Method for evaluating effect of drug on tumor and tumor-related cells

Abstract

A method for evaluating the effect of a drug on a tumor and tumor-related cells. The tumor and the tumor-related cells are cultured in a culture medium containing a to-be-tested drug and a metabolic marker containing deuterium, the tumor and the tumor-related cells are imaged by means of coherent Raman scattering microscopy, and a metabolic product signal of the metabolic marker in an image is quantified, so as to evaluate the effect of the to-be-tested drug on the tumor and the tumor-related cells. According to the provided method, by means of quantifying unique chemical bonds that are newly generated after cellular metabolism, different cell samples can be subjected to drug effect detection based on the differences in metabolic inhibition by means of the same detection scheme. The method is simple and rapid, requiring a short culture time, which avoids the influence of prolonged culture on various properties of cells. The method has a better universality while reducing the detection cost.

Description

Method for evaluating effect of drug on tumor and tumor-related cells TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a method for evaluating the effect of a drug on tumor and tumor-related cells. BACKGROUND

[0002] Tumor is a special neoplasm, which is a space-occupying mass protruding from the human body affected by carcinogenic factors, and is divided into benign tumor and malignant tumor. Malignant tumor, due to its fast growth rate and low differentiation degree, is now the main cause of human death, causing great harm to human society.

[0003] Tumor is a complex disease, and its prevention and treatment needs to be considered from multiple aspects. The type, stage, genetic background and in vivo environment of each patient's tumor are unique, so the response to drugs will also be different. For different cancer types and subtypes of tumors of specific types and stages, drug efficacy research and guidance for the development of new anti-tumor drugs can more effectively kill or inhibit the growth of tumor and tumor-related cells, thereby delaying disease progression and prolonging the survival of patients. The detection results of the effect of tumor drugs can provide an important reference for drug development. By understanding the response of different types and stages of tumors and tumor-related cells to drug action, researchers can more targetedly develop new drugs or optimize the formulation of existing drugs to meet the treatment needs of different patients. Therefore, detecting and evaluating the effect of tumor and tumor-related cells on drugs is of great significance in the field of tumor research and drug development.

[0004] The experimental models for evaluating the effect of tumor drugs at present mainly include in vivo detection models and in vitro detection models. The in vivo drug testing method, such as the nude mouse inhibition method, simulates the internal environment of the human body, retains the activity of tumor tissue cells in the body and interprets the mechanism, and has the characteristics of higher accuracy, sensitivity and the like. However, the nude mouse transplantation method has the problems of long time period and high cost. The in vitro testing method for evaluating the effect of tumor drugs has also been developed to some extent, and the most commonly used methods at present include the adenosine triphosphate bioluminescence method (ATP-TCA) and the three-dimensional tumor cell primary culture chemotherapy drug effect detection method (CD-DST) and the like. The principle of the ATP-TCA method is that the endogenous ATP content can reflect the cell activity, and the ATP level in the cell metabolism process is in a relatively stable dynamic balance. The tumor cells are cultured under the condition of containing a series of concentrations of chemotherapy drugs for 2-5 days, after the culture is completed, the ATP concentration in the culture hole is extracted and detected, and the luciferin-luciferase system is used to detect the ATP. Under aerobic conditions, the luminous intensity is proportional to the ATP concentration, and the ATP concentration reflects the number of living cells in the culture hole, and the higher the content is, the more the living cells are, and the higher the resistance to chemotherapy drugs is. The method is subject to the metabolic characteristics of tumor cells, and the processing is complex, and appropriate reagents and buffers are also needed to ensure the accuracy and reliability of the reaction, so there are certain difficulties in wide application and promotion. The CD-DST technology is a new type of testing drug effect method combining collagen gel coating culture and image analysis system, and the mouse breast cancer epithelial cells are planted into the collagen gel matrix, and the growth state of the tumor cells in the gel is very close to the growth form of the tumor cells in the biological body. This method also has the problems of complex operation and difficult processing. In addition, different tumors and tumor-related cells will have different situation phenotypes and applicable drugs, and whether the existing technology is based on gene sequencing or based on target protein expression, the detection reagent, process and analysis need to be changed according to the difference of the sample type, the cost of detection is high, the process is complex and responsible, and the effect difference is large.

