CN111912826B - Method for evaluating efficacy of antitumor drug at cellular level - Google Patents
Method for evaluating efficacy of antitumor drug at cellular level Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N2021/653—Coherent methods [CARS]
- G01N2021/655—Stimulated Raman
Abstract
The invention relates to a method for evaluating the efficacy of an anti-tumor drug at the cellular level, which comprises the steps of setting an anti-tumor drug experimental group and a control group with different concentrations, wherein a group with the anti-tumor drug concentration of 0 and heavy water added is used as a positive control group (pos), and a group with the anti-tumor drug concentration of 0 and no heavy water added is used as a negative control group (neg); and (3) after the tumor cells cultured in the experimental group and the control group are eliminated, centrifugally cleaning, and then dripping the tumor cells into a low-Raman background chip for Raman detection, respectively calculating C-D/(C-D+C-H) of the experimental group and the control group according to the obtained Raman spectra, and determining whether the antitumor drug to be detected is effective on the tumor cells according to the C-D/(C-D+C-H) of the experimental group and the control group. Compared with the prior art, the method for evaluating the effectiveness of the anti-tumor drug from the cell metabolism level can evaluate some anti-tumor drugs which only inhibit cell growth but not inhibit cell metabolism more accurately than the method for evaluating the effectiveness of the drug by calculating the cell number in the traditional method, so as to guide the drug administration.
Description
Technical Field
The invention belongs to the technical field of drug screening, and particularly relates to a method for evaluating the drug effect of an anti-tumor drug at the cellular level.
Background
In vitro researches of antitumor drugs, commonly used drug effect evaluation methods mainly comprise CCK8, MTT, SRB methods and the like, wherein the methods generally comprise the steps of co-culturing the antitumor drugs with cells before testing, wherein the co-culturing time is generally 48-72 hours, then adding a reaction reagent for continuous culturing for a plurality of hours, and calculating half effective concentration of the drugs by utilizing the principle that the color reaction between the reagent and cell components and the OD value of the solution after the reaction are in linear relation with the number of the cells, but the methods have the defects of complicated operation, high price, unstable results and the like.
Disclosure of Invention
The present invention aims to overcome the above-mentioned drawbacks of the prior art and to provide a simple method for evaluating the efficacy of antitumor drugs at the cellular level.
The aim of the invention can be achieved by the following technical scheme:
the invention provides a method for evaluating the efficacy of an anti-tumor drug at the cellular level, which detects the metabolic activity of cells after the effect of the anti-tumor drug by using a Raman-heavy water mark combination technology and evaluates the efficacy of the anti-tumor drug from the metabolic level.
Further, the method for evaluating the efficacy of the antitumor drug at the cellular level comprises the following steps:
setting an anti-tumor drug experimental group and a control group with different concentrations, wherein a group with the anti-tumor drug concentration of 0 and the heavy water added is used as a positive control group (pos), and a group with the anti-tumor drug concentration of 0 and the heavy water not added is used as a negative control group (neg);
in the experimental group, after the digested tumor cells are cultured to the cell wall, the antitumor drugs with different concentrations are respectively added into the positive control group and the negative control group, after the digested tumor cells are cultured to the cell wall, the antitumor drugs are not added,
adding heavy water after the experiment group and the positive control group continue to be cultured for a first period, adding no heavy water after the negative control group continues to be cultured for a first period, dissolving cells after the negative control group continues to be incubated for a second period, centrifugally cleaning, then dripping the cells on a low-Raman background chip for Raman detection, and respectively calculating C-D/(C-D+C-H) of the experiment group and the control group according to the obtained Raman spectrum.
According to the invention, whether the antitumor drug to be detected is effective on tumor cells or not is determined according to the C-D/(C-D+C-H) of the experimental group and the control group.
In one embodiment of the invention, the drug concentration settings in the experimental group were 0.5. Mu.M, 1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, respectively.
