CN111118099A - Method for screening leukemia high-flux drugs - Google Patents
Method for screening leukemia high-flux drugs Download PDFInfo
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- CN111118099A CN111118099A CN201811279792.5A CN201811279792A CN111118099A CN 111118099 A CN111118099 A CN 111118099A CN 201811279792 A CN201811279792 A CN 201811279792A CN 111118099 A CN111118099 A CN 111118099A
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- cells
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
Abstract
The invention provides a method for screening leukemia high-flux drugs, which comprises the following steps: separating, extracting, identifying and culturing the blood tumor stem cells: collecting a bone marrow sample; carrying out the separation of the mononuclear cells; separating leukemia stem cell population by immunomagnetic bead method; carrying out subculture on the cells after the magnetic beads are separated and purified; establishing a drug library: according to the pharmaceutical specification and pharmacokinetics, the concentration of the drug is selected, the drug is mixed with the blood tumor stem cells for culture, and the inhibition effect of the drug on the blood tumor stem cells is analyzed. The high-throughput drug screening method can provide proper treatment scheme suggestions for patients in a personalized and accurate manner, strive for precious treatment opportunity for patients who relapse refractory, reduce the risk of reagent, increase the curative effect of the drug to the greatest extent, reduce the side effect of the drug, can be used as a drug combination, and can be used for making a comprehensive score for a clinical common chemotherapeutic drug combination, thereby providing a reference for doctors in the aspect of making clinical combined drug for the patients.
Description
Technical Field
The invention relates to a method for screening leukemia high-flux drugs.
Background
John Dick isolated and purified CD34 in Acute Myeloid Leukemia (AML) patients+/CD38-Phenotypic AML cells, which can be used to generate human-like leukemia by inoculating NOD/SCID mice, CD34-/CD38+Phenotypic leukemia cells are not prone to tumor formation, so CD34 is presumed+/CD38-The phenotypic leukemia cells are AML stem cells. After the 21 st century, cancer stem cells were isolated and validated in solid tumors as well (breast and brain tumors at the earliest), completely announcing the arrival of the cancer stem cell era. Tumor stem cells refer to tumor cells with stem cell characteristics, and have the capacity of self-renewal and multi-differentiation, and generally, such cells are considered to have the potential of forming tumors and developing cancers, particularly, the source of new cancers generated after the cancer is transferred out, and the cells are seeds for the recurrence of the tumors. In functional experiments are defined as cells that form tumors and self-renew when transplanted into immunodeficient mice. There are studies showing that: when murine leukemia cells were transplanted into mice of the same strain, only 1% to 4% of the transplanted cells were found to be able to form intrasplenic clones, indicating that only a portion of the tumor cells were tumorigenic. In leukemia, only one in ten thousand cells have this potential for unlimited proliferation, but they are small in number but have the characteristics of stem cells, perhaps the only tumor cells that remain proliferating, and are called tumor stem cells.
The tumor cells are variant cells and cause cancer, the cells can also continuously proliferate and differentiate to form relatively mature tumor cells which can not proliferate any more and can not form cell colonies, or the tumor cells can only divide for a few times, in vitro clonal culture can only form a few or dozens of cells which are transiently differentiated and finally die, and the tumor stem cells can continuously proliferate to form cell colonies which play a decisive role in the formation and growth processes of tumors.
In basic research on cancer treatment, the original research on cancer is a theoretical foundation to overcome cancer. The clonal evolution theory and the cancer stem cell theory are two major lines explaining the origin of cancer. The clonal evolution theory emphasizes the ability of cells to randomly acquire cumulative mutations with persistent proliferation, while the cancer stem cell theory emphasizes the hierarchy in tumor tissues-only a few primitive mutant cells have persistent proliferation ability. However, we must recognize that the two teachings are not mutually compatible. The clonal evolutionary theory may be based on the cancer stem cell theory. We can say that the cell has the ability to proliferate widely by natural selection, and this cell becomes a cancer stem cell. And the natural selection can be established on any possible factors of a plurality of factors of the ecological complexity of the organization. At the heart of the clonal evolutionary theory, is the "variation". The genotype and phenotype of cancer stem cells also change continuously during the progression of cancer, and its only invariant feature is the ability to self-renew extensively. Many treatment regimens simply provide another direction for cancer stem cells to survive and proliferate. Therefore, we believe on the one hand that the cancer stem cell theory is rationalized in explaining the problem of cancer origin, and on the other hand also realize that cancer stem cells have a complex genetic diversity, which requires particular attention in the choice of treatment regimen.
