CN111912826A - Method for evaluating drug effect of anti-tumor drug at cellular level - Google Patents
Method for evaluating drug effect of anti-tumor drug at cellular level Download PDFInfo
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- 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
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- 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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- 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]
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Abstract
The invention relates to a method for evaluating the drug effect of an anti-tumor drug at a cellular level, which comprises the steps of setting an anti-tumor drug experimental group and a control group with different concentrations, wherein the anti-tumor drug concentration is 0, the group added with heavy water is used as a positive control group (pos), and the anti-tumor drug concentration is 0, and the group not added with heavy water is used as a negative control group (neg); digesting the tumor cells cultured in the experimental group and the control group, centrifuging, cleaning, and then dripping the cells on 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 medicament from the cell metabolism level can evaluate certain anti-tumor medicaments which only inhibit cell growth but not cell metabolism more accurately to guide medicament administration compared with the method for evaluating the medicament effectiveness by calculating the cell number in the traditional method.
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 a cellular level.
Background
In the in vitro research of antitumor drugs, the commonly used drug effect evaluation methods mainly include the methods of CCK8, MTT, SRB and the like, which are generally characterized in that before the test, the antitumor drug and cells are co-cultured, the co-culture time is generally 48-72h, then a reaction reagent is added for continuous culture for several hours, and the half effective concentration of the drug is calculated by utilizing the principle that the color reaction between the reagent and the cell components and the linear relation between the OD value of the solution after the reaction and the number of the cells exist, but the methods have the defects of complicated operation, high price, unstable result and the like.
Disclosure of Invention
The present invention aims at overcoming the defects of the prior art and providing a simple method for evaluating the drug effect of an antitumor drug at a cellular level.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a method for evaluating the drug effect of an anti-tumor drug at a cellular level, which detects the metabolic activity of cells after the anti-tumor drug acts by a Raman-deuterium labeling combined technology and evaluates the drug effect of the anti-tumor drug from the metabolic level.
Further, the method for evaluating the drug effect of the anti-tumor drug at a cellular level specifically comprises the following steps:
setting an anti-tumor drug experimental group and a control group with different concentrations, wherein the anti-tumor drug concentration is 0, one group added with heavy water is used as a positive control group (pos), and the anti-tumor drug concentration is 0, and the other group not added with heavy water is used as a negative control group (neg);
in the experimental group, the digested tumor cells are cultured until the cells adhere to the wall, the anti-tumor drugs with different concentrations are respectively added into the positive control group and the negative control group, the digested tumor cells are cultured until the cells adhere to the wall, the anti-tumor drugs are not added,
and (3) adding heavy water into the experimental group and the positive control group after continuously culturing for a first time period, adding no heavy water into the negative control group after continuously culturing for the first time period, digesting the cells after continuously incubating for a second time period, centrifugally cleaning, dropwise adding the cells on a low-Raman background chip for Raman detection, and respectively calculating C-D/(C-D + C-H) of the experimental group and the control group according to the obtained Raman spectrums.
According to the invention, whether the antitumor drug to be detected is effective on tumor cells is determined according to C-D/(C-D + C-H) of an experimental group and a control group.
In one embodiment of the present invention, the drug concentration is 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, in the experimental group.
In one embodiment of the present invention, the cell culture in the experimental group and the control group uses DMEM medium.
In one embodiment of the present invention, the experimental group and the positive control group are further cultured for a first period of time and then added with heavy water 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 48 h.
In one embodiment of the invention, the low raman background chip is available from shanghai deuterium peak medical instruments ltd, Cat No 1001.
In one embodiment of the invention, a WITEC-alpha300 Raman spectrometer is used for collecting a Raman spectrum of cells, the grating is 600g/mm, the spectrum center is set to be 2300, the laser is selected to be 532nm, and the light spot size is 350 nm; when the Raman spectrum of the cell is collected, a 100-time objective lens is used for finding the cell in a visual field, then a light spot is focused to the center of the cell, the laser power is set to be 7-9 mW, and the accumulation time is set to be 3 s; collecting Raman spectra of 20-30 cells in each group; intercepting 1770-3400 cm during data processing-1Performing atlas, and then performing background removal and normalization processing on the data by using self-contained software of the instrument; the C-D peak and the C-H peak are respectively positioned at 2000-2300cm-1And 2800-3100cm-1(ii) a The area ratio of the C-D peak to the C-D peak plus the C-H peak, i.e., 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 antitumor drug. The inhibition rate of the anti-tumor drug obtained by the Raman-heavy water labeling combination method on cells is in phase with the lethality rate obtained by the CCK-8 methodCorrelation analysis, the correlation between the two methods is very high (R)20.9822). This shows that the effect of the anti-tumor drug can be effectively evaluated by combining Raman with the heavy water labeling technology.
