CN103289957A - Methods and kit for preparing stem cell-like lung cancer cells and loading dendritic cells on antigen composition thereof - Google Patents

Methods and kit for preparing stem cell-like lung cancer cells and loading dendritic cells on antigen composition thereof Download PDF

Info

Publication number
CN103289957A
CN103289957A CN2013101740827A CN201310174082A CN103289957A CN 103289957 A CN103289957 A CN 103289957A CN 2013101740827 A CN2013101740827 A CN 2013101740827A CN 201310174082 A CN201310174082 A CN 201310174082A CN 103289957 A CN103289957 A CN 103289957A
Authority
CN
China
Prior art keywords
cell
lung carcinoma
stem
carcinoma cell
cell lung
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN2013101740827A
Other languages
Chinese (zh)
Other versions
CN103289957B (en
Inventor
黄启明
李一民
徐世洪
李一汉
陈宗明
连日平
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangxi Austria Biological Technology Co. Ltd.
Original Assignee
BEIJING XIPUSHENG INTERNATIONAL BIOTECHNOLOGY Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BEIJING XIPUSHENG INTERNATIONAL BIOTECHNOLOGY Co Ltd filed Critical BEIJING XIPUSHENG INTERNATIONAL BIOTECHNOLOGY Co Ltd
Priority to CN201310174082.7A priority Critical patent/CN103289957B/en
Publication of CN103289957A publication Critical patent/CN103289957A/en
Application granted granted Critical
Publication of CN103289957B publication Critical patent/CN103289957B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention provides a method for preparing stem cell-like lung cancer cells. The method comprises a step of authenticating whether the stem cell-like lung cancer cells exist in a lung cancer cell specimen to be detected or not, namely, detecting whether at least two of the following differential proteins are specifically expressed in the lung cancer cell specimen to be detected, wherein the differential proteins include protein kinase Ciota, five-time transmembrane single-stranded glycoprotein Prominin1, acetaldehyde dehydrogenase 1 or a breast cancer medicine resistance-protein ATP (Adenosine Triphosphate)-binding cassette transporter G superfamily member 2. The invention further provides a method for preparing the antigen dendritic cells loaded by the stem cell-like lung cancer cells, and a dendritic cell vaccine obtained via the method. According to the methods provided by the invention, the stem cell-like lung cancer cells can be efficiently and accurately obtained only by detecting at least two of the proteins; and the stem cell-like lung cancer cells obtained on the basis of the methods are high in purity and easy to enrich.

