CN111233873B - Application of methyl taurocten in preparation of drugs for inhibiting proliferation and inducing differentiation of leukemia cells and leukemia stem cells - Google Patents
Application of methyl taurocten in preparation of drugs for inhibiting proliferation and inducing differentiation of leukemia cells and leukemia stem cells Download PDFInfo
- Publication number
- CN111233873B CN111233873B CN202010065701.9A CN202010065701A CN111233873B CN 111233873 B CN111233873 B CN 111233873B CN 202010065701 A CN202010065701 A CN 202010065701A CN 111233873 B CN111233873 B CN 111233873B
- Authority
- CN
- China
- Prior art keywords
- cells
- leukemia
- stem cells
- mla
- cell
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/08—Bridged systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C59/235—Saturated compounds containing more than one carboxyl group
- C07C59/245—Saturated compounds containing more than one carboxyl group containing hydroxy or O-metal groups
- C07C59/265—Citric acid
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Hematology (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses an application of methyl bullactatin in preparing drugs for inhibiting the proliferation of leukemia cells and leukemia stem cells and inducing the differentiation of the leukemia cells and the leukemia stem cells, which can not only induce the apoptosis of AML cell lines KG-1 and HL-60 with high CHRNA7 expression, but also induce the differentiation of the AML cell lines and can be used as an effective drug for treating primary diagnosis and relapse refractory AML.
Description
Technical Field
The invention relates to a leukemia treatment drug, in particular to an application of methyl taurocten in preparing drugs for inhibiting the proliferation and inducing the differentiation of leukemia cells and leukemia stem cells, belonging to the technical field of medicine.
Background
Acute Myeloid Leukemia (AML) is a malignant tumor formed by malignant proliferation of bone marrow hematopoietic stem cells, and the morbidity and mortality rate of AML account for about 35% and 49% of the total leukemia, respectively. The data of the investigation of Chinese tumors in 2015 showed that the mortality rate of leukemia in China ranks 9 th in all tumors, while the prevalence of AML patients (Zhengrongshou, sungkexin, zhangwei, zhang hongmei, xiaonong, chenru, guo Xiuwen, weiwen Qiang, he Jie) in 2015 are mainly analyzed in other types than M3 [ J]J. Zhonghua tumor, 2019,41 (1): 19-28). AML can occur at any age, with an acute onset, with rapid progression and natural survival within 6 months to 1 year. Currently, combination chemotherapy is still the most commonly used means for AML treatment, but chemotherapy resistance and the susceptibility to relapse after drug withdrawal in AML patients are one of the key factors affecting AML treatment efficacy. In addition, leukemia stem cells have been found to be the source of chemotherapy resistance and recurrence in AML patients (Zeijlemaker W, grob) T, Meijer R, Hanekamp D, Kelder A, Carbaat-Ham JC, Oussoren-Brockhoff YJM, Snel AN. CD34 + CD38 − Leukemia cell frequency to predict output time in the ingredient muscle tissue, leukemia 2019, 33 (5): 1102-1112). Thus, initiation of leukemic stem cell apoptosis or/and differentiation pathways will be more likely to cure AML. The search for a drug capable of inhibiting the proliferation of leukemia cells and leukemia stem cells has become a common pursuit of the goal of scientists.
Disclosure of Invention
In order to solve the problems, the invention provides the application of the methyloxepin in preparing the medicines for inhibiting the proliferation of leukemia cells and leukemia stem cells and inducing the differentiation of the leukemia cells and the leukemia stem cells.
The technical scheme of the invention is as follows:
application of methyl taurocten in preparing medicine for inhibiting proliferation and inducing differentiation of leukemia cells and leukemia stem cells.
The Methylomacetin (MLA) is an extract from seeds of a Chinese medicinal herb delphinium and has a chemical formula as follows:
is a specific inhibitor of CHRNA7 (alpha 7 nicotinic acetylcholine receptor, nicotinic acetylcholine receptor alpha 7), the binding site of which in CHRNA7 is similar to alpha-gold cobratoxin, the low dose can improve the cognitive function of animals, and can inhibit the release of TNF-alpha from microglia. The methyl oxepin not only induces apoptosis of AML cell lines KG-1 and HL-60 with high CHRNA7 expression, but also induces differentiation. Meanwhile, methyloxletin concentrations as high as 50 μ M had no effect on the survival of normal human PBMCs.
