CN104087556A - Preparation method of breast cancer stem cells as well as antigen composition thereof and anti-tumor application of antigen composition - Google Patents
Preparation method of breast cancer stem cells as well as antigen composition thereof and anti-tumor application of antigen composition Download PDFInfo
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- CN104087556A CN104087556A CN201410300288.4A CN201410300288A CN104087556A CN 104087556 A CN104087556 A CN 104087556A CN 201410300288 A CN201410300288 A CN 201410300288A CN 104087556 A CN104087556 A CN 104087556A
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Abstract
The invention provides a method for preparing breast cancer stem cells. The method comprises a step of identifying whether breast cancer stem cells exist in a to-be-detected breast cancer cell specimen or not, namely detecting whether at least two of the following differential proteins are specifically expressed or not in the to-be-detected breast cancer cell specimen: an ATP (adenosine triphosphate)-binding cassette subfamily G component 2, quintic transmembrane single-stranded glycoprotein PROM1, acetaldehyde dehydrogenase-1, and a phosphatase and tensin homologue. The invention further provides an antigen composition for preparing the breast cancer stem cells, an anti-tumor application of the antigen composition. a dendritic cell vaccine obtained by loading the antigen composition with dendritic cells, a kit for preparing the dendritic cell vaccines as well as an anti-tumor application of the dendritic cell vaccine.
Description
Technical field
The present invention relates to prepare tumor stem cell and antigen composition thereof and antitumor application, described antigen composition loaded dendritic cell prepares dendritic cell vaccine, prepares the antitumor application of test kit and the described dendritic cell vaccine of described dendritic cell vaccine.Especially, the present invention relates to prepare the antitumor application that breast carcinoma stem cell and antigen composition and antitumor application, described antigen composition loaded dendritic cell thereof prepare dendritic cell vaccine, prepare test kit and the described dendritic cell vaccine of described dendritic cell vaccine.
Background technology
At present, the tumorigenesis breast cancer cell with stem/progenitor cells characteristic has been identified and be separated to prior art in mammary cancer knurl.For example, " Isolation and In vitro Propagation of Tumorigenic Breast Cancer Cells with Stem/Progenitor Cell Properties (thering is separation and the external breeding of the tumorigenesis breast cancer cell of stem/progenitor cells characteristic) ", the people such as Dario Ponti, Cancer Research (cancer research), the 65th the 13rd phase of volume, 5506-5511 page, disclosed in 1 day July in 2005, and it finds CD44
+and CD24
-/lowcell, keep specially carcinogenic active and demonstrated stem-like cell characteristic.Be that prior art can, by detecting CD44 and the CD24 of cell of the doubtful breast carcinoma stem cell of cancer knurl place's separation, be identified breast carcinoma stem cell.
But the character that also has same CD44 and CD24 in normal stem cell, therefore the method for prior art cannot be by the distinguishing of the breast carcinoma stem cell of cancer knurl place's separation and normal stem cell efficiently and accurately, thereby enrichment quickly and effectively external breeding breast carcinoma stem cell.For example, specific expressed CD133 albumen in breast carcinoma stem cell is not expressed in breast cancer cell, but is having expression in normal hemopoietic stem cell yet.
How to obtain efficiently and accurately in sum breast carcinoma stem cell, and can utilize the breast carcinoma stem cell of gained to prepare the vaccine that resists mammary cancer in donor, remain problem demanding prompt solution.
Summary of the invention
The present invention has found a kind of method of preparing breast carcinoma stem cell by research, the method at short notice efficiently and accurately obtains the high and enrichment of purity and is easy to breast carcinoma stem cell, solves prior art and is difficult to efficiently and accurately at short notice and obtains the problem that the high and enrichment of purity is easy to breast carcinoma stem cell.
Second object of the present invention is to provide a kind of breast carcinoma stem cell antigen composition and antitumor application thereof.
The 3rd object of the present invention is to provide the test kit that uses breast carcinoma stem cell antigen composition loaded dendritic cell of the present invention to prepare dendritic cell vaccine and prepare this dendritic cell vaccine.
The 4th object of the present invention is to provide the dendritic cell vaccine antineoplastic application clinically obtaining by the method for the invention.
The invention provides a kind of method of preparing breast carcinoma stem cell, wherein, described method comprises identifies the step that whether has breast carcinoma stem cell in breast cancer cell sample to be measured:
Described step comprises and detects in described breast cancer cell sample to be measured at least two kinds of the following differential protein of specifically expressing whether: ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues.
The present invention also provides a kind of test kit of preparing breast carcinoma stem cell, and wherein, described test kit comprises:
1) breast carcinoma stem cell basic medium;
2) at least two kinds of ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues;
3) enzyme of peptic cell;
4) cytokine and;
5) working instructions;
Wherein, described working instructions comprise the method for preparing breast carcinoma stem cell of the present invention.
The present invention also provides a kind of preparation method of above-mentioned breast carcinoma stem cell antigen composition, and wherein, the method comprises the following steps:
1) prepare according to the method described in the present invention breast carcinoma stem cell,
2) with physiological saline washing resuspended described breast carcinoma stem cell,
3) breast carcinoma stem cell described in cracking.
The present invention also provides a kind of preparation method of dendritic cell of breast carcinoma stem cell antigen load, wherein, the method comprises: by cracking breast carcinoma stem cell, obtain breast carcinoma stem cell antigen composition, normal dendritic cell is contacted to the anti-breast cancer dendritic cell that obtains load tumour antigen with breast carcinoma stem cell antigen composition, wherein, described method comprises uses method of the present invention to prepare breast carcinoma stem cell, or uses method of the present invention to prepare breast carcinoma stem cell antigen composition.
The present invention also provides a kind of dendritic cell vaccine of the breast carcinoma stem cell antigen load being obtained by above-mentioned method, described dendritic cell vaccine comprises dendritic cell, wherein, described dendritic cell is the dendritic cell of breast carcinoma stem cell antigen load of the present invention.
The present invention also provides a kind of test kit of preparing the dendritic cell of breast carcinoma stem cell antigen load, and wherein, described test kit comprises:
1) breast carcinoma stem cell basic medium;
2) at least two kinds of ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues;
3) enzyme of peptic cell;
4) B27 or N2, epithelical cell growth factor, Prostatropin and Regular Insulin;
5) dendritic cell basic medium;
6) macrosome cell-phagocyte G CFS;
7) interleukin-4, interferon alpha and tumor necrosis factor alpha; And
6) working instructions;
Wherein, described working instructions comprise the method for preparing the dendritic cell of breast carcinoma stem cell antigen load of the present invention.
By method of the present invention, only by detecting ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues at least two kinds, just can obtain efficiently and accurately breast carcinoma stem cell, high and enrichment is easy based on described method gained breast carcinoma stem cell purity.Medicine prepared by the dendritic cell of the breast carcinoma stem cell being obtained by method provided by the invention and the antigen composition being obtained by this breast carcinoma stem cell, this antigen of load, can kill with external initiation immunne response the breast carcinoma stem cell that causes breast cancer relapse in vivo, thereby provide possibility for overcoming recurrence and the transfer of mammary cancer tolerance, radical cure mammary cancer.