The invention utilizes the change of the metabolic activity of the tumor cells under the action of the anti-tumor drug to evaluate the drug effect of the anti-tumor drug.
The working principle of the invention is as follows:
during the reduction of intracellular NAD/NADP, hydrogen in water is converted into biomass, especially biomacromolecules such as lipids and proteins. The C-H bonds in these macromolecules have characteristic peaks in the Raman spectrum (2800-3100 cm)-1In between). Deuterium (D) in heavy water is used to synthesize important biological macromolecules as it is metabolized by cells, thereby enabling the C-H peak (2800-3000 cm) in Raman spectra when heavy water is present-1) Shift to generate C-D peak (2000-2300 cm)-1) The intensity of the C-D peak reflects the metabolic activity of the cell. 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 strength difference of C-D peaks.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the detection process does not use toxic reagents, and only uses D which is nontoxic to human bodies and cells2O。
2. Compared with the traditional method for evaluating the effectiveness of the anti-tumor medicament by calculating the number of cells, the method for evaluating the effectiveness of the anti-tumor medicament by the cell metabolism level can more accurately evaluate certain anti-tumor medicaments which only inhibit cell growth but not cell metabolism, and further guides medication.
3. More effective information can be provided, and when the Raman-deuterium labeling combined technology is used for antitumor drug evaluation, the change of cell metabolic activity before and after drug action can be reflected, the change of molecular substances of cells before and after drug action can be researched by observing the change of a cell Raman spectrum fingerprint area, and the action mechanism of the drug can be further researched.
Drawings
FIG. 1 shows the results of Raman-heavy water labeling coupled technology for breast cancer drug assessment.
Fig. 1 includes fig. 1A and 1B.
FIG. 1A shows the mean value of Raman spectra of cells of each concentration group after the antitumor drug GSK233470 acts on breast cancer cells MCF-748 h, the shaded part is standard deviation (n is 20-30), and the peak of C-D is located at 2000-2300cm-1In the meantime.
The different lines in FIG. 1A represent different concentrations, 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 drug action, pos group is a positive control group without drug addition with heavy water, and neg is a negative control group without drug addition with heavy water.
FIG. 2 is a CCK-8 method for testing the effect of the antitumor drug GSK233470 on the activity of breast cancer cells MCF-7.
FIG. 3 is the mean value of Raman spectrum fingerprint of cells of different concentration groups after the antitumor drug GSK233470 acts on breast cancer cells MCF-748 h.
The different lines in FIG. 3 represent different concentrations, 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 drug effect of an anti-tumor drug at a cellular level, which detects the metabolic activity of cells after the anti-tumor drug acts by a Raman-deuterium labeling combined technology and evaluates the drug effect 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 the anti-tumor drug concentration is 0, one group added with heavy water is used as a positive control group (pos), and the anti-tumor drug concentration is 0, and the other group not added with heavy water is used as a negative control group (neg);
in the experimental group, the digested tumor cells are cultured and then added with anti-tumor drugs with different concentrations respectively,
in the positive control group and the negative control group, after the digested tumor cells are cultured, no anti-tumor medicine is added,
and (3) continuously culturing the experimental group and the positive control group for 24H, adding heavy water, continuously culturing the negative control group for 24H, adding no heavy water, continuously incubating for 48H, digesting the cells, centrifugally cleaning, dropwise adding the cells on 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.
In one embodiment of the present invention, the drug concentration is 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, in the experimental group.
In one embodiment of the present invention, the cell culture in the experimental group and the control group uses DMEM medium.
In one embodiment of the invention, the proportion of the heavy water added to the experimental group and the positive control group is 30% (v/v) after the culture is continued for 24 hours.
In one embodiment of the invention, the low raman background chip is available from shanghai deuterium peak medical instruments ltd, Cat No 1001.
In one embodiment of the invention, Raman spectra of cells are collected using WITEC-alpha 300.
In one embodiment of the invention, when the Raman spectrum of the cell is collected, the grating is 600g/mm, the spectrum center is set to be 2300, the laser is selected to be 532nm, and the spot size is 350 nm; when the Raman spectrum of the cell is collected, a 100-time objective lens is used for finding the cell in a visual field, then a light spot is focused to the center of the cell, the laser power is set to be 7-9 mW, and the accumulation time is set to be 3 s; collecting Raman spectra of 20-30 cells in each group; intercepting 1770-3400 cm during data processing-1Performing atlas, and then performing background removal and normalization processing on the data by using self-contained software of the instrument; the C-D peak and the C-H peak are respectively positioned at 2000-2300cm-1And 2800-3100cm-1(ii) a The area ratio of the C-D peak to the C-D peak plus the C-H peak, i.e., C-D/(C-D + C-H), was calculated using R studio software.