A large number of researches show that a group of trace cell groups, which account for about 0.1% -1% of all leukemia cells, exist in the body of a leukemia patient, and the cells are generally insensitive to chemotherapeutic drugs and are the chief culprit of causing the leukemia to relapse. They can cause and maintain leukemia, both in long-term cell culture and in animal models, and this population of leukemia cells is called Leukemia Stem Cells (LSCs). Leukemia, a hematologic malignancy originating from hematopoietic stem cells, has a tendency to increase year by year. Although current treatments provide complete remission in most patients, there has been no breakthrough in efficacy and approximately 70% of patients still experience relapse. The underlying cause of recurrence is the presence of LSC. Therefore, LSC is a key factor affecting the efficacy and long-term survival of AML patients, and effective clearance of LSC is a difficult point and bottleneck in the current treatment of AML.
Disclosure of Invention
The technical problem to be solved is as follows: the invention is based on the leukemia stem cell tumor stem cell theory, through collecting the bone marrow of a hemopathy patient who relapses and is difficult to treat, separating and extracting the tumor stem cells of the patient through a laboratory, amplifying and culturing, and then carrying out drug sensitivity test analysis on the cultured tumor stem cells through a drug screening platform, the purpose of personalized and accurate drug administration is realized. The invention constructs a high-throughput drug screening method aiming at hematological tumors, which comprises chemotherapy drugs, traditional Chinese medicines and small molecular targeted drugs which are clinically listed and aim at leukemia treatment and under-study drugs which are not yet listed.
The technical scheme is as follows: a method for leukemia high throughput drug screening, comprising the steps of:
step 1: separating, extracting, identifying and culturing the blood tumor stem cells: (1) collecting a bone marrow sample; (2) separating mononuclear cells by adopting a bone marrow mononuclear cell separating medium kit; (3) separation of leukemia CD34 by immunomagnetic bead method+/CD38-A population of stem cells; (4) separating and purifying magnetic beads, inoculating the cells to DMEM medium containing 20ng/ml epidermal growth factor, 100u/ml penicillin, 100u/ml streptomycin and 10 wt% fetal calf serum, and culturing at 37 deg.C with 5% CO2Culturing in an incubator, determining the adherent condition of cells after 12h, changing liquid, and subculturing;
step 2: establishing a drug library: according to the pharmaceutical specification and pharmacokinetics, the concentration of the drug is selected, the drug is mixed with the blood tumor stem cells for culture, and the inhibition effect of the drug on the blood tumor stem cells is analyzed.
Further, the step 1 of separating mononuclear cells by using the kit for separating bone marrow mononuclear cells comprises the following specific steps:
1) adding a proper amount of LF (low frequency) liquid into collected bone marrow cells, uniformly mixing, centrifuging at a rotating speed of 500g for 20min, removing supernatant, repeatedly washing precipitates twice with F liquid, and suspending with whole blood containing 20 wt% fetal calf serum and tissue diluent for later use; in the present invention, the LF liquid is physiological saline, and the F liquid is phosphate buffer.