Description

Method and the test kit of the preparation of stem-like cell lung carcinoma cell and antigen composition loaded dendritic cell thereof
Technical field
The present invention relates to prepare stem-like cell tumour cell, its antigen composition, this antigen dendritic cell of load method, use the test kit of described method and the dendritic cell that obtain by described method and vaccine especially, the present invention relates to prepare stem-like cell lung carcinoma cell and this antigen dendritic cell of antigen composition load thereof method, use the test kit of described method and the dendritic cell vaccine by described method acquisition.Background technology
At present, the tumorigenesis lung carcinoma cell with stem/progenitor cells characteristic has been identified and be separated to prior art in lung cancer tumor.For example, " Side population in human lung cancer cell lines and tumors is enriched with stem-like cancer cells (being rich in the stem-like cell tumour cell in human lung cancer cell line and the tumour in the subgroup) ", people such as Maria M.Ho, Cancer Research (cancer research), the 67th the 10th phase of volume, the 4827-4833 page or leaf, disclose in 15 days Mays in 2007, the cell that it finds mammary cancer drug-resistant protein ATP binding transport Protein G superfamily member 2 (ABCG2) keeps carcinogenic activity specially and has demonstrated the stem-like cell characteristic.Be that prior art can be identified the stem-like cell lung carcinoma cell by detecting ABCG2 or the Prominin1 of the doubtful stem-like cell lung carcinoma cell that separates at cancer knurl place.
But the character that same ABCG2 and Prominin1 are also arranged in normal cell, therefore distinguishing of the method for the prior art stem-like cell lung carcinoma cell that cancer knurl place can't be separated and normal cell efficiently and accurately, thereby enrichment quickly and effectively and external breeding stem-like cell lung carcinoma cell.For example, specific expressed protein kinase C iota in the stem-like cell lung carcinoma cell does not express in lung carcinoma cell, but in normal pulmonary alveolar macrophage expression is being arranged also.
To sum up how to obtain the stem-like cell lung carcinoma cell efficiently and accurately, and can utilize the stem-like cell lung carcinoma cell of gained to prepare the vaccine that resists lung cancer in the donor, remain problem demanding prompt solution.
Summary of the invention
In order to overcome the problem that the very difficult efficiently and accurately at short notice of prior art obtains purity height and the easy stem-like cell lung carcinoma cell of enrichment, the purpose of this invention is to provide a kind of method for preparing the stem-like cell lung carcinoma cell, this method efficiently and accurately at short notice obtains purity height and the easy stem-like cell lung carcinoma cell of enrichment.
Of the present invention the provides the method for a kind of stem-like cell lung carcinoma cell antigen composition and this antigen dendritic cell of load thereof, and the test kit that uses the method for the invention is provided, the dendritic cell vaccine that obtains by the method for the invention.
The invention provides a kind of method for preparing the stem-like cell lung carcinoma cell, wherein, described method comprises identifies the step that whether has the stem-like cell lung carcinoma cell in the lung carcinoma cell sample to be measured:
Described step comprises at least two kinds that detect in the described lung carcinoma cell sample to be measured the following differential protein of specifically expressing whether: protein kinase C iota, stride film strand glycoprotein Prominin1, acetaldehyde dehydrogenase 1 or mammary cancer drug-resistant protein ATP binding transport Protein G superfamily member 2 for five times.
The present invention also provides a kind of preparation method of above-mentioned stem-like cell lung carcinoma cell antigen composition, and wherein, this method may further comprise the steps:
1) prepare the stem-like cell lung carcinoma cell according to the method described in the present invention,
2) with physiological saline washing and resuspended described stem-like cell lung carcinoma cell,
3) the described stem-like cell lung carcinoma cell of cracking.
The present invention also provides a kind of preparation method of dendritic cell of stem-like cell lung carcinoma cell antigen load, wherein, this method comprises: obtain stem-like cell lung carcinoma cell antigen composition by cracking stem-like cell lung carcinoma cell, normal dendritic cell is contacted the anti-lung cancer dendritic cell that obtains the load tumour antigen with stem-like cell lung carcinoma cell antigen composition, wherein, described method comprises uses method of the present invention to prepare the stem-like cell lung carcinoma cell, perhaps uses method of the present invention to prepare stem-like cell lung carcinoma cell antigen composition.
The present invention also provides a kind of dendritic cell vaccine, and described dendritic cell vaccine comprises dendritic cell, and wherein, described dendritic cell is the dendritic cell of stem-like cell lung carcinoma cell antigen load of the present invention.
The present invention also provides a kind of test kit for preparing the dendritic cell vaccine of stem-like cell lung carcinoma cell antigen load, and wherein, described test kit comprises:
1) stem-like cell lung carcinoma cell basic medium;
2) protein kinase C iota, stride film strand glycoprotein Prominin1, acetaldehyde dehydrogenase 1 or mammary cancer drug-resistant protein ATP binding transport Protein G superfamily member 2 at least two kinds for five times;
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 the dendritic cell of preparation stem-like cell lung carcinoma cell antigen load of the present invention.
By method of the present invention, only by detecting protein kinase C iota, striding film strand glycoprotein Prominin1, acetaldehyde dehydrogenase 1 or mammary cancer drug-resistant protein ATP binding transport Protein G superfamily member 2 at least two kinds for five times, just can obtain the stem-like cell lung carcinoma cell efficiently and accurately, easy based on described method gained stem-like cell lung carcinoma cell purity height and enrichment.The medicine of the dendritic cell preparation of the stem-like cell lung carcinoma cell that is obtained by method provided by the invention and the antigen composition that is obtained by this stem-like cell lung carcinoma cell, this antigen of load, can kill the stem-like cell lung carcinoma cell that causes the lung cancer recurrence with external initiation immunne response in vivo, thereby provide possibility for overcoming lung cancer tolerance, the recurrence of effecting a radical cure lung cancer and transfer.
Description of drawings
Fig. 1 represents by embodiment 1 or/and the stem-like cell lung carcinoma cell antigen composition loaded dendritic cell phenotypic map of 2 preparation PKC iota, PROM1, ALDH1 and ABCG2 four positives.
Fig. 2 represents by embodiment 1 or/and the response result figure to PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1 that the special stem-like cell lung carcinoma cell antigen composition loaded dendritic cell that reaches more than the two species diversity albumen of 2 gained tables produces.
Fig. 3 represent by embodiment 1 or/and the two positive stem-like cell lung carcinoma cell antigen composition loaded dendritic cells of 2 gained PKC iota and PROM1 with by preparation example 1,4 and three kinds of antigen compositions of Comparative Examples 1 gained loaded dendritic cell respectively, cause the response result comparison diagram that PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1 are produced.
Fig. 4 represents by embodiment 1 or/and the two positive stem-like cell lung carcinoma cell antigen composition loaded dendritic cells of 2 gained PKC iota and PROM1, and by preparation example 1,4 and three kinds of antigen compositions of Comparative Examples 1 gained respectively loaded dendritic cell cause the response result comparison diagram that stem-like cell lung carcinoma cell that Comparative Examples 1 is obtained produces.
Embodiment
The invention provides a kind of method for preparing the stem-like cell lung carcinoma cell, wherein, described method comprises identifies the step that whether has the stem-like cell lung carcinoma cell in the lung carcinoma cell sample to be measured:
Described step comprises at least two kinds that detect in the described lung carcinoma cell sample to be measured the following differential protein of specifically expressing whether: protein kinase C iota (Protein kinase C iota, PKC iota), stride for five times film strand glycoprotein Prominin1 (PROM1), acetaldehyde dehydrogenase 1 (aldehyde dehydrogenase1, ALDH1) or ABCG2.Wherein, protein kinase C iota is the indispensable gene of lung cancer stem cells hyperplasia, chemicotherapy is being produced opposing, transforms growth and is invading the central role of playing the part of aspect the profit.
Preferably, it is positive that described specifically expressing differential protein refers to use the result of the described four species diversity albumen at least two kinds (containing protein kinase C iota interior) in the described lung carcinoma cell sample of protein immunoblot technology for detection;
Wherein, with the negative contrast of known pure lung carcinoma cell, with 100 parts of described negative controls of protein immunoblot technology for detection, every part contains 5 * 10 6Individual cell records the gray scale of every species diversity albumen and the gray level ratio of standard confidential reference items β Actin muscle, and mean value and the standard variance sum of this species diversity albumen gray level ratio of survey is defined as cut off value;
Get 100 parts of lung carcinoma cell samples to be measured, every part of sample contains 5 * 10 6Individual cell records the mean value of surveying differential protein gray level ratio of the same race with negative control, and the mean value of this gray level ratio is more than or equal to described cut off value, and namely the protein immunoblot technology for detection result of this species diversity albumen of this lung carcinoma cell sample to be measured is positive.
Further preferably, described specifically expressing differential protein refers to use the result of the described four species diversity albumen in the described lung carcinoma cell sample of protein immunoblot technology for detection all positive.Preferably, when having the stem-like cell lung carcinoma cell in identifying described lung carcinoma cell sample to be measured, this method also comprises:
Use the step of the stem-like cell lung carcinoma cell in immunomagnetic beads method and/or the described cell to be measured of selected by flow cytometry apoptosis method enrichment;
Wherein, utilization is at obtaining protein kinase C iota (Protein kinase C iota, PKC iota), stride for five times film strand glycoprotein Prominin1 (PROM1), acetaldehyde dehydrogenase 1 (aldehyde dehydrogenase1, ALDH1) or at least two kinds the antibody of ABCG2.
Described method can also comprise separate stem cells sample lung carcinoma cell from exsomatize lung cancer tumor tissues and/or lung cancer cell line.The lung cancer tumor tissues that operation is taken out in patient's body belongs to discarded stripped lung cancer tumor tissues.The present invention can be suitable for conventional method and obtain lung carcinoma cell.In addition, also can therefrom isolate corresponding stem-like cell lung carcinoma cell by the existing commercial cloned culture that can buy.For example, A549 people's non-small cell lung cancer cell can buy and can therefrom isolate the cell strain system of corresponding stem-like cell lung carcinoma cell exactly from U.S. typical case thing preservation center (ATCC).
The present invention also provides a kind of preparation method of above-mentioned stem-like cell lung carcinoma cell antigen composition, and wherein, this method may further comprise the steps:
1) prepare the stem-like cell lung carcinoma cell according to the method described in the present invention,
2) with physiological saline washing and resuspended described stem-like cell lung carcinoma cell,
3) the described stem-like cell lung carcinoma cell of cracking.
