, hatch with anti-CD90 antibody and fixed cell, 4 ℃ of night incubation, wash away not binding antibody with PBS again, add afterwards the anti-DAPI of reaching of FITC-two, after incubated at room 30min～120min, wash away not binding antibody and DAPI with PBS again, mounting after drying at room temperature.Laser confocal microscope detects, and the quantity that positive cell detected is more than one.

Result: adopt method of the present invention can identify stem cell and further can carry out purifying (Fig. 3) to it from liver cancer tissue.

Test kit and the application method thereof of embodiment 4, identification, purifying liver stem cells and Lung stem cells

Described test kit comprises:

1) cell pretreatment reagent A: ammonium chloride (1 *);

2) identification, purified reagent B: the PT of gelatin combination and RC;

3) container: be used for holding 1), 2) the circular reagent bottle of two kinds of solution;

Described test kit also comprises wash-out sugar N-acetyl-glucosamine, semi-lactosi reagent.

Other experiment materials: with embodiment 1.

Application method:

a) add isopyknic NH4Cl lysate (solution A) and cell mass mixing, room temperature is placed 2～3min, at 4 ℃, with the centrifugal 3～5min of 200g, add 4 ℃ of precoolings of 1ml to contain the PBS re-suspended cell precipitation of 2%FBS after abandoning supernatant, use afterwards blood counting instrument counting cells quantity, cell is divided into two parts, portion adds the lectin of gelatin combination, wherein the concentration of lectin is 4.8mg/ml, standing 30～45min under 37 ℃ of conditions, afterwards, take off the confluent monolayer cells precipitation, identification, the cell mass of purifying can further be used N-acetyl-glucosamine, two kinds of wash-out sugar of semi-lactosi elute the lectin binding substances from cytolemma, at 4 ℃, with the centrifugal 2～3min of 220g, add 4 ℃ of precoolings to contain the PBS mixing of 1%FBS after abandoning supernatant.Subsequently, at 4 ℃, after the centrifugal 3～5min of 220g, abandon supernatant, add the PBS re-suspended cell that contains 1%FBS of 4 ℃ of precoolings, thereby obtain not contain any foreign labeled cell mass, further can add physiological saline to regulate concentration and the volume of stem cell.Adopt the quantity of flow cytometer identification and analysis positive cell group, result shows that positive cell quantity is respectively 2.6% (Fig. 4 .B), 4.3% (Fig. 4 .B '), and the liver stem cells of purifying and Lung stem cells purity are respectively 80%, 89%.Before analysis, interpolation propidium iodide (PI) comes the dead cell in Exclusion analysis.

b) 4% paraformaldehyde solution fixed cell 15～30mim, after room temperature is placed 30min～60min, again successively through (55 ℃～75 ℃ of antigen retrieval, 30min～80min), room temperature is cooling (adds permeable membrane damping fluid and fixed cell to hatch after 30min～60min), incubation time is 20～40min, add afterwards BSA sealing (room temperature, 30min～60min), PBS washes away residue not in conjunction with after BSA, with anti--Alb antibody, anti--c-Kit antibody respectively with fixedly contain liver stem cells, the slide glass of Lung stem cells is hatched, after 4 ℃ of overnight incubation, wash away not binding antibody with PBS again, the slide glass that contains liver stem cells adds corresponding lectin, the anti-DAPI that reaches of TRITC-two, the slide glass that contains Lung stem cells adds corresponding lectin, the anti-DAPI that reaches of FITC-two, all after incubated at room 30min～120min, wash away not binding antibody and DAPI with PBS again, mounting after drying at room temperature.Laser confocal microscope detects, and the quantity that positive cell detected is more than one.

Result: adopt test kit of the present invention can identify liver stem cells and Lung stem cells and further can carry out purifying (Fig. 4) to it from mixed cellularity group.

Test kit and the application method thereof of embodiment 5, identification, purifying cardiac stem cells

Described test kit comprises:

1) cell pretreatment reagent A: 0.2% sodium-chlor;

2) identification, purified reagent B:FITC-SJ lectin;

3) container: be used for holding 1), 2) the square reagent bottle of two kinds of solution;

Described test kit also comprises wash-out sugar N-acetyl-glucosamine reagent.

Other experiment materials: with embodiment 1.

