CN103908452A - Composite drug and medicinal preparation for inhibiting lung cancer cell metastasis and detection method for lung cancer cell metastasis - Google Patents
Composite drug and medicinal preparation for inhibiting lung cancer cell metastasis and detection method for lung cancer cell metastasis Download PDFInfo
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- CN103908452A CN103908452A CN201410055267.0A CN201410055267A CN103908452A CN 103908452 A CN103908452 A CN 103908452A CN 201410055267 A CN201410055267 A CN 201410055267A CN 103908452 A CN103908452 A CN 103908452A
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Abstract
The invention provides a composite drug and a medicinal preparation for inhibiting lung cancer cell metastasis and a detection method for lung cancer cell metastasis. The method comprises the following steps: selecting lung cancer cells in an exponential growth phase and treating the lung cancer cells with the composite drug composed of a component 1 and a component 2 so as to obtain a plurality of contrast samples, wherein the concentrations of the component 1 of the composite drug in the plurality of contrast samples are different, the component 1 is 3-[2,4-bis((3S)-3-methylmorpholine-4-yl)pyridino-[5,6-e]pyrimidine-7-yl]-N-methylbenzamide, the component 2 is ABT737, and the component 1 functions as an inhibitor for the mammalian target of rapamycin in the process of lung cancer cell metastasis; digesting the lung cancer cells with pancreatin and resuspending the cells with a medium containing no fetal calf serum; carrying out cell counting and diluting a cell suspension after counting; and adding the cell suspension into a Transwell cabin, adding a medium containing fetal calf serum into a culture hole below the Transwell cabin, carrying out culture in a 5% carbon dioxide incubator at a temperature of 37 DEG C, then carrying out dyeing with methylrosanilinium chloride and microscopically shooting and detecting metastasis of the lung cancer cells.
Description
Technical field
The present invention relates to medical treatment and field of medicaments, particularly relate to a kind of composition of medicine that lung carcinoma cell shifts that suppresses, a kind of pharmaceutical preparation of lung carcinoma cell transfer and detection method that a kind of lung carcinoma cell shifts of suppressing.
Background technology
Pulmonary carcinoma is a kind of common pulmonary malignant tumour, malignant tumor is the cellularity disease of harm humans life and health, tumor cell is converted into malignant cell from normal cell in organism, all can there is profound change in its form, function, metabolism and propagation etc., its that can be regarded as that the abnormal differentiation of cell causes has immortality.
Most pulmonary carcinoma originates from bronchial mucosa epithelium, in recent years, along with the impact of smoking and various environmental factorss, its mortality rate of M & M increase year after year of pulmonary carcinoma has accounted for first of cancer mortality, countries in the world are industrially developed country particularly, the sickness rate of pulmonary carcinoma and case fatality rate all rise rapidly, die from pulmonary carcinoma in the male patient of carninomatosis and rank first.At present, on market, do not have extraordinary medicine inhibition tumor cell to shift.How the unlimited transfer ability of inhibition tumor cell, is a matter of utmost importance in oncotherapy.
Summary of the invention
Technical problem to be solved by this invention is to provide the mechanism that a kind of novel inhibition lung carcinoma cell shifts.
In order to address the above problem, the invention discloses a kind of composition of medicine that lung carcinoma cell shifts that suppresses, described lung carcinoma cell is selected from non-small cell lung cancer cell strain A549, described composition of medicine is by 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl]-N-methyl-benzamide and ABT737 composition, described 3-[2, two ((3S)-3-methyl morpholine-4-yl) pyrido [5, the 6-e] pyrimidin-7-yls of 4-]-N-methyl-benzamide in the transfer process of lung carcinoma cell as the inhibitor of mammal rapamycin target protein.
Preferably, 3-[2 described in described composition of medicine, two ((3S)-3-methyl morpholine-4-yl) pyrido [5,6-e] pyrimidin-7-yls of 4-] molar concentration rate of-N-methyl-benzamide and described ABT737 is 1:1.
The invention also discloses a kind of pharmaceutical preparation that lung carcinoma cell shifts that suppresses, to suppress composition of medicine that lung carcinoma cell shifts as active component, and be aided with acceptable with carrier;
Described lung carcinoma cell is selected from non-small cell lung cancer cell strain A549;
Described composition of medicine is by 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl]-N-methyl-benzamide and ABT737 composition, described 3-[2, two ((3S)-3-methyl morpholine-4-yl) pyrido [5, the 6-e] pyrimidin-7-yls of 4-]-N-methyl-benzamide in the transfer process of lung carcinoma cell as the inhibitor of mammal rapamycin target protein.