Accompanying drawing explanation
Fig. 1 is expressed as mammary cancer knurl stem cell balling-up growth figure and the flow cytometer detected result figure of acquisition;
Fig. 2 is expressed as by embodiment 6 gained ABCG2 and PROM1 two positive, ABCG2, PROM1 and ALDH1 tri-positives, the cytotoxic T lymphocyte (CTL) that the breast carcinoma stem cell antigen composition loaded dendritic cell of ABCG2, PROM1, ALDH1 and PTEN tetra-positives obtains, the response result figure of the breast carcinoma stem cell respectively embodiment 3 being obtained;
Fig. 3 represents by preparation example 1,3 and three kinds of antigen compositions difference loaded dendritic cells of comparative example 4 gained, and embodiment 6 gained ABCG2 and the PROM1 pair of cytotoxic T lymphocyte (CTL) that positive breast cancer stem cell antigen composition loaded dendritic cells obtain, respectively the response result comparison diagram to ABCG2 and the two positive breast cancer stem cells generations of PROM1.
Fig. 4 shows by preparation example 1,3 and three kinds of antigen compositions of comparative example 4 gained cytotoxic T lymphocyte (CTL) that loaded dendritic cell and embodiment 6 gained ABCG2 and the two positive breast cancer stem cell antigen composition loaded dendritic cells of PROM1 obtain respectively, the response result comparison diagram that the breast carcinoma stem cell respectively comparative example 4 being obtained produces.
Embodiment
The invention provides a kind of method of preparing breast carcinoma stem cell, wherein, described method comprises identifies the step that whether has breast carcinoma stem cell in breast cancer cell sample to be measured:
Described step comprises and detects in described breast cancer cell sample to be measured at least two kinds of the following differential protein of specifically expressing whether: ATP linking frame subfamily G composition 2 (ATP-binding cassette superfamily G member2, ABCG2), five cross-film strand glycoprotein (Prominin1, PROM1), acetaldehyde dehydrogenase-1 (Aldehyde dehydrogenase-1, ALDH1), Phosphoric acid esterase and tensin homologue (phosphatase and tensin homolog, PTEN).Wherein, Prominin-1 is the first member of 5 transmembrane protein families, containing 865 amino acid, has the outer N end of born of the same parents, C end in born of the same parents, and two large born of the same parents' outer shrouds are containing 8 glycosylated sites.
Preferably, described specifically expressing differential protein refers to that to use the result of at least two kinds, the described four species diversity albumen in breast cancer cell sample described in double-colored pair of fusion probe interphase fluorescence in situ hybridization technology for detection positive;
Wherein, with the negative contrast of known pure breast cancer cell, with 100 parts of described negative controls of double-colored pair of fusion probe interphase fluorescence in situ hybridization technology for detection, every part containing 10
3individual cell, record percentage that every species diversity albumen false positive signal number of cells accounts for every part of total cellular score by the false positive rate of this species diversity albumen of survey, the mean value of this species diversity albumen false positive rate of survey and standard variance sum are defined as to cut off value;
Get 100 parts of breast cancer cell samples to be measured, every part of sample is containing 10
3individual cell, recording and surveying with negative control the percentage that differential protein positive signal number of cells of the same race accounts for every part of total cellular score is the mean value of the positive rate of institute's error of measurement M-band, this positive rate mean value is more than or equal to described cut off value, and double-colored pair of fusion probe interphase fluorescence in situ hybridization technology for detection result of this species diversity albumen of this breast cancer cell sample to be measured is positive.
Further preferably, described specifically expressing differential protein refers to that to use the result of the described four species diversity albumen in breast cancer cell sample described in double-colored pair of fusion probe interphase fluorescence in situ hybridization technology for detection all positive.
Preferably, while there is breast carcinoma stem cell in identifying described breast cancer cell sample to be measured, the method also comprises:
The mammary cancer knurl stem cell balling-up growth of described acquisition, detects CD44 through flow cytometer
+and CD24
-/lowmammary cancer knurl stem cell account for the more than 30% of total cell count;
The step of the breast carcinoma stem cell described in use immunomagnetic beads method and/or the enrichment of selected by flow cytometry apoptosis method in cell to be measured;
Wherein, utilize the antibody of at least two kinds for the ATP linking frame subfamily G composition 2 that obtains specifically expressing, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues.
Described method can also comprise separating mammary cancer stem cell from vitro breast cancer tumour tissue and/or breast cancer cell line.The breast cancer tumour tissue of operation from taking out in patient body, belongs to discarded in vitro breast cancer tumour tissue.The present invention can be suitable for conventional method and obtain breast cancer cell.In addition, by the existing commercial cloned culture that can buy, also can therefrom isolate corresponding breast carcinoma stem cell.For example, the cell strain system of corresponding breast carcinoma stem cell can buy and can therefrom isolate from U.S. typical case thing preservation center (ATCC) to MCF-7 people's breast adenocarcinoma cell exactly.
The present invention also provides a kind of test kit of preparing breast carcinoma stem cell, and wherein, described test kit comprises:
1) breast carcinoma stem cell basic medium;
2) at least two kinds of ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues; Preferably include described four kinds of albumen.
3) enzyme of peptic cell;
4) cytokine and;
5) working instructions;
Wherein, described working instructions comprise the method for preparing breast carcinoma stem cell of the present invention.
Preferably, described test kit also comprises the antibody of at least two kinds for described ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues; Described working instructions comprise the method for preparing breast carcinoma stem cell of the present invention.
Wherein, described breast carcinoma stem cell basic medium can be NS-basal, DMEM/F12 (1: 1) etc.Described breast carcinoma stem cell culture system comprises the breast carcinoma stem cell basic medium of breast carcinoma stem cell and 5mL-50mL, at 37 ℃, 5%CO
2in incubator, cultivate.
The enzyme of described peptic cell is preferably pancreatin and/or A Ou replaces this enzyme (Accutase).Wherein, when breast carcinoma stem cell basic medium reaches 5mL-50mL, described pancreatin and/or Accutase, preferably as tumor tissues separating mammary reagent that cancer stem cell is used, can be used the consumption of this area routine, and the 0.5mL-10mL that for example general consumption is 1 times of concentration carrys out peptic cell.
Described cytokine is B27 or N2, epithelical cell growth factor, Prostatropin and Regular Insulin.Wherein, described B27 or N2, when cultivating breast carcinoma stem cell, for example, add in breast carcinoma stem cell basic medium (NS-basal), and reaching final concentration is 1 times.Wherein said B27 and N2 are the commercial cell cultures additives supplying (being cytokine), for example, and B27 and the N2 of 50 times that U.S. Gibco company produces.Described epithelical cell growth factor (EGF), when cultivating breast carcinoma stem cell, for example, adds in breast carcinoma stem cell basic medium (NS-basal), and reaching final concentration is 10ng/mL-50ng/mL.Described Prostatropin (bFGF), when cultivating breast carcinoma stem cell, for example, adds in breast carcinoma stem cell basic medium (NS-basal), and reaching final concentration is 10ng/mL-50ng/mL.Described Regular Insulin, when cultivating breast carcinoma stem cell, for example, adds in breast carcinoma stem cell basic medium (NS-basal), and reaching final concentration is 0.5mg/mL-10mg/mL.
The present invention also provides a kind of preparation method of above-mentioned breast carcinoma stem cell antigen composition, and wherein, the method comprises the following steps:
1) prepare according to the method described in the present invention breast carcinoma stem cell,
2) with physiological saline washing resuspended described breast carcinoma stem cell,
3) breast carcinoma stem cell described in cracking.
Wherein, breast carcinoma stem cell antigen composition method of the present invention being made residual nutrient media components and residual chemotherapeutics of possibility when being administered to patient can not cause that patient is uncomfortable.