[0005] Therefore, at present, there is still a lack of a low-cost, high-efficiency, fast and accurate method for evaluating the effect of drugs on tumors and tumor-related cells. SUMMARY

[0006] In order to solve the above technical problems, the application provides a method for evaluating the effect of drugs on tumors and tumor-related cells based on cell metabolism imaging and application, tumor and tumor-related cells are cultured in a culture medium containing a to-be-tested drug and a deuterium-containing metabolic marker, metabolic products of the metabolic marker in the tumor and tumor-related cells are detected by coherent Raman microscopic imaging technology, cell morphology and cell state are detected and quantitatively analyzed by high-throughput chemical component image, and cell metabolism intensity is quantitatively analyzed by signal intensity of cell metabolic products.

[0007] The first object of the present application is to provide a method for evaluating the effect of a drug on tumor and tumor-related cells based on cell metabolic imaging, wherein tumor and tumor-related cells are cultured in a culture medium containing a drug to be tested and a metabolic marker containing deuterium, the tumor and tumor-related cells are imaged by coherent Raman microscopic imaging technology, and the metabolic product signal of the metabolic marker is quantified, and the cell sample is subjected to drug effect detection based on metabolic inhibition by the same detection scheme, thereby evaluating the effect of the drug to be tested on tumor and tumor-related cells.

[0008] Further, the tumor and tumor-related cells are cultured in the first culture medium containing the drug for a first duration, and the metabolic marker containing deuterium is added, so that the tumor and tumor-related cells are cultured in the second culture medium containing the drug to be tested and the metabolic marker for a second duration.

[0009] In an embodiment of the present application, 1640 culture medium is selected as the initial culture medium, and the first culture medium and the second culture medium are obtained by adding the drug and the metabolic marker containing deuterium to the initial culture medium.

[0010] Further, the first duration and the second duration are 0-48 hours, and the sum of the first duration and the second duration is 24-96 hours.

[0011] Preferably, the first duration and the second duration are 24 hours.

[0012] Further, the coherent Raman microscopic imaging technology includes one or more of stimulated Raman scattering microscopic imaging and coherent anti-Stokes microscopic imaging technology.

[0013] In an embodiment of the present application, stimulated Raman scattering microscopic imaging is selected for detection.

[0014] Further, the metabolic marker includes one or more of heavy water, deuterated palmitic acid, deuterated oleic acid, deuterated amino acid, deuterated glucose, and deuterated cholesterol.

[0015] Further, the final concentration of the heavy water in the culture medium is 30-50%.

[0016] Further, cell skeleton imaging based on carbon-hydrogen signals is performed in the 2800 cm -1 to 3000 cm -1 band.

[0017] Further, metabolic marker metabolic product imaging based on carbon-deuterium signals is performed in the 2050 cm -1 to 2250 cm -1 band.

[0018] Further, the detection process further comprises collecting non-resonant signals at 1902cm -1 Wave number to collect non-resonant signals for removing non-resonant signals in post-processing.

[0019] In one embodiment of the present application, at 2912cm -1 and 2133cm -1 Wave number to collect carbon-hydrogen and carbon-deuterium signals respectively.

[0020] In one embodiment of the present application, at 2930cm -1 and 2850cm -1 Wave number to collect CH P and CH L signals respectively, and at 2177cm -1 and 2135cm -1 to detect CD P and CD L signals respectively.

[0021] Further, when the coherent Raman optical microscopic imaging technique is used for detection, the culture seeding density of the tumor and tumor-related cells to be detected is (0.05-1)×10 5 cells / cm 2 .

[0022] Further, the amount of the culture medium added is (100-500)μL / cm 2 .

[0023] The second object of the present application is to provide a kit for detecting the effect of a drug on tumor and tumor-related cells, which comprises the reagents used in the above-mentioned evaluation method.

[0024] The third object of the present application is to provide the use of the above-mentioned detection kit in evaluating the effect of a drug on tumor and tumor-related cells.