Wherein, the washing times of described physiological saline can residual nutrient media components decides in the elutant by measuring.Stem-like cell lung carcinoma cell antigen composition residual nutrient media components and residual chemotherapeutics of possibility when the patient is given in injection that method of the present invention is made can not cause that patient's discomfort gets final product.
Cracking stem-like cell lung carcinoma cell of the present invention can use the various cleavage methods of this area routine to realize.For the consideration that does not increase new material behind cracking stem-like cell lung carcinoma cell, the step of preferred described cracking stem-like cell lung carcinoma cell comprises heat-shocked and/or multigelation; The condition of described heat-shocked cracking is included in 37～45 ℃ and keeps 1h-6h down, is preferably included in 38～43 ℃ and keeps 2h-4h down; The number of times of described multigelation is 3-5 time, be 10min-2h per twice pitch time, be preferably 30min-1h, described freezing temperature range is subzero 120 ℃-subzero 196 ℃ (can use liquid nitrogen, dry ice to wait to realize), preferred subzero 150 ℃-subzero 196 ℃, the temperature range of described thawing is 10 ℃-37 ℃, is preferably 20 ℃-37 ℃.Because after heat-shocked, stem-like cell lung carcinoma cell of the present invention can produce heat shock protein(HSP), still has described heat shock protein(HSP) after the cracking.The dendritic cell vaccination that the having of heat shock protein(HSP) helps after the load is replied.Therefore, more preferably the step of described cracking stem-like cell lung carcinoma cell comprises heat-shocked and multigelation; The condition of described heat-shocked cracking is included in 37-45 ℃ and keeps 1h-6h down; The number of times of described multigelation is that be 10min-2h 3-5 time twice pitch time, and described freezing temperature range is subzero 120 ℃-subzero 196 ℃, and the temperature range of described thawing is 20 ℃-37 ℃.
After cracking is finished, usually remove cell debris excessive in the lysate by step centrifugal and that filter, for example, remove by the centrifugal 1min of 600rpm, supernatant is with 0.45 μ m membrane filtration then.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 the stem-like cell lung carcinoma cell of preparation gained is 1 * 10 in the described step 1) 5To 1 * 10 8Cell/mL.
Wherein, described stem-like cell lung carcinoma cell basic medium can be any substratum that can be used in culturing stem cells sample lung carcinoma cell in this area, for example DMEM/F12 etc.Described stem-like cell lung carcinoma cell culture system comprises the stem-like cell lung carcinoma cell basic medium of stem-like cell lung carcinoma cell and 3mL-50mL, at 37 ℃, 5%CO 2Cultivate in the incubator.
The enzyme of described peptic cell can be the enzyme of the peptic cell of this area routine, is preferably pancreatin and/or Ah especially for this enzyme.Wherein, when stem-like cell lung carcinoma cell basic medium reaches 3mL-50mL, described pancreatin and/or Ah especially replace this enzyme (Accutase) preferably as tumor tissues separate stem cells sample reagent that lung carcinoma cell uses, can use the consumption of this area routine, for example general consumption is that the 0.5mL-10mL of 1 times of concentration comes peptic cell, namely 1/6 of the basic medium volume to 1/5.
Described cytokine is B27 or N2, epithelical cell growth factor, Prostatropin and Regular Insulin.Wherein, described B27 adds in stem-like cell lung carcinoma cell basic medium (for example DMEM/F12) when culturing stem cells sample lung carcinoma cell, and reaching final concentration is 2ng/mL-50ng/mL.Described N2 adds in stem-like cell lung carcinoma cell basic medium (for example DMEM/F12) when culturing stem cells sample lung carcinoma cell, and reaching final concentration is 2ng/mL-50ng/mL.Wherein said B27 and N2 are the commercial cell cultures additives that supplies (being cytokine), for example, and B27 and N2 that U.S. biotech company, U.S. Gibco company produce.Described epithelical cell growth factor adds in stem-like cell lung carcinoma cell basic medium (for example DMEM/F12) when culturing stem cells sample lung carcinoma cell, and reaching final concentration is 5ng/mL-100ng/mL.Described Prostatropin adds in stem-like cell lung carcinoma cell basic medium (for example DMEM/F12) when culturing stem cells sample lung carcinoma cell, and reaching final concentration is 5ng/mL-100ng/mL.Described Regular Insulin adds in stem-like cell lung carcinoma cell basic medium (for example DMEM/F12) when culturing stem cells sample lung carcinoma cell, and reaching final concentration is 1mg/mL-50mg/mL.
Described test kit can also comprise non-essential animal serum.Described animal serum can be human serum and/or bovine serum.Described serum can be used as the terminator of digestive ferment, its consumption is as the conventional amount used of digestive ferment terminator in the prior art, for example, work as 0.1mL-10mL with the enzyme liquid phase of peptic cell, namely the volume ratio of the enzyme liquid of animal serum and peptic cell is 1: 1 to 1: 5.
The present invention also provides a kind of preparation method of dendritic cell of stem-like cell lung carcinoma cell antigen load, wherein, this method comprises: obtain stem-like cell lung carcinoma cell antigen composition by cracking stem-like cell lung carcinoma cell, normal dendritic cell is contacted the anti-lung cancer dendritic cell that obtains the load tumour antigen with stem-like cell lung carcinoma cell antigen composition, wherein, described method comprises uses method of the present invention to prepare the stem-like cell lung carcinoma cell, perhaps uses method of the present invention to prepare stem-like cell lung carcinoma cell antigen composition.The stem-like cell lung carcinoma cell of preferred described normal dendritic cell and preparation stem-like cell lung carcinoma cell antigen composition is from same individuality.Can avoid the side effect that causes owing to immunological rejection so to greatest extent.Certainly, if described normal dendritic cell is not from same individuality with the stem-like cell lung carcinoma cell for preparing stem-like cell lung carcinoma cell antigen composition, still can use the medicine of this area anti-immunological rejection commonly used to reduce side effect.The medicine of described anti-immunological rejection is dexamethasone etc. for example.
The present invention also provides a kind of dendritic cell vaccine, 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 stem-like cell lung carcinoma cell antigen load of the present invention.
Before the preparation dendritic cell vaccine, with the dendritic cell of physiological saline washing and resuspended described stem-like cell lung carcinoma cell antigen load.The washing times of described physiological saline can residual nutrient media components decides in the elutant by measuring.Dendritic cell vaccine of the present invention residual nutrient media components and residual chemotherapeutics of possibility when the patient is given in injection can not caused that patient's discomfort gets final product.Described vaccine preferably includes 1 * 10 5To 5 * 10 7The dendritic cell of individual/mL stem-like cell lung carcinoma cell of the present invention antigen load; More preferably 5 * 10 5To 1 * 10 7Individual/mL.Described vaccine also comprises human serum albumin; Wherein, be benchmark with described vaccine, the content of described human serum albumin is preferably 1-5 bulking value %, more preferably 2-3 bulking value %.
The present invention also provides a kind of test kit for preparing the dendritic cell vaccine of stem-like cell lung carcinoma cell antigen load, and wherein, described test kit comprises:
1) stem-like cell lung carcinoma cell basic medium;
2) protein kinase C iota (Protein kinase C iota, PKC iota), stride for five times film strand glycoprotein Prominin1 (PROM1), acetaldehyde dehydrogenase 1 (aldehyde dehydrogenase1, ALDH1) or at least two kinds of ABCG2; 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;
7) interleukin-4, interferon alpha and tumor necrosis factor alpha; And
6) working instructions;
Wherein, described working instructions comprise the preparation method of the dendritic cell of stem-like cell lung carcinoma cell antigen load of the present invention.
Preferably, described test kit also comprises (the Protein kinase C iota at described protein kinase C iota, PKC iota), stride for five times film strand glycoprotein Prominin1 (PROM1), acetaldehyde dehydrogenase 1 (aldehyde dehydrogenase1, ALDH1) or at least two kinds the antibody of ABCG2.Utilize described antibody, use immunomagnetic beads method and/or the flow cytometer stem-like cell lung carcinoma cell in can the described cell to be measured of separating method enrichment.
Wherein, described stem-like cell lung carcinoma cell basic medium can be any substratum that can be used in culturing stem cells sample lung carcinoma cell in this area, for example DMEM/F12 etc.Described stem-like cell lung carcinoma cell culture system comprises the stem-like cell lung carcinoma cell basic medium of stem-like cell lung carcinoma cell and 3mL-50mL, at 37 ℃, 5%CO 2Cultivate in the incubator.
The enzyme of described peptic cell can be the enzyme of the peptic cell of this area routine, is preferably pancreatin and/or Ah especially for this enzyme.Wherein, when stem-like cell lung carcinoma cell basic medium reaches 3mL-50mL, described pancreatin and/or Ah especially replace this enzyme (Accutase) preferably as tumor tissues separate stem cells sample reagent that lung carcinoma cell uses, can use the consumption of this area routine, for example general consumption is that the 0.5mL-10mL of 1 times of concentration comes peptic cell, namely 1/6 of the basic medium volume to 1/5.
Wherein, described B27 adds in stem-like cell lung carcinoma cell basic medium (for example DMEM/F12) when culturing stem cells sample lung carcinoma cell, and reaching final concentration is 2ng/mL-50ng/mL.Described N2 adds in stem-like cell lung carcinoma cell basic medium (for example DMEM/F12) when culturing stem cells sample lung carcinoma cell, and reaching final concentration is 2ng/mL-50ng/mL.Described epithelical cell growth factor adds in stem-like cell lung carcinoma cell basic medium (for example DMEM/F12) when culturing stem cells sample lung carcinoma cell, and reaching final concentration is 5ng/mL-100ng/mL.Described Prostatropin adds in stem-like cell lung carcinoma cell basic medium (for example DMEM/F12) when culturing stem cells sample lung carcinoma cell, and reaching final concentration is 5ng/mL-100ng/mL.Described Regular Insulin adds in stem-like cell lung carcinoma cell basic medium (for example DMEM/F12) when culturing stem cells sample lung carcinoma cell, and reaching final concentration is 1mg/mL-50mg/mL.
Described test kit can also comprise non-essential animal serum.Described animal serum can be human serum and/or bovine serum.Described serum can be used as the terminator of digestive ferment, its consumption be in the prior art as the conventional amount used of digestive ferment terminator, for example, work as 0.1mL-10mL with the enzyme liquid phase of peptic cell.
The resulting stem-like cell lung carcinoma cell of the method antigen composition that is prepared stem-like cell lung carcinoma cell antigen composition by the present invention is preferably 1 * 10 from cell concn 5To 1 * 10 8Cell/mL, more preferably 1 * 10 6To 5 * 10 7Cell/mL prepares the resulting stem-like cell lung carcinoma cell of method of stem-like cell lung carcinoma cell by the present invention.Described cell concn is excessive to cause waste, otherwise cell concn is too small, and then deficiency is so that enough antigen in the dendritic cell load.
Described dendritic cell basic medium can be any substratum of cultivating dendritic cell that can be used in this area, for example RPMI/1640 etc.Described dendritic cell culture system comprises the dendritic cell basic medium of stem-like cell lung carcinoma cell and 1mL-100mL, preferred 30-80mL, at 37 ℃, 5%CO 2Cultivate in the incubator.Wherein, described macrosome cell-phagocyte G CFS (GM-CSF) adds in the dendritic cell basic medium final concentration 100IU/mL-2500IU/mL to when stem-like cell lung carcinoma cell antigen-loaded dendritic cell.Described interleukin-4 adds in the dendritic cell basic medium final concentration 100IU/mL-1500IU/mL to when stem-like cell lung carcinoma cell antigen-loaded dendritic cell.Described interferon alpha adds in the dendritic cell basic medium final concentration 100IU/mL-2000IU/mL to when stem-like cell lung carcinoma cell antigen-loaded dendritic cell.Described tumor necrosis factor alpha adds in the dendritic cell basic medium final concentration 0.5ng/mL-100ng/mL to when stem-like cell lung carcinoma cell antigen-loaded dendritic cell.