In one embodiment of the invention, the culture of cells in the experimental and control groups uses DMEM medium.
In one embodiment of the present invention, the experimental group and the positive control group were further cultured for a first period of time, and then the heavy water was added at a ratio of 30% (v/v).
In one embodiment of the present invention, the first time period is 24h, and the second time period is 48h.
In one embodiment of the invention, the low raman background chip is purchased from Shanghai deuterium peak medical instruments, inc., cat No 1001.
In one embodiment of the invention, a WITEC-alpha300 Raman spectrometer is used for collecting Raman spectra of cells, a grating is 600g/mm, the spectrum center is set to 2300, a laser is 532nm, and the spot size is 350nm; when the Raman spectrum of the cell is collected, a 100-time objective lens is used, the cell is found in the field of view, then a light spot is focused at the center of the cell, the laser power is set to 7-9 mW, and the accumulation time is 3s; collecting Raman spectra of 20-30 cells in each group; intercepting 1770-3400 cm during data processing -1 The map is used for carrying out background removal and normalization processing on the data by using the self-contained software of the instrument; the C-D peak and the C-H peak are respectively located at 2000-2300cm -1 And 2800-3100cm -1 The method comprises the steps of carrying out a first treatment on the surface of the The ratio of the areas of the C-D peak to the C-D peak plus the C-H peak, C-D/(C-D+C-H), was calculated using R studio software.
The invention also uses CCK-8 method to measure the half-lethal concentration of the anti-tumor drug. The anti-tumor drug obtained by the combination method of Raman and heavy water labeling has high correlation (R 2 = 0.9822). This suggests that raman-binding heavy water labeling techniques can effectively evaluate the effect of antitumor drugs.
The invention utilizes the metabolic activity of tumor cells under the action of anti-tumor drugs to evaluate the drug effect of the anti-tumor drugs.
The working principle of the invention is as follows:
during the intracellular NAD/NADP reduction, hydrogen in water is converted into biomass, especially biomacromolecules such as lipids and proteins. The C-H bonds in these macromolecules are in Raman spectrumHas characteristic peaks (2800-3100 cm) -1 Between). Deuterium (D) in heavy water, when present, is used to synthesize important biomacromolecules with cellular metabolism, thereby allowing C-H peaks (2800-3000 cm -1 ) Is shifted to form C-D peak (2000-2300 cm -1 ) The C-D peak intensity reflects the metabolic activity of the cells. After the antitumor drugs with different concentrations act for a period of time, the cell metabolism is inhibited, and whether the drugs are effective on tumor cells can be obtained according to the existence and the intensity difference of the C-D peak.
Compared with the prior art, the invention has the following advantages:
1. the detection process does not use toxic reagent, only uses D which is non-toxic to human body and cells 2 O。
2. The effectiveness of the antitumor drug is evaluated from the level of cellular metabolism, and compared with the method for evaluating the effectiveness of the drug by calculating the number of cells in the conventional method, the method can evaluate some antitumor drugs which only inhibit the growth of cells but not inhibit the cellular metabolism more accurately, thereby guiding the administration.
3. The method can provide more effective information, and the Raman-heavy water labeling combined technology is used for evaluating the antitumor drug, so that not only can the change of the metabolic activity of the cells before and after the drug action be reflected, but also the change of the molecular substances of the cells before and after the drug action can be studied by observing the change of the fingerprint area of the Raman spectrum of the cells, and the action mechanism of the drug can be further studied.
Drawings
FIG. 1 is a graph showing the results of Raman-heavy water labeling combined technique for breast cancer drug evaluation.
Fig. 1 includes fig. 1A and 1B.
FIG. 1A shows the mean value of Raman spectra of cells in each concentration group after an antitumor drug GSK233470 acts on breast cancer cells MCF-7 48h, the shadow part is standard deviation (n=20-30), and the C-D peak is positioned at 2000-2300cm -1 Between them.