IC of 2 cell lines KG-1 and HL-60 obtained by calculation by using CCK-8 test after MLA treatment for 96h 50 The (half inhibitory concentration) values were 22.24. Mu.M and 13.72. Mu.M, respectively. With IC 50 For reference, the subsequent apoptosis test and cell differentiation test were performed, and the time for treating the cells with MLA was 96 hours. Therefore, it can be used as an effective drug for treating AML that is refractory to the initial diagnosis and recurrence.
As is inferred from animal experimental results, the dose of MLA applied to humans is about 1.1mg/kg, and administration is performed by intravenous route. The required dose of MLA at 110mg/ml was withdrawn under sterile conditions, placed in a sterile pyrogen-free infusion bag containing 0.9% physiological saline, the concentration of MLA after dilution was 1.1mg/kg, and the solution was mixed by gently inverting the bag. Before intravenous injection, the injection is observed to have particles or change color.
The invention has the beneficial effects that: can be used as an effective medicine for treating primary diagnosis and relapse refractory AML.
The invention is further illustrated by the following examples in conjunction with the drawings.
Drawings
FIG. 1 is a graph showing CHRNA7 expression in AML cell lines KG-1, HL-60 and K562 according to example of the present invention;
FIG. 2 is a graph showing the flow results of MLA induced apoptosis of CHRNA 7-highly expressed AML cell lines KG-1 and HL-60 in the present invention;
FIG. 3 is a graph showing the statistical results of KG-1 and HL-60 cell apoptosis induced by MLA in accordance with the present invention
FIG. 4 is a graph showing the results of Wright's staining of KG-1 and HL-60 cells induced by MLA according to the present invention;
FIG. 5 is a diagram showing the results of flow-based assays of KG-1 and HL-60 cell differentiation induced by MLA according to the present invention;
FIG. 6 is a graph showing CHRNA7 expression in myeloid-derived leukemia stem cells of AML patients according to an embodiment of the present invention;
FIG. 7 is a graph showing the flow results of MLA inducing apoptosis of AML leukemia stem cells in accordance with an embodiment of the present invention;
FIG. 8 is a graph showing the statistical results of the induction of apoptosis of AML leukemia stem cells by MLA in accordance with the present invention;
FIG. 9 is a graph showing the results of the Swiss staining of the MLA-induced differentiation of AML leukemia stem cells in the example of the present invention;
FIG. 10 is a graph showing the flow-type assay results of the MLA-induced differentiation of AML leukemia stem cells according to the embodiment of the present invention;
FIG. 11 is a Kaplan-Meier survival curve for mice according to an embodiment of the present invention.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.
Example 1
A small molecule for inhibiting the proliferation and inducing the differentiation of leukemia cells and leukemia stem cells is methyloxepin.
The Methylomacetin (MLA) is an extract from seeds of a Chinese medicinal herb delphinium and has a chemical formula as follows:
the methyloxletin is a specific inhibitor of CHRNA7 (alpha 7 nicotinic acetylcholine receptor, nicotinic acetylcholine receptor alpha 7), the binding site of the methyloxletin in CHRNA7 is similar to that of alpha-bungarotoxin, the cognitive function of animals can be improved at low dose, and the release of TNF-alpha by microglia can be inhibited. Experiments prove that the methyl oxepin not only induces apoptosis of AML cell lines KG-1 and HL-60 with high CHRNA7 expression, but also induces differentiation of the AML cell lines KG-1 and HL-60. Meanwhile, it was confirmed that methyloxletin concentrations up to 50. Mu.M had no effect on the survival of normal human PBMCs.
Experimental example I
Experiment on killing effect of MLA on leukemia cells and leukemia stem cells
(1) Culture of leukemia cell lines
Culturing cells in RPMI 1640 culture medium containing 10% fetal calf serum, collecting and counting the cells when the cells enter logarithmic growth phase, and performing subsequent experiments including cell proliferation test, cell apoptosis test and cell differentiation test.