2) Adding a separation solution with the same amount as the bone marrow single cell suspension into a 15mL centrifuge tube, wherein the volume of the separation solution is more than or equal to 7 mL;
3) carefully sucking the marrow single cell suspension with a pipette, adding the marrow single cell suspension on the liquid level of the separation liquid, and centrifuging at the rotation speed of 400-;
4) after centrifugation, the cells in the centrifuge tube are divided into four layers from top to bottom at the moment, wherein the first layer is a dilution liquid layer, the second layer is an annular milky mononuclear cell layer, the third layer is a transparent separation liquid layer, the fourth layer is a red blood cell layer, the second layer of the annular milky mononuclear cell layer is carefully sucked into another 15mL centrifuge tube by a suction tube, 10mL of cleaning solution is added into the centrifuge tube, and the cells are uniformly mixed;
5) centrifuging at 250g for 10min, and removing supernatant;
6) resuspending the cells in 5-10 mL wash with a pipette;
7) centrifuging at 250g for 10min, and removing supernatant;
8) 0.5mL of the corresponding liquid required for the subsequent experiment was taken to resuspend the cells for use.
Further, the method for separating leukemia CD34 by using immunomagnetic beads in the step 1+/CD38-The specific steps of the stem cell population are as follows:
1)CD38-negative sorting: magnetic markers, each 107Adding 800ul buffer solution into the total cells for resuspension, adding 200ul biotin antibody, mixing uniformly, and incubating for 15min at 4 ℃; washing the cells with a buffer solution, centrifuging at 1200rpm/min for 10min, and removing the supernatant; adding 200ul of biotin antibody-containing micro-strain buffer solution, mixing, and incubating at 4 deg.C for 15 min; washing the cells with a buffer solution, centrifuging at 1200rpm/min for 10min, and removing the supernatant; every 10 th7Adding 800ul buffer solution into the total cells for resuspension; placing the cell suspension into a MACS sorter, slowly adding 2ml of buffer solution into an LD type sorting column, and then adding the resuspended cell suspension into an LD type analytical column for negative selection; collecting all negative components and counting;
2)CD34+positive sorting: magnetic labeling, resuspending cells in 200ul buffer solution, adding 100ul of blocker and 200ul of CD34 magnetic beads, mixing uniformly, and incubating at 4 ℃ for 30 min; use slowlyWashing cells with the flushing liquid, centrifuging at 1200rpm/min for 10min, and removing supernatant; every 10 th7Adding 800ul buffer solution into the total cells for resuspension; magnetic bead sorting, setting into MACS sorter, adding 2ml buffer solution into LD type sorting column slowly, negative cell flowing out first, adding 500ul buffer solution into empty original specimen test tube, adding into LD type sorting column, repeating for 3 times, moving LD type sorting column out of sorter, adding 1000ul buffer solution into LD type sorting column, and pushing out marked cell rapidly by piston equipped with sorting column.
Collection of 50ml bone marrow from patient 3X 107-7×107Individual bone marrow mononuclear cells, separated and purified by MACS and then subjected to CD34+/CD38-The purity of the cells reaches 73 +/-5.4 percent.
Further, the basic drug spectrum for leukemia therapy in the drug library is selected from the following: vincristine, vinblastine, etoposide VP-16, teniposide, irinotecan, gemcitabine, methotrexate, arabinoside, 5-fluorouracil, cyclophosphamide, ifosfamide, ledapas, busulfan, melphalan, chlorambucil, cisplatin, carboplatin, bleomycin, lomustine, carmustine, dacarbazine, hydroxyureaamycin, epirubicin, daunorubicin, mitoxantrone, pirarubicin, imatinib, nilotinib, dasatinib, bortezomib, decitabine, thalidomide, lenalidomide, dexamethasone, prednisone, hydrocortisone, cinobufagin, bexarotene, pavine formate, homoharringtonine, hydroxycamptothecin.
Further, in step 2, the screening doses of 1.1 μ M and 1.0 μ M are calculated in step 2 according to the drug specification and pharmacokinetics, and the inhibition rate of the drug on the tumor stem cells is calculated, that is, in the high-throughput drug in vitro test, the killing effect of the screening dose of the drug on the tumor cells of the patient is calculated, and the calculation formula of the inhibition rate is as follows: inhibition (%) × 100% (1-survival number of tumor cells in drug-screened group/survival number of tumor cells in blank control group); the medicines with the inhibition rate of more than 65 percent can be used as clinical optional medicines. .