The present invention also provides a kind of method for cancer for the treatment of, and it comprises to the patient uses the medicine that is selected from by in the stem-like cell lung carcinoma cell antigen composition of the inventive method preparation, the group that dendritic cell vaccine of the present invention is formed.
The preferred described mode of using is injection.Particularly, the mode of using stem-like cell lung carcinoma cell antigen composition is selected from by in injection in the tumor tissues, intravenous injection, subcutaneous injection and the intradermal injection one or more; The mode of using described dendritic cell vaccine is the subcutaneous or intradermal injection at the 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 stem-like cell lung carcinoma cell antigen composition is 1 * 10 from cell concn preferably 5To 1 * 10 8The stem-like cell lung carcinoma cell of the present invention of cell/mL, its significant quantity is the 0.1mL-5mL/ individuality; Described dendritic cell vaccine preferably includes 1 * 10 5To 5 * 10 7Individual/mL anticancer dendritic cell of the present invention, its significant quantity is the 0.1mL-2mL/ individuality.More preferably described stem-like cell lung carcinoma cell antigen composition is 1 * 10 from cell concn preferably 5To 1 * 10 8The stem-like cell lung carcinoma cell of the present invention of cell/rmL, its significant quantity is the 0.2mL-2mL/ individuality; Described dendritic cell vaccine preferably includes 1 * 10 5To 5 * 10 7Individual/mL anticancer dendritic cell of the present invention, its significant quantity is the 0.5mL-2mL/ individuality.
The above stem-like cell lung carcinoma cell antigen composition and described dendritic cell vaccine all are the dosage of a shot, four injections course for the treatment of, a week at interval.Stem-like cell lung carcinoma cell antigen composition and described dendritic cell vaccine generally the 3rd day after having injected first pin to onset in the 7th day.Described effective standard is: play the cytokine of immunological enhancement such as the rising of interleukin-22, interleukin 12, interferon-gamma and tumor necrosis factor alpha etc.; Play the reduction of immunosuppressant cytokine such as interleukin 6, interleukin-11 0, transforming growth factor-beta and epithelical cell growth factor etc.; And the t lymphocyte subset group changes i.e.: the rising of the positive rate of CD3, CD4 and CD8 or improvement, the rising of CD16/56 positive rate, and the reduction of CD4CD25FoxP3 three positive rates.After finish a course for the treatment of, can continue administration after three months at interval, can treat two or four courses for the treatment of; If necessary, be also can continue to treat in six months or 1 year pitch time again, as long as the patient is survived always.
Wherein, described effective standard can also be divided into following Three Estate:
1. blood samples of patients cytokines measurement:
Play the cytokine of immunological enhancement such as the rising of interleukin-22, interleukin 12, interferon-gamma and tumor necrosis factor alpha etc.; Play the reduction of immunosuppressant cytokine such as interleukin 6, interleukin-11 0, transforming growth factor-beta and epithelical cell growth factor etc.; And t lymphocyte subset group variation, that is: the rising of the positive rate of CD3, CD4 and CD8 or improvement, the rising of CD16/56 positive rate, and the reduction of CD4CD25FoxP3 three positive rates etc.
2. patient's lung cancer tumor marker detection as the reduction of the expression of lung cancer specific antigens such as alpha-fetoprotein, carcinomebryonic antigen, glycoprotein, mainly is to exempt from and chemiluminescence detection etc. by enzyme.
3, iconography detects, and detects as CT, nucleus magnetic resonance and B ultrasonic, can detect treatment front and back lung cancer tumor cell and dwindle.
Preferred described stem-like cell lung carcinoma cell and/or described dendritic cell come from same patient, like this side effect that can avoid immunological rejection to cause to greatest extent.The lung cancer tumor tissue that the operation that the present invention mentions is taken out in patient's body belongs to discarded stripped lung cancer tumor tissue, is not the lung cancer tumor tissue for realizing that the present invention specially takes out in patient's body.In addition, the cell mentioned of the present invention can be the commercial clone that supplies.And the technology of existing long-term frozen preservation cell is very ripe, such as freeze-stored cell under liquid nitrogen (subzero 196 ℃), for example cord blood cell preservation.Lung cancer tumor tissue, tumor cell line and dendritic cell that those skilled in the art can utilize above-mentioned technology preservation the present invention relates to fully, 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 differential protein detection of expression of preparation example 1 human lung carcinoma cell
Will be available from the human lung carcinoma cell (A549) of U.S. ATCC company, in 10mLRPMI1640 substratum (containing volume ratio 10% foetal calf serum and 1 * penicillin and Streptomycin sulphate mixed solution, Beijing Suo Laibao company), 75cm 2In the culturing bottle, place 37 ℃, 5%CO 2Cultivate in the incubator, when cell growth reaches 90% of culturing bottle bottom surface and converges, after 2mL pancreatin (Beijing Suo Laibao 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.
Get 100 part of 50 μ L (5 * 10 6Individual) above-mentioned cell suspension adds 500 μ L precooling instant total proteins and extracts lysate (RIPA, Beijing hundred Imtech) (contain volume ratio 1% proteinase inhibitor, Switzerland Roche company), hatch 20 minutes on ice after, per minute 13000 changes, and 4 ℃ centrifugal 20 minutes.Get supernatant, packing-80 ℃ preservation is standby.According to the operation of bicinchoninic acid (BCA) protein quantification test kit (Wuhan doctor's moral company) operation instruction, measure protein concentration.Adjust protein concentration with RIPA, the sample final concentration is 1.0 μ g/ μ L, adds 5 * protein sample damping fluid, and 95 ℃ following 5 minutes.
According to the molecular weight of target protein, prepare 10% separation gel, concentrated gum concentration is 5%.Protein sample applied sample amount to be detected: 20 μ g/ holes.Deposition condition: concentrate glue constant voltage 90V, about 20 minutes; Separation gel constant voltage 120V dyes albumen in advance by standard molecular weight and determines the electrophoresis stand-by time.Wet commentaries on classics method is changeed the film condition: the 300mA constant current; 0.45 μ m aperture pvdf membrane changes 80 minutes film time.The ponceau staining reagent dyeed to film after the commentaries on classics film was finished, and observed and changeed the film effect.The sealing: with film be immersed in fully 5% (mg/mL) bovine serum albumin (BSA) commentaries on classics film damping fluid (contain volume ratio 3% tween 20, TBST) in, horizontal shaking table was hatched 1 hour under the room temperature.
Primary antibodie is hatched: 5% (mg/mL) BSA-TBST dilution primary antibodie is (as following table 1, all available from U.S. Santa company; β Actin muscle, Beta actin are the confidential reference items contrasts), 4 ℃ of horizontal shaking table overnight incubation.
The film numbering The primary antibodie title The source Dilution ratio Dilution back volume
1 PKC?iota Rabbit 1∶200 4mL
2 PROM1 Mouse 1∶200 4mL
3 ALDH1 Rabbit 1∶200 4mL
4 ABCG2 Mouse 1∶200 4mL
5 Beta?actin Mouse 1∶1000 4mL
Next day, wash film: TBST washes 3 times, each 10 minutes.Two anti-hatching: 5% (mg/mL) BSA-TBST dilution two is anti-, goat anti-rabbit igg (H+L) horseradish peroxidase (HRP) and mountain sheep anti-mouse igg (H+L) HRP1: 10000, and incubated at room 40 minutes.Wash film: TBST washes film 3 times, each 10 minutes.Chemical luminous substrate ECL is added drop-wise to the protein powder of film, reacts 3-5 minute; Exposure: 10 seconds-5 minutes (time shutter is adjusted with the different light intensity degree), developed photographic fixing 2 minutes.
The film scanner scanning is the TIF format picture greater than 300DPI, use Quantity One analysis software (Bio-Rad company) to carry out gray analysis, the result shows that the mean value of PKC iota, PROM1, ALDH1 and ABCG2 and confidential reference items β-actin gray level ratio is respectively 0.35,0.46,0.47 and 0.59, and standard deviation is respectively 0.17,0.21,0.20 and 0.27.
Differential protein is expressed cut off value by the mean value+3 times standard deviation value of 100 parts of human lung carcinoma cell protein immunoblot technology for detection gained gray level ratios, be that PKC iota cut off value is 0.86, the PROM1 cut off value be 1.09, ALDH1 cut off value be 1.07 and the ABCG2 cut off value be 1.40.
The differential protein detection of expression of preparation example 2 normal people's alveolar epithelial cellss
People's alveolar epithelial cells (HPAEpiC) that will be available from the normal cell of U.S. ScienCell company, (contain 20% foetal calf serum and 1 * penicillin and Streptomycin sulphate mixed solution at 10mL Dole Becquerel improvement Iger (DMEM)/high glucose medium, Beijing Suo Laibao company) in, 75cm 2In the culturing bottle, place 37 ℃, 5%CO 2Cultivate in the incubator, when cell growth reaches 90% of culturing bottle bottom surface and converges, after 2mL pancreatin (Beijing Suo Laibao company) digestion, tongue is expected blue counting, with fresh DMEM/ high glucose medium (containing 20% foetal calf serum and 1 * penicillin and the Streptomycin sulphate mixed solution) cultivation of going down to posterity.
Differential protein expression level according to 100 parts of above-mentioned cells of protein immunoblot technology for detection in the preparation example 1, through gray analysis, the result shows that the mean value of PKC iota, PROM1, ALDH1 and ABCG2 and confidential reference items β-actin gray level ratio is respectively 0.00,0.53,0.61 and 0.35, all is lower than the cut off value that differential protein is expressed.
The differential protein detection of expression of preparation example 3 normal people's alveolar stem cells
People's lung mescenchymal stem cell (HPMSC) that will be available from the normal cell of U.S. ATCC company, (contain volume ratio 10% foetal calf serum and 1 * penicillin and Streptomycin sulphate mixed solution at 10mL Dole Becquerel improvement Iger (DMEM)/F12 (1: 1) substratum, Beijing Suo Laibao company) in, 75cm 2In the culturing bottle, place 37 ℃, 5%CO 2Cultivate in the incubator, when cell growth reaches 90% of culturing bottle bottom surface and converges, after 2mL pancreatin (Beijing Suo Laibao company) digestion, tongue is expected blue counting, with fresh DMEM/F12 (1: 1) substratum (containing volume ratio 10% foetal calf serum and 1 * penicillin and the Streptomycin sulphate mixed solution) cultivation of going down to posterity.
Differential protein expression level according to 100 parts of above-mentioned cells of protein immunoblot technology for detection in the preparation example 1, through gray analysis, the result shows that the mean value of PKC iota, PROM1, ALDH1 and ABCG2 and confidential reference items β-actin gray level ratio is respectively 0.00,0.98,1.01 and 0.24, all is lower than the expression cut off value.
The differential protein detection of expression of the tissue-derived stem-like cell lung carcinoma cell of preparation example 4 patients with lung cancer
Gather when cancerous lung tissue comes comfortable patient to undergo surgery, the patient is diagnosed as lung cancer through pathologic finding, and signs Informed Consent Form with the patient, patient information such as following table three.
Patient's sequence number Age Sex The pathology somatotype Whether sign Informed Consent Form
1 41 The man The non-small cell adenocarcinoma of lung Be
2 36 The man Low differentiation lung squamous cancer Be
3 52 The woman The non-small cell adenocarcinoma of lung Be
4 46 The man Small cell lung cancer Be
5 57 The woman The non-small cell adenocarcinoma of lung Be