For do not increase the consideration of new material after cracking breast carcinoma stem cell, the step of preferred described cracking breast carcinoma stem cell comprises ultrasonic degradation and/or multigelation; During the condition of described ultrasonic degradation is included on ice or mixture of ice and water bathes, 45 watts, 1min, 5sec/l time, then gets ultrasonic rear suspension in the cytoclastic efficiency of Microscopic observation.At 4 ℃, keep 1h-6h, be preferably included at 38～43 ℃ and keep 2h-4h; The number of times of described multigelation is 3-5 time, be 10min-2h the interval time of every twice, be preferably 30min-1h, described freezing temperature range is subzero 120 ℃-subzero 196 ℃ (can realize with liquid nitrogen, dry ice etc.), preferably subzero 150 ℃-subzero 196 ℃, the temperature range of described thawing is 10 ℃-37 ℃, is preferably 20 ℃-37 ℃.
After cracking completes, conventionally by step centrifugal and that filter, remove cell debris excessive in lysate, for example, by the centrifugal 1min of 800rpm, remove, then 0.45 μ m membrane filtration for supernatant.The basal component of described composition comprises physiological saline.The antigen composition of the present invention that cracking obtains can be preserved in-80 ℃.
Preferably, the concentration of preparing the breast carcinoma stem cell of gained described step 1) is 1 * 10
6to 5 * 10
7cell/mL, as primary immune response treatment, is enough to cause in vivo immunne response effect.
Further preferably, described method also comprises step 4) add human serum albumin; Wherein, the described breast carcinoma stem cell antigen composition of take is benchmark, and the content of described human serum albumin is 1-5 bulking value %.
A kind of method that the present invention also provides breast carcinoma stem cell antigen-loaded dendritic cell to prepare, wherein, the method comprises: by cracking breast carcinoma stem cell, obtain breast carcinoma stem cell antigen composition, immature dendritic cell is contacted to the anti-breast cancer dendritic cell that obtains load tumour antigen with breast carcinoma stem cell antigen composition, wherein, described method comprises and uses method of the present invention to prepare breast carcinoma stem cell antigen composition.Preferred described immature dendritic cell with the breast carcinoma stem cell of preparing breast carcinoma stem cell antigen composition from same individuality.Can avoid to greatest extent like this side effect causing due to immunological rejection.Certainly, if described immature dendritic cell is not from same individuality with the breast carcinoma stem cell of preparing breast carcinoma stem cell antigen composition, still can reduce side effect with the medicine of the conventional anti-immunological rejection in this area.The medicine of described anti-immunological rejection such as dexamethasone etc.
The present invention also provides a kind of dendritic cell vaccine of the breast carcinoma stem cell antigen composition load being obtained by above-mentioned method, described dendritic cell vaccine comprises dendritic cell, can derive from peripheral blood, marrow and Cord blood etc., wherein, described dendritic cell is the dendritic cell of breast carcinoma stem cell antigen composition of the present invention load.
Before preparing dendritic cell vaccine, by the dendritic cell of physiological saline washing the load of resuspended described breast carcinoma stem cell antigen composition.Dendritic cell vaccine of the present invention residual nutrient media components and residual chemotherapeutics of possibility when being administered to patient can not caused to patient is uncomfortable.Described vaccine comprises 1 * 10
6to 5 * 10
7the dendritic cell of individual/mL breast carcinoma stem cell antigen composition of the present invention load, treats as primary immune response.Described vaccine also comprises human serum albumin; Wherein, take described vaccine as benchmark, the content of described human serum albumin is 2-3 bulking value %.
The present invention also provides a kind of test kit of preparing the dendritic cell of breast carcinoma stem cell antigen load, and wherein, described test kit comprises:
1) breast carcinoma stem cell basic medium;
2) at least two kinds of ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues; Preferably include described four kinds of albumen.
3) enzyme of peptic cell;
4) B27 or N2, epithelical cell growth factor, Prostatropin and Regular Insulin;
5) dendritic cell basic medium;
6) macrosome cell-phagocyte G CFS and interleukin-4;
7) tumor necrosis factor alpha; And
6) working instructions;
Wherein, described working instructions comprise the preparation method of the dendritic cell of breast carcinoma stem cell antigen load of the present invention.
Preferably, described test kit also comprises the antibody of at least two kinds for described ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues.Utilize described antibody, use immunomagnetic beads method and/or flow cytometer can separating method enrichment described in breast carcinoma stem cell in cell to be measured.
Wherein, described breast carcinoma stem cell basic medium can be NS-basal, DMEM/F12 (1: 1) etc.Described breast carcinoma stem cell culture system comprises the breast carcinoma stem cell basic medium of breast carcinoma stem cell and 5mL-50mL, at 37 ℃, 5%CO
2in incubator, cultivate.
The enzyme of described peptic cell is preferably pancreatin and/or Accutase.Wherein, when breast carcinoma stem cell basic medium reaches 5mL-50mL, described pancreatin and/or Accutase, preferably as tumor tissues separating mammary reagent that cancer stem cell is used, can be used the consumption of this area routine, and the 0.5mL-10mL that for example general consumption is 1 times of concentration carrys out peptic cell.
Wherein, described B27 or N2, when cultivating breast carcinoma stem cell, for example, add in breast carcinoma stem cell basic medium (NS-basal), and reaching final concentration is 1 times.Described EGF, when cultivating breast carcinoma stem cell, for example, adds in breast carcinoma stem cell basic medium (NS-basal), and reaching final concentration is 10ng/mL-50ng/mL.Described bFGF, when cultivating breast carcinoma stem cell, for example, adds in breast carcinoma stem cell basic medium (NS-basal), and reaching final concentration is 10ng/mL-50ng/mL.Described Regular Insulin, when cultivating breast carcinoma stem cell, for example, adds in breast carcinoma stem cell basic medium (NS-basal), and reaching final concentration is 0.5mg/mL-10mg/mL.
The resulting breast carcinoma stem cell antigen composition of method of being prepared breast carcinoma stem cell antigen composition by the present invention is 1 * 10 from cell concn
6to 5 * 10
7the resulting breast carcinoma stem cell of method that cell/mL prepares breast carcinoma stem cell by the present invention, treats as primary immune response.Described cell concn is excessive causes waste, otherwise cell concn is too small, is not enough to cause the antineoplastic immunne response effect of dendritic cell.
Described dendritic cell basic medium can be X-VIVO15, X-VIVO20, AIM-V, RPMI/1640 etc.Described dendritic cell culture system comprises the dendritic cell basic medium of breast carcinoma stem cell and 1mL-100mL, preferred 30-80mL, at 37 ℃, 5%CO
2in incubator, cultivate.Wherein, described macrosome cell-phagocyte G CFS (GM-CSF) adds in dendritic cell basic medium when breast carcinoma stem cell antigen composition loaded dendritic cell, final concentration 5001U/mL-2000IU/mL.Described interleukin-4 adds in dendritic cell basic medium when breast carcinoma stem cell antigen composition loaded dendritic cell, final concentration 100IU/mL-1000IU/mL.Described tumor necrosis factor alpha adds in dendritic cell basic medium when breast carcinoma stem cell antigen composition loaded dendritic cell, final concentration 5ng/mL-100ng/mL.
The present invention also provides a kind of method for the treatment of cancer, and it comprises to patient uses the medicine in the group that the breast carcinoma stem cell antigen composition that selects free the inventive method to prepare, anticancer dendritic cell of the present invention and dendritic cell vaccine of the present invention form.