The different lines in FIG. 1A represent different concentrations of 0.5. Mu.M, 1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, respectively.
FIG. 1B shows the ratio of C-D/(C-H+C-D) of each drug concentration group after the drug action, pos group is a positive control group without adding heavy water and no drug, neg is a negative control group without adding heavy water and no drug.
FIG. 2 shows the effect of CCK-8 method on the activity of breast cancer cell MCF-7 of anti-tumor GSK233470.
FIG. 3 shows the mean value of the Raman spectrum fingerprint areas of the cells in different concentration groups after the anti-tumor drug GSK233470 acts on the breast cancer cells MCF-7 for 48 hours.
The different lines in FIG. 3 represent different concentrations of 0.5. Mu.M, 1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, respectively.
Detailed Description
The invention provides a method for evaluating the efficacy of an anti-tumor drug at a cellular level, which detects the metabolic activity of cells after the effect of the anti-tumor drug by using a Raman-heavy water mark combination technology and evaluates the efficacy of the anti-tumor drug from the metabolic level, and specifically comprises the following steps:
setting an anti-tumor drug experimental group and a control group with different concentrations, wherein a group with the anti-tumor drug concentration of 0 and the heavy water added is used as a positive control group (pos), and a group with the anti-tumor drug concentration of 0 and the heavy water not added is used as a negative control group (neg);
in the experimental group, after the digested tumor cells are cultured, antitumor drugs with different concentrations are respectively added,
in the positive control group and the negative control group, after the digested tumor cells are cultured, no anti-tumor medicine is added,
adding heavy water after the experiment group and the positive control group are continuously cultured for 24 hours, adding no heavy water after the negative control group is continuously cultured for 24 hours, then carrying out centrifugal cleaning after the cells are continuously incubated for 48 hours, then dripping the cells on a low-Raman background chip for Raman detection, respectively calculating C-D/(C-D+C-H) of the experiment group and the control group according to the obtained Raman spectrum, and determining whether the antitumor drug to be detected is effective on tumor cells according to the C-D/(C-D+C-H) of the experiment group and the control group.
In one embodiment of the invention, the drug concentration settings in the experimental group were 0.5. Mu.M, 1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, respectively.
In one embodiment of the invention, the culture of cells in the experimental and control groups uses DMEM medium.
In one embodiment of the present invention, the ratio of heavy water added after the experimental group and the positive control group were further cultured for 24 hours was 30% (v/v).
In one embodiment of the invention, the low raman background chip is purchased from Shanghai deuterium peak medical instruments, inc., cat No 1001.
In one embodiment of the invention, the Raman spectrum of the cells is acquired using WITEC-alpha 300.
In one embodiment of the invention, when the Raman spectrum of the cell is acquired, the grating is 600g/mm, the spectrum center is set to 2300, the laser is 532nm, and the light spot size is 350nm; when the Raman spectrum of the cell is collected, a 100-time objective lens is used, the cell is found in the field of view, then a light spot is focused at the center of the cell, the laser power is set to 7-9 mW, and the accumulation time is 3s; collecting Raman spectra of 20-30 cells in each group; intercepting 1770-3400 cm during data processing -1 The map is used for carrying out background removal and normalization processing on the data by using the self-contained software of the instrument; the C-D peak and the C-H peak are respectively located at 2000-2300cm -1 And 2800-3100cm -1 The method comprises the steps of carrying out a first treatment on the surface of the The ratio of the areas of the C-D peak to the C-D peak plus the C-H peak, C-D/(C-D+C-H), was calculated using R studio software.
The invention will now be described in detail with reference to the drawings and specific examples.
Example 1
In this example, the evaluation of antitumor drug of breast cancer cell MCF-7 is taken as an example, and the antitumor drug is PDK-1 inhibitor GSK233470.