6 43 The man The non-small cell adenocarcinoma of lung Be
7 36 The woman Low differentiation lung squamous cancer Be
8 27 The woman The non-small cell lung adenosquamous carcinoma Be
9 67 The woman Small cell lung cancer Be
10 70 The man The non-small cell adenocarcinoma of lung Be
With eye scissors the lung cancer tumor tissue being shredded becomes about 1mm 3Fritter.The fritter of organizing that gained shreds restrains with 1 with 0.25% pancreatin solution: the weightmeasurement ratio digestion of 0.5mL is after 5 minutes, the calf serum (U.S. Hyclone company) that adds pancreatin liquor capacity 1/5 stops, digest the good fritter of organizing with the centrifugal collection of 1000rpm, abandon supernatant, (Ah especially is for this enzyme with Accutase in gained centrifugation, U.S. Sigma company) with 1 gram: the volume ratio of 1mL was 37 ℃ of following mixing digestion 10 minutes, centrifugal 5min collecting cell under 1000rpm is resuspended with Dole's Becquerel improvement Iger-F12 substratum in order to the weightmeasurement ratio of 1 gram: 1mL then.The resuspended liquid of gained filters with 40 μ m molecular sieves (U.S. company BD).With the molecular sieve filtration in described aperture, what hold back is large stretch of cell debris and the cell mass that do not digested, and what leach is cell suspension.
Get 1 part of 50 μ L (2 * 10 6Individual) the above-mentioned cell suspension that obtains, according to the protein immunoblot technology, protein immunoblot technology in the preferred preparation example 1, detect 10 patients differential protein expression level of 10 parts of above-mentioned cells respectively, through gray analysis, the result shows that the mean value of PKC iota, PROM1, ALDH1 and ABCG2 and confidential reference items β-actin gray level ratio is respectively 0.00,0.80,0.91 and 1.27, and differential protein all is lower than the expression cut off value.
After collecting remaining described cell suspension, to wherein contain 5ng/mL B27,40ng/mL human epidermal growth factor (epidermal growth factor, EGF), Dole's Becquerel improvement Iger-F12 of 30ng/mL Prostatropin (bEGF) and 30mg/mL Regular Insulin (Dulbecco ' s modified Eagle ' s medium-F12) (DMEM/F12) substratum (U.S. Hyclone company) make concentration of cell suspension be diluted as 2 * 10 6Cell/mL.Cell suspension with every 5mL gained dilution is transferred to 25cm then 2In the sterilization plastic culture bottle at 37 ℃, 5%CO 2Cultivate 24h in the incubator.
Cultivate the gained cell and be into neural spherical suspension growth, draw substratum in the 15mL centrifuge tube, per minute 1000 changes, 4 ℃ of centrifugal 10 minutes collection suspension cells, abandon supernatant, sedimentation cell adds fresh Dole's Becquerel improvement Iger-F12 (DMEM/F12) substratum that contains 8ng/mL B27,30ng/mL human epidermal growth factor, 20ng/mL Prostatropin and 20mg/mL Regular Insulin.When the growth of cell ball reaches the 1 μ m left and right sides, with Accutase be digested to unicellular after, going down to posterity with Dole's Becquerel improvement Iger-F12 (DMEM/F12) substratum that contains 8ng/mLB27,30ng/mL human epidermal growth factor, 20ng/mL Prostatropin and 20mg/mL Regular Insulin, (1 bottle passes several bottles, and every bottle of initial cultured cells density is 2 * 10 in cultivations 6Cell/mL).Passed for 4 generations again according to the above-mentioned condition that goes down to posterity, obtain into neural spherical cell.
Get 1 part of 50 μ L (2 * 10 6Individual) the neural ball of above-mentioned one-tenth cultivates the stem-like cell lung carcinoma cell suspension that obtains, differential protein expression level according to 10 parts of above-mentioned cells of above-mentioned 10 patients difference of protein immunoblot technology for detection in the preparation example 1, through gray analysis, the result shows that the mean value of PKC iota, PROM1, ALDH1 and ABCG2 and confidential reference items β-actin gray level ratio is respectively 2.31,3.46,7.97 and 6.48, differential protein all is higher than the expression cut off value, shows to cultivate this 5 species diversity albumen of stem-like cell lung carcinoma cell specifically expressing that obtains.
Comparative Examples 1 utilizes the method for people's records such as Maria M.Ho to separate and preparation stem-like cell lung carcinoma cell and antigen composition thereof
1 * phosphoric acid buffer rinsing of patients with lung cancer tumor specimen usefulness 5mL 3 times, after using aseptic grinding rod mechanical process grinding in no mycetocyte plate, the DMEM substratum that adds 2mL (contains 0.1 collagenase activities Wu ¨ nsch units/mL, Switzerland Roche diagnostic companies) at 37 ℃, digestion is 4 hours in the wave and culture case.Digestive system is further removed agglomerate with the 18.5G needle tubing, and sieves with 100 μ m cells sieve, obtains single cell suspension.Single cell suspension is with 70% and 40% cellular segregation liquid Percoll gradient centrifugation, and per minute 2500 left the heart 20 minutes under the room temperature, was collected in the cell at 70%/40% interface.Cell is resuspended to 106/mL with DMEM substratum (American I nvitrogen Life Technologies, Inc.) (containing volume ratio 2% calf serum and 10mmol/LHEPES damping fluid), add final concentration fluorescence dye Hoechst33342 to final concentration 5 μ g/mL, under 37 ℃, hatched 90 minutes, and shook up once in 10 minutes at interval.Cell with cold HBSS damping fluid (containing volume ratio 2% calf serum and 10mmol/L HEPES damping fluid) washing once then, and 4 ℃ of following per minutes 1000 leave 5 minutes collecting cells of the heart, and resuspended with cold HBSS damping fluid (containing volume ratio 2% calf serum and 10mmol/L HEPES damping fluid).Cross 40 μ m cells sieve before the cell sorting and obtain cell suspension, use U.S. company BD flow cytometer FACSVantage SE sorting then, obtain the side group cell (SP) that Hoechst33342 dyes, i.e. stem-like cell lung carcinoma cell according to instrument working instructions operation sorting.
Get 1 part of 50 μ L (2 * 10 6Individual) the above-mentioned cell suspension that obtains, differential protein expression level according to 10 parts of above-mentioned cells of 10 patients' difference of protein immunoblot technology for detection in the preparation example 1, through gray analysis, the result shows that the mean value of PKC iota, PROM1, ALDH1 and ABCG2 and confidential reference items β-actin gray level ratio is respectively 0.05,0.98,0.61 and 2.27, express the cut off value except the ABCG2 gray level ratio is higher than, remaining differential protein all is lower than the expression cut off value.
The stem-like cell lung carcinoma cell of embodiment 1 immunomagnetic beads method enrichment specifically expressing differential protein
Using goes down to posterity in the preparation example 4 to cultivate obtains 10 8Individual cell, with the abundant mixing cell of 0.5mL Incubating Solution PBE (gas in the pH7.21 * PBS that contains 0.5%BSA and 0.08%EDTA, vacuum filtration degerming and liquid), add primary antibodie (the anti-people PKC of the rabbit iota antibody of 20 μ g/ml final concentrations, U.S. Santa company), hatched 30 minutes for 4 ℃.
Wash cell once with 20 times of volume PBE, after adding the abundant mixing cell of 0.3mL PBE again, the ultra micro magnetic bead (American I nvitrogen Life Technologies, Inc.) that adds 50 μ L, two anti-goat anti-rabbit iggs (H+L) bag quilt, mixing was hatched 15 minutes for rearmounted 8 ℃, add 5 times of volume PBE then and wash cell 3 times, use the abundant suspendible cell of 0.3mL PBE again.
Separator column is fit in the magnetic separator, adds 0.5ml PBE, under action of gravity, flow to end the pre-treatment separator column naturally.Add the PBE suspension of 0.3mL cell in the separator column, under action of gravity, flow to end PBE naturally.Separator column is taken off from magnetic separator, draw the washing fluid pressurization with special syringe and inject the PKC iota positive cell that adsorbs under the wash-out.
Use primary antibodie to be the antibody of mouse anti human PROM1, the anti-people ALDH1 of rabbit and mouse anti human ABCG2, and the ultra micro magnetic bead of the IgG (H+L) of two corresponding anti-mountain sheep anti mouses and goat antirabbit bag quilt, but continuous sorting goes out to express among PKC iota, PROM1, ALDH1 and the ABCG2 at least two kinds stem-like cell lung carcinoma cell.
The stem-like cell lung carcinoma cell of embodiment 2 selected by flow cytometry apoptosis method enrichment specifically expressing differential proteins
Using goes down to posterity in the preparation example 4 to cultivate obtains 10 8Individual cell, resuspended to 2 * 10 with 1 * PBS (pH7.2) (containing volume ratio 2% foetal calf serum) 6/ mL adds the anti-people PKC of the rabbit iota antibody of 20 μ g/mL final concentrations, hatches under 37 ℃ 90 minutes, and shakes up once in 10 minutes at interval.Cell with 1 cold * PBS (pH7.2) (containing volume ratio 2% foetal calf serum) washing once then, and 4 ℃ of following per minutes 1000 leave 5 minutes collecting cells of the heart, adding 5mL contains the 1 cold * PBS (pH7.2) of two anti-goat anti-rabbit iggs (H+L) of 50 μ LFITC marks, mixing was hatched 30 minutes for rearmounted 4 ℃, and with 1 cold * PBS (pH7.2) (containing volume ratio 2% calf serum) washing 3 times, be resuspended among 1 * PBS (pH7.2) (containing volume ratio 2% calf serum) of 5mL.Cell suspension uses U.S. company BD flow cytometer FACSVantage SE sorting then, obtains PKC iota positive cell according to instrument working instructions operation sorting.
Use primary antibodie to be the antibody of mouse anti human PROM1, the anti-people ALDH1 of rabbit and mouse anti human ABCG2, and the IgG (H+L) of two anti-mountain sheep anti mouses of corresponding FITC mark and goat antirabbit, use the continuous enrichment of U.S. company BD flow cytometer FACSVantage SE express among PKC iota, PROM1, ALDH1 and the ABCG2 at least two kinds the stem-like cell lung carcinoma cell.
Also can use paramagnetic particle method enrichment PKC iota positive cell earlier, use other differential protein positive cells of flow cytometer enrichment then, so also can obtain the stem-like cell lung carcinoma cell of the two or more differential proteins of enrichment specifically expressing.
The preparation of embodiment 3 stem-like cell lung carcinoma cell antigen compositions
Utilize method of the present invention to separate and preparation stem-like cell lung carcinoma cell and antigen composition thereof.
Get among the lung carcinoma cell of above-mentioned preparation example 1 and 4 gained and stem-like cell lung carcinoma cell and above-described embodiment 1-2 at least two kinds stem-like cell lung carcinoma cell among enrichment specifically expressing PKC iota, PROM1, ALDH1 and the ABCG2, selective enrichment PKC iota and PROM1 are two positive, PKC iota, PROM1 and ALDH1 three positives, the stem-like cell lung carcinoma cell of PKC iota, PROM1, ALDH1 and ABCG2 four positives; And get the stem-like cell lung carcinoma cell that Comparative Examples 1 obtains.
Multigelation is 5 times in liquid nitrogen and under the room temperature (25 ℃), detects the complete cracking of described cell by inverted microscope.The gained lysate is centrifugal 1min under 600rpm, collects supernatant with 0.45 μ m membrane filtration, collects filtrate.Described lysate is the antigen composition of stem-like cell lung carcinoma cell, is stored in-80 ℃ standby.
Embodiment 4 dendritic cell are obtained
Get the 100mL peripheral blood through Ficoll-Hypaque density gradient centrifugation, obtain mononuclearcell.Peripheral blood mononuclear cell is resuspended with the RPMI1640 substratum, and adds in 6 orifice plates adherent.Hatch 90min in 37 ℃, 5%CO2 incubator after, the attached cell washing is not collected, for inducing the CIK cell.Attached cell adds perfect medium RPMI1640 inducing culture, contains 5% huge G CFS and the 1500IU/mL IL-4 of biting of autoserum, 2000IU/mL recombinant humangranulocyte.Cell is changed fresh culture (containing 2000IU/mL GM-CSF and 1500IU/mL IL-4) when cultivating by the 3rd day.Cultivation to the five days results immature dendritic cell (DCs), mature DCs adopt complete RPMI1640 substratum to obtain being induced to the 7th day, contain 0.1 μ g/mL lipopolysaccharides and 20ng/mL tumor necrosis factor alpha.In the time of the 5th day, add special antigen (as PKC iota, PROM1, ALDH1 and ABCG2 four positive stem-like cell lung carcinoma cell antigens), load DCs.