The mode of using is preferably injection.Particularly, the mode of using breast carcinoma stem cell antigen composition is selected one or more in injection in free tumor tissues, intravenous injection, subcutaneous injection and intradermal injection; The mode of using described dendritic cell vaccine is the subcutaneous or intradermal injection at lymphoglandula position.Preferably use the mode of described dendritic cell vaccine for to carry out subcutaneous or intradermal injection at belly ditch or oxter.
Described breast carcinoma stem cell antigen composition is preferably 1 * 10 from cell concn
6to 5 * 10
7the breast carcinoma stem cell of the present invention of cell/mL, its significant quantity is that 0.1mL-5mL/ is individual; Described dendritic cell vaccine preferably includes 1 * 10
6to 5 * 10
7individual/mL anticancer dendritic cell of the present invention, its significant quantity is that 0.1mL-2mL/ is individual.More preferably described breast carcinoma stem cell antigen composition is preferably 1 * 10 from cell concn
6to 1 * 10
7the breast carcinoma stem cell of the present invention of cell/mL, its significant quantity is that 0.5mL-1mL/ is individual; Described dendritic cell vaccine preferably includes 5 * 10
6to 5 * 10
7individual/mL anticancer dendritic cell of the present invention, its significant quantity is that 1mL-2mL/ is individual.The above breast carcinoma stem cell antigen composition and described dendritic cell vaccine are all the dosage of a shot, four injections course for the treatment of, one week, interval.
Described breast carcinoma stem cell and/or described dendritic cell come from same patient, the side effect that can avoid to greatest extent immunological rejection to cause like this.The mammary cancer tumor tissue of the operation that the present invention mentions from taking out in patient body, belongs to discarded in vitro mammary cancer tumor tissue, not for realizing the specially mammary cancer tumor tissue from taking out in patient body of the present invention.In addition the cell that, the present invention mentions can be the commercial clone supplying.And the technology of existing long-term frozen preservation cell is very ripe, for example, such as freeze-stored cell, the preservation of Cord Blood-Derived hemopoietic stem cell under liquid nitrogen (subzero 196 ℃).Mammary cancer tumor tissue, tumour cell, tumor stem cell and dendritic cell that those skilled in the art can utilize above-mentioned technology preservation the present invention relates to completely, dendritic cell can originate peripheral blood, marrow and Cord blood etc.
Below, specific embodiments of the invention are described, but technical scope of the present invention is not limited to these examples.
The detection of tumor stem cell in embodiment 1 human breast cancer cell
By the human breast cancer cell purchased from U.S. ATCC company (MCF-7), in 10mLRPMI1640 substratum (containing volume ratio 10% foetal calf serum, U.S. Gibco company and 1 * penicillin and Streptomycin sulphate mixed solution, U.S. Hyclone company), 75cm
2in culturing bottle, place 37 ℃, 5%CO
2in incubator, cultivate, when Growth of Cells reaches 90% of culturing bottle bottom surface and converges, with after 2mL pancreatin (U.S. Hyclone company) digestion, tongue is expected blue counting, with fresh RPMI1640 substratum (containing volume ratio 10% foetal calf serum and 1 * penicillin and the Streptomycin sulphate mixed solution) cultivation of going down to posterity.
At the bottom of MCF-7 passage grows to bottle, 90% when converge, and gets 1 part of 50 μ L (2 * 10 after digestion
6individual) cell suspension adds respectively one to and contains 20 μ L FITC-CD44 (U.S. company BD) and 20 μ LPE-CD24 (U.S. company BD), in another 1ml centrifuge tube that contains 20 μ L FITC-IgG1 (U.S. company BD) and 20 μ L PE-IgG1 (U.S. company BD).Mix and be placed on 4 ℃ of refrigerator dyeing 30 minutes, then use 1 * phosphate buffered saline buffer (PBS) of 1mL to wash three times, finally use the cell after the resuspended washing of 1 * PBS of 0.5mL, cell after gained streaming antibody staining detects (U.S. company BD) with FACSCalibur basic model flow cytometer, and result does not detect CD44
+and CD24
-/lowbreast carcinoma stem cell.
The detection of tumor stem cell in the stem cell in embodiment 2 normal breast sources
By the people's mammary gland stem cell (MaSC) purchased from Canadian Stemcell company, at 10mL NS-basal substratum (Canadian Stemcell company, NeuroCult
tM) in (containing 10ng/mL human epidermal growth factor (epidermal growth factor, EGF), 10ng/mL Prostatropin (bEGF), U.S. peprotech company), 75cm
2in culturing bottle, place 37 ℃, 5%CO
2in incubator, cultivate, when cell balling-up growth reaches 10 μ m, with 10ml transfer pipet, get cell culture fluid in 15ml centrifuge tube, the centrifugal 5min of 1000rpm, abandon supernatant with after 2mL pancreatin (U.S. Hyclone company) peptic cell precipitation, tongue is expected blue counting, with fresh NS-basal substratum (containing 10ng/mL EGF), 10ng/mL bEGF) cultivation of going down to posterity.
After MaSC digestion, get 1 part of 50 μ L (2 * 10
6individual) cell suspension adds respectively one to and contains 20 μ LFITC-CD44 (U.S. company BD) and 20 μ L PE-CD24 (U.S. company BD), in another 1ml centrifuge tube that contains 20 μ L FITC-IgG1 (U.S. company BD) and 20 μ L PE-IgG1 (U.S. company BD).Mix and be placed on 4 ℃ of refrigerator dyeing 30 minutes, then use 1 * phosphate buffered saline buffer (PBS) of 1mL to wash three times, finally use the cell after the resuspended washing of 1 * PBS of 0.5mL, cell after gained streaming antibody staining detects (U.S. company BD) with FACSCalibur basic model flow cytometer, and result does not detect CD44
+and CD24
-/lowbreast carcinoma stem cell.
The detection of tumor stem cell in the tissue-derived breast carcinoma stem cell of preparation example 3 patient with breast cancer
Breast cancer tissue gathers while coming comfortable patient to perform the operation, and patient is diagnosed as mammary cancer cancer through pathologic finding, and signs Informed Consent Form with patient, and patient information is as following table one.
| Patient's numbering | Age | Sex | Pathological | Whether sign Informed Consent Form |
| BSC-001 | 24 | Female | Expressions of ER +++PR ++Her2/neu -- | Be |
| BSC-002 | 26 | Female | Expressions of ER -PR -Her2/neu - | Be |
| BSC-003 | 32 | Female | Expressions of ER -PR -Her2/neu - | Be |
| BSC-004 | 42 | Female | Expressions of ER -PR ++Her2/neu - | Be |
| BSC-005 | 54 | Female | Expressions of ER -PR -Her2/neu - | Be |
| BSC-006 | 51 | Female | Expressions of ER ++PR +Her2/neu - | Be |
| BSC-007 | 32 | Female | Expressions of ER -PR -Her2/neu -- | Be |
Note: hormone receptor (Estrogen receptor, ER), progesterone receptor (progesterone receptor, PR) and ErbB-2 (human epidermal growth factor receptor2, Her2/neu)
With eye scissors, mammary cancer tumor tissue is shredded and becomes about 1mm
3fritter.Gained shreds organize fritter with 1000IU/ml type i collagen enzyme (U.S. sigma company) and 100IU/ml Unidasa (U.S. sigma company) RPMI1640 cultivation and in 1 gram: the weightmeasurement ratio of 2mL, at 37 ℃, hatch 4h, sucking-off cell suspension, by 40 μ m screen filtrations for cell suspension, filtered liquid is collected in 15ml centrifuge tube, with the RPMI1640 containing 10%FBS, rinse after 1 time, with PBS, rinse 2 times.Then centrifugal 5min collecting cell under 1000rpm, in order to 1 gram: the weightmeasurement ratio of 1mL is resuspended with NS-basal substratum.