In this example, the concentrations of the antitumor drugs in the experimental group were set to 0.5. Mu.M, 1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, respectively;
a group with an antitumor drug concentration of 0 and heavy water added was used as a positive control group (pos);
a group with an antitumor drug concentration of 0 and without heavy water added was used as a negative control group (neg);
the specific operation method comprises the following steps:
in the experimental group, after the digested tumor cells are cultured (using DMEM culture medium), anti-tumor drugs with different concentrations are respectively added, in the positive control group and the negative control group, after the digested tumor cells are cultured, no anti-tumor drugs are added,
adding heavy water after the experiment group and the positive control group are continuously cultured for 24 hours, obtaining the proper heavy water adding proportion of 30% (v/v) used in the invention through a heavy water cytotoxicity test, adding no heavy water after the negative control group is continuously cultured for 24 hours, and after the negative control group is continuously incubated for 48 hours, carrying out cell digestion, carrying out centrifugal cleaning, wherein the centrifugal washing step is provided with the rotating speed of a centrifugal machine of 1000rpm for 5 minutes.
And then dripping the glass slide glass on a low-Raman background chip for Raman detection, wherein the low-Raman background chip is purchased from Shanghai deuterium peak medical instrument Co., ltd., cat No. 1001, and is a glass slide glass containing a metal coating.
Collecting Raman spectrum of the cell by using WITEC-alpha300, setting the spectrum center as 2300, selecting 532nm by a laser, and enabling the light spot size to be 350nm; when the Raman spectrum of the cell is collected, a 100-time objective lens is used, the cell is found in the field of view, then a light spot is focused at the center of the cell, the laser power is set to 7-9 mW, and the accumulation time is 3s; collecting Raman spectra of 20-30 cells in each group; intercepting 1770-3400 cm during data processing -1 Atlas, then use the instrument to carry on background removal and normalization (/ area) to deal with the data from the software; the C-D peak and the C-H peak are respectively located at 2000-2300cm -1 And 2800-3100cm -1 The method comprises the steps of carrying out a first treatment on the surface of the The ratio of the areas of the C-D peak to the C-D peak plus the C-H peak, C-D/(C-D+C-H), was calculated using R studio software.
FIG. 1 shows the results of the Raman-heavy water labeling combined technique for breast cancer drug evaluation.
FIG. 1A shows the mean value of Raman spectra of cells in each concentration group after an antitumor drug GSK233470 acts on breast cancer cells MCF-7 48h, the shadow part is standard deviation (n=20-30), and the C-D peak is positioned at 2000-2300cm -1 Between them.
FIG. 1B shows the ratio of C-D/(C-H+C-D) of each drug concentration group after the drug action, pos group is a positive control group without adding heavy water and no drug, neg is a negative control group without adding heavy water and no drug.
The bar graph according to fig. 1B shows that the C-D/(C-h+c-D) ratio of the drug-added group is significantly lower than that of the pos control group (p < 0.001), and that the C-D/(C-h+c-D) ratio is lower as the drug concentration increases.
The present example also measured the median lethal concentration of the antitumor drug using CCK-8. FIG. 2 shows the effect of CCK-8 method on the activity of breast cancer cell MCF-7 of anti-tumor GSK233470. After MCF-7 cells were incubated with different concentrations of GSK233470 for two days, cell viability was determined using the CCK-8 kit. As can be seen from fig. 2, in the additive drug group, the cell viability was lower and lower as the drug concentration was increased.
The anti-tumor drug obtained by the Raman-heavy water labeling combined technical method has high correlation (R 2 = 0.9822). This suggests that the raman-heavy water labeling combined technique can effectively evaluate the effect of the antitumor drug.
In addition, after the anti-tumor drug GSK233470 acts on breast cancer cells MCF-7 for 48 hours, the average value of the cell Raman spectrum fingerprint regions of different concentration groups is shown in figure 3, and the difference of the different groups at the positions of the marked Raman peaks in the figure can be seen visually, and the substances represented by the peak positions are shown in table 1.