The invention is described in detail below with reference to the figures and specific embodiments.
Example 1
In the present example, the antitumor drug evaluation of breast cancer cell MCF-7 is taken as an example, and the antitumor drug used is PDK-1 inhibitor GSK 233470.
In this example, the concentrations of the antitumor drugs in the experimental groups were set to 0.5. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, and 20. mu.M, respectively;
one group with the concentration of the antitumor drug of 0 and added with heavy water is used as a positive control group (pos);
one group with the concentration of the antitumor drug of 0 and without adding heavy water is used as a negative control group (neg);
the specific operation method comprises the following steps:
in the experimental group, the digested tumor cells are cultured (by using DMEM culture medium), then the antitumor drugs with different concentrations are respectively added into the positive control group and the negative control group, the digested tumor cells are cultured, then the antitumor drugs are not added,
and (3) continuously culturing the experimental group and the positive control group for 24 hours, adding heavy water, obtaining the proper heavy water addition ratio of 30% (v/v) for the method through a heavy water cytotoxicity test, continuously culturing the negative control group for 24 hours, adding no heavy water, continuously incubating for 48 hours, digesting the cells, and centrifugally cleaning, wherein the centrifugal rotation speed of the centrifugal machine is 1000rpm in the centrifugal washing step, and the time is 5 min.
Then dropwise adding the metal-coated 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 limited company, Cat No 1001 and is a glass slide glass containing a metal coating.
Collecting a Raman spectrum of cells by using WITEC-alpha300, wherein the grating is 600g/mm, the spectrum center is set to be 2300, the laser is selected to be 532nm, and the light spot size is 350 nm; when the Raman spectrum of the cell is collected, a 100-time objective lens is used for finding the cell in a visual field, then a light spot is focused to the center of the cell, the laser power is set to be 7-9 mW, and the accumulation time is set to be 3 s; collecting Raman spectra of 20-30 cells in each group; intercepting 1770-3400 cm during data processing-1Mapping, and then performing background removal and normalization (/ area) processing on the data by using self-contained software of the instrument; the C-D peak and the C-H peak are respectively positioned at 2000-2300cm-1And 2800-3100cm-1(ii) a C-D peak calculation Using R studio softwareAnd the area ratio of the C-D peak to the C-H peak is C-D/(C-D + C-H).
FIG. 1 shows the results of the Raman-rehydration marker combination technique used for breast cancer drug assessment in this example.
FIG. 1A shows the mean value of Raman spectra of cells of each concentration group after the antitumor drug GSK233470 acts on breast cancer cells MCF-748 h, the shaded part is standard deviation (n is 20-30), and the peak of C-D is located at 2000-2300cm-1In the meantime.
FIG. 1B shows the ratio of C-D/(C-H + C-D) of each drug concentration group after drug action, pos group is a positive control group without drug addition with heavy water, and neg is a negative control group without drug addition with heavy water.
The bar graph according to FIG. 1B shows that the C-D/(C-H + C-D) ratio is significantly lower in the drug-added group than in the pos control group (p <0.001), and the C-D/(C-H + C-D) ratio becomes lower as the drug concentration increases.
This example also measured the median lethal concentration of the antitumor drug by the CCK-8 method. FIG. 2 is a CCK-8 method for testing the effect of the antitumor drug GSK233470 on the activity of breast cancer cells MCF-7. MCF-7 cells were cultured for two days by adding GSK233470 at different concentrations, and then the cell viability was measured by using a CCK-8 kit. As can be seen from fig. 2, the cell viability of the drug-added group becomes lower as the concentration of the drug increases.
The correlation analysis is carried out on the inhibition rate of the antitumor drug obtained by the Raman-heavy water labeling combination technology method on cells and the lethality rate obtained by the CCK-8 method, and the correlation of the two methods is very high (R is a high correlation value)20.9822). This indicates that the Raman-deuterium labeling combined technology can effectively evaluate the effect of the antitumor drug.
In addition, the mean value of the cell Raman spectrum fingerprint area of different concentration groups after the antitumor drug GSK233470 acts on the breast cancer cells MCF-748 h refers to fig. 3, the difference of different groups at the marked Raman peak position in the figure can be seen by naked eyes, and the substances represented by each peak position are shown in table 1.