Get 1 part of 50 μ L (2 * 10
6individual) the above-mentioned cell suspension that obtains adds to respectively in the centrifuge tube that has 20 μ L FITC-CD44 and 20 μ L PE-CD24 (BD company).Be placed in 4 ℃ of refrigerators dyeing 30 minutes, then use 1 * phosphate buffered saline buffer (PBS) washing three times of 1mL, finally use the cell after the resuspended washing of 1 * PBS of 0.5mL, the cell after gained washing detects with FACSCalibur basic model flow cytometer.Result shows, CD44
+and CD24
-/lowmammary cancer knurl stem cell account for 0.6% of cell after gained washing.
Collect after remaining described cell suspension, add wherein and contain 1 times of B27,20ng/mL human epidermal growth factor (epidermal growth factor, EGF), the NS-basal perfect medium of 20ng/mL Prostatropin (bEGF) (U.S. peprotech company) and 10mg/mL Regular Insulin (U.S. sigma company), make concentration of cell suspension be diluted as 2 * 10
5cell/mL.Then the cell suspension of every 5mL gained dilution is transferred in 25cm2 Tissue Culture Flask at 37 ℃, 5%CO
2in incubator, cultivate 48h.
Cultivate gained cell and be neural spherical growth, with 5ml transfer pipet, cell culture fluid is drawn in 15ml centrifuge tube, the centrifugal 3min of 800rpm, abandon supernatant, add the fresh NS-basal perfect medium that contains 1 times of B27,20ng/mL EGF, 20ng/mL bFGF and 10mg/mL Regular Insulin resuspended gently, join 25cm
2in Tissue Culture Flask, at 37 ℃, 5%CO
2in incubator, continue to cultivate.When cell balling-up growth reaches 100 μ m, take out cell culture fluid and repeat the above-mentioned centrifugal cell precipitation that obtains, add after 3ml Accutase digestion, the centrifugal 5min of 1000rpm, abandon supernatant, obtain unicellular add NS-basal perfect medium that fresh use contains 1 times of B27,20ng/mL EGF, 20ng/mL bFGF and 20mg/mL Regular Insulin to go down to posterity (1 bottle passes 2 bottles, and every bottle of initial cultured cells density is 2 * 10 in cultivation
5cell/mL).According to the above-mentioned condition that goes down to posterity, passed again for 4 generations, obtain neural spherical breast carcinoma stem cell.
Get 1 bottle of cultivation to the breast carcinoma stem cell in 5 generations, through accutase digestion, centrifugal rear cell precipitation is resuspended with 1mL1 * PBS, counting.Get 1 part of 50 μ L (2 * 10
6individual) above-mentioned going down to posterity obtain cell suspension and add to respectively in the centrifuge tube that has 20 μ L FITC-CD44 (BD company) and 20 μ L PE-CD24 (BD company).Be placed in 4 ℃ of refrigerator dyeing 30 minutes, then use 1 * phosphate buffered saline buffer (PBS) of 1mL to wash three times, finally use the cell after the resuspended washing of 1 * PBS of 0.5mL, cell after gained washing detects with FACSCalibur basic model flow cytometer, the result that obtains the detection of breast carcinoma stem cell flow cytometer is as follows, in Table two.
| Patient's numbering | Streaming phenotype | Fluidic cell detected result |
| BSC-001 | CD44 +/CD24 -/low | 45.99％ |
| BSC-002 | CD44 +/CD24 -/low | 33.25％ |
| BSC-003 | CD44 +/CD24 -/low | 34.98％ |
| BSC-004 | CD44 +/CD24 -/low | 31.75％ |
| BSC-005 | CD44 +/CD24 -/low | 43.18％ |
| BSC-006 | CD44 +/CD24 -/low | 39.65％ |
| BSC-007 | CD44 +/CD24 -/low | 46.76％ |
In Figure 1A, be shown as patient and number the mammary cancer knurl stem cell balling-up growth figure that BSC002 obtains, and in Figure 1B, be flow cytometer detected result figure, show CD44
+and CD24
-/lowmammary cancer knurl stem cell account for 33.25% of cell after gained washing.
Comparative example 4 is utilized the method separation of people's records such as Dario Ponti and is prepared breast carcinoma stem cell
The fresh tumor specimen mechanical shear of patient with breast cancer is broken into fragment of tissue, and the collagenase and the Unidasa molten night (being Switzerland Roche diagnostic companies) that add 1: 1 digest 2 hours in 37 ℃ of incubators.Digestive system sieves with 30 μ m cell sieves, obtains single cell suspension.DMEM/F12 substratum for single cell suspension (Belgian Cambrex BioScience company) (containing 10ng/mL bFGF, 20ng/mL EGF, 5Ag/mL Regular Insulin and 0.4% bovine serum albumin, all buy from Sigma) is resuspended to 10
3cell/mL, at 37 ℃, 5%CO
2in incubator, continue to cultivate non-adherent balling-up growth.The every cultivation of cell digests in 37 ℃ of incubators and digests 2min with mass volume ratio 0.25% pancreas enzyme-EDTA for three days afterwards, then add DMEM/F12 substratum (containing 10ng/mL bFGF, 20ng/mL EGF, 5Ag/mL Regular Insulin and 0.4% bovine serum albumin) continue to cultivate, obtain breast carcinoma stem cell.
Embodiment 5 breast carcinoma stem cell differential gene expressions detect
The DNA specific probe of Biotin mark and the DNA specific probe of Digoxigenin mark are purchased from rich peaceful medical science genetic research center, Beijing.
The operations such as wash-out are undertaken after sample disposal, sex change, hybridization, hybridization by double-colored two operation instructionss that merge fluorescence in situ hybridization (DD-FISH) test kits (U.S. Vysis company) and provide.Under NikonE600 fluorescent microscope, by three look optical filtering pieces (DAPI/TRITC/FITC), observe hybridization signal, orange (orange, O) signal is BCR, green (green, G) be ABL, merging signal (fusion, F) is BCR-ABL fusion gene, and background chromatin is blue.
By COHU high resolution, CCD carries out image capture, by Macprobe410 fluorescence image processing system (Britain PSI company), carries out image analysis; 1000 cells of every part of sample counting.
FISH result judging criterion: O signal is defined as to F signal with G signal overlap or while contacting; D-FISH: 4 hybridization signals that the visible 2O2G of normal cell interphasic nucleus is separated from one another, have typical t (9; 22) in the interphasic nucleus of transposition, visible 1O1G2F or 2O2G1F hybridization signal.
The normal cut off value that D-FISH detects: (x ± 3SD) value that normal cut off value is pressed gained positive cell number in control group.Positive cell rate be greater than cut off value sample we be defined as extremely.With D-FISH, positive cell rate being detected is 0%～0.5%, mean value 0.25%, standard deviation 0.16%, cut off value D=0.25% ± 3 * 0.16%=0.73%.