Table 1 substances represented by the Raman peaks in FIG. 3
According to the embodiment, after the antitumor drugs with different concentrations act for a period of time, the cell metabolism is inhibited, and whether the antitumor drugs are effective on tumor cells or not can be obtained according to the difference of the existence and the intensity of the C-D peak, so that the method provided by the invention evaluates the effectiveness of the antitumor drugs from the cell metabolism level, and compared with the traditional method in which the effectiveness of the antitumor drugs is evaluated by calculating the number of cells, the method provided by the invention can evaluate certain antitumor drugs which only inhibit the growth of cells but not inhibit the metabolism of cells more accurately, and further guide the medication. The detection process of the invention does not existUsing toxic agents, only D which is non-toxic to humans and cells 2 O. The method can provide more effective information, and the Raman-heavy water labeling combined technology is used for evaluating the anti-tumor drugs, so that not only can the metabolic activity change of cells before and after the drug action be reflected, but also the change of molecular substances of cells before and after the drug action can be studied by observing the change of the cell Raman spectrum fingerprint region, and the action mechanism of the drugs can be further studied.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (1)
1. A method for evaluating the efficacy of an anti-tumor drug at the cellular level, which is characterized in that the metabolic activity of the cells after the effect of the anti-tumor drug is detected by a Raman-heavy water labeling combined technology, and the efficacy of the anti-tumor drug is evaluated from the metabolic level;
the method comprises the following steps:
setting an anti-tumor drug experimental group and a control group with different concentrations, wherein a group with the anti-tumor drug concentration of 0 and heavy water added is used as a positive control group, and a group with the anti-tumor drug concentration of 0 and no heavy water added is used as a negative control group;
in the experimental group, after the digested tumor cells are cultured to the cell wall, the antitumor drugs with different concentrations are respectively added into the positive control group and the negative control group, after the digested tumor cells are cultured to the cell wall, the antitumor drugs are not added,
adding heavy water after the experiment group and the positive control group are continuously cultured for a first time period, adding no heavy water after the negative control group is continuously cultured for a first time period, carrying out centrifugal cleaning after continuously incubating for a second time period, then dripping the cells on a low Raman background chip for Raman detection, respectively calculating C-D/(C-D+C-H) of the experiment group and the control group according to the obtained Raman spectrum, and determining whether the antitumor drug to be detected is effective on tumor cells according to the C-D/(C-D+C-H) of the experiment group and the control group;
the experimental group and the positive control group are continuously cultured for a first period of time, and then the adding proportion of heavy water is 30% v/v;
collecting a Raman spectrum of the cell by using a WITEC-alpha300 Raman spectrometer;
grating 600g/mm, spectrum center set to 2300, laser selected 532nm, spot size 350nm; when the Raman spectrum of the cell is collected, a 100-time objective lens is used, the cell is found in the field of view, then a light spot is focused at the center of the cell, the laser power is set to 7-9 mW, and the accumulation time is 3s; collecting Raman spectra of 20-30 cells in each group; intercepting 1770-3400 cm during data processing -1 The map is used for carrying out background removal and normalization processing on the data by using the self-contained software of the instrument; the C-D peak and the C-H peak are respectively located at 2000-2300cm -1 And 2800-3100cm -1 The method comprises the steps of carrying out a first treatment on the surface of the Calculating the area ratio of the C-D peak to the C-D peak plus the C-H peak, namely C-D/(C-D+C-H), by using R studio software;
in the experimental group, the drug concentration settings were 0.5. Mu.M, 1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 15. Mu.M, 20. Mu.M, respectively;
the cell culture uses DMEM culture medium;
the first time period is 24 hours, and the second time period is 48 hours;
the low Raman background chip is purchased from Shanghai deuterium peak medical instrument limited company, cat No 1001;
the antitumor drug is a PDK-1 inhibitor GSK2334470.
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