Application method:

a) add 0.2% sodium-chlor (solution A) and cell mass mixing, room temperature adds 1.6% sodium-chlor recovery solution to ooze to waiting after placing 30second～1min, at 4 ℃, with the centrifugal 3～5min of 200g, add 4 ℃ of precoolings of 1ml to contain the PBS re-suspended cell precipitation of 2%FBS after abandoning supernatant, use afterwards blood counting instrument counting cells quantity, cell is divided into two parts, portion adds lectin, wherein, the concentration of lectin is 7.6mg/ml, portion adds isopyknic excessive homotype contrast, two parts of cells equal lucifuge under 16 ℃ of conditions is hatched 45～60min, afterwards, at 4 ℃, after the centrifugal 3～5mm of 200g, abandon supernatant, the PBS re-suspended cell that adds 4 ℃ of precoolings, selected by flow cytometry apoptosis lectin positive cell group, the stem cell purity of purifying is 89%.Before analysis, interpolation propidium iodide (PI) comes the dead cell in Exclusion analysis.Collecting described stem cell further adds wash-out sugar N-Z acyl glycosamine described lectin binding substances wash-out from described cell mass, thereby obtain not to be with any foreign labeled cell sample, wherein, can further add physiological saline to regulate volume and the concentration of sample.

b) 4% paraformaldehyde solution fixed cell 15～30mim, after room temperature is placed 30min～60min, again successively through (55 ℃～75 ℃ of antigen retrieval, 30min～80min), room temperature is cooling (adds permeable membrane damping fluid and fixed cell to hatch after 30min～60min), incubation time is 30～60min, (the room temperature of BSA sealing afterwards, 30min～60min), PBS washes away residue not in conjunction with after BSA, hatch with anti-Nkx2.5 antibody and fixed cell, 4 ℃ of overnight incubation, wash away unconjugated antibody with PBS again, after adding again the anti-DAPI incubated at room 60min～120min of reaching of TRITC-two, wash away not binding antibody and DAPI with PBS again, mounting after drying at room temperature.Laser confocal microscope detects, and the quantity that positive cell detected is more than one.

Result: adopt test kit of the present invention can identify cardiac stem cells and further can carry out purifying (Fig. 5) to it from mixed cellularity group.

Industrial applicibility

as mentioned above, according to method of the present invention, test kit, can be with a kind of simple, quick and economic method, accurately and effectively identify on the one hand adult stem cell, on the other hand can be high with yield ground purifying adult stem cell, and the cell liquid that contains that obtains thus need not subsequently loaded down with trivial details cell suspension preparation process, can directly carry out cryopreservation, and the described sample that contains stem cell can be with any external mark, so that it is used for the fundamental research industry relevant with medical applications, as stem cell self mechanism and correlation technique research, stem cell is used for the treatment of, repair impaired or pathological tissues, the stem cell transplantation technical field, immunotherapy field and drug screening etc.

Claims (15)

1. lectin is used for the method for identification, purifying stem cell, it is characterized in that, it comprises:

(1) make described cell mass and cell pretreatment reagent in advance under contact conditions, make a kind of lectin of sample contact, the latter is contained: (a) at least a lectin, it can identify glycoprotein and/or glycolipid sugar-chain end specific site, and this lectin directly or indirectly is connected with (b) at least a binding substances; With

(2) will separate from described sample with the cell mass that described lectin binding substances is combined, obtain to be rich in the sample of stem cell, wherein, on described cell, existence shows that with the acceptor of described lectin pathoklisis they are stem cells.

2. method according to claim 1, is characterized in that, method that can be by adding wash-out sugar is lectin binding substances wash-out from the described cell mass, thereby obtain not contain any foreign labeled sample that is rich in stem cell.

3. the cell pretreatment process choosing adopts the erythrocytic reagent in precipitation or cracking process precipitation or lysing cell group, and it is any of 0.1～20% hydroxyethylamyle, gelatin, dextran, polyvinylpyrrolidone, methylcellulose gum, carboxymethyl starch that described reagent may be selected to be mass concentration; Or may be selected to be any less than 0.9% normal saline solution of ammonium chloride erythrocyte cracked liquid, mass concentration; Maybe can select any cell pretreatment that carries out of Sodium Diatrizoate-dextran, HITOPAQUE, Ficoll, wherein, described reagent repeats to contact described cell mass once at least.

4. method according to claim 1, it is characterized in that, lectin in described lectin binding substances comprises one or more combination of phytohemagglutinin natural or chemosynthesis, zoo-agglutinin or derivatives thereof, the concentration of described lectin is 0.001～40mg/ml, the time that contacts with described cell mass, incubation temperature was for being less than or equal to 38 ℃ in order to be less than or equal to 120 minutes.

5. according to claim 1 or 4 described methods, it is characterized in that, binding substances in described lectin binding substances can be selected in advance to carry out combination with any of biomacromolecule of fluorescence dye, magnetic microsphere or high molecular, or selects to have aggegation is have a kind of of affinity antibodies or antibody derivatives or antibody fragment.