(2) Isolation and culture of acute myelogenous leukemia stem cells
Collecting AML patient bone marrow specimen, diluting with PBS in equal volume, mixing, adding into appropriate amount of Ficoll lymphocyte separation liquid, centrifuging at 2000rpm for 25min, sucking out mononuclear cell, and washing with PBS for 2 times to obtain bone marrow mononuclear cell; adding mouse anti-human CD34-PE and CD38-FITC antibodies into the cell specimen, and separating CD34+ CD 38-cells from the cell specimen by using a flow cytometry sorter, wherein the cells are leukemia stem cells; the cells can be used for evaluating the anti-leukemia effect of MLA.
(3) CCK8 method for detecting cell proliferation
The experiments were divided into 2 major groups: DMSO group and MLA group. Each large group was set with 6 different doses, each dose was 3 replicates. Inoculating cells to a 96-well plate, wherein 5000 cells are per well, the final volume is 100 mu L per well, culturing for 24h, 48h and 72h at 37 ℃ in 5% CO2 respectively, adding 10 mu L CCK-8 into each well within 4h before the culture is finished, placing the culture plate in an incubator for incubation for 2-4 h, measuring OD450nm by using a microplate reader, calculating IC50, and selecting the processing time corresponding to the minimum value of IC50 as the processing time of a subsequent experiment.
(4) Detection of apoptosis by flow cytometry
KG-1, HL-60 and K562 cells in a logarithmic growth phase or leukemia stem cells (CD 34+ CD 38-) separated from AML patient bone marrow are inoculated into a 6-well plate, MLA is added, meanwhile, a blank control and a compound well are set up, after culture is carried out for 72h at 37 ℃ and 5% CO2, the cells are collected, washing is carried out by using a binding buffer solution, 5 mu L of FITC-Annexin V and PI are respectively added, incubation is carried out for 15min in a dark place, and detection is carried out by an up-flow cytometer, so that the apoptosis condition is determined.
(5) Effect on differentiation
(1) Rui-giemsa staining: KG-1, HL-60, K562 cells in logarithmic growth phase or leukemia stem cells (CD 34) isolated from the bone marrow of AML patients + CD38 - ) Inoculating to 6-well plate, adding MLA, setting blank and multiple wells at 37 deg.C with 5% CO 2 Culturing for 96h, collecting cells, staining with Raynaud-Giemsa staining solution for 1min, adding equal amount of Raynaud-Giemsa buffer solution, mixing, staining for 10-15min, and observing cell differentiation under optical microscope;
(2) FCM: the cells were treated and collected according to the method of (1), 5. Mu.L of human FITC-CD14 and FITC-CD15 antibodies were added to each sample, incubated on ice for 30min, washed 1 time with PBS, and then detected by an up-flow cytometer to detect the differentiation of the cells.
Through the five steps, the apoptosis and differentiation conditions of the leukemia cells and the leukemia stem cells before and after MLA treatment are observed, and the killing effect of the MLA on the leukemia cells and the leukemia stem cells is determined.
Experimental example two
Expression of CHRNA7 in AML cell lines KG-1, HL-60 and K562
The specific method comprises the following steps:
CHRNA7 expression profiles (flow cytometry) in AML cell lines KG-1, HL-60 and K562 are shown in FIG. 1.
Materials and reagents: rabbit anti-human CHRNA7 antibody (purchased from Proteitech corporation), fetal bovine serum, PBS containing 3% fetal bovine serum, and goat anti-rabbit IgG secondary antibody labeled with Alexa Fluor647
The operation steps are as follows: KG-1, HL-60, K562 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, when they entered the logarithmic growth phase, the cells were collected, centrifuged at 1000rpm for 5min, the supernatant was discarded, 1ml PBS containing 3% fetal bovine serum was added to the cell pellet, the cells were resuspended, centrifuged at 1000rpm for 5min, the supernatant was discarded, the cells were counted, 1X 106 cells were removed therefrom, 10. Mu.L rabbit anti-human CHRNA7 antibody (Proteintetech, 1.
The experimental results are as follows: as can be seen from FIG. 1, the percentage of KG-1 cells carrying fluorescence in the experimental group was 94%, while the percentage of KG-1 cells carrying fluorescence in the ctrl and isotype groups was only 0.01% and 7.71%, so the percentage of KG-1 cells carrying fluorescence in the experimental group was significantly higher than that in the control group, and the CHRNA7 protein on the surface of KG-1 cells was highly expressed. Similarly, the percentage of HL-60 cells carrying fluorescence (98.82%) in the experimental group is obviously higher than that in the control group (0.67% and 4.16%), so the CHRNA7 protein on the surface of the HL-60 cells is also highly expressed; the percentage of the K562 cells carrying fluorescence (2.16%) in the experimental group is obviously higher than that in the control group (0.09% and 3.91%), so that the CHRNA7 protein on the surface of the K562 cells is not expressed.