Preferably, 3-[2 described in described composition of medicine, two ((3S)-3-methyl morpholine-4-yl) pyrido [5,6-e] pyrimidin-7-yls of 4-] molar concentration rate of-N-methyl-benzamide and described ABT737 is 1:1.
Preferably, described preparation is oral liquid, soft gelatin capsule, oral administration mixed suspension, solid dispersion, capsule, slow releasing agent, granule, tablet, injection, ointment, patch or implant.
The invention also discloses the detection method that a kind of lung carcinoma cell shifts, described lung carcinoma cell is selected from non-small cell lung cancer cell strain A549, and described method comprises:
Select the lung carcinoma cell of exponential phase of growth, adopt the composition of medicine being formed by component 1 and component 2 to process and form many group comparative sample described lung carcinoma cell, wherein, the concentration difference of the component 1 of many group samples, the concentration difference of the component 2 of many group samples, described component 1 is 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl]-N-methyl-benzamide, described component 2 is ABT737, described component 1 in the transfer process of lung carcinoma cell as the inhibitor of mammal rapamycin target protein;
Adopt lung carcinoma cell described in trypsinization, and with the culture medium re-suspended cell that does not contain hyclone;
Carry out cell counting, after counting, cell suspension is diluted;
Cell suspension is added to Transwell cell, and add containing the culture medium of hyclone to the culture hole of Transwell cell below, after cultivating in 37 DEG C and 5% CO2 gas incubator, with violet stain, under microscope, take pictures and detect the transfer of described lung carcinoma cell.
Preferably, 3-[2 described in described composition of medicine, two ((3S)-3-methyl morpholine-4-yl) pyrido [5,6-e] pyrimidin-7-yls of 4-] molar concentration rate of-N-methyl-benzamide and described ABT737 is 1:1.
Compared with prior art, the present invention includes following advantage:
According to the embodiment of the present invention, adopt 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl] composition of medicine of-N-methyl-benzamide and ABT737 composition acts on the lung carcinoma cell that is selected from non-small cell lung cancer cell strain A549, checking by experiment, the present invention innovates after the composition of medicine drug combination of proposition than two kinds of medicine individual processing, and its transfer ability to lung carcinoma cell has remarkable coordinate repression.
Brief description of the drawings
Fig. 1 is the flow chart of the detection embodiment of a kind of lung carcinoma cell transfer of the present invention;
Fig. 2 be variable concentrations in example of the present invention composition of medicine to lung carcinoma cell shift testing result.
Detailed description of the invention
For above-mentioned purpose of the present invention, feature and advantage can be become apparent more, below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
The ultimate principle of cell migration (cell migration) is that cell is receiving migration signal or experiencing the movement producing after the Concentraton gradient of Cucumber.In transition process, cell is constantly repeating forwards to stretch out prominent foot, the then cyclic process of tractive cell space.Cancer cell metastasis refers to shift and refers to that tumor cell invades lymphatic vessel from original site, blood vessel or other are by way of being brought to its place's continued growth, form the tumor with original site tumor same type, there is great harm, in the direction for the treatment of pulmonary carcinoma, how to suppress lung carcinoma cell and shift, just become a key issue in oncotherapy.
The invention provides a kind of composition of medicine that lung carcinoma cell shifts that suppresses, described lung carcinoma cell is selected from non-small cell lung cancer cell strain A549, described composition of medicine is by 3-[2, two ((3S)-3-methyl morpholine-4-yl) pyrido [5, the 6-e] pyrimidin-7-yls of 4-]-N-methyl-benzamide and ABT737 composition.
A549 cell line is to transplant culture by D.J.Griad by cancerous lung tissue, and patient is 58 years old white man male.A549 can be by the synthetic lecithin that contains high-load unsaturated fatty acid of cytidine diphosphocholine approach.24% cell is hypo-triploid cell, contains 66 chromosomes, and, producing 64,65,67 somatic frequencies of dyeing also very high, most cells all has 2 X and 2 Y chromosomes.
Wherein, 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl]-N-methyl-benzamide is AZD2014, mTOR(ammalian target of rapamycin, mammal rapamycin target protein) complex mTORC1(ammalian target of rapamycin complex1) and mTORC2(ammalian target of rapamycin complex2) double inhibitor, there is potential anti-tumor activity amine.ABT737 is R-(-) enantiomer of gossypol acetic acid (Gossypol acetic acid), with Bcl-2 (B cell lymphoma/lewkmia-2B cell lymphoma/leukemia-2,), Bcl-xL (the Bcl-2-like survival factors) and Mcl-1 (Myeloid cell leukemia-1, myelocytic leukemia gene-1) combination.ABT737 is 4-[4-[(4'-Chloro[1,1'-biphenyl]-2-yl) methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio) methyl] propyl] amino]-3-nitrophenyl] sulfonyl]-benzamide, it is a kind BH3 inhibitor, can suppress Bcl-xL, Bcl-2 and Bcl-w simultaneously.