Table three breast carcinoma stem cell and tumour cell D-FISH detected result
The breast carcinoma stem cell of embodiment 6 immunomagnetic beads method enrichment specifically expressing differential proteins
Use in preparation example 3 and go down to posterity and cultivate acquisition 10
8individual cell, with 0.5mL Incubating Solution PBE (containing pH7.21 * PBS of 0.5%BSA and 0.08%EDTA, gas in vacuum filtration degerming and liquid), fully mix cell, add primary antibodie (the anti-human ABCG2 antibody of rabbit of 20 μ g/ml final concentrations, U.S. Santa company), hatch 30 minutes for 4 ℃.
With 20 times of volume PBE, wash cell once, adding 0.3mL PBE fully mixes after cell again, add the coated ultra micro magnetic bead of the anti-goat anti-rabbit igg of 50 μ L bis-(H+L) (American I nvitrogen Life Technologies, Inc.), mixing rearmounted 8 ℃ hatches 15 minutes, then add 5 times of volume PBE to wash cell 3 times, then use the abundant suspendible cell of 0.3mL PBE.
Separator column is fit in magnetic separator, adds 0.5ml PBE, under action of gravity, naturally flow to end, pre-treatment separator column.PBE suspension to adding 0.3mL cell in separator column naturally flows to end PBE under action of gravity.Separator column is taken off from magnetic separator, with special syringe, draw washing fluid pressurization and inject, the ABCG2 positive cell adsorbing under wash-out.
The antibody that use primary antibodie is mouse anti human PROM1, the anti-human ALDH1 of rabbit and mouse anti human PTEN, and the coated ultra micro magnetic bead of IgG (H+L) of two corresponding anti-mountain sheep anti mouses and goat antirabbit, can continuous separate select the breast carcinoma stem cell of expressing in ABCG2, PROM1, ALDH1 and PTEN at least two kinds.
Embodiment 7 breast carcinoma stem cell antigen composition preparations
Utilize method separation of the present invention and prepare breast carcinoma stem cell and antigen composition thereof.
Get in the breast cancer cell of above-mentioned preparation example 1 and 3 gained and breast carcinoma stem cell and above-described embodiment 6 breast carcinoma stem cell of at least two kinds in enrichment specifically expressing ABCG2, PROM1, ALDH1 and PTEN, selective enrichment ABCG2 and PROM1 are two positive, ABCG2, PROM1 and ALDH1 tri-positives, the breast carcinoma stem cell of ABCG2, PROM1, ALDH1 and PTEN tetra-positives; And get the breast carcinoma stem cell that comparative example 4 obtains.
200 μ l cell suspensions are on ice or during mixture of ice and water bathes, with 45 watts of cell Ultrasonic Cell Disruptor operating powers, ultrasonic time 5 seconds, 10 seconds off times, work times 20 times, then get ultrasonic rear suspension in the cytoclastic efficiency of Microscopic observation.Cross the centrifugal 1min of 800rpm and remove, then 0.45 μ m membrane filtration for supernatant.The basal component of described composition comprises physiological saline, and described lysate is the antigen composition of breast carcinoma stem cell, is stored in-80 ℃ standby.
Obtaining of embodiment 8 mammary cancer knurl stem cell antigen loaded dendritic cell vaccines
Get 100mL peripheral blood through lymphocyte separation medium density gradient centrifugation, obtain mononuclearcell.Peripheral blood mononuclear cell is resuspended with RPMI1640 substratum, and adds in 6 orifice plates adherent.At 37 ℃, 5%CO
2in incubator, hatch after 90min, attached cell washing is not collected.Attached cell adds perfect medium RPMI1640 inducing culture, contains 5% huge G CFS (GM-CSF) and the 500IU/mL interleukin-4 (IL-4) bitten of autoserum, 1000IU/mL recombinant humangranulocyte.When cultivating by the 3rd day, cell is changed fresh culture (containing 1000IU/mL GM-CSF and 500IU/mL IL-4).Cultivation to the five days results immature dendritic cell (DCs), mature DCs adopts complete RPMI1640 substratum to obtain being induced to the 7th day, containing 0.1 μ g/mL lipopolysaccharides and 20ng/mL tumor necrosis factor alpha.In the time of the 5th day, add special antigen (as breast carcinoma stem cell antigen), load DCs.
Cultivate after 5 days 3 * 10
6individual immature DC s (derives from 1 * 10 with the breast carcinoma stem cell antigen composition described in embodiment 3 and comparative example 4
6breast carcinoma stem cell) at 37 ℃, 5%CO
2in incubator, load is 4 hours.The DCs of antigen load obtains by centrifugal (10min, 1000rpm) results.Gained DCs is through physiological saline washing 3 times, after be resuspended in physiological saline, to concentration be 3 * 10
6ripe DC/mL, and add the human serum albumin that final concentration is mass volume ratio 2%, thus be prepared into the DC vaccine of breast carcinoma stem cell antigen composition load.
Enrichment specifically expressing ABCG2 in the breast cancer cell of above-mentioned preparation example 1 and 3 gained and breast carcinoma stem cell and above-described embodiment 6, PROM1, the breast carcinoma stem cell of at least two kinds in ALDH1 and PTEN, and the breast carcinoma stem cell of comparative example 4 acquisitions, its antigen composition loaded dendritic cell passes through CD86-FITC, CD80-FITC, CD83-PE and CD11c-Percp (U.S. company BD) dyeing, after flow cytometer detects load, DC phenotype changes, dendritic cell CD11c/CD83, two positive rates of CD11c/CD86 and CD11c/CD80 are all higher than more than 91%, meet DC cell-specific phenotype, and high expression level costimulatory molecules.
The external knurl of killing of breast carcinoma stem cell antigen load DC vaccine of effect illustrative examples 1: embodiment 1 and 3, embodiment 6 and comparative example 4 gained is tested
In 6 orifice plates, the RPMI1640 of still take containing 5% calf serum is substratum, in the mature dendritic cell of above-mentioned antigen load: T cell=1: 20 ratio is mixed mature dendritic cell and the T cell of above-mentioned antigen load, add IL-2 (final concentration is 100IU/mL) simultaneously, cultivate altogether 7 days, every 1 day, all half amount was changed liquid (the RPMI1640 substratum of the IL-2 that is 100U/mL containing 5% calf serum, final concentration) during this time, obtained CTL cell.
Select corresponding embodiment 1 and 3, embodiment 6 and comparative example 4 breast carcinoma stem cells as target cell, use
51cr mark, target cell (2 * 10
6/ mL) by with 300 μ Ci
51cr in RPMI1640 substratum 37 ℃ hatch 2 hours.1 * PBS washing three times for target cell that mark is good, and finally use RPMI1640 (containing 10% calf serum) to be resuspended to 2 * 10
5concentration.With every hole 2 * 10
4the target cell of individual mark (0.1mL) is added in the hole of 96 orifice plates.
By the CTL cell (effector cell) of above-mentioned generation respectively with the effect target of 2.5: 1,5: 1,10: 1,20: 1 and 40: 1 than adding in corresponding hole, at 37 ℃, hatch 4 hours.After hatching, get γ radiometer rolling counters forward for supernatant 75 μ L components.Special
51the per-cent that Cr discharges calculates according to following formula.
Wherein, the per minute umber of pulse of spontaneous release obtains by single culture target cell (not adding effector cell), the maximum per minute umber of pulse discharging is to obtain after the single culture target cell of processing with final concentration 2%NP-40 (tensio-active agent, the raw work in Shanghai).The per minute umber of pulse detecting is cultivated and is obtained by adding effector cell's target cell.