Experimental example III
MLA induced apoptosis and differentiation of CHRNA 7-highly expressed AML cell lines KG-1 and HL-60 as shown in FIG. 2, FIG. 3, FIG. 4 and FIG. 5.
Materials and reagents: MLA solution (dissolved in DMSO to a final concentration of 10mM, purchased from MCE), RPMI 1640 medium, fetal bovine serum, annexin-V-FITC/PI apoptosis kit (purchased from BD Bioscience), FITC labeled mouse anti-human CD14 and CD15 antibodies (purchased from BD Bioscience), 15mL sterile centrifuge tubes, 1.5mL centrifuge tubes, 10. Mu.L, 200. Mu.L and 1mL sterile tips, 6-well cell culture plates.
The operation steps are as follows: culturing KG-1 and HL-60 cells in RPMI 1640 medium containing 10% fetal bovine serum, collecting and counting the cells when they entered the logarithmic growth phase, inoculating them in 6-well plates at 50X 106/well, adding MLA to final concentrations of 5. Mu.M and 10. Mu.M, and using DMSO-added group as a control, incubating at 37 ℃ for 5% CO 2 Culturing for 96 hours in the cell culture box, collecting cells, dividing the cells into 3 parts, centrifuging the first part of cells for 5min at 1000rpm, discarding supernatant, adding 1ml of 1 × Binding Buffer into cell sediment, re-suspending the cell sediment, respectively adding 5 μ L of Annexin-V-FITC and PI, dyeing for 15min on ice in a dark place, and detecting by using a flow cytometer; centrifuging the second cell at 1000rpm for 5min, discarding the supernatant, smearing, air drying, staining with Reishi-Giemsa staining solution for 1min, adding equivalent Reishi-Giemsa buffer solution, mixing, staining for 10-15min, and observing cell differentiation under optical microscope; centrifuging the third cell at 1000rpm for 5min, discarding supernatant, adding 1ml PBS containing 3% fetal calf serum, equally dividing into 2 tubes, centrifuging at 1000rpm for 5min, discarding supernatant, respectively adding 5 μ L mouse anti-human CD14-FITC and CD15-FITC antibodies, incubating on ice for 30min, adding 1ml PBS containing 3% fetal calf serum, resuspending cells, centrifuging at 1000rpm for 5min, discardingAnd (6) supernatant fluid.
The experimental results are as follows: as can be seen from the results of FIGS. 2 and 3, the apoptosis rates of KG-1 cells were 27.5% + -1.37% and 42.39% + -0.99%, respectively, significantly higher than that of the non-drug combination (12.68% + -1.36%) at MLA concentrations of 5. Mu.M and 10. Mu.M; the apoptosis rates of HL-60 cells are respectively 25.72% + -1.42% and 35.16% + -1.22%, which are obviously higher than those of non-added medicines (11.64% + -1.52%, P > 0.05%). As can be seen from the results in FIG. 4, part of KG-1 nuclei in the MLA-added group were increased in lobular division and part of nuclei-to-cytoplasmic ratio were decreased, compared with the non-added group; in HL-60 cells, the number of nuclear lobe-increased cells is also obviously increased. The flow detection result in fig. 5 shows that, compared with the group without drugs, after the FITC-labeled mouse anti-human CD14 antibody is added, the KG-1 cell fluorescence intensity of the MLA group is obviously enhanced, which indicates that MLA can induce the expression of the mature monocyte marker CD14 on the surface of KG-1 cell; meanwhile, compared with the group without the drug, after the FITC-labeled mouse anti-human CD15 antibody is added, the fluorescence intensity of the HL-60 cells of the group with the MLA is obviously enhanced, which indicates that the MLA can induce the expression of the mature marrow cell marker CD15 on the surface of the HL-60 cells.
From the above data, it can be seen that MLA induced not only apoptosis of the high-expression AML cell lines KG-1 and HL-60 of CHRNA7 (fig. 2, fig. 3), but also differentiation thereof (fig. 4, fig. 5).