Further, Table 1 shows the detection spectrum of the drugs commonly used for leukemia
TABLE 1
Has the advantages that:
1. the separation and identification of the tumor stem cells in a laboratory can effectively analyze the etiology analysis of the relapsed refractory leukemia, and provides disease typing and treatment reference for clinicians.
2. The tumor stem cell drug screening method can give a proper treatment scheme suggestion aiming at the individuation and the precision of a patient, provide reference for a clinician, strive for precious treatment opportunity for a relapse refractory patient, reduce the risk of reagent, increase the curative effect of the drug to the maximum extent and reduce the side effect of the drug.
3. The medicine which accords with the individualized curative effect of a patient can be effectively found through a high-flux large-scale drug sensitivity test of the tumor stem cells, a medicine library comprises chemotherapy medicines, traditional Chinese medicines, small-molecule targeted medicines and medicines which are not yet researched in the market, the medicine screening range is wide, and the appropriate medicines with good curative effect and small side effect can be screened out by utilizing the method.
4. The tumor stem cell drug screening method can not only screen single drugs, but also can be used as drug combinations, especially the effect combinations of common chemotherapeutic drugs, can be used for making a comprehensive score aiming at the combination of the common clinical chemotherapeutic drugs, and provides a powerful reference for doctors to establish clinical combined medication for patients.
5. Compared with the construction of a mouse tumor model, the high-flux drug screening method has the advantages of short time, low cost, more contribution to large-scale technical application and the like.
6. The high-flux drug screening method has the advantages of short culture time and high-flux and wide screening, can be used as a preliminary screening standard in an accurate solid tumor drug model, and can be verified by a humanized mouse tumor model after preliminary screening to improve the accuracy of the model.
7. The high-throughput drug screening method and the high-throughput sequencing model can be verified mutually to improve the accuracy of the method.
Detailed Description
Drug screening example 1:
patient, female 30 years old
And (3) clinical diagnosis: similar to leukemia hemogram, the leukemia-like reaction is marked by the increase of leucocytes after serious infection
And (4) screening results: the drug screening results recommend the following medications: daunorubicin, mitoxantrone, homoharringtonine, Dinaciclib, cinobufagin. Among them, Dinaciclib is a potent Cyclin Dependent Kinase (CDK) inhibitor.
The inhibition rate (%) is the killing effect of the drug screening dose on the tumor cells of the patient in a high-throughput drug in-vitro test.
Inhibition (%). ratio (1-survival number of tumor cells in drug-screened group/survival number of tumor cells in blank control group). 100%
Drug screening example 2:
patient male 18 years old
And (3) clinical diagnosis: acute leukemia (double series, Ph (+); and (3) carrying out BCR/ABL quantitative detection: p210(-), P190 (+); BCR-ABL copy number 3.87X 108ABL copy number 2.38X 108，BCR-ABL/ABL＝1.62
The drug screening results recommend the following medications:
chemotherapy drugs: daunorubicin, epirubicin, aclarubicin, mitoxantrone, pirarubicin, vincristine, vinblastine, hydroxycamptothecin, homoharringtonine, cinobufagin
Targeting preparation: dasatinib, Ponatinib
The inhibition rate (%) is the killing effect of the drug screening dose on the tumor cells of the patient in a high-throughput drug in-vitro test.