Embodiment 5 lung carcinoma cell antigen load DCs
Cultivate after 5 days 3 * 10 6Individual immature DC s (derives from 1 * 10 with preparation example 1 and 4, embodiment 1-2 and Comparative Examples 1 described stem-like cell lung carcinoma cell cracking antigen composition 6The stem-like cell lung carcinoma cell) at 37 ℃, 5%CO 2Load is 4 hours in the incubator.The DCs of antigen load by centrifugal (10min, 1000rpm) results obtain.Gained DCs is through physiological saline washing 3 times, after be resuspended in the physiological saline, to concentration be 3 * 10 6Ripe DC/mL, and to add final concentration be the human serum albumin of mass volume ratio 2%, thus be prepared into the DC vaccine of stem-like cell lung carcinoma cell cracking antigen composition load.
Embodiment 1 is or/and 2 PKC iota, the PROM1 that prepare, ALDH1 and ABCG2 four positive stem-like cell lung carcinoma cell antigen-loaded dendritic cells dye by CD86-PE, CD80-PE, CD40-FITC, CD83-PE, CD11c-FITC and HLA-DR-PerCP (BD company), and the DC phenotype changed after flow cytometer detected load.Fig. 1 shows that the DC phenotype of preparation is that CD11c+/HLA-DR+ is 99.0%, CD11c+/CD83+ be 98.5%, CD86+/HLA-DR+ be 99.6%, CD80+/HLA-DR+ be 99.1% and CD40+/HLA-DR+ be 99.7%, meet DC cell-specific phenotype, and the high expression level costimulatory molecules.
Effect illustrative examples 1: the external knurl of killing of the DC vaccine of the lung carcinoma cell antigen load of preparation example 1 and 4, embodiment 1-2 and Comparative Examples 1 gained is tested
In 6 orifice plates, be substratum with the RPMI1640 that contains 5% calf serum still, by the mature dendritic cell of above-mentioned antigen load: mature dendritic cell and the T cell of the above-mentioned antigen load of mixed of T cell=1: 5, add GM-CSF (final concentration is 800U/mL) and IL-4 (final concentration is 20ng/mL) simultaneously, cultivated altogether 7 days, during this time every 1 day all half amount change liquid (contain 5% calf serum, final concentration is the GM-CSF of 800U/mL and the RPMI1640 substratum of the IL-4 that final concentration is 20ng/mL), obtain the CTL cell.
Select corresponding lung carcinoma cell as target cell, use 51The Cr mark, namely target cell (2 * 10 6/ mL) by with 300 μ Ci 51Cr in the RPMI1640 substratum 37 ℃ hatched 2 hours.The target cell that mark is good is washed three times with 1 * PBS, and finally uses 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.
With 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 the corresponding hole, hatched 4 hours at 37 ℃.After hatching, get the γ radiometer rolling counters forward of supernatant 75 μ L components.Special 51The per-cent that Cr discharges calculates according to following formula.
Figure BSA00000893448800181
Wherein, the per minute umber of pulse of spontaneous release obtains by single culture target cell (not adding the effector cell), and the maximum per minute umber of pulse that discharges is to obtain behind the single culture target cell of using final concentration 2%NP-40 (tensio-active agent, the worker is given birth in Shanghai) to handle.The per minute umber of pulse that detects obtains by the target cell cultivation of adding the effector cell.
Fig. 2 has shown by embodiment 1 or/and 2 gained PKC iota and PROM1 are two positive, PKC iota, PROM1 and ALDH1 three positives, the stem-like cell lung carcinoma cell antigen composition loaded dendritic cell of PKC iota, PROM1, ALDH1 and ABCG2 four positives, obtain cytotoxic T lymphocyte (CTL), by the response result to PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1 of this CTL cell generation.As shown in Figure 2, improve constantly along with imitating the target ratio, the CTL that the stem-like cell lung carcinoma cell is produced replys also special rising, and good by the embodiment effect of four kinds of positive differential proteins of enrichment.Wherein, PP-DC1 is expressed as the dendritic cell initiation of PKC iota and the two positive stem-like cell lung carcinoma cell antigen loads of PROM1 to the toxicity activity of PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1; PPA-DC2 is expressed as the dendritic cell initiation of PKC iota, PROM1 and ALDH1 three positive stem-like cell lung carcinoma cell antigen loads to the toxicity activity of PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1; PPAA-DC3 is expressed as the dendritic cell initiation of PKC iota, PROM1, ALDH1 and ABCG2 four positive stem-like cell lung carcinoma cell antigen loads to the toxicity activity of PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1.The two positive stem-like cell lung carcinoma cells of described PKC iota and PROM1 according to embodiment 1 or/and 2 preparations.
Fig. 3 has shown by embodiment 1 or/and the two positive stem-like cell lung carcinoma cell antigen composition loaded dendritic cells (PP-DC1) of 2 gained PKC iota and PROM1, and by preparation example 1,4 and three kinds of antigen compositions of Comparative Examples 1 gained loaded dendritic cell respectively, obtain four kinds of cytotoxic T lymphocytes (CTL), the response result comparison diagram that respectively the two positive stem-like cell lung carcinoma cells of PKC iota and PROM1 is produced by these four kinds of CTL cells.Wherein, PP-DC1 is expressed as the dendritic cell initiation of PKC iota and the two positive stem-like cell lung carcinoma cell antigen loads of PROM1 to the toxicity activity of PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1; The dendritic cell of preparation example 1 lung cell A549 antigen load of representing A549-DC4 causes the toxicity activity to PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1, LSC-DC5 represents dendritic cell that the lung cancer of preparation example 4 becomes the neurosphere cell antigen load to the toxicity activity of PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1, and DLSC-DC6 represents that the dendritic cell of stem-like cell lung carcinoma cell antigen load of Comparative Examples 1 is to the toxicity activity of PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1.
As shown in Figure 3 by embodiment 1 or/and the two positive stem-like cell lung carcinoma cell antigen composition loaded dendritic cells (PP-DC1) of 2 gained PKC iota and PROM1 cause cellular cytoxicity activity to PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1 will be higher than A549-DC4, LSC-DC5 and DLSC-DC6 respectively and cause cellular cytoxicity activity to PKC iota and the two positive stem-like cell lung carcinoma cells of PROM1.The two positive stem-like cell lung carcinoma cells of described PKC iota and PROM1 are according to embodiment 1 or 2 preparations.
Fig. 4 has shown by embodiment 1 or/and the two positive stem-like cell lung carcinoma cell antigen composition loaded dendritic cells of 2 gained PKC iota and PROM1, and by preparation example 1,4 and three kinds of antigen compositions of Comparative Examples 1 gained loaded dendritic cell respectively, obtain four kinds of cytotoxic T lymphocytes (CTL), the response result comparison diagram that the stem-like cell lung carcinoma cell that respectively Comparative Examples 1 is obtained by these four kinds of CTL cells produces.Wherein, PP-DC1 ' is expressed as the dendritic cell initiation of PKC iota and the two positive stem-like cell lung carcinoma cell antigen loads of PROM1 to the toxicity activity of the stem-like cell lung carcinoma cell of Comparative Examples 1 acquisition; The dendritic cell of A549-DC4 ' expression lung cell A549 antigen load causes the toxicity activity of the stem-like cell lung carcinoma cell that Comparative Examples 1 is obtained, the lung cancer of LSC-DC5 ' expression preparation example 4 becomes the dendritic cell of neurosphere cell antigen load to the toxicity activity of the stem-like cell lung carcinoma cell of Comparative Examples 1 acquisition, and the dendritic cell of the stem-like cell lung carcinoma cell antigen load of DLSC-DC6 ' expression Comparative Examples 1 is to the toxicity activity of the stem-like cell lung carcinoma cell of Comparative Examples 1 acquisition.
Or/and causing the cellular cytoxicity activity of the stem-like cell lung carcinoma cell that Comparative Examples 1 is obtained, the two positive stem-like cell lung carcinoma cell antigen composition loaded dendritic cells (PP-DC1) of 2 gained PKC iota and PROM1 to be higher than the cellular cytoxicity activity that A549-DC4, LSC-DC5 and DLSC-DC6 cause the stem-like cell lung carcinoma cell that Comparative Examples 1 is obtained by embodiment 1 respectively as shown in Figure 4.
Effect illustrative examples 2: the lung carcinoma cell of preparation example 1 and 4, embodiment 1-2 and Comparative Examples 1 gained kills the effect in the knurl experiment in vivo
Be substratum with the RPMI1640 that contains 5% calf serum, with 3 * 10 6Individual immature dendritic cell (derives from 1 * 10 with the lung carcinoma cell cracking antigen composition of above-mentioned preparation example 1 and 4, embodiment 1-2 and Comparative Examples 1 gained 6The stem-like cell lung carcinoma cell) at 37 ℃, 5%CO 2Load is 4 hours in the incubator.The mature dendritic cell of antigen load obtains by the centrifugal 10min of 1000rpm.The mature dendritic cell of described antigen load is washed 3 times with physiological saline, resuspended to 1 * 10 then 6The concentration of individual/mL is preserved standby down for 4 ℃.
The stem-like cell lung carcinoma cell is set up lotus lung cancer tumor mouse model (referring to " growth of human colon cancer cell energy induced tumor in immunodeficient mouse ", people such as O ' Brien CA, nature, the 445th volume, 106-110 page or leaf, on February 3rd, 2008; O ' Brien CA, Pollett A, Gallinger S, et al.A human colon cancer cell capable of initiating tumour growth in immunodeficient mice.Nature, 2007,445 (7123): 106-110.), be divided into A, B, C, D, E, F and G7 group at random, 6 every group: the A group is the dendritic cell treatment group of the stem-like cell lung carcinoma cell antigen load of enrichment PKC iota and the two positives of PROM1; The B group is the dendritic cell treatment group of the stem-like cell lung carcinoma cell antigen load of PKC iota, PROM1 and ALDH1 three positives; The C group is the dendritic cell treatment group of the stem-like cell lung carcinoma cell antigen load of PKC iota, PROM1, ALDH1 and ABCG2 four positives; The D group is the dendritic cell comparative group (injection preparation is identical with the A group) of the lung cell A549 antigen load of preparation example 1 preparation; The E group is the dendritic cell comparative group (injection preparation is identical with the A group) of the stem-like cell lung carcinoma cell antigen load of preparation example 4 preparations; F group for Comparative Examples 1 obtain the dendritic cell of stem-like cell lung carcinoma cell antigen load be comparative group (injection preparation is organized identical with A); The G group is for organizing with volume physiological saline is blank.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/the mL dendritic cell, each 0.5mL, same dosage immunity 1 time again behind the two weeks.Injection back respectively at 7,14,21,28,35d calculates mouse model lotus knurl size, G organizes in identical time point at subcutaneous injection 0.5mL physiological saline and calculate mouse model lotus knurl size.
Through the dendritic cell vaccine of the stem-like cell lung carcinoma cell antigen load inhibition growing state (mm to the mice with tumor model 3,
Figure BSA00000893448800211
) see Table 3.
Table 3
Figure BSA00000893448800212
Annotate: the blank group of * is compared P much smaller than 0.01 with the treatment group; #Comparative group is compared P＜0.01 with the treatment group.
In addition, relevant embodiment 1 is or/and kill tumor activity in the body that the above stem-like cell lung carcinoma cell load DC of 2 enrichment gained, two species diversity protein positives causes, be better than far away relevant preparation example 1,4 and the body of the lung carcinoma cell of Comparative Examples 1 in kill tumor activity.In addition about embodiment 5 or/and kill tumor activity in the body of the stem-like cell lung carcinoma cell of 6 gained PKC iota, PROM1, ALDH1 and ABCG2 four positives, be better than relevant embodiment 1 or/and kill tumor activity in the body of the stem-like cell lung carcinoma cell of 2 gained, two species diversity protein positives and three species diversity protein positives.

