In-vitro cytotoxicity analysis method based on combination of cis-platinum and ciclopirox olamine

Abstract

The invention discloses an in-vitro cytotoxicity analysis method based on combination of cis-platinum and ciclopirox olamine. The method comprises the following steps: step A1, preparing a test reagent; the test reagent is a cis-platinum combined ciclopirox olamine solution; the concentration ratio of cis-platinum to ciclopirox olamine in the cis-platinum and ciclopirox olamine combined solution is 1: 10; a2, inoculating and culturing the cells; the cells comprise human normal liver cells, human umbilical vein endothelial cells, breast cancer cells, cervical cancer cells, colon cancer cells and non-small cell lung cancer; and step A3, MTT cytotoxicity detection to finally obtain the optimal concentration ratio. The cisplatin and ciclopirox olamine compound has the advantages that the cisplatin and the ciclopirox olamine are mixed according to different concentrations, the cell toxicity is analyzed by using an MTT method, a cell line most sensitive to the two drugs is screened out, the combined drug effect of the drugs is analyzed by using a Chou-Tallay method, and finally the optimal concentration ratio that more cancer cells can be killed while normal cells are not damaged as far as possible is obtained. Therefore, the optimal scheme of drug combination is determined.

Description

Technical Field

[0001] The present invention relates to a method for analyzing in vitro cytotoxicity, in particular to a method for analyzing in vitro cytotoxicity based on cisplatin combined with ciclopirox olamine, and belongs to the technical field of anti-cancer drug preparation. Background Art

[0002] With the development of science and technology, new anti-tumor drugs have emerged continuously; although anti-tumor drugs have made great progress clinically, a series of tumor drugs still have strong side effects on the human body. Making the cell signal transduction pathways of tumor cell differentiation and proliferation as the target has become the focus of research and development of anti-tumor drugs.

[0003] Due to the existence of chloride ion groups in traditional anti-cancer drug cisplatin, a large amount of oxygen free radicals will be generated during the hydration metabolism process, which will cause damage to the normal body to a great extent. Cisplatin, as the first-generation platinum-based anti-tumor drug, has a divalent platinum at its structural center, connecting two chlorine groups, and at the same time having two ligands as amino groups, which has an important impact on the generation of oxygen free radicals. The activity of cisplatin mainly depends on the concentration of chloride ion accumulation caused by it in cells. After cisplatin is injected intravenously, it can reach the vicinity of cells, and its Cl - groups are rapidly hydrated in cells, migrate to the DNA in the nucleus, crosslink with the purine bases on the DNA, deform the DNA strand, thereby destroying or restricting the unwinding, replication and transcription of the DNA strand, and achieving the purpose of inhibiting the proliferation of tumor cells. Cisplatin can quickly enter tissues and is mainly distributed in the liver, prostate, kidney and other parts, but it cannot pass through the blood-brain barrier, so the therapeutic effect on brain metastasis lesions is poor. Cisplatin is a cell cycle non-specific drug, so it also has cytotoxicity to normal cells and shows relatively strong toxic and side effects clinically.

[0004] Ciclopirox olamine is a broad-spectrum antifungal drug of pyridone, which is different from other known antifungal drugs such as azoles and allylamines in terms of structure and mechanism of action. The antibacterial mechanism of ciclopirox olamine is mainly to inhibit fungi by changing the integrity of the fungal cell membrane, causing the outflow of intracellular substances and blocking the uptake of protein precursors.

[0005] The MTT (thiazolyl blue) method is a method widely used to detect cell survival and growth. Its detection principle is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan and deposit in cells, while dead cells do not have this function. Dissolving formazan in cells with dimethyl sulfoxide and measuring its light absorption value can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystal formation is proportional to the number of cells. Compared with the neutral red method, this method can omit some washing steps, has high sensitivity and is easy to operate.