Inhibition (%). ratio (1-survival number of tumor cells in drug-screened group/survival number of tumor cells in blank control group). 100%
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (5)
1. A method for high throughput drug screening for leukemia, comprising: the method comprises the following steps:
step 1: separating, extracting, identifying and culturing the blood tumor stem cells: (1) collecting a bone marrow sample; (2) separating mononuclear cells by adopting a bone marrow mononuclear cell separating medium kit; (3) separation of leukemia CD34 by immunomagnetic bead method+/CD38-A population of stem cells; (4) separating and purifying magnetic beads, inoculating the cells to DMEM medium containing 20ng/ml epidermal growth factor, 100u/ml penicillin, 100u/ml streptomycin and 10 wt% fetal calf serum, and culturing at 37 deg.C with 5% CO2Culturing in an incubator, determining the adherent condition of cells after 12h, changing liquid, and subculturing;
step 2: establishing a drug library: according to the pharmaceutical specification and pharmacokinetics, the concentration of the drug is selected, the drug is mixed with the blood tumor stem cells for culture, and the inhibition effect of the drug on the blood tumor stem cells is analyzed.
2. The method for leukemia high-throughput drug screening according to claim 1, wherein: the method for separating the mononuclear cells by adopting the bone marrow mononuclear cell separating medium kit in the step 1 comprises the following specific steps:
1) adding a proper amount of LF (low frequency) liquid into collected bone marrow cells, uniformly mixing, centrifuging at a rotating speed of 500g for 20min, removing supernatant, repeatedly washing precipitates twice with F liquid, and suspending with whole blood containing 20 wt% fetal calf serum and tissue diluent for later use;
2) adding a separation solution with the same amount as the bone marrow single cell suspension into a 15mL centrifuge tube, wherein the volume of the separation solution is more than or equal to 7 mL;
3) carefully sucking the marrow single cell suspension with a pipette, adding the marrow single cell suspension on the liquid level of the separation liquid, and centrifuging at the rotation speed of 400-;
4) after centrifugation, the cells in the centrifuge tube are divided into four layers from top to bottom at the moment, wherein the first layer is a dilution liquid layer, the second layer is an annular milky mononuclear cell layer, the third layer is a transparent separation liquid layer, the fourth layer is a red blood cell layer, the second layer of the annular milky mononuclear cell layer is carefully sucked into another 15mL centrifuge tube by a suction tube, 10mL of cleaning solution is added into the centrifuge tube, and the cells are uniformly mixed;
5) centrifuging at 250g for 10min, and removing supernatant;
6) resuspending the cells in 5-10 mL wash with a pipette;
7) centrifuging at 250g for 10min, and removing supernatant;
8) 0.5mL of the corresponding liquid required for the subsequent experiment was taken to resuspend the cells for use.
3. The method for leukemia high-throughput drug screening according to claim 1, wherein: the method for separating leukemia CD34 by using immunomagnetic beads in the step 1+/CD38-The specific steps of the stem cell population are as follows:
1)CD38-negative sorting: magnetic markers, each 107Adding 800ul buffer solution into the total cells for resuspension, adding 200ul biotin antibody, mixing uniformly, and incubating for 15min at 4 ℃; washing the cells with a buffer solution, centrifuging at 1200rpm/min for 10min, and removing the supernatant; adding 200ul of biotin antibody-containing micro-strain buffer solution, mixing, and incubating at 4 deg.C for 15 min; washing the fine particles with bufferCentrifuging at 1200rpm/min for 10min, and discarding the supernatant; every 10 th7Adding 800ul buffer solution into the total cells for resuspension; placing the cell suspension into a MACS sorter, slowly adding 2ml of buffer solution into an LD type sorting column, and then adding the resuspended cell suspension into an LD type analytical column for negative selection; collecting all negative components and counting;
2)CD34+positive sorting: magnetic labeling, resuspending cells in 200ul buffer solution, adding 100ul of blocker and 200ul of magnetic beads of CD34, mixing, and incubating at 4 ℃ for 30 min; washing the cells with a buffer solution, centrifuging at 1200rpm/min for 10min, and removing the supernatant; every 10 th7Adding 800ul buffer solution into the total cells for resuspension; magnetic bead sorting, setting into MACS sorter, adding 2ml buffer solution into LD type sorting column slowly, negative cell flowing out first, adding 500ul buffer solution into empty original specimen test tube, adding into LD type sorting column, repeating for 3 times, moving LD type sorting column out of sorter, adding 1000ul buffer solution into LD type sorting column, and pushing out marked cell rapidly by piston equipped with sorting column.