[0025] The beneficial effects of the present application are:

[0026] The application provides a tumor and tumor-related cell drug effect evaluation method. The method can detect a specific chemical bond newly generated after cell metabolism, and can detect a to-be-detected sample after simple treatment and short-time culture, thereby maximizing the consistency of the to-be-detected tumor and tumor-related cells in vivo, minimizing cell variation, and improving detection accuracy. The culture process is shortened, so that the to-be-detected sample can be obtained without adding small factor reagents and other reagents, thereby reducing the culture cost. The to-be-detected sample is imaged and detected based on the coherent Raman microscopic imaging technology, so that the cell morphology and cell state can be directly and clearly observed, and in addition, the cell metabolism can be quantitatively analyzed according to the signal intensity of the metabolic product. By adjusting the imaging wave number, the specific chemical composition of the to-be-detected cell can be quantitatively imaged and spatially positioned in different channels, thereby further widening the application scenarios. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in conjunction with the drawings, in which

[0028] Fig. 1 is a detection imaging diagram of GES-1 cells after 6 hours, 12 hours and 24 hours of culture under a 30% D2O condition in the embodiment 3 of the application;

[0029] Fig. 2 is a detection imaging diagram of GES-1 cells after 6 hours, 12 hours and 24 hours of culture under a 50% D2O condition in the embodiment 3 of the application;

[0030] Fig. 3 is a CH and CD signal intensity analysis of GES-1 cells under 30% D2O and 50% D2O culture conditions in the embodiment 3 of the application, wherein A is the CH signal intensity analysis, B is the CD signal intensity analysis, and C is the CD / CH signal intensity ratio analysis;

[0031] Fig. 4 is a detection imaging diagram of GES-1 cells under 30% D2O and GC4 culture conditions in the embodiment 4 of the application;

[0032] Fig. 5 is a CH and CD signal intensity analysis of GES-1 cells under 30% D2O and GC4 culture conditions in the embodiment 4 of the application, wherein A is the CH signal intensity analysis, B is the CD signal intensity analysis, and C is the CD / CH signal intensity ratio analysis;

[0033] Fig. 6 is a detection imaging diagram of GES-1 cells under different concentration gradients of drugs without distinguishing protein and lipid signals in the embodiment 5 of the application;

[0034] Fig. 7 is a CH signal intensity trend diagram of GES-1 cells under 30% D2O and different concentration gradients of drugs in the embodiment 5 of the application;

[0035] Figure 8 is a graph of the trend of CD signal intensity of GES-1 cells in 30% D2O and different concentration gradients of the drug according to Example 5 of the present application;

[0036] Figure 9 is a detection imaging diagram of GES-1 cells in 30% D2O and different concentration gradients of the drug according to Example 5 of the present application in the case of distinguishing protein and lipid signals;

[0037] Figure 10 is a graph of the trend of CH signal intensity of GES-1 cells in 30% D2O and different concentration gradients of the drug according to Example 5 of the present application; P

[0038] Figure 11 is a graph of the trend of CD signal intensity of GES-1 cells in 50% D2O and different concentration gradients of the drug according to Example 5 of the present application; P

[0039] Figure 12 is a graph of the trend of CH signal intensity of GES-1 cells in 50% D2O and different concentration gradients of the drug according to Example 5 of the present application; L

[0040] Figure 13 is a graph of the trend of CD signal intensity of GES-1 cells in 50% D2O and different concentration gradients of the drug according to Example 5 of the present application; L

[0041] Figure 14 is a detection imaging diagram of GES-1 cells in 50% D2O and different concentration gradients of the drug according to Example 5 of the present application in the case of distinguishing protein and lipid signals;

[0042] Figure 15 is a detection imaging diagram of GES-1 cells in 50% D2O and different concentration gradients of the drug according to Example 5 of the present application in the case of distinguishing protein and lipid signals. DETAILED DESCRIPTION

[0043] The present application will be further described below in conjunction with the drawings and specific embodiments so that those skilled in the art can better understand and implement the present application, but the embodiments are not intended to limit the present application.

[0044] ​​​​GES-1 cell line is selected for experiment. GES-1 cell is gastric epithelial cell, which is homologous to tumor cell transformed from gastric epithelial cell and has symbiotic relationship with gastric cancer cell in tumor tissue. GES-1 cell can simulate the biological characteristics and functions of gastric epithelial cell, study the molecular mechanism of gastric cancer occurrence, including cell signal transduction, gene expression regulation, cell cycle control, etc. GES-1 cell can be used to test the potential toxicity and therapeutic effect of drug on gastric epithelial cell, so as to screen drug with selective toxicity to gastric cancer cell, and has good stability and consistency, which is an ideal model for evaluating the effect of gastric cancer drug.