[0006] At present, the prior art discloses a Chinese invention patent with the publication number CN111956807B, entitled Preparation of a Portulaca Oleracea Polysaccharide and Cisplatin Complex and Study on Its Synergistic Antitumor Activity, which specifically discloses the determination of the cell half-lethal number (IC50) by the MTT method. 50 ), and its immune effect was determined by cell immunofluorescence staining. The POP-ALN-CIS-Pt conjugate prepared by it has mild synthesis conditions and can be easily repeated in the preparation process. It can inhibit the growth of cervical cancer cells and has an immunomodulatory effect on them. However, there is no systematic determination of the therapeutic effect of the POP-ALN-CIS-Pt conjugate on other cancer cells. Therefore, it is urgent to develop an in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an optimal concentration ratio that can kill more cancer cells while minimizing the damage to normal cells, thereby determining an in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine for drug combination.

[0008] In order to solve the above technical problems, the in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine of the present invention comprises the following steps:

[0009] Step A1: preparing test reagents;

[0010] The test reagent is a cisplatin combined with ciclopirox olamine solution; the concentration ratio of cisplatin to ciclopirox olamine in the cisplatin combined with ciclopirox olamine solution is 1:10;

[0011] Step A2: Cell inoculation and culture;

[0012] The cells include normal human liver cells, human umbilical vein endothelial cells, breast cancer cells, cervical cancer cells, colon cancer cells and non-small cell lung cancer;

[0013] Step A3: MTT cytotoxicity assay;

[0014] In this step, the cytotoxicity of various cancer cells in vitro was analyzed to screen out the cell lines most sensitive to the two drugs, and the combined efficacy of the drugs was analyzed by the Chou-Talalay method to finally obtain the optimal concentration ratio.

[0015] The steps of cell inoculation and culture in step A2 are as follows:

[0016] The amplified and cultured human normal hepatocytes, human umbilical vein endothelial cells, breast cancer cells, cervical cancer cells, colon cancer cells, and non-small cell lung cancer cells in the logarithmic growth phase were digested with trypsin solution and centrifuged to obtain cell suspensions. After counting, they were respectively inoculated into 96-well cell culture plates and incubated in a constant temperature incubator for 24 h, and 100 μL of test reagent was added to each well.

[0017] The preparation method of the trypsin solution is as follows:

[0018] Weigh 0.1 - 0.3 g of trypsin, add 200 - 220 mL of phosphate buffered saline solution, stir in an ice bath for 30 - 35 min, sterilize with a 0.2 - 0.22 μm microporous filter, aliquot, and store at -20 °C; the dosage ratio of trypsin to phosphate buffered saline solution is 0.1 - 0.3 g:200 - 220 mL.

[0019] The preparation method of the phosphate buffered saline solution is as follows:

[0020] Take KC1, KH 2 PO 4 、NaC1 and Na 2 HPO 4 ·12H 2 O, add ultrapure water, adjust the pH to 6.9 - 7.2, and sterilize by autoclaving to obtain the phosphate buffered saline solution;

[0021] The dosage ratio of KC1, KH 2 PO 4 、NaC1、Na 2 HPO 4 ·12H 2 O and ultrapure water is 0.2 - 0.4 g:0.2 - 0.4 g:8 - 10 g:2.5 - 3.5 g:1 L.

[0022] Each well in the 96-well cell culture plate contains 1×10 4 cells; 0.1 mL / well of cell culture medium is added to the 96-well cell culture plate; the cell culture medium is RPMI - 1640 culture medium supplemented with 10% FBS; the environmental conditions of the constant temperature incubator are a temperature of 37 °C and containing 5% CO 2 .

[0023] The steps of MTT cytotoxicity detection in step A3 are as follows:

[0024] Directly add MTT solution to the cell culture plate, incubate in an incubator for 3 - 5 h, then discard the solution in the culture plate, add dimethyl sulfoxide solution, place it in a microplate shaker and shake for 10 - 20 min, and detect the absorbance value of each well at a wavelength of 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the IC 50Values ​​and CI values.

[0025] The amount of the MTT solution is 20 μL / well; the amount of the dimethyl sulfoxide solution is 150 μL / well.

[0026] The preparation method of the MTT solution is as follows:

[0027] Weigh MTT, put it into a small beaker, add phosphate buffered saline solution and stir on an electromagnetic stirrer for 30-35 minutes, sterilize with a 0.2-0.22 μm microporous filter, divide into portions, and store at -20°C.

[0028] The dosage ratio of the MTT and phosphate buffered saline solution is 250-255 mg: 50-55 mL.