In the embodiment of the present invention, preferably, 3-[2 described in described composition of medicine, two ((3S)-3-methyl morpholine-4-yl) pyrido [5, the 6-e] pyrimidin-7-yls of 4-] molar concentration rate of-N-methyl-benzamide and described ABT737 is 1:1.
Accordingly, the present invention also provides a kind of pharmaceutical preparation that lung carcinoma cell shifts that suppresses, and to suppress composition of medicine that lung carcinoma cell shifts as active component, and is aided with the acceptable carrier of using.
Wherein, described lung carcinoma cell is selected from non-small cell lung cancer cell strain A549, described composition of medicine is by 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl]-N-methyl-benzamide and ABT737 composition, described 3-[2, two ((3S)-3-methyl morpholine-4-yl) pyrido [5, the 6-e] pyrimidin-7-yls of 4-]-N-methyl-benzamide in the transfer process of lung carcinoma cell as the inhibitor of mammal rapamycin target protein.
In the embodiment of the present invention, preferably, 3-[2 described in described composition of medicine, two ((3S)-3-methyl morpholine-4-yl) pyrido [5, the 6-e] pyrimidin-7-yls of 4-] molar concentration rate of-N-methyl-benzamide and described ABT737 is 1:1.
In the embodiment of the present invention, preferably, described preparation can be any one among oral liquid, soft gelatin capsule, oral administration mixed suspension, solid dispersion, capsule, slow releasing agent, granule, tablet, injection, ointment, patch or implant.
Accordingly, the detection method that the present invention also provides a kind of lung carcinoma cell to shift, described lung carcinoma cell is selected from non-small cell lung cancer cell strain A549, and described method comprises:
Step 101, select the lung carcinoma cell of exponential phase of growth, adopt the composition of medicine being formed by component 1 and component 2 to process and form many group comparative sample described lung carcinoma cell, wherein, the concentration difference of the component 1 of many group samples, the concentration difference of the component 2 of many group samples, described component 1 is 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl]-N-methyl-benzamide, described component 2 is ABT737, described component 1 in the transfer process of lung carcinoma cell as the inhibitor of mammal rapamycin target protein.
In the embodiment of the present invention, first choose lung carcinoma cell from non-small cell lung cancer cell strain A549, select the lung carcinoma cell of exponential phase of growth.
In the embodiment of the present invention, adopt composition of medicine to process lung carcinoma cell, and the transfer of lung carcinoma cell is detected, composition of medicine is made up of component 1 and component 2, component 1 is 3-[2, two ((3S)-3-methyl morpholine-4-yl) pyrido [5, the 6-e] pyrimidin-7-yls of 4-]-N-methyl-benzamide, component 2 is ABT737, component 1 in the transfer process of lung carcinoma cell as the inhibitor of mammal rapamycin target protein.
The effect of checking composition of medicine, can adopt lung carcinoma cell described in the sample treatment of multiple various combination, in many group samples, the concentration difference of component 1, the concentration of component 1 is also different respectively, thereby form comparison, can show that the addition of component 1 and component 2 is on suppressing the impact of proliferation of lung cancer cells effect.
Step 102, adopt lung carcinoma cell described in trypsinization, and with the culture medium re-suspended cell that does not contain hyclone.
Adopt trypsinization lung carcinoma cell, and further adopt the not culture medium re-suspended cell containing hyclone (FBS).
Step 103, carry out cell counting, after counting, cell suspension is diluted.
Further can also carry out cell counting, after counting, cell suspension be diluted, final concentration maintains finite concentration.
Step 104, cell suspension is added to Transwell cell, and add containing the culture medium of hyclone to the culture hole of Transwell cell below, after cultivating in 37 DEG C and 5% CO2 gas incubator, with violet stain, under microscope, take pictures and detect the transfer of described lung carcinoma cell.
The meaning such as this root of Trans-has transfer, transhipment, pass, well has the meaning of cell, can understanding from literal, this is the device that a class has the cup-shaped of permeability, according to the introduction in the Transwell description of Corning company, can think that this is a kind of membrane filter (Membrane filters), also can think a kind of support (permeable supports) that has permeability.Transwell cell is put into culture plate, in cell, deserve to be called chamber, claim lower chamber in culture plate, upper indoor splendid attire upper strata culture fluid, lower indoor splendid attire lower floor culture fluid, levels culture fluid is separated by with polycarbonate membrane.We are by cell kind upper indoor, and because polycarbonate membrane has permeability, the composition in lower floor's culture fluid can have influence on indoor cell, thereby can study the impact of composition cell growth, motion etc. in lower floor's culture fluid.