TABLE 1 materials represented by respective Raman peaks in FIG. 3
It can be seen from the above examples that after the antitumor drugs with different concentrations act for a period of time, the cell metabolism is inhibited, and whether the drug is effective on tumor cells can be obtained according to the existence and intensity difference of the C-D peak, so that the method of the present invention estimates the effectiveness of the antitumor drugs from the cell metabolism level. The detection process of the invention does not use toxic reagents, and only uses D which is nontoxic to human bodies and cells2And O. The method can provide more effective information, and when the Raman-deuterium labeling combined technology is used for antitumor drug evaluation, the method not only can reflect the change of the cell metabolic activity before and after the drug action, but also can research the change of molecular substances of cells before and after the drug action by observing the change of the Raman spectrum fingerprint area of the cells, and can further research the action mechanism of the drug.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, 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 embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A method for evaluating the drug effect of an anti-tumor drug at a cellular level is characterized in that the cell metabolic activity of the anti-tumor drug after the action is detected by a Raman-deuterium labeling combined technology, and the drug effect of the anti-tumor drug is evaluated from the metabolic level.
2. The method for evaluating the pharmacodynamic action of an antitumor drug at a cellular level according to claim 1, comprising the steps of:
setting an anti-tumor drug experimental group and a control group with different concentrations, wherein the anti-tumor drug concentration is 0, the group added with heavy water is used as a positive control group, and the group not added with heavy water is used as a negative control group;
in the experimental group, the digested tumor cells are cultured until the cells adhere to the wall, the anti-tumor drugs with different concentrations are respectively added into the positive control group and the negative control group, the digested tumor cells are cultured until the cells adhere to the wall, the anti-tumor drugs are not added,
adding heavy water into the experimental group and the positive control group after the experimental group and the positive control group are continuously cultured for the first time period, adding no heavy water into the negative control group after the negative control group is continuously cultured for the first time period, digesting the cells after the incubation for the second time period, centrifugally cleaning the cells, then dropwise adding the cells on 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 spectrums, 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.
3. The method for evaluating the pharmacological effect of an antitumor drug according to claim 2, wherein the concentrations of the drugs in the experimental groups are set to 0.5. mu.M, 1.25. mu.M, 2.5. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, and 20. mu.M, respectively.
4. The method for evaluating the pharmacological effect of an antitumor drug according to claim 2, wherein the cell culture uses DMEM medium.
5. The method for evaluating the pharmaceutical effect of an antitumor drug at a cellular level as claimed in claim 2, wherein the ratio of the heavy water added to the experimental group and the positive control group after the culture is continued for the first period is 30% v/v.
6. The method for evaluating the pharmacodynamic action of an antitumor drug at a cellular level according to claim 2, wherein the first time period is 24 hours and the second time period is 48 hours.
7. The method for evaluating the drug effect of an antitumor drug at a cellular level as claimed in claim 2, wherein the low Raman background chip is purchased from Shanghai deuterium peak medical instruments, Inc., Cat No 1001.
8. The method for evaluating the pharmacodynamic action of an antitumor drug at a cellular level as claimed in claim 2, wherein the Raman spectrum of the cell is collected by WITEC-alpha300 Raman spectrometer.
9. The method for evaluating the pharmacological effect of an antitumor drug according to claim 8, wherein the grating is 600g/mm, the center of the spectrum is 2300, the laser is 532nm, and the spot size is 350 nm; when the Raman spectrum of the cell is collected, a 100-time objective lens is used for finding the cell in a visual field, then a light spot is focused to the center of the cell, the laser power is set to be 7-9 mW, and the accumulation time is set to be 3 s; and collecting Raman spectra of 20-30 cells in each group.
10. The method for evaluating the pharmacological effect of an antitumor drug according to claim 8, wherein 1770 to 3400cm is intercepted during data processing-1Performing atlas, and then performing background removal and normalization processing on the data by using self-contained software of the instrument; the C-D peak and the C-H peak are respectively positioned at 2000-2300cm-1And 2800-3100cm-1(ii) a The area ratio of the C-D peak to the C-D peak plus the C-H peak, i.e., C-D/(C-D + C-H), was calculated using R studio software.
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| WO2021239121A1 (en) * | 2020-05-29 | 2021-12-02 | 中国科学院青岛生物能源与过程研究所 | Method for screening or evaluating medicament |
| US11358984B2 (en) | 2018-08-27 | 2022-06-14 | Regeneran Pharmaceuticals, Inc. | Use of Raman spectroscopy in downstream purification |
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| CN113075192A (en) * | 2021-03-19 | 2021-07-06 | 中国科学院苏州生物医学工程技术研究所 | Multi-drug resistant tumor cell identification method based on Raman spectrum |
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