Experimental example four
CHRNA7 expression in leukemic stem cells, as shown in FIG. 6.
Materials and reagents: ficoll lymphocyte separation, PBS, mouse anti-human CD34 and CD38 antibodies, FITC labeled mouse anti-human CD38 antibody and PE labeled mouse anti-human CD34 antibody (purchased from BD Bioscience), rabbit anti-human CHRNA7 antibody, alexa Fluor647 labeled goat anti-rabbit IgG secondary antibody, 15mL sterile centrifuge tube, 1.5mL centrifuge tube, 10. Mu.L, 200. Mu.L and 1mL sterile tip.
The method comprises the following operation steps:
1. isolation of bone marrow mononuclear cells: collecting bone marrow of an acute myelogenous leukemia patient, diluting the bone marrow with PBS of the same amount, adding the bone marrow into the upper layer of a Ficoll lymphocyte separation liquid, centrifuging the mixture at 2000rpm for 20min, sucking out a mononuclear cell layer, adding the mononuclear cell layer into a new centrifuge tube, adding a proper amount of PBS containing 3% fetal calf serum, centrifuging the mixture at 1500rpm for 5min, discarding the supernatant, and resuspending the cells with PBS containing 3% fetal calf serum and counting the cells.
2、CD34 + CD38 - Sorting the cells: adding mouse anti-human CD34 antibody to the human bone marrow mononuclear cells separated in step 1, every 10 th 6 Adding 5 μ L of the extract into cells, mixing, incubating on ice for 30min, and sorting with cell sorter (BD FACSARIA II) to obtain CD34 + CD38 - The cells are Leukemia Stem Cells (LSCs).
3. Expression of CHRNA7 on the surface of leukemic stem cells: the 100 ten thousand cells separated from 2 were divided into 3 parts on average: the first cell was added with rabbit anti-human CHRNA7 antibody, incubated on ice for 2h, washed 2 times with PBS containing 3% fetal calf serum, added with 2. Mu.L of Alexa Fluor 647-labeled goat anti-rabbit IgG secondary antibody, FITC-labeled mouse anti-human CD38 antibody and PE-labeled mouse anti-human CD34 antibody, incubated on ice for 30min, added with 1ml of PBS containing 3% fetal calf serum, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were subjected to detection by an up-flow cytometer. FITC-labeled mouse anti-human CD38 antibody is added to the second cell, the cells are incubated on ice for 30min, 1ml of PBS containing 3% fetal bovine serum is added, the cells are centrifuged at 1000rpm for 5min, and then the supernatant is discarded and the cells are detected by an up-flow cytometer. PE-labeled mouse anti-human CD34 antibody in the third cell was incubated on ice for 30min, 1ml of PBS containing 3% fetal bovine serum was added, centrifuged at 1000rpm for 5min, the supernatant was discarded, and the cells were examined by flow cytometry.
The experimental results are as follows: as can be seen from the results in FIG. 6, leukemia stem cells, namely CD34 + CD38 - CHRNA7 positive cells account for about 68.62%, indicating that CHRNA7 is highly expressed in leukemia stem cells.
Experimental example five
The effect of MLA on leukemia stem cell apoptosis is shown in fig. 7, fig. 8, fig. 9 and fig. 10.
Materials and reagents: MLA solution (dissolved in DMSO to a final concentration of 10mM, purchased from MCE), RPMI 1640 medium, fetal bovine serum, annexin-V-FITC/PI apoptosis kit (purchased from BD Bioscience), FITC-labeled murine anti-human CD14 and CD15 antibodies (purchased from BD Bioscience), 15mL sterile centrifuge tubes, 1.5mL centrifuge tubes, 10. Mu.L, 200. Mu.L and 1mL sterile tips, 12-well cell culture plates.