[0029] The preparation method of the phosphate buffered saline solution is as follows:

[0030] Take KC1, KH 2 PO 4 , NaCl and Na 2 HPO 4 12H 2 O, add ultrapure water, adjust the pH to 6.9-7.2, and sterilize by high pressure to obtain a phosphate buffered saline solution; the KCl, KH 2 PO 4 、NaCl、Na 2 HPO 4 12H 2 The usage ratio of O and ultrapure water is 0.2-0.4g:0.2-0.4g:8-10g:2.5-3.5g:1L.

[0031] The advantages of the present invention are:

[0032] By mixing cisplatin and ciclopirox at different concentrations, the cytotoxicity of various cancer cells in vitro was analyzed using the MTT method, the cell lines most sensitive to the two drugs were screened, and the combined efficacy of the drugs was analyzed by the Chou-Talalay method. Ultimately, the optimal concentration ratio that can kill more cancer cells while minimizing damage to normal cells was obtained, thereby determining the best drug combination plan. DETAILED DESCRIPTION

[0033] The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine of the present invention is further described in detail below in conjunction with specific embodiments.

[0034] The sources and properties of some of the raw materials used in the present invention are as follows:

[0035] Cisplatin was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; ciclopirox olamine was purchased from Hubei Resentek Bio-Technology Co., Ltd.; trypsin was purchased from Beijing Solarbio Science & Technology Co., Ltd.; FBS was purchased from Absin (Shanghai) BioScience Co., Ltd.; RPMI-1640 culture medium was purchased from Shanghai Guduo Bio-Technology Co., Ltd.; MTT was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0036] Example 1:

[0037] The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine in this example includes the following steps:

[0038] S1. Preheat the normal saline to 60°C, slowly add cisplatin powder, and stir to obtain a cisplatin solution with a concentration of 100 μM. Add ciclopirox olamine to methanol and stir to obtain a ciclopirox olamine solution with a concentration of 100 μM. Mix the cisplatin solution and the ciclopirox olamine solution to obtain a cisplatin combined with ciclopirox olamine solution, where the concentration ratio of the cisplatin solution to the ciclopirox olamine solution in the cisplatin combined with ciclopirox olamine solution is 1:1;

[0039] S2. Take 0.2 g of KC1, 0.2 g of KH 2 PO 4 4, 8 g of NaC1 and 2.5 g of Na 2 2HPO 4 4·12H 2 2O, add 1 L of ultrapure water, adjust the pH to 6.9, and autoclave to obtain a phosphate buffered saline solution;

[0040] S3. Weigh 0.1 g of trypsin, add it to 200 mL of phosphate buffered saline solution, stir in an ice bath for 30 min, sterilize it with a 0.2 μm microporous filter, aliquot it, and store it at -20°C to obtain a trypsin solution;

[0041] S4. Weigh 250 mg of MTT, put it into a small beaker, add 50 mL of phosphate buffered saline solution, stir on a magnetic stirrer for 30 min, sterilize it with a 0.2 μm microporous filter, aliquot it, and store it at -20°C to obtain an MTT solution;

[0042] S5. The amplified and cultured human normal liver cells, human umbilical vein endothelial cells, breast cancer cells, cervical cancer cells, colon cancer cells and non-small cell lung cancer cells in the logarithmic growth phase are digested and centrifuged with trypsin solution respectively to obtain cell suspensions. After counting, they are respectively inoculated into 96-well cell culture plates so that each well has 1×10 4Cells were placed in an incubator at a constant temperature for 24 h, and 100 μL of cisplatin combined with ciclopirox olamine solution was added to each. Among them, 0.1 mL / well of cell culture medium was added to the 96-well cell culture plate. The cell culture medium was RPMI-1640 culture medium supplemented with 10% FBS. The environmental conditions of the incubator were a temperature of 37 °C and containing 5% CO 2 ;

[0043] S6. 20 μL / well of MTT solution was directly added to the 96-well cell culture plate. After incubation in the incubator for 4 h, the solution in the culture plate was discarded, 150 μL / well of dimethyl sulfoxide solution was added, and it was placed in a microplate oscillator and shaken for 15 min. The absorbance value of each well at a wavelength of 490 nm was measured on an enzyme-linked immunosorbent assay (ELISA) reader, and the IC 50 value was calculated.