[0045] Complete medium: 44.5 mL DMEM / 1640 RPMI + 5 mL fetal bovine serum + 500 μL penicillin-streptomycin double antibody.

[0046] Example 1: Preparation of detection cell

[0047] (1) Melt pipetting: put the frozen GES-1 cells into a 37°C constant temperature water bath, shake constantly to thaw, and keep the mouth of the frozen tube not immersed in water. After observing the frozen cells to the liquid state, quickly disinfect with alcohol and place in a clean bench, take 20 μL cells to an EP tube, add 20 μL 0.4% trypan blue, and detect whether the cells are alive. After sufficient blowing and sucking, take 20 μL of the mixture to a cell counting plate, and use a pipette to transfer 1 mL of the remaining cells (add 4 mL of corresponding complete medium, a total of 5 mL) to a centrifuge tube prepared in advance.

[0048] (2) Centrifugal counting: centrifuge the cell suspension at 1000 rpm under room temperature for 3 min, and count the GES-1 cells added to the cell counting plate from the upper, middle and lower three areas.

[0049] (3) Transfer culture: calculate the total amount of GES-1 cells (number of cells per milliliter x total number of milliliters) according to the data obtained after counting, aspirate the supernatant after centrifugation to remove the waste liquid, and prepare different amounts of T25 culture bottles according to the total number of cells, so that the number of cells in each culture bottle is maintained at 7 x 10 5 to 1 x 10 6 . Add 6 mL of complete medium to each T25 culture bottle, take the same number of milliliters of complete medium as the number of culture bottles (for example, 4 x 10 6 cells are detected by cell counting, 4 T25 culture bottles are needed, and 4 mL of complete medium is added to the centrifuge tube with the supernatant removed in advance) Blow and suck the cells to ensure uniformity, add 1 mL of cells to each bottle, gently shake back and forth to ensure uniform distribution of cells in the bottle, and place horizontally in a cell culture box with the condition set to 37°C and 5% CO2.

[0050] (4) Observation: After overnight (~12h), observe the state of the recovered cells, replace the complete medium every 48h, the steps are to pour out the supernatant, wash twice with 1 mL of sterile PBS, pour out, add 1-2 mL of trypsin according to the ease of detachment of adherent cells, take out the cells and observe under a microscope, and when the state of cell detachment is good, take it out and gently tap the walls of the culture bottle until most of the cells are detached. If there is no detachment, continue to add 1 mL of trypsin for about 1 min. Add the termination solution before the cells are completely detached to avoid excessive damage to the cells. Add the termination solution to 5 mL. Count 20 μL of the mixed solution to ensure that the cell viability is ≥90% (the amount of cells per cryopreservation tube is generally 2 x 10 6 , re-centrifuge and pour out the supernatant, and add about 1 mL of cryopreservation solution). If the cell state is not good, continue to replace the bottle according to the steps of (3) transfer culture.

[0051] (5) Cell passage: Observe the cell culture bottle under a microscope, digest at an appropriate density, terminate, count, and make the cells cover the field of view but not particularly crowded. The total amount of GES-1 cells after counting (cell density x cell volume) is (2-3) x 10 6 , the cells in the cell culture bottle are transferred to a 15 mL centrifuge tube, and the centrifuge tube containing the collected cells is placed in a centrifuge at 1000 rpm for 3 minutes. During centrifugation, prepare new T25 cell culture bottles, and according to the number of cells, pass 2 or 3 bottles per 1 bottle. The amount of cells in the cell culture bottle after passage is generally (0.8-1) x 10 6 , the total amount of cells corresponding to a six-well plate is (0.25-0.35) x 10 6 . Add 6 mL of complete medium to each bottle in the clean bench, discard the supernatant after the cells are centrifuged, resuspend the cells with complete medium, calculate the number of bottles that can be passed, resuspend each bottle with 1 mL of medium, and after resuspension, draw the cells into new culture bottles with a pipette. After mixing the cells, place them horizontally in a cell culture incubator at 37°C, 5% CO2, and after the cells grow to sufficient amount, perform subsequent drug addition and differential culture experiments. The cell survival rate is more than 85%, and the amount of complete medium added per unit area is 300-400 μL.