4. The method for leukemia high-throughput drug screening according to claim 1, wherein: the basic drug spectrum selection of leukemia treatment in the drug library is as follows: vincristine, vinblastine, etoposide VP-16, teniposide, irinotecan, gemcitabine, methotrexate, arabinoside, 5-fluorouracil, cyclophosphamide, ifosfamide, ledapas, busulfan, melphalan, chlorambucil, cisplatin, carboplatin, bleomycin, lomustine, carmustine, dacarbazine, hydroxyureaamycin, epirubicin, daunorubicin, mitoxantrone, pirarubicin, imatinib, nilotinib, dasatinib, bortezomib, decitabine, thalidomide, lenalidomide, dexamethasone, prednisone, hydrocortisone, cinobufagin, bexarotene, pavine formate, homoharringtonine, hydroxycamptothecin.
5. The method for leukemia high-throughput drug screening according to claim 1, wherein: in the step 2, the screening doses of 1.1 mu M and 1.0 mu M are calculated according to the drug specification and the pharmacokinetics, and the inhibition rate of the drug on the tumor stem cells is calculated, namely in the high-throughput drug in-vitro test, the killing effect of the screening doses of the drug on the tumor cells of the patient is calculated, and the calculation formula of the inhibition rate is as follows: inhibition rate (1-survival number of tumor cells in drug screening group/survival number of tumor cells in blank control group) × 100%; the medicines with the inhibition rate of more than 65 percent can be used as clinical optional medicines.
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| CN201811279792.5A CN111118099A (en) | 2018-10-30 | 2018-10-30 | Method for screening leukemia high-flux drugs |
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080187950A1 (en) * | 2006-12-07 | 2008-08-07 | Weissman Irving L | Identification and isolation of acute myeloid leukemia stem cells |
| 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 |
| US20100008896A1 (en) * | 2008-07-08 | 2010-01-14 | Ji Zhang | Use of Fenretinide or Bioactive Derivatives Thereof and Pharmaceutical Compositions Comprising the Same |
| CN102220282A (en) * | 2011-04-26 | 2011-10-19 | 广东省人民医院 | Method for separating leukemia stem cells from leukemia cell strain |
| CN102409026A (en) * | 2010-09-25 | 2012-04-11 | 殷勤伟 | Novel method capable of enriching and amplifying suspended blood cancer stem cells, novel culture medium and application thereof to drug screening |
| KR20130001108A (en) * | 2010-05-19 | 2013-01-03 | 비비아 바이오테크 에스.엘. | Methods for providing personalized medicine tests ex vivo for hematological neoplasms |
| CN105056236A (en) * | 2015-08-13 | 2015-11-18 | 暨南大学 | Application of ORP4L (Oxsterol binding protein-related protein, 4L) in preparation of product for treating acute myeloid leukemia |
| US20160175331A1 (en) * | 2014-12-23 | 2016-06-23 | University Of Maryland, Baltimore | Treatment of leukemia with artemisinin derivatives and combinations with other antineoplastic agents |
| WO2016159301A1 (en) * | 2015-04-01 | 2016-10-06 | 国立大学法人広島大学 | Therapeutic agent for chronic myeloid leukemia |
| CN107898804A (en) * | 2017-12-21 | 2018-04-13 | 大连理工大学 | A kind of application of anti-tumor agent comprising salmosin in human leukemia cell |