[0044] Example 2:

[0045] The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine in this example includes the following steps:

[0046] S1. Normal saline was preheated to 62 °C, cisplatin powder was slowly added and stirred to obtain a cisplatin solution with a concentration of 200 μM. Ciclopirox olamine was added to methanol and stirred to obtain a ciclopirox olamine solution with a concentration of 200 μM. The cisplatin solution and the ciclopirox olamine solution were mixed to obtain a cisplatin combined with ciclopirox olamine solution, where the concentration ratio of the cisplatin solution to the ciclopirox olamine solution in the cisplatin combined with ciclopirox olamine solution was 1:1;

[0047] S2. 0.3 g of KC1, 0.3 g of KH 2 PO 4 , 9 g of NaC1 and 2.8 g of Na 2 HPO 4 ·12H 2 O were taken, 1 L of ultrapure water was added, the pH was adjusted to 7, and it was autoclaved to obtain phosphate buffered saline (PBS);

[0048] S3. 0.2 g of trypsin was weighed, added to 204 mL of phosphate buffered saline and stirred in an ice bath for 32 min, sterilized with a 0.21 μm microporous filter, aliquoted, and stored at -20 °C to obtain trypsin solution;

[0049] S4. 252 mg of MTT was weighed, placed in a small beaker, 52 mL of phosphate buffered saline was added and stirred on a magnetic stirrer for 32 min, sterilized with a 0.21 μm microporous filter, aliquoted, and stored at -20 °C to obtain MTT solution;

[0050] S5. Digest the amplified and cultured human normal hepatocytes, human umbilical vein endothelial cells, breast cancer cells, cervical cancer cells, colon cancer cells, and non-small cell lung cancer cells in the logarithmic growth phase with trypsin solution, and centrifuge to obtain cell suspensions. After counting, inoculate them into 96-well cell culture plates respectively, so that each well has 1×10 4 cells, incubate in a constant temperature incubator for 24 h, and add 100 μL of cisplatin combined with ciclopirox olamine solution to each well. Among them, 0.1 mL / well of cell culture medium is added to the 96-well cell culture plate. The cell culture medium is RPMI-1640 culture medium supplemented with 10% FBS. The environmental conditions of the constant temperature incubator are a temperature of 37 °C and containing 5% CO 2 ;

[0051] S6. Directly add 20 μL / well of MTT solution to the 96-well cell culture plate, incubate in the incubator for 4 h, discard the solution in the culture plate, add 150 μL / well of dimethyl sulfoxide solution, place it in a microplate oscillator and shake for 15 min, and detect the absorbance value of each well at a wavelength of 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the IC 50 value.

[0052] Example 3:

[0053] The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine in this example includes the following steps:

[0054] S1. Preheat the normal saline to 64 °C, slowly add cisplatin powder, and stir to obtain a cisplatin solution with a concentration of 400 μM. Add ciclopirox olamine to methanol and stir to obtain a ciclopirox olamine solution with a concentration of 400 μM. Mix the cisplatin solution and the ciclopirox olamine solution to obtain a cisplatin combined with ciclopirox olamine solution, where the concentration ratio of the cisplatin solution to the ciclopirox olamine solution in the cisplatin combined with ciclopirox olamine solution is 1:1;

[0055] S2. Take 0.3 g of KC1, 0.3 g of KH 2 PO 4 , 9 g of NaC1, and 3.2 g of Na 2 HPO 4 ·12H 2 O, add 1 L of ultrapure water, adjust the pH to 7.1, and autoclave to obtain a phosphate buffered saline solution;

[0056] S3. Weigh 0.2 g of trypsin, add it to 214 mL of phosphate buffered saline solution, stir in an ice bath for 34 min, sterilize it with a 0.21 μm microporous filter, dispense it, and store it at -20 °C to obtain a trypsin solution;

[0057] S4. Weigh 254 mg of MTT and place it in a small beaker. Add 54 mL of phosphate buffered saline solution and stir on a magnetic stirrer for 34 min. Sterilize it with a 0.21 μm microporous filter, aliquot it, and store it at -20 °C to obtain the MTT solution;