Fig. 2 has shown by embodiment 6 gained ABCG2 and PROM1 two positive, ABCG2, PROM1 and ALDH1 tri-positives, the breast carcinoma stem cell antigen composition loaded dendritic cell of ABCG2, PROM1, ALDH1 and PTEN tetra-positives, obtain cytotoxic T lymphocyte (CTL), the response result of the breast carcinoma stem cell that embodiment 3 is obtained being produced by this CTL cell.As shown in Figure 2, along with effect target ratio improves constantly, the CTL that breast carcinoma stem cell is produced replys also special rising, and better by the embodiment effect of four kinds of positive differential proteins of enrichment.Wherein, AP-DC1 is expressed as the toxicity activity of the dendritic cell initiation of ABCG2 and the two positive breast cancer stem cell antigen loads of PROM1 to the breast carcinoma stem cell of embodiment 3 acquisitions; The dendritic cell that APA-DC2 is expressed as ABCG2, PROM1 and ALDH1 tri-positive breast cancer stem cell antigen loads causes the toxicity of the breast carcinoma stem cell of embodiment 3 acquisitions active; The dendritic cell that APAP-DC3 is expressed as ABCG2, PROM1, ALDH1 and PTEN tetra-positive breast cancer stem cell antigen loads causes the toxicity of the breast carcinoma stem cell of embodiment 3 acquisitions active.
Fig. 3 has shown by preparation example 1,3 and three kinds of antigen compositions difference loaded dendritic cells of comparative example 4 gained, and embodiment 6 gained ABCG2 and the two positive breast cancer stem cell antigen composition loaded dendritic cells of PROM1, obtain four kinds of cytotoxic T lymphocytes (CTL), the response result the comparison diagram respectively two positive breast cancer stem cells of ABCG2 and PROM1 being produced by these four kinds of CTL cells.Wherein, AP-DC1 is expressed as the toxicity activity of the dendritic cell initiation of ABCG2 and the two positive breast cancer stem cell antigen loads of PROM1 to ABCG2 and PROM1 pair of positive breast cancer stem cells; The dendritic cell of preparation example 1 breast cancer cell MCF-7 antigen load that represents MCF-DC causes the toxicity of ABCG2 and the two positive breast cancer stem cells of PROM1 active, BSC-DC represents that the dendritic cell that the mammary cancer of preparation example 3 becomes neurosphere cell antigen load is active to the toxicity of ABCG2 and the two positive breast cancer stem cells of PROM1, and it is active to the toxicity of ABCG2 and the two positive breast cancer stem cells of PROM1 that DBSC-DC represents to contrast the dendritic cell of breast carcinoma stem cell antigen load of side 4.
By embodiment 6 gained ABCG2 and the two positive breast cancer stem cell antigen composition loaded dendritic cells (AP-DC1) of PROM1, cause and will higher than MCF-DC, BSC-DC and DBSC-DC, cause the cellular cytoxicity activity to ABCG2 and the two positive breast cancer stem cells of PROM1 respectively to the cellular cytoxicity activity of ABCG2 and the two positive breast cancer stem cells of PROM1 as shown in Figure 3.The two positive breast cancer stem cells of described ABCG2 and PROM1 are according to embodiment 6 preparations.
Fig. 4 has shown by preparation example 1,3 and three kinds of antigen compositions difference loaded dendritic cells of comparative example 4 gained and embodiment 6 gained ABCG2 and the two positive breast cancer stem cell antigen composition loaded dendritic cells of PROM1, obtain four kinds of cytotoxic T lymphocytes (CTL), the response result comparison diagram that the breast carcinoma stem cell respectively comparative example 4 being obtained by these four kinds of CTL cells produces.Wherein, DAP-DC1 is expressed as the toxicity activity of the dendritic cell initiation of ABCG2 and the two positive breast cancer stem cell antigen loads of PROM1 to the breast carcinoma stem cell of comparative example 4 acquisitions; The dendritic cell of breast cancer cell MCF-7 antigen load that represents DMCF-DC causes the toxicity activity of the breast carcinoma stem cell that comparative example 4 is obtained, DBSC-DC represents that the mammary cancer of preparation example 4 becomes the dendritic cell of neurosphere cell antigen load active to the toxicity of the breast carcinoma stem cell of comparative example 4 acquisitions, and DDBSC-DC represents that the toxicity of the breast carcinoma stem cell that the dendritic cell of the breast carcinoma stem cell antigen load of comparative example 4 obtains comparative example 4 is active.
By embodiment 6 gained ABCG2 and the two positive breast cancer stem cell antigen composition loaded dendritic cells (DAP-DC1) of PROM1, cause and will higher than DMCF-DC, DBSC-DC and DDBSC-DC, cause the cellular cytoxicity activity to the breast carcinoma stem cell of comparative example 4 acquisitions respectively to the cellular cytoxicity activity of the breast carcinoma stem cell of comparative example 4 acquisitions as shown in Figure 4.
Effect illustrative examples 2: embodiment 1 and 3, embodiment 6 and comparative example 4 breast carcinoma stem cells kill the effect in knurl experiment in vivo
The RPMI1640 of take containing 5% calf serum is substratum, by 3 * 10
6individual immature dendritic cell with above-described embodiment 6 gained without or through screening breast carcinoma stem cell schizolysis antigen composition (derive from 1 * 10
6breast carcinoma stem cell) at 37 ℃, 5%CO
2in incubator, load is 4 hours.The mature dendritic cell of antigen load obtains by the centrifugal 10min of 1000rpm.By physiological saline washing 3 times for the mature dendritic cell of described antigen load, then resuspended to 1 * 10
6the concentration of individual/mL, saves backup at 4 ℃.
Breast carcinoma stem cell is set up lotus mammary cancer knurl mouse model (referring to " the assessment new model of targeted human tumor vessel and human cancer stem-cell therapy ", the people such as Burgos-Ojeda D, cancer research, the 445th volume, 3555-3565 page, on July 15th, 2013; A novel model for evaluating therapies targeting human tumor vasculature and human cancer stem-like cells.Cancer Res.2013; 73 (12): 3555-3565.), be divided at random A, B, C, D, E, F and G7 group, 6 every group: A group is the dendritic cells in treatment group of enrichment ABCG2 and the two positive breast carcinoma stem cell antigen loads of PROM1; The dendritic cells in treatment group that B group is the breast carcinoma stem cell antigen load of ABCG2, PROM1 and ALDH1 tri-positives; The dendritic cells in treatment group that C group is the breast carcinoma stem cell antigen load of ABCG2, PROM1, ALDH1 and PTEN tetra-positives; The dendritic cell comparative group that D group is the breast cancer cell MCF-7 antigen load of preparation example 1 preparation (injection preparation is identical with A group); The dendritic cell comparative group that E group is the breast carcinoma stem cell antigen load of preparation example 3 preparations (injection preparation is identical with A group); The dendritic cell that F group is the breast carcinoma stem cell antigen load of comparative example 4 acquisitions is comparative group (injection preparation is identical with A group); G group is the blank group of same volume physiological saline.Six groups of A, B, C, D, E and F were the 0th day mice with tumor model h inf 0.5-2 * 10
6individual/mL dendritic cell, each 0.5mL, same dosage immunity 1 time again after two weeks.After injection respectively at 7,14,21,28,35d calculates mouse model lotus knurl size, G group is in same time point at subcutaneous injection 0.5mL physiological saline and to calculate mouse model lotus knurl big or small.
Inhibition growing state (mm through the dendritic cell vaccine of breast carcinoma stem cell antigen load to mice with tumor model
3,
) in Table four.