Claims (10)

1. a method for preparing the stem-like cell lung carcinoma cell is characterized in that, described method comprises identifies the step that whether has the stem-like cell lung carcinoma cell in the lung carcinoma cell sample to be measured:
Described step comprises at least two kinds that detect in the described lung carcinoma cell sample to be measured the following differential protein of specifically expressing whether: protein kinase C iota, stride film strand glycoprotein Prominin1, acetaldehyde dehydrogenase 1 or mammary cancer drug-resistant protein ATP binding transport Protein G superfamily member 2 for five times.
2. method according to claim 1, wherein, described specifically expressing differential protein refers to use the result of the described differential protein in the described lung carcinoma cell sample of protein immunoblot technology for detection positive;
Wherein, with the negative contrast of known pure lung carcinoma cell, with 100 parts of described negative controls of protein immunoblot technology for detection, every part contains 5 * 10 6Individual cell records the gray scale of every species diversity albumen and the gray level ratio of standard confidential reference items β Actin muscle, and mean value and the standard variance sum of this species diversity albumen gray level ratio of survey is defined as cut off value;
Get 100 parts of lung carcinoma cell samples to be measured, every part of sample contains 5 * 10 6Individual cell records the mean value of surveying differential protein gray level ratio of the same race with negative control, and the mean value of this gray level ratio is more than or equal to described cut off value, and namely the protein immunoblot technology for detection result of this species diversity albumen of this lung carcinoma cell sample to be measured is positive.
3. method according to claim 1 and 2, when having the stem-like cell lung carcinoma cell in identifying described lung carcinoma cell sample to be measured, this method also comprises:
Use the step of the stem-like cell lung carcinoma cell in immunomagnetic beads method and/or the described cell to be measured of selected by flow cytometry apoptosis method enrichment;
Wherein, utilize at the protein kinase C iota that obtains specifically expressing, stride at least two kinds antibody of above differential protein in film strand glycoprotein Prominin1, acetaldehyde dehydrogenase 1 or the mammary cancer drug-resistant protein ATP binding transport Protein G superfamily member 2 for five times.
4. the preparation method of a stem-like cell lung carcinoma cell antigen composition is characterized in that, this method may further comprise the steps:
1) prepare the stem-like cell lung carcinoma cell according to any described method among the claim 1-3,
2) with physiological saline washing and resuspended described stem-like cell lung carcinoma cell,
3) the described stem-like cell lung carcinoma cell of cracking;
Wherein, described step 3) comprises by heat-shocked and/or multigelation and comes cracking stem-like cell lung carcinoma cell; The condition of described heat-shocked cracking is included in 37～45 ℃ and keeps 1h-6h down; The number of times of described multigelation is that be 10min-2h 3-5 time pitch time, and described freezing temperature range is subzero 120 ℃-subzero 196 ℃, and the temperature range of described thawing is 20 ℃-37 ℃.
5. the preparation method of the dendritic cell of a stem-like cell lung carcinoma cell antigen load, it is characterized in that, this method comprises: obtain stem-like cell lung carcinoma cell antigen composition by cracking stem-like cell lung carcinoma cell, normal dendritic cell is contacted the anti-lung cancer dendritic cell that obtains the load tumour antigen with stem-like cell lung carcinoma cell antigen composition, it is characterized in that, described method comprises that any described method prepares the stem-like cell lung carcinoma cell among the right to use requirement 1-3, and perhaps any described method prepares stem-like cell lung carcinoma cell antigen composition in the right to use requirement 4.
6. the dendritic cell of a stem-like cell lung carcinoma cell antigen load that is obtained by the described method of claim 5.
7. dendritic cell vaccine, described dendritic cell vaccine comprises dendritic cell, it is characterized in that, described dendritic cell is the dendritic cell of the described stem-like cell lung carcinoma cell of claim 6 antigen load.
8. test kit for preparing the dendritic cell vaccine of stem-like cell lung carcinoma cell antigen load is characterized in that described test kit comprises:
1) stem-like cell lung carcinoma cell basic medium;
2) protein kinase C iota, stride film strand glycoprotein Prominin1, acetaldehyde dehydrogenase 1 or mammary cancer drug-resistant protein ATP binding transport Protein G superfamily member 2 at least two kinds for five times;
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 described method of claim 5.
9. test kit according to claim 8, wherein, described test kit also comprises at described protein kinase C iota, strides at least two kinds antibody of film strand glycoprotein Prominin1, acetaldehyde dehydrogenase 1 or mammary cancer drug-resistant protein ATP binding transport Protein G superfamily member 2 for five times.
10. the preparation method of the DC vaccine of stem-like cell lung carcinoma cell cracking antigen composition load is characterized in that, with eye scissors the lung cancer tumor tissue being shredded becomes about 1mm 3Fritter; The fritter of organizing that gained shreds restrains with 1 with 0.25% pancreatin solution: the weightmeasurement ratio digestion of 0.5mL is after 5 minutes, the calf serum that adds pancreatin liquor capacity 1/5 stops, digest the good fritter of organizing with the centrifugal collection of 1000rpm, abandon supernatant, gained centrifugation restrains with 1 for this enzyme with Ah especially: the volume ratio of 1mL was 37 ℃ of following mixings digestion 10 minutes, centrifugal 5min collecting cell under 1000rpm is resuspended with Dole's Becquerel improvement Iger-F12 substratum in order to the weightmeasurement ratio of 1 gram: 1mL then; The resuspended liquid of gained is with 40 μ m molecular sieve filtrations; With the molecular sieve filtration in described aperture, what hold back is large stretch of cell debris and the cell mass that do not digested, and what leach is cell suspension;
Collect described cell suspension, and make concentration of cell suspension be diluted as 2 * 10 to the Dole's Becquerel improvement Iger-F12 substratum that wherein contains 5ng/mL B27,40ng/mL human epidermal growth factor, 30ng/mL Prostatropin and 30mg/mL Regular Insulin 6Cell/mL; Cell suspension with every 5mL gained dilution is transferred to 25cm then 2In the sterilization plastic culture bottle at 37 ℃, 5%CO 2Cultivate 24h in the incubator;
Cultivate the gained cell and be into neural spherical suspension growth, draw substratum in the 15mL centrifuge tube, per minute 1000 changes, 4 ℃ of centrifugal 10 minutes collection suspension cells, abandon supernatant, sedimentation cell adds the fresh Dole's Becquerel improvement Iger-F12 substratum that contains 8ng/mL B27,30ng/mL human epidermal growth factor, 20ng/mL Prostatropin and 20mg/mL Regular Insulin; When the growth of cell ball reaches the 1 μ m left and right sides, with Accutase be digested to unicellular after, with the Dole's Becquerel improvement Iger-F12 substratum that contains 8ng/mL B27,30ng/mL human epidermal growth factor, 20ng/mL Prostatropin and the 20mg/mL Regular Insulin cultivation of going down to posterity; Passed for 4 generations again according to the above-mentioned condition that goes down to posterity, obtain into neural spherical cell;
Use into neural ball cultivation and obtain 10 8The stem-like cell lung carcinoma cell of individual cell with the abundant mixing cell of 0.5mL Incubating Solution PBE, adds primary antibodie, hatches 30 minutes for 4 ℃; Wherein, described Incubating Solution PBE is the pH7.21 * PBS that contains 0.5%BSA and 0.08%EDTA, gas in vacuum filtration degerming and the liquid; Described primary antibodie is the anti-people PKC of the rabbit iota antibody of 20 μ g/ml final concentrations;
Wash cell once with 20 times of volume PBE, after adding the abundant mixing cell of 0.3mL PBE again, add the ultra micro magnetic bead of 50 μ L, two anti-goat anti-rabbit iggs (H+L) bag quilt, mixing was hatched 15 minutes for rearmounted 8 ℃, add 5 times of volume PBE then and wash cell 3 times, use the abundant suspendible cell of 0.3mL PBE again;
Separator column is fit in the magnetic separator, adds 0.5ml PBE, under action of gravity, flow to end the pre-treatment separator column naturally; Add the PBE suspension of 0.3mL cell in the separator column, under action of gravity, flow to end PBE naturally; Separator column is taken off from magnetic separator, draw the washing fluid pressurization with syringe and inject the PKC iota positive cell that adsorbs under the wash-out;
Use primary antibodie to be the antibody of mouse anti human PROM1, the anti-people ALDH1 of rabbit and mouse anti human ABCG2, and the ultra micro magnetic bead of the IgG (H+L) of two corresponding anti-mountain sheep anti mouses and goat antirabbit bag quilt, but continuous sorting goes out to express among PKC iota, PROM1, ALDH1 and the ABCG2 at least two kinds stem-like cell lung carcinoma cell;
Use above-mentioned going down to posterity to cultivate acquisition 10 8Individual cell, resuspended to 2 * 10 with 1 * PBS 6/ mL adds the anti-people PKC of the rabbit iota antibody of 20 μ g/mL final concentrations, hatches under 37 ℃ 90 minutes, and shakes up once in 10 minutes at interval; Cell with cold 1 * PBS washing once then, and 4 ℃ of following per minutes 1000 leave 5 minutes collecting cells of the heart, adding 5mL contains the 1 cold * PBS of two anti-goat anti-rabbit iggs (H+L) of 50 μ L FITC marks, mixing was hatched 30 minutes for rearmounted 4 ℃, and with cold 1 * PBS washing 3 times, be resuspended among 1 * PBS of 5mL; Cell suspension uses flow cytometer FACSVantage SE sorting then, obtains PKC iota positive cell according to instrument working instructions operation sorting;
Use primary antibodie to be the antibody of mouse anti human PROM1, the anti-people ALDH1 of rabbit and mouse anti human ABCG2, and the IgG (H+L) of two anti-mountain sheep anti mouses of corresponding FITC mark and goat antirabbit, use the continuous enrichment of flow cytometer FACSVantage SE express among PKC iota, PROM1, ALDH1 and the ABCG2 at least two kinds the stem-like cell lung carcinoma cell;
Or use paramagnetic particle method enrichment PKC iota positive cell earlier, and use other differential protein positive cells of flow cytometer enrichment then, so also can obtain the stem-like cell lung carcinoma cell of the two or more differential proteins of enrichment specifically expressing;
Get among above-mentioned gained enrichment specifically expressing PKC iota, PROM1, ALDH1 and the ABCG2 at least two kinds stem-like cell lung carcinoma cell, selective enrichment PKC iota and PROM1 are two positive, PKC iota, PROM1 and ALDH1 three positives, the stem-like cell lung carcinoma cell of PKC iota, PROM1, ALDH1 and ABCG2 four positives; In liquid nitrogen and 25 ℃ of following multigelations of room temperature 5 times, detect the complete cracking of described cell by inverted microscope; The gained lysate is centrifugal 1min under 600rpm, collects supernatant with 0.45 μ m membrane filtration, collects filtrate; Described lysate is the antigen composition of stem-like cell lung carcinoma cell, is stored in-80 ℃ standby;
Get the 100mL peripheral blood through Ficoll-Hypaque density gradient centrifugation, obtain mononuclearcell; Peripheral blood mononuclear cell is resuspended with the RPMI1640 substratum, and adds in 6 orifice plates adherent; At 37 ℃, 5%CO 2After hatching 90min in the incubator, the attached cell washing is not collected, and is used for inducing the CIK cell; Attached cell adds perfect medium RPMI1640 inducing culture, contains 5% huge G CFS and the 1500IU/mL IL-4 of biting of autoserum, 2000IU/mL recombinant humangranulocyte; Cell is changed fresh culture when cultivating by the 3rd day; Cultivation to the five days results immature dendritic cell DCs, mature DCs adopts complete RPMI1640 substratum to obtain being induced to the 7th day, contains 0.1 μ g/mL lipopolysaccharides and 20ng/mL tumor necrosis factor alpha; In the time of the 5th day, add special antigen, load DCs; Wherein, described fresh culture contains 2000IU/mL GM-CSF and 1500IU/mL IL-4; Special antigen is PKC iota, PROM1, ALDH1 and ABCG2 four positive stem-like cell lung carcinoma cell antigens;
Cultivate after 5 days 3 * 10 6Individual immature DC s with the stem-like cell lung carcinoma cell cracking antigen composition of above-mentioned preparation at 37 ℃, 5%CO 2Load is 4 hours in the incubator; The DCs of antigen load obtains by centrifugal results; Gained DCs is through physiological saline washing 3 times, after be resuspended in the physiological saline, to concentration be 3 * 10 6Ripe DC/mL, and to add final concentration be the human serum albumin of mass volume ratio 2%, thus be prepared into the DC vaccine of stem-like cell lung carcinoma cell cracking antigen composition load.
CN201310174082.7A 2013-05-13 2013-05-13 Methods and kit for preparing stem cell-like lung cancer cells and loading dendritic cells on antigen composition thereof Active CN103289957B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201310174082.7A CN103289957B (en) 2013-05-13 2013-05-13 Methods and kit for preparing stem cell-like lung cancer cells and loading dendritic cells on antigen composition thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201310174082.7A CN103289957B (en) 2013-05-13 2013-05-13 Methods and kit for preparing stem cell-like lung cancer cells and loading dendritic cells on antigen composition thereof

Publications (2)

Publication Number Publication Date
CN103289957A true CN103289957A (en) 2013-09-11
CN103289957B CN103289957B (en) 2017-03-22

Family

ID=49091494

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201310174082.7A Active CN103289957B (en) 2013-05-13 2013-05-13 Methods and kit for preparing stem cell-like lung cancer cells and loading dendritic cells on antigen composition thereof

Country Status (1)

Country Link
CN (1) CN103289957B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104087556A (en) * 2014-06-30 2014-10-08 北京弘润源生物技术有限公司 Preparation method of breast cancer stem cells as well as antigen composition thereof and anti-tumor application of antigen composition
CN104324368A (en) * 2014-09-26 2015-02-04 广州复大医疗股份有限公司复大肿瘤医院 Lung cancer stem cell vaccine, preparation method and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101538554A (en) * 2009-04-30 2009-09-23 北京弘润天源生物技术有限公司 Survivability-screened cancer stem cells, preparation method, application and kit as well as antigen composite thereof, preparation method, application and kit
CN101560496A (en) * 2009-04-30 2009-10-21 北京弘润天源生物技术有限公司 Dendritic cell carried by tumor stem cell antigen and subjected to tolerance screening, preparation method and application thereof, kit and vaccine comprising dendritic cell

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101538554A (en) * 2009-04-30 2009-09-23 北京弘润天源生物技术有限公司 Survivability-screened cancer stem cells, preparation method, application and kit as well as antigen composite thereof, preparation method, application and kit
CN101560496A (en) * 2009-04-30 2009-10-21 北京弘润天源生物技术有限公司 Dendritic cell carried by tumor stem cell antigen and subjected to tolerance screening, preparation method and application thereof, kit and vaccine comprising dendritic cell

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
RODERICK P.REGALA，ET AL: "Atypical Protein Kinase Ci Is Required for Bronchioalveolar Stem Cell Expansion and Lung Tumorigenesis", 《CANCER RESEARCH》 *
刘文文 等: "肺癌干细胞标志物的研究进展", 《吉林医学》 *
闫秀萍: "肺癌干细胞的分选及鉴定实验研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104087556A (en) * 2014-06-30 2014-10-08 北京弘润源生物技术有限公司 Preparation method of breast cancer stem cells as well as antigen composition thereof and anti-tumor application of antigen composition
CN104324368A (en) * 2014-09-26 2015-02-04 广州复大医疗股份有限公司复大肿瘤医院 Lung cancer stem cell vaccine, preparation method and application thereof

Also Published As

Publication number Publication date
CN103289957B (en) 2017-03-22

Similar Documents

Publication Publication Date Title
CN103597072B (en) Mononuclear cell proliferation agent, mononuclear cell proliferation culture medium, the manufacturing method of monocyte, the manufacturing method of the manufacturing method of Dendritic Cells and dendritic cell vaccine
CN105087487B (en) A kind of method of efficient amplification CIK
CN103627673B (en) A kind of human glioma cell line and its method for building up and application
CN102876631B (en) Method for separating immune cells from blood and application of method to disease treatment
CN104845934B (en) Bleeding of the umbilicus CD34+Derived from hematopoietic precursor cells Dendritic Cells is prepared on a large scale method
CN101538554A (en) Survivability-screened cancer stem cells, preparation method, application and kit as well as antigen composite thereof, preparation method, application and kit
CN104711221A (en) Method for automatically separating immune cells and extracting PRP from adult peripheral blood
CN104694473B (en) The method that immunocyte is extracted in automation from adult peripheral blood
CN103301449B (en) A kind of preparation method of large-scale culture dendritic cell vaccine and application thereof
CN102643784A (en) Expansion system in vitro for hematopoietic stem/progenitor cell
CN103784950A (en) Preparation method of breast cancer-specific epitope polypeptide-loaded dendritic cell vaccine and kit thereof
CN109456940A (en) The method for obtaining NK cell or dendritic cells
CN106554942A (en) A kind of efficient clinical grade CD56+The preparation method of group's immunocyte
CN101560496A (en) Dendritic cell carried by tumor stem cell antigen and subjected to tolerance screening, preparation method and application thereof, kit and vaccine comprising dendritic cell
CN105296427A (en) In-vitro multiplication culture method of hematopoietic stem cells
CN106434552A (en) Novel NKT-like cell subpopulation and application thereof in tumor treatment
CN107109366A (en) Tolerogenic dendritic cells, its generation method and application thereof
CN103800897A (en) Preparation method and kit for dendritic cell vaccine loaded by tumor specific antigenic epitope polypeptide
CN106047798A (en) Aminotic cell culture medium for high-density cell culture system
CN102154208A (en) Preparation method and use of cord blood-derived (CD)133 and brain glioma stem cell antigen carrying dendritic cells
CN104745530A (en) Human hepatocellular carcinoma cell line, and establishing method and application thereof
CN103289957A (en) Methods and kit for preparing stem cell-like lung cancer cells and loading dendritic cells on antigen composition thereof
CN107641617B (en) System for efficiently preparing megakaryocytes and platelets of non-human primates in vitro and application of system
CN103509101A (en) Cell factor for amplifying umbilical cord blood hematopoietic stem cells and culture medium thereof
CN105233280A (en) DC-based glioma holoantigen vaccine and preparation method thereof

Legal Events

Date Code Title Description
C06 Publication
DD01 Delivery of document by public notice

Addressee: Beijing Xipusheng International Biotechnology Co., Ltd.

Document name: Notification of Passing Preliminary Examination of the Application for Invention

PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
DD01 Delivery of document by public notice

Addressee: Li Yimin

Document name: Notification of Publication of the Application for Invention

C14 Grant of patent or utility model
GR01 Patent grant
TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20170802

Address after: 330096, Jiangxi Nanchang Nanchang hi tech Industrial Development Zone, AI Sihu North Road No. 3 workshop No. 688, west section of the 2

Patentee after: Jiangxi Austria Biological Technology Co. Ltd.

Address before: 100079 Beijing city Fengtai District East 249 Building No. 2 room 103

Patentee before: Beijing Xipusheng International Biotechnology Co., Ltd.