[0058] S5. The human normal hepatocytes, human umbilical vein endothelial cells, breast cancer cells, cervical cancer cells, colon cancer cells, and non-small cell lung cancer cells in the logarithmic growth phase after amplification culture are digested with trypsin solution and centrifuged respectively to obtain cell suspensions. After counting, they are inoculated into 96-well cell culture plates respectively, so that each well contains 1×10 4 cells, and incubated in a constant temperature incubator for 24 h. Then, 100 μL of cisplatin combined with ciclopirox olamine solution is added to each well. Among them, 0.1 mL / well of cell culture medium is added to the 96-well cell culture plate. The cell culture medium is RPMI-1640 culture medium supplemented with 10% FBS. The environmental conditions of the constant temperature incubator are a temperature of 37 °C and containing 5% CO 2 ;

[0059] S6. Directly add 20 μL / well of MTT solution to the 96-well cell culture plate. After incubating in the incubator for 4 h, discard the solution in the culture plate, add 150 μL / well of dimethyl sulfoxide solution, place it in a microplate oscillator and shake for 15 min. Detect the absorbance value of each well at a wavelength of 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the IC 50 value.

[0060] Example 4:

[0061] The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine in this example includes the following steps:

[0062] S1. Preheat the normal saline to 65 °C, slowly add cisplatin powder and stir to obtain a cisplatin solution with a concentration of 500 μM. Add ciclopirox olamine to methanol and stir to obtain a ciclopirox olamine solution with a concentration of 500 μM. Mix the cisplatin solution and the ciclopirox olamine solution to obtain a cisplatin combined with ciclopirox olamine solution, where the concentration ratio of the cisplatin solution to the ciclopirox olamine solution in the cisplatin combined with ciclopirox olamine solution is 1:1;

[0063] S2. Take 0.2 g of KC1, 0.2 g of KH 2 PO 4 , 8 g of NaC1 and 2.5 g of Na 2 HPO 4 ·12H 2 O, add 1 L of ultrapure water, adjust the pH to 7.2, and autoclave to obtain the phosphate buffered saline solution;

[0064] S3. Weigh 0.3 g of trypsin, add it to 220 mL of phosphate buffered saline solution, stir in an ice-water bath for 35 min, sterilize it with a 0.22-μm microporous filter, dispense it into aliquots, and store it at -20 °C to obtain trypsin solution;

[0065] S4. Weigh 255 mg of MTT, put it into a small beaker, add 55 mL of phosphate buffered saline solution, stir it on a magnetic stirrer for 35 min, sterilize it with a 0.22-μm microporous filter, dispense it into aliquots, and store it at -20 °C to obtain MTT solution;

[0066] S5. The human normal hepatocytes, human umbilical vein endothelial cells, breast cancer cells, cervical cancer cells, colon cancer cells, and non-small cell lung cancer cells in the logarithmic growth phase after amplification culture are digested with trypsin solution and centrifuged respectively to obtain cell suspensions. After counting, they are respectively inoculated into 96-well cell culture plates, with 1×10 4 cells per well, incubated in a constant temperature incubator for 24 h, and 100 μL of cisplatin combined with ciclopirox olamine solution is added to each well. Among them, 0.1 mL / well of cell culture medium is added to the 96-well cell culture plate. The cell culture medium is RPMI-1640 culture medium supplemented with 10% FBS. The environmental conditions of the constant temperature incubator are a temperature of 37 °C and containing 5% CO 2 ;

[0067] S6. Directly add 20 μL / well of MTT solution to the 96-well cell culture plate, incubate it in the incubator for 4 h, discard the solution in the culture plate, add 150 μL / well of dimethyl sulfoxide solution, place it in a microplate shaker and shake for 15 min, and detect the absorbance value of each well at a wavelength of 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the IC 50 value.

[0068] Comparative Example 1:

[0069] In this comparative example, compared with Example 1, only "cisplatin combined with ciclopirox olamine solution" is replaced with "cisplatin solution", and the remaining steps and parameters are the same. This comparative example will not be repeated here, and the results are shown in Table 1.