The method comprises the following operation steps: the leukemia stem cells obtained by sorting were seeded into 12-well plates at 50X 10 6 /well, adding MLA to final concentrations of 5. Mu.M and 10. Mu.M, respectively, using DMSO-added group as a control, and 5% CO at 37% 2 Culturing for 96h in the cell culture box, collecting cells, dividing the cells into 3 parts, centrifuging the first part of cells for 5min at 1000rpm, discarding supernatant, adding 1ml of 1 × Binding Buffer into cell precipitates, re-suspending the cell precipitates, respectively adding 5 μ L of Annexin-V-FITC and PI, dyeing for 15min on ice in a dark place, and detecting by using a flow cytometer; centrifuging the second cell at 1000rpm for 5min, discarding the supernatant, smearing, air drying, dyeing for 1min by using a Ruhry-Giemsa staining solution, adding an equal amount of Ruhry-Giemsa buffer solution, uniformly mixing, dyeing for 10 to 15min, and observing the cell differentiation condition under an optical microscope; the third cell is centrifuged at 1000rpm for 5min, the supernatant is discarded, 1ml of PBS containing 3% fetal calf serum is added, the cells are averagely divided into 2 tubes, centrifuged at 1000rpm for 5min, the supernatant is discarded, 5 muL of mouse anti-human CD15-FITC antibody is respectively added, the cells are incubated on ice for 30min, 1ml of PBS containing 3% fetal calf serum is added for resuspension of the cells, centrifuged at 1000rpm for 5min, and the supernatant is discarded.
The experimental results are as follows: as can be seen from the results of FIGS. 7 and 8, the leukemia stem cell apoptosis rates were 42.22% + -4.37% and 71.11% + -3.99% respectively, which are significantly higher than those of the non-drug group (13.45% + -1.56%) when the MLA concentration was 5. Mu.M and 10. Mu.M. As can be seen from the results of FIG. 9, the number of nuclear lobes of some leukemic stem cells in the MLA-treated group was increased and the cytoplasmic ratio of some leukemic stem cells was decreased, compared to the non-treated group. The flow detection results in fig. 10 show that, compared with the group without drugs, after FITC-labeled mouse anti-human CD15 antibody is added, the fluorescence intensity of leukemia stem cells in the MLA group is significantly enhanced, indicating that MLA can induce the expression of the mature myeloid lineage cell marker CD15 on the surface of leukemia stem cells.
As can be seen from the above data, MLA induced not only apoptosis of leukemic stem cells (fig. 7, fig. 8), but also differentiation thereof (fig. 9, fig. 10).
Experimental example five
Materials and reagents: MLA solution (dissolved in DMSO to a final concentration of 50mg, purchased from MCE), RPMI 1640 medium, fetal bovine serum, cell counting plate
Experimental animals: NSG mice
The method comprises the following operation steps: culturing KG-1 cells in RPMI 1640 medium containing 10% fetal calf serum, collecting and counting the cells when they have entered the logarithmic phase, inoculating 6~8 week old NSG mice, 5X 10 6 For each cell, MLA was administered by intravenous injection (10 mg/kg) starting 3 days later, 1 time every 3 days, and the behavior, body weight, survival status, etc. of the mice were observed every day.
The experimental results are as follows: as can be seen from FIG. 11, the mean survival of the MLA-injected NSG mice was 63.5 days, which was significantly longer than that of the placebo group (mean survival of 34.5 days) and the solvent group (mean survival of 34 days) ((ii))P=0.016）。
Claims (2)
1. The application of the methyloxletin in preparing the medicine for inhibiting the proliferation of acute myeloid leukemia cells and leukemia stem cells and inducing the differentiation of the cells;
the chemical formula of the methyl oxfordine is as follows:
。
2. the use of claim 1, wherein: use of mefacitene for the manufacture of a medicament effective for the treatment of incipient wetness and relapsed refractory AML.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010065701.9A CN111233873B (en) | 2020-01-20 | 2020-01-20 | Application of methyl taurocten in preparation of drugs for inhibiting proliferation and inducing differentiation of leukemia cells and leukemia stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010065701.9A CN111233873B (en) | 2020-01-20 | 2020-01-20 | Application of methyl taurocten in preparation of drugs for inhibiting proliferation and inducing differentiation of leukemia cells and leukemia stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111233873A CN111233873A (en) | 2020-06-05 |