Table four
The blank group of note: * is compared P much smaller than 0.01 with treatment group;
#comparative group is compared P < 0.01 with treatment group.
In addition, in the body that breast carcinoma stem cell load DC more than relevant embodiment 6 enrichment gained two species diversity gene masculines causes, kill tumor activity, be better than far away in the body of breast carcinoma stem cell of relevant preparation example 1,3 and comparative example 4 and kill tumor activity.In addition in the body about the breast carcinoma stem cell of embodiment 6 gained ABCG2, PROM1, ALDH1 and PTEN tetra-positives, kill tumor activity, be better than in the body of breast carcinoma stem cell of relevant embodiment 6 gained two species diversity protein positives and three species diversity gene masculines and kill tumor activity.
Claims (10)
1. a method of preparing breast carcinoma stem cell, is characterized in that, described method comprises identifies the step that whether has breast carcinoma stem cell in breast cancer cell sample to be measured:
Described step comprises and detects in described breast cancer cell sample to be measured at least two kinds of the following differential protein of specifically expressing whether: ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues;
While there is breast carcinoma stem cell in identifying described breast cancer cell sample to be measured, the method also comprises: the step of the breast carcinoma stem cell described in use immunomagnetic beads method and/or the enrichment of selected by flow cytometry apoptosis method in cell to be measured;
The mammary cancer knurl stem cell balling-up growth of described acquisition, detects CD44 through flow cytometer
+and CD24
-/lowmammary cancer knurl stem cell account for the more than 30% of total cell count.
2. method according to claim 1, wherein, described specifically expressing differential protein refers to that to use the result of at least two kinds, the described four species diversity albumen in breast cancer cell sample described in double-colored pair of fusion probe interphase fluorescence in situ hybridization technology for detection positive;
Wherein, with the negative contrast of known pure breast cancer cell, with 100 parts of described negative controls of double-colored pair of fusion probe interphase fluorescence in situ hybridization technology for detection, every part containing 10
3individual cell, record percentage that every species diversity albumen false positive signal number of cells accounts for every part of total cellular score by the false positive rate of this species diversity albumen of survey, the mean value of this species diversity albumen false positive rate of survey and standard variance sum are defined as to cut off value;
Get 100 parts of breast cancer cell samples to be measured, every part of sample is containing 10
3individual cell, recording and surveying with negative control the percentage that differential protein positive signal number of cells of the same race accounts for every part of total cellular score is the mean value of the positive rate of institute's error of measurement M-band, this positive rate mean value is more than or equal to described cut off value, shows that the double-colored pair of fusion probe interphase fluorescence in situ hybridization technology for detection result of this species diversity albumen of this breast cancer cell sample to be measured is positive.
3. method according to claim 1 and 2, while there is breast carcinoma stem cell in identifying described breast cancer cell sample to be measured, the method also comprises:
The step of the breast carcinoma stem cell described in use immunomagnetic beads method and/or the enrichment of selected by flow cytometry apoptosis method in cell to be measured;
Wherein, utilize the antibody of at least two kinds for four species diversity albumen such as the ATP linking frame subfamily G composition 2 that obtains specifically expressing, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues.
4. a test kit of preparing breast carcinoma stem cell, is characterized in that, described test kit comprises:
1) breast carcinoma stem cell basic medium;
2) at least two kinds of ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1, Phosphoric acid esterase and tensin homologues;
3) enzyme of peptic cell;
4) cytokine and;
5) working instructions;
Wherein, described working instructions comprise the method described in claim 1 or 2.
5. test kit according to claim 4, wherein, described test kit also comprises the antibody of at least two kinds for described ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues; Described working instructions comprise method claimed in claim 3.
6. a preparation method for breast carcinoma stem cell antigen composition, is characterized in that, the method comprises the following steps:
1) breast carcinoma stem cell described in any one in claim 1-3;
2) with physiological saline washing resuspended described 1 * 10
5to 1 * 10
8cell/mL breast carcinoma stem cell;
3) breast carcinoma stem cell described in cracking;
Wherein, described step 3) comprise by heat-shocked and/or multigelation and carry out cracking breast carcinoma stem cell; The condition of described heat-shocked cracking is included at 37～45 ℃ and keeps 1h-6h; The number of times of described multigelation is 3-5 time, and be 10min-2h the interval time of twice, and described freezing temperature range is subzero 120 ℃-subzero 196 ℃, and the temperature range of described thawing is 10 ℃-37 ℃.
7. the preparation method of the dendritic cell of breast carcinoma stem cell antigen composition load, it is characterized in that, the method comprises: by cracking breast carcinoma stem cell, obtain breast carcinoma stem cell antigen composition, normal dendritic cell is contacted to the anti-breast cancer dendritic cell that obtains load tumour antigen with breast carcinoma stem cell antigen composition, it is characterized in that, described breast carcinoma stem cell is the breast carcinoma stem cell described in any one in claim 1-4; Described breast carcinoma stem cell antigen composition is breast carcinoma stem cell antigen composition claimed in claim 6.
8. a test kit of preparing the dendritic cell of breast carcinoma stem cell antigen load, is characterized in that, described test kit comprises:
1) breast carcinoma stem cell basic medium;
2) at least two kinds of ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues;
3) enzyme of peptic cell;
4) B27 or N2, epithelical cell growth factor, Prostatropin and Regular Insulin;
5) dendritic cell basic medium;
6) macrosome cell-phagocyte G CFS;
7) interleukin-4, interferon alpha and tumor necrosis factor alpha; And
6) working instructions;
Wherein, described working instructions comprise method claimed in claim 7.
9. test kit according to claim 8, wherein, described test kit also comprises the antibody of at least two kinds for described ATP linking frame subfamily G composition 2, five cross-film strand glycoprotein PROM1, acetaldehyde dehydrogenase-1 and Phosphoric acid esterase and tensin homologues.
10. the application of dendritic cell described in breast carcinoma stem cell antigen composition or claim 7-9 described in claim 6.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103289957A (en) * | 2013-05-13 | 2013-09-11 | 北京希普生国际生物医学研究院 | Methods and kit for preparing stem cell-like lung cancer cells and loading dendritic cells on antigen composition thereof |
| CN103396995A (en) * | 2013-07-16 | 2013-11-20 | 广州赛哲生物科技有限公司 | Three-dimensional culture method for screening breast cancer stem cells |
-
2014
- 2014-06-30 CN CN201410300288.4A patent/CN104087556A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103289957A (en) * | 2013-05-13 | 2013-09-11 | 北京希普生国际生物医学研究院 | Methods and kit for preparing stem cell-like lung cancer cells and loading dendritic cells on antigen composition thereof |
| CN103396995A (en) * | 2013-07-16 | 2013-11-20 | 广州赛哲生物科技有限公司 | Three-dimensional culture method for screening breast cancer stem cells |
Non-Patent Citations (4)
| Title |
|---|
| 牛畅等: "乳腺癌干细胞和乳腺癌", 《生物技术通讯》 * |
| 田玉玲等: "《免疫优化算法、模型及应用》", 28 February 2013, 国防工业出版社 * |
| 陆士新: "《干细胞与肿瘤》", 31 July 2009, 中国协和医科大学出版社 * |
| 陈德荣等: "乳腺癌干细胞临床与基础研究的进展", 《中国肿瘤临床》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219731A (en) * | 2015-09-01 | 2016-01-06 | 东南大学 | A kind of ovarian cancer stem cell vaccine and preparation method thereof |
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