[0070] Comparative Example 2:

[0071] In this comparative example, compared with Example 1, only "cisplatin combined with ciclopirox olamine solution" is replaced with "ciclopirox olamine solution", and the remaining steps and parameters are the same. This comparative example will not be repeated here, and the results are shown in Table 1.

[0072] Performance test:

[0073] Further explore the drug combination regimen of cisplatin and ciclopirox olamine by the Chou-Talalay method;

[0074] Preheat the normal saline to 65°C, slowly add cisplatin powder, and stir to obtain a cisplatin solution with a concentration of 100 μM. Add ciclopirox olamine to methanol and stir to obtain ciclopirox olamine solutions A, B, and C with concentrations of 500 μM, 1000 μM, and 2000 μM respectively. Mix the cisplatin solution with the ciclopirox olamine solutions to obtain cisplatin combined with ciclopirox olamine solutions A, B, and C, where the concentration ratios of the cisplatin solution to the ciclopirox olamine solution in the cisplatin combined with ciclopirox olamine solutions are 1:5, 1:10, and 1:20 respectively;

[0075] Take 0.2 g of KC1, 0.2 g of KH 2 PO 4 、8 g of NaC1 and 2.5 g of Na 2 HPO 4 ·12H 2 O, add 1 L of ultrapure water, adjust the pH to 7.2, and autoclave to obtain a phosphate buffer solution;

[0076] Weigh 0.3 g of trypsin, add it to 220 mL of phosphate buffer solution, stir in an ice bath for 35 min, sterilize it with a 0.22 μm microporous filter, aliquot it, and store it at -20°C to obtain a trypsin solution;

[0077] Weigh 255 mg of MTT, put it into a small beaker, add 55 mL of phosphate buffer solution, stir it on a magnetic stirrer for 35 min, sterilize it with a 0.22 μm microporous filter, aliquot it, and store it at -20°C to obtain an MTT solution;

[0078] Digest the amplified and cultured non-small cell lung cancer in the logarithmic growth phase with the trypsin solution, centrifuge it to obtain a cell suspension, and after counting, inoculate it into a 96-well cell culture plate respectively, so that each well has 1×10 4 cells, incubate it in a constant temperature incubator for 24 h, repeat the preparation 7 times, and add 100 μL of cisplatin solution, 100 μL of ciclopirox olamine solution A, 100 μL of ciclopirox olamine solution B, 100 μL of ciclopirox olamine solution C, 100 μL of cisplatin combined with ciclopirox olamine solution A, 100 μL of cisplatin combined with ciclopirox olamine solution B, and 100 μL of cisplatin combined with ciclopirox olamine solution C respectively. Among them, 0.1 mL / well of cell culture medium is added to the 96-well cell culture plate, and the cell culture medium is RPMI-1640 culture medium supplemented with 10% FBS. The environmental conditions of the constant temperature incubator are a temperature of 37°C and containing 5% CO 2 ;

[0079] Add 20 μL / well of MTT solution directly into a 96-well cell culture plate. After incubating in an incubator for 4 h, discard the solution in the culture plate, add 150 μL / well of dimethyl sulfoxide solution, place it in a microplate oscillator and oscillate for 15 min, and detect the absorbance value of each well at a wavelength of 490 nm on an enzyme-linked immunosorbent assay (ELISA) reader to calculate the IC 50 value, and the results are shown in Table 2.

[0080] Through the median-effect equation model, the effects achieved by the drugs at the three ratios are concentration-dependent. Analyze the combination index (CI) of the specific concentrations of the drug combination. The specific operation steps are as shown in Example 1, and the results are shown in Table 3.

[0081] Through the median-effect equation model, calculate the drug combination index and the median effective dose of different ratios of cisplatin and ciclopirox olamine in combination at a specific effect, and the results are shown in Table 4.