Cell suspension is added to after Transwell cell, add the culture medium that contains hyclone (FBS) to the culture hole of Transwell cell below, and cultivate in 37 DEG C and 5% CO2 gas incubator, cultivate after a period of time, can adopt alcohol fixation, then use violet stain, under microscope, take pictures and detect the transfer of described lung carcinoma cell.
In order to make those skilled in the art understand better the present invention, detection method lung carcinoma cell of the present invention being shifted by concrete example describes.
The present invention processes A549 cell strain by selecting the concentration of ABT737 to be respectively 0,1,2,5,10,15,20,30 and 40 μ mol/L early stage, select the concentration of AZD2014 to be respectively 0,2,5,10,15,20 and 30 μ mol/L processing A549 cell strains, the processing time is respectively 24h and 48h.
By MTT(3-(4,5-dimethylthiazole-2)-2,5-diphenyl tetrazole bromine salt) analyze, selecting respectively ABT737 is the concentration of 20 μ mol/L, AZD201420 μ mol/L.(MTT colorimetry is a kind of method that detects cell survival and growth.Its detection principle is that the succinate dehydrogenase in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) energy dissolved cell, measures its absorbance value with enzyme-linked immunosorbent assay instrument at 490nm wavelength place, can indirectly reflect living cells quantity.MTT result shows that the associating inhibition index of these two kinds of medicines is less than 1 in the time that ABT737 is 20 μ mol/L, AZD201420 μ mol/L, so select these two combined concentration to carry out subsequent experimental.
Select the A549 cell of exponential phase of growth, plant 6 orifice plates, cell density is about 70%, after cell attachment, uses respectively these two kinds of medicine individual processing and Combined Treatment A549 cell.
Process after 24h, with 0.25% trypsin digestion cell, and with the 1640 culture medium re-suspended cells that do not contain FBS, carry out respectively cell counting, after counting, cell suspension is diluted, final concentration is 1 × 105/ml.
Add 100 μ L cell suspension to Transwell cell, and add 250 μ L containing 1640 culture medium of 10%FBS to the culture hole of Transwell cell below, be placed in 37 DEG C, in 5%CO2 incubator, cultivate, after 48h, 75% alcohol fixation, then use violet stain, the observation of taking pictures under microscope.
Fig. 2 be variable concentrations in example composition of medicine to lung carcinoma cell shift testing result.Wherein, concentration comprises: the concentration of ABT737 is 0uM, and the concentration of AZD2014 is 0uM; The concentration of ABT737 is 20uM, and the concentration of AZD2014 is 0uM; The concentration of ABT737 is 0uM, and the concentration of AZD2014 is 20uM; The concentration of ABT737 is 20uM, and the concentration of AZD2014 is 20uM.
The ultimate principle of Transwell is that transwell cell is put into culture plate, is called chamber in cell, is called lower chamber in culture plate, upper indoor interpolation upper strata culture fluid, and lower indoor interpolation lower floor culture fluid, levels culture fluid is separated by with film.What in figure, show is to process after lung cell A549 with the composition of medicine of variable concentrations, A549 is through the number of the little ventricular cell of Transwell, in figure, show, the number that cell of treated with medicaments does not pass the little ventricular cell of Transwell is in 170 left and right, after only processing with 20uM ABT737, pass Transwell cell A549 cell number approximately 135, after only processing with 20uM AZD2014, pass Transwell cell A549 cell number 95 left and right, after 20uM ABT737 and 20uMAZD2014 Combined Treatment, be 40 left and right through Transwell cell A549 cell number, drug combination, , after drug treating, be obviously less than independent medication cell number after treatment through Transwell cell A549 cell number.
In sum, after the ABT737 of the above-mentioned testing result demonstration embodiment of the present invention and AZD2014 drug combination, than two kinds of medicine individual processing, its transfer ability to lung cell A549 has remarkable coordinate repression.
According to the embodiment of the present invention, adopt 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl] composition of medicine of-N-methyl-benzamide and ABT737 composition acts on the lung carcinoma cell that is selected from non-small cell lung cancer cell strain A549, checking by experiment, the present invention innovates after the composition of medicine drug combination of proposition than two kinds of medicine individual processing, and its transfer ability to lung carcinoma cell has remarkable coordinate repression.