| CN108251374A (en) * | 2018-01-25 | 2018-07-06 | 四川大学华西第二医院 | A kind of people's B-lineage Acute Lymphocyte Leukemia cell strain and its application |
-
2018
- 2018-10-30 CN CN201811279792.5A patent/CN111118099A/en active Pending
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080187950A1 (en) * | 2006-12-07 | 2008-08-07 | Weissman Irving L | Identification and isolation of acute myeloid leukemia stem cells |
| US20100008896A1 (en) * | 2008-07-08 | 2010-01-14 | Ji Zhang | Use of Fenretinide or Bioactive Derivatives Thereof and Pharmaceutical Compositions Comprising the Same |
| 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 |
| KR20130001108A (en) * | 2010-05-19 | 2013-01-03 | 비비아 바이오테크 에스.엘. | Methods for providing personalized medicine tests ex vivo for hematological neoplasms |
| CN102409026A (en) * | 2010-09-25 | 2012-04-11 | 殷勤伟 | Novel method capable of enriching and amplifying suspended blood cancer stem cells, novel culture medium and application thereof to drug screening |
| CN102220282A (en) * | 2011-04-26 | 2011-10-19 | 广东省人民医院 | Method for separating leukemia stem cells from leukemia cell strain |
| US20160175331A1 (en) * | 2014-12-23 | 2016-06-23 | University Of Maryland, Baltimore | Treatment of leukemia with artemisinin derivatives and combinations with other antineoplastic agents |
| WO2016159301A1 (en) * | 2015-04-01 | 2016-10-06 | 国立大学法人広島大学 | Therapeutic agent for chronic myeloid leukemia |
| JP2016193870A (en) * | 2015-04-01 | 2016-11-17 | 国立大学法人広島大学 | Therapeutic agent for chronic myelogenous leukemia |
| CN105056236A (en) * | 2015-08-13 | 2015-11-18 | 暨南大学 | Application of ORP4L (Oxsterol binding protein-related protein, 4L) in preparation of product for treating acute myeloid leukemia |
| CN107898804A (en) * | 2017-12-21 | 2018-04-13 | 大连理工大学 | A kind of application of anti-tumor agent comprising salmosin in human leukemia cell |
| CN108251374A (en) * | 2018-01-25 | 2018-07-06 | 四川大学华西第二医院 | A kind of people's B-lineage Acute Lymphocyte Leukemia cell strain and its application |
Non-Patent Citations (8)
| Title |
|---|
| DENG CH等: "Leukemia stem cells in drug resistance and metastasis", 《CHIN MED J (ENGL)》, vol. 123, no. 07, pages 954 - 960 * |
| DOW LW等: "Correlation of drug sensitivity in vitro with clinical responses in childhood acute myeloid leukemia", 《BLOOD》, vol. 68, no. 02, pages 400 - 405 * |
| HONGO T等: "In vitro drug sensitivity testing can predict induction failure and early relapse of childhood acute lymphoblastic leukemia", 《BLOOD》, vol. 89, no. 08, pages 2959 - 2965 * |
| 周忠海;许文荣;王兴忠;邱国强;谢晓宝;周洪兴;钱晖;阴晴;朱伟;徐静;司煜安;: "髓系白血病干细胞培养及其特征鉴定", 临床检验杂志, no. 04, pages 249 - 252 * |
| 徐瑞荣;王敬毅;崔兴;王琰;胡述博;李丽珍;: "噻唑蓝法检测中药复方对人白血病CD34+CD38-干细胞群的影响", 中国药物与临床, no. 09, pages 696 - 698 * |
| 徐瑞荣等: "免疫磁珠分选系统在急性髓系白血病干细胞分离中的应用", 《浙江中西医结合杂志》, no. 10, pages 610 - 612 * |
| 王国征等: "免疫磁珠分选白血病KG1a细胞中CD34+CD38-干细胞及其特性研究", 《新乡医学院学报》, vol. 30, no. 03, pages 181 - 184 * |
| 韩慧娟: "吉西他滨对CD34~+CD38~-髓系白血病干细胞的生物学效应", 中国优秀硕士学位论文全文数据库医药卫生科技辑(月刊), no. 03, pages 072 - 74 * |
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