[0082] Table 1 Cytotoxic effects of cisplatin and ciclopirox olamine on different human cell lines

[0083]

[0084] Table 2 Cytotoxic effects of different ratios of cisplatin and ciclopirox olamine in combination on non-small cell lung cancer

[0085]

[0086] Table 3 Drug combination index of different concentrations of cisplatin and ciclopirox olamine in combination on non-small cell lung cancer

[0087]

[0088] Table 4 Drug combination index and median effective dose of different ratios of cisplatin and ciclopirox olamine in combination at a specific effect

[0089]

[0090] Note: ED50, ED75, and ED90 respectively represent the drug amounts when 50%, 75%, and 90% of the experimental subjects show positive reactions; Dm represents the median effective dose, and m is the slope in the median-effect equation, indicating the sensitivity to the drug.

[0091] Data analysis:

[0092] As can be seen from Table 1, among all cell lines, cisplatin has the strongest cytotoxicity to breast cancer cells and the weakest cytotoxicity to colon cancer cells. Secondly, it is human normal liver cells, and the difference between the two is not significant. Furthermore, ciclopirox olamine has the strongest cytotoxicity to non-small cell lung cancer and the weakest toxicity to human normal liver cells. The IC 50The values are all between 16 and 30 μM, and the inhibitory effect of ciclopirox olamine on these 6 cell lines is weaker than that of cisplatin. After mixing cisplatin and ciclopirox olamine in equal ratios, the cytotoxicity against non-small cell lung cancer is the strongest, which is 2 times lower than that of using cisplatin alone and about 5 times lower than that of using ciclopirox olamine alone. In summary, non-small cell lung cancer will be selected for the study of the anti-tumor mechanism of drug combination in subsequent experiments.

[0093] As can be seen from Table 2, the IC 50 value of cisplatin alone is about 5.61 μM. In contrast, after drug combination, the IC 50 value of cisplatin decreases, showing a negative dose-dependence. Among them, the cytotoxicity of cisplatin in the third group is the strongest at a molar ratio of 1:20, and the IC 50 value is about 0.36 μM, which is reduced by 15 times. In addition, the same trend is also shown after the combination of ciclopirox olamine. When using ciclopirox olamine alone, the IC 50 value in the third group is the lowest, about 7.38 μM, and the highest is in the second group, with the IC 50 value about 18.58 μM, and there is no dose-dependence. After drug combination, the IC 50 value of ciclopirox olamine in the second group is the lowest, about 6.31 μM, which is nearly 3 times lower than that of using ciclopirox olamine alone. This shows that after the combination of cisplatin and ciclopirox olamine, the cytotoxicity is enhanced compared with the individual action of each drug, and the IC 50 value decreases significantly, and the effect is the best at the ratios of 1:10 and 1:20;

[0094] It can be found through the median-effect equation model that the effects achieved by the drugs at the three ratios are all concentration-dependent. Analyzing the combination index (CI) of the specific concentrations of the drug combination, the results are statistically shown in Table 3. When cisplatin and ciclopirox olamine are combined at 1:1, 1:5, and 1:20, there is a synergistic effect, but at some combined concentrations, the CI value is greater than 1, that is, there is an antagonistic effect. When combined at 1:10, the CI values are all less than 1, that is, there is only a synergistic effect. The statistical results in Table 4 show that when the ratio of cisplatin to ciclopirox olamine is 1:10, its CI values are the smallest and less than 1, which are 0.53, 0.49, and 0.48 respectively.

[0095] In addition, the value of m in the medium-effect equation represents the sensitivity of cells to the drug. The larger the value of m, the stronger the sensitivity. As can be seen from Table 4, the value of m is the largest at a ratio of 1:20, which is 2.08. When the dose of ciclopirox olamine is reduced to make the ratio of the two 1:10, the value of m is 1.18, a decrease of 0.9. This indicates that there is a slight difference in the sensitivity of cells to the combination of the two groups of drugs. However, the median effective dose Dm of 1:10 is only 0.08 more than the minimum value of 0.63 of 1:20, which is almost negligible. The above results show that when cisplatin is combined with ciclopirox olamine at a concentration ratio of 1:10 and acts on cells, there is a strong synergistic cytotoxic effect.