For embodiment of the method, for simple description, therefore it is all expressed as to a series of combination of actions, but those skilled in the art should know, the present invention is not subject to the restriction of described sequence of movement, because according to the present invention, some step can adopt other orders or carry out simultaneously.Secondly, those skilled in the art also should know, the embodiment described in description all belongs to preferred embodiment, and related action and parts might not be that the present invention is necessary.
Above to a kind of composition of medicine that lung carcinoma cell shifts that suppresses provided by the present invention, a kind of suppress lung carcinoma cell shift pharmaceutical preparation and a kind of lung carcinoma cell shift detection method be described in detail, applied specific case herein principle of the present invention and embodiment are set forth, the explanation of above embodiment is just for helping to understand method of the present invention and core concept thereof; , for one of ordinary skill in the art, according to thought of the present invention, all will change in specific embodiments and applications, in sum, this description should not be construed as limitation of the present invention meanwhile.
Claims (7)
1. one kind is suppressed the composition of medicine that lung carcinoma cell shifts, it is characterized in that, described lung carcinoma cell is selected from non-small cell lung cancer cell strain A549, described composition of medicine is by 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl]-N-methyl-benzamide and ABT737 composition, described 3-[2, two ((3S)-3-methyl morpholine-4-yl) pyrido [5, the 6-e] pyrimidin-7-yls of 4-]-N-methyl-benzamide in the transfer process of lung carcinoma cell as the inhibitor of mammal rapamycin target protein.
2. composition of medicine as claimed in claim 1, it is characterized in that, 3-[2 described in described composition of medicine, two ((3S)-3-methyl morpholine-4-yl) pyrido [5,6-e] pyrimidin-7-yls of 4-] molar concentration rate of-N-methyl-benzamide and described ABT737 is 1:1.
3. suppress the pharmaceutical preparation that lung carcinoma cell shifts, it is characterized in that, to suppress composition of medicine that lung carcinoma cell shifts as active component, and be aided with acceptable with carrier;
Described lung carcinoma cell is selected from non-small cell lung cancer cell strain A549;
Described composition of medicine is by 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl]-N-methyl-benzamide and ABT737 composition, described 3-[2, two ((3S)-3-methyl morpholine-4-yl) pyrido [5, the 6-e] pyrimidin-7-yls of 4-]-N-methyl-benzamide in the transfer process of lung carcinoma cell as the inhibitor of mammal rapamycin target protein.
4. pharmaceutical preparation as claimed in claim 3, it is characterized in that, 3-[2 described in described composition of medicine, two ((3S)-3-methyl morpholine-4-yl) pyrido [5,6-e] pyrimidin-7-yls of 4-] molar concentration rate of-N-methyl-benzamide and described ABT737 is 1:1.
5. pharmaceutical preparation as claimed in claim 3, is characterized in that, described preparation is oral liquid, soft gelatin capsule, oral administration mixed suspension, solid dispersion, capsule, slow releasing agent, granule, tablet, injection, ointment, patch or implant.
6. the detection method that lung carcinoma cell shifts, is characterized in that, described lung carcinoma cell is selected from non-small cell lung cancer cell strain A549, and described method comprises:
Select the lung carcinoma cell of exponential phase of growth, adopt the composition of medicine being formed by component 1 and component 2 to process and form many group comparative sample described lung carcinoma cell, wherein, the concentration difference of the component 1 of many group samples, the concentration difference of the component 2 of many group samples, described component 1 is 3-[2, two ((the 3S)-3-methyl morpholine-4-yl) pyridos [5 of 4-, 6-e] pyrimidin-7-yl]-N-methyl-benzamide, described component 2 is ABT737, described component 1 in the transfer process of lung carcinoma cell as the inhibitor of mammal rapamycin target protein;
Adopt lung carcinoma cell described in trypsinization, and with the culture medium re-suspended cell that does not contain hyclone;
Carry out cell counting, after counting, cell suspension is diluted;
Cell suspension is added to Transwell cell, and add containing the culture medium of hyclone to the culture hole of Transwell cell below, after cultivating in 37 DEG C and 5% CO2 gas incubator, with violet stain, under microscope, take pictures and detect the transfer of described lung carcinoma cell.
7. combined method as claimed in claim 6, it is characterized in that, 3-[2 described in described composition of medicine, two ((3S)-3-methyl morpholine-4-yl) pyrido [5,6-e] pyrimidin-7-yls of 4-] molar concentration rate of-N-methyl-benzamide and described ABT737 is 1:1.
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Cited By (1)
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| CN109294990A (en) * | 2018-10-23 | 2019-02-01 | 昆明医科大学第附属医院 | A kind of cell model method for building up of simulated lung carcinogenesis mechanism and application |
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