[0052] Example 2: Cell culture inoculation density experiment

[0053] The high cell density will cause the cell to produce self-protection mechanism, thus causing certain deviation and error to the cell in the toxicity experiment, increasing the uncertainty of the experimental results, and some cells will be connected with each other through the extracellular matrix (mainly protein filaments and adhesion structure), and the displacement of any cell will generate a pulling force on the cells around it. Unlike other cells, this group of cells at the head of the cell layer will produce a significant high pressure stress response under such layout arrangement. This stress response eventually triggers apoptosis and overflow of nearby cells. The low cell density will affect the growth rate of the cells, resulting in low sample size, thus prolonging the experimental period, increasing the cost, increasing the probability of genetic mutation, reducing the similarity of the parent cells, and reducing the reliability of the experiment. In order to prevent the above two points from causing result distortion due to the cell itself, the most suitable cell density per unit area and the corresponding amount of culture medium are selected according to the results of the control experiment and cell passage. Moreover, the state, density and viability of the cells will affect the accuracy of the detection, and for the culture of experimental cells, the amount of cells per unit area (1 cm 2 ) at the time of inoculation of experimental cells is between (0.05-1) x 10 5 / cm 2 . The cell viability is more than 85%, and the amount of complete culture medium added per unit area (1 cm 2 ) is between 100 μL and 500 μL. This condition takes into account the comprehensive results of cell proliferation, cell density, imaging saturation, etc., and reduces the data distortion caused by the cell itself as much as possible.

[0054] In this experiment, six cell inoculation density gradients and four heavy water concentration experimental groups were set. After 24 hours of plating culture, the viability was detected, and then the ALAMBLUE activity detection reagent was incubated for 12 h for activity detection to confirm the best cell culture conditions (six-well plate inoculation area of 8 cm 2 ), and the viability results were obtained according to 0% / 30% / 50% / 70% heavy water concentration, 0.01 / 0.05 / 0.25 / 0.5 / 1 / 2 x 10 5 / cm 2 The cell inoculation density was used as the culture experimental group, and the viability and cell density after culture were measured, and the results are shown in Table 1.

[0055] Table 1 Cell viability under different heavy water concentrations and different inoculation densities

[0056] In summary, too low cell number will affect cell growth rate, resulting in low sample volume, reduced accuracy, prolonged test period, increased cost, increased probability of genetic mutation, reduced similarity to parent cells, and decreased experimental reliability. Too high cell number will result in poor cell state, low cell activity, and low viability, so appropriate cell culture inoculation density has a direct impact on the reliability of metabolic detection results directly related to cell activity. According to the cell viability detection results above, when the cell culture is performed in a 30-50% heavy water concentration medium, the cell culture inoculation density for detection is between 0.05x10 5 and 1x10 5 / cm 2 , the single cell viability is high and the activity is strong, and the appropriate cell metabolism visualization coherent Raman single cell metabolism detection is suitable.

[0057] Example 3: Heavy water culture conditions

[0058] The GES-1 cells obtained by culture in Example 1 were cultured in 1640 medium containing 30% D2O or 50% D2O, respectively, and divided into 3 control groups, each cultured for 6 hours, 12 hours and 24 hours. After the culture was completed, the cells were fixed and detected. The specific culture conditions are as follows:

[0059] (1) Add the prepared cell solution to three small culture dishes, respectively, and label them as dish 1, dish 2 and dish 3. Centrifuge the prepared cell solution into three parts;

[0060] (2) Add 2 mL of 1640 medium added with 30% / 50% D2O to dish 1, dish 2 and dish 3 to resuspend the cells;

[0061] (3) After 6 hours, remove the heavy water-containing medium from dish 1, add 1 mL of 4% formaldehyde for fixation, and place it in a 4°C refrigerator for half an hour. Then remove the fixing solution, add 2 mL of PBS buffer, and store it in a 4°C refrigerator;

[0062] (4) After 12 hours, remove the heavy water-containing medium from dish 2, add 1 mL of 4% formaldehyde for fixation, and place it in a 4°C refrigerator for half an hour. Then remove the fixing solution, add 2 mL of PBS buffer, and store it in a 4°C refrigerator;

[0063] (5) Remove the cell solution from dish 3, add 1 mL of 4% formaldehyde for fixation, and place it in a 4°C refrigerator for half an hour. Then remove the fixing solution, add 2 mL of PBS buffer, and store it in a 4°C refrigerator.

[0064] Perform SRS imaging on the fixed blank control group and drug-added sample group. CH (carbon hydrogen) and