[0096] Those of ordinary skill in the art should understand that the discussion of any of the above embodiments is exemplary only and is not intended to imply that the scope of the present invention is limited to these examples; under the concept of the present invention, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of the present invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. An in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine, characterized in that: The following steps are involved: Step A1: preparing test reagents; The test reagent is a cisplatin combined with ciclopirox olamine solution; the concentration ratio of cisplatin to ciclopirox olamine in the cisplatin combined with ciclopirox olamine solution is 1:1-10; Step A2: Cell inoculation and culture; The cells include normal human liver cells, human umbilical vein endothelial cells, breast cancer cells, cervical cancer cells, colon cancer cells and non-small cell lung cancer; Step A3: MTT cytotoxicity assay; In this step, the cytotoxicity of various cancer cells in vitro was analyzed to screen out the cell lines most sensitive to the two drugs, and the combined efficacy of the drugs was analyzed by the Chou-Talalay method to finally obtain the optimal concentration ratio.

2. The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine according to claim 1, characterized in that: The steps of cell inoculation and culture in step A2 are as follows: Normal human liver cells, human umbilical vein endothelial cells, breast cancer cells, cervical cancer cells, colon cancer cells and non-small cell lung cancer cells in the logarithmic growth phase of the amplified culture were digested with trypsin solution and centrifuged to obtain cell suspensions. After counting, they were inoculated into 96-well cell culture plates and incubated in a constant temperature incubator for 24 hours, and 100 μL of test reagent was added to each well.

3. The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine according to claim 2, characterized in that: The preparation method of the trypsin solution is as follows: Weigh 0.1-0.3 g of trypsin, add 200-220 mL of phosphate buffered saline solution, stir in an ice water bath for 30-35 min, sterilize with a 0.2-0.22 μm microporous filter, divide into portions, and store at -20°C; the usage ratio of the trypsin and phosphate buffered saline solution is 0.1-0.3 g: 200-220 mL.

4. The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine according to claim 3, characterized in that: The preparation method of the phosphate buffered saline solution is as follows: Take KC1, KH2PO4, NaC1 and Na2HPO4·12H2O, add ultrapure water, adjust the pH to 6.9-7.2, and sterilize by high pressure to obtain phosphate buffered saline solution; The usage ratio of KC1, KH2PO4, NaC1, Na2HPO4·12H2O and ultrapure water is 0.2-0.4g: 0.2-0.4g: 8-10g: 2.5-3.5g: 1L.

5. The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine according to claim 4, characterized in that: Each well of the 96-well cell culture plate has 1×10 4 cells; 0.1 mL / well of cell culture medium was added to the 96-well cell culture plate; the cell culture medium was RPMI-1640 culture medium supplemented with 10% FBS; the environmental conditions of the constant temperature incubator were 37° C. and 5% CO2.

6. The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine according to claim 1, characterized in that: The steps of MTT cytotoxicity detection in step A3 are as follows: Add MTT solution directly to the cell culture plate, incubate in an incubator for 3-5 hours, discard the solution in the culture plate, add dimethyl sulfoxide solution, place in a microplate oscillator and oscillate for 10-20 minutes, detect the absorbance of each well at a wavelength of 490nm on an ELISA reader, and calculate the IC 50 Values ​​and CI values.

7. According to claim 6, the in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine is characterized in that: The amount of the MTT solution is 20 μL / well; the amount of the dimethyl sulfoxide solution is 150 μL / well.

8. The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine according to claim 7, characterized in that: The preparation method of the MTT solution is as follows: Weigh MTT, put it into a small beaker, add phosphate buffered saline solution and stir on an electromagnetic stirrer for 30-35 minutes, sterilize with a 0.2-0.22 μm microporous filter, divide into portions, and store at -20°C.

9. The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine according to claim 8, characterized in that: The dosage ratio of the MTT and phosphate buffered saline solution is 250-255 mg: 50-55 mL.

10. The in vitro cytotoxicity analysis method based on cisplatin combined with ciclopirox olamine according to claim 9, characterized in that: The preparation method of the phosphate buffered saline solution is as follows: Take KC1, KH2PO4, NaC1 and Na2HPO4·12H2O, add ultrapure water, adjust the pH to 6.9-7.2, and sterilize by high pressure to obtain a phosphate buffered saline solution; the dosage ratio of KC1, KH2PO4, NaC1, Na2HPO4·12H2O and ultrapure water is 0.2-0.4g: 0.2-0.4g: 8-10g: 2.5-3.5g: 1L.

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