6. method according to claim 5, it is characterized in that, to between 800,000 dalton (Mw), the biomacromolecule of wherein said high molecular may be selected to be any of bovine serum albumin, hydroxyethylamyle, gelatin, methylcellulose gum, carboxymethyl starch, dextran, polyvinylpyrrolidone to the molecular weight of the biomacromolecule of described high molecular of being combined with lectin 10,000 dalton (Mw).

7. method according to claim 1, it is characterized in that, the described cell mass that contains stem cell derives from tissue before rodent, the mankind or other mammiferous adult tissues, lesion/cancer, lesion/cancer tissue, metastatic tumor/cancerous tissue or other potential combinations that comprises one or more tissues of stem cell, or selection is from primary, the passage cell group's of described tissue one or more combination.

8. lectin is used for test kit and the application method thereof of identification, purifying stem cell, and wherein, described lectin can be used for also determining that in tissue, cell mass, stem cell exists and content, and it is in the purposes that is used for from described cell mass enrichment, purification stem cell.

According to claim 8 for identification, the purifying stem cell test kit, it is characterized in that: described test kit comprises:

1) cell pretreatment reagent A;

2) identification, purified reagent B;

3) and optional container;

Wherein, to may be selected to be mass concentration be any of 01～20% hydroxyethylamyle, gelatin, dextran, polyvinylpyrrolidone, methylcellulose gum, carboxymethyl starch to described cell pretreatment reagent A; Or may be selected to be any less than 0.9% normal saline solution of ammonium chloride erythrocyte cracked liquid, mass concentration; Cell pretreatment reagent or may be selected to be any of Sodium Diatrizoate-dextran, HITOPAQUE, Ficoll, wherein, described reagent repeats to contact described cell mass once at least.

wherein, identification, purified reagent B is the lectin binding substances, include but not limited to phytohemagglutinin natural or synthetic, the combination of one or more of zoo-agglutinin or derivatives thereof, binding substances in wherein said lectin binding substances can be selected in advance and fluorescence dye, the biomacromolecule of magnetic microsphere or high molecular any carries out combination, or select to have aggegation is have a kind of of affinity antibodies or antibody derivatives or antibody fragment, the concentration of described lectin is 0.001～40mg/ml, the time that contacts with described cell mass is for being less than or equal to 120 minutes, incubation temperature is for being less than or equal to 38 ℃.

10. test kit according to claim 9, it is characterized in that, the described cell mass that contains stem cell be rodent, the mankind or other Mammalss sources normal adult and or pathology, tumour, cancer before, carcinous, metastasis of cancer tissue, or from primary, the passage cell group's of described tissue one or more combination.

11. according to claim 1 or 9, arbitrary claim is described, wherein, can select subsequently to adopt fluorescent activation cell sorting method for the identification of with the cell mass of sorting binding lectin binding compounds or the described cell mass of selective precipitation method separation and combination lectin, further, the described stem cell that separates, it comprises the acceptor that can be combined with lectin on cytolemma.

12. the stem cell of the purifying that according to claim 1 or 9, the method for arbitrary claim, test kit obtain, it is characterized in that, method by adding wash-out sugar is described lectin binding substances wash-out from the described cell, thereby makes the stem cell of acquisition not be with any external marker.

13. according to claim 1, arbitrary claim is described in 10,11 or 12, the stem cell of wherein said identification, purifying comprises myeloid-lymphoid stem cell, multipotential stem cell, special energy stem cell, adult stem cell, tumor stem cell, cancer stem cell, further, it contacts with known stem cell markers.

14. according to claim 1 or 9 arbitrary claims are described, the stem cell of acquisition also can add physiological saline, PBS damping fluid, HBSS damping fluid to adjust concentration and the volume of stem cell.

15. test kit application method according to claim 8 is characterized in that, its application method comprises the following steps:

(1) described cell sample contacts with the cell pretreatment reagent A: as add the erythroprecipitin agent, select to get supernatant; As add erythrocyte cracked liquid, select to abandon supernatant after centrifugal and get cell precipitation;

(2) the described cell mass through step (1) is contacted with described identification, purified reagent B, the concentration of described lectin is 0.001～40mg/ml, the time that contacts with described cell mass, incubation temperature was for being less than or equal to 38 ℃ in order to be less than or equal to 120 minutes;

(3) separate the cell mass that obtains the lectin positive: can select to adopt fluorescent activation cell sorting, magnetic bead sorting method or staticly settle method;

(4) add wash-out sugar described lectin binding substances from wash-out on the described cell of step (3) acquisition, thereby the stem cell that makes acquisition is not with any external marker, further, the stem cell of acquisition also can add physiological saline, PBS damping fluid, HBSS damping fluid to adjust concentration and the volume of stem cell.

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