| CN111233873B true CN111233873B (en) | 2023-01-24 |
Family
ID=70876225
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010065701.9A Active CN111233873B (en) | 2020-01-20 | 2020-01-20 | Application of methyl taurocten in preparation of drugs for inhibiting proliferation and inducing differentiation of leukemia cells and leukemia stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111233873B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6416735B1 (en) * | 1999-11-08 | 2002-07-09 | Research Triangle Institute | Ligands for α-7 nicotinic acetylcholine receptors based on methyllcaconitine |
| CA2513433A1 (en) * | 2003-01-22 | 2004-08-05 | Pharmacia & Upjohn Company Llc | Treatment of diseases with alpha-7 nach receptor full agonists |
-
2020
- 2020-01-20 CN CN202010065701.9A patent/CN111233873B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| Variation of nicotinic subtype α7 and muscarinic subtype M3 acetylcholine receptor expression in three main types of leukemia;TAWIT SURIYO et al.;《ONCOLOGY LETTERS》;20191231;第17卷;摘要，Figure 1，第1360-1361页 * |
| 糖皮质激素对小胶质细胞内钙的影响;何舒翘等;《中国药理学通报》;20170525;第33卷(第6期);第878页摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111233873A (en) | 2020-06-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dall'Era et al. | Adoptive Treg cell therapy in a patient with systemic lupus erythematosus | |
| CN106222201B (en) | A kind of method for preparing CAR T cells and obtained CAR T cells and its application | |
| Ahmad et al. | Immunostimulatory effects of the standardized extract of Tinospora crispa on innate immune responses in Wistar Kyoto rats | |
| Saito et al. | Effect of Notch expression in glioma stem cells on therapeutic response to chemo-radiotherapy in recurrent glioblastoma | |
| Dong et al. | Antitumor effects of artesunate on human breast carcinoma MCF-7 cells and IGF-IR expression in nude mice xenografts | |
| He et al. | Atractylodin may induce ferroptosis of human hepatocellular carcinoma cells | |
| Qian et al. | Ginsenoside-Rh2 inhibits proliferation and induces apoptosis of human gastric cancer SGC-7901 side population cells | |
| CN103555666A (en) | Culture method for increasing amplification efficiency and activity of Vgamma9Vdelta2T cells | |
| Pei et al. | α-Cyperone inhibits the proliferation of human cervical cancer HeLa cells via ROS-mediated PI3K/Akt/mTOR signaling pathway | |
| Gao et al. | Solanine inhibits immune escape mediated by hepatoma Treg cells via the tgfβ/smad signaling pathway | |
| Yan et al. | Effects of wogonoside on invasion and migration of lung cancer A549 cells and angiogenesis in xenograft tumors of nude mice | |
| Wang et al. | EV71-infected CD14+ cells modulate the immune activity of T lymphocytes in rhesus monkeys | |
| CN111233873B (en) | Application of methyl taurocten in preparation of drugs for inhibiting proliferation and inducing differentiation of leukemia cells and leukemia stem cells | |
| CN106551945A (en) | Purposes of the Calycosin-7-O-BETA-D-glucoside in asthmatic medicament is prepared | |
| Wu et al. | Downregulation of AKT/mTOR signaling pathway for Salmonella-mediated autophagy in human anaplastic thyroid cancer | |
| Dash et al. | Stimulation of immune function activity by the alcoholic root extract of Heracleum nepalense D. Don | |
| CN109875998B (en) | Application of amide alkaloid in preparation of tumor chemotherapy drug sensitizer and anti-tumor drug composition | |
| CN113444765A (en) | Drug-resistant breast cancer treatment drug and screening method thereof | |
| CN106834226A (en) | A kind of method of modifying of ion vitro immunization cell preparation and its application | |
| CN111053773A (en) | Chemical small molecule for inhibiting proliferation and inducing differentiation of leukemia cells and leukemia stem cells | |
| CN113025571B (en) | Treatment method of human peripheral blood T lymphocytes, immunogen preparation and application | |
| CN104622864A (en) | Application of chlorogenic acid in preparing drugs for prevention and treatment of primary cutaneous T-cell lymphoma | |
| Ghazvinian et al. | Chemo-immune cell therapy by intratumoral injection of adoptive NK cells with capecitabine in gastric cancer xenograft model | |
| Dlouhy et al. | Interleukin-1 receptor associated kinase 1/4 and bromodomain and extra-terminal inhibitions converge on NF-κB blockade and display synergistic antitumoral activity in activated B-cell subset of diffuse large B-cell lymphoma with MYD88 L265P mutation | |
| CN109078188A (en) | A kind of action target spot and anti-tumor drug of anti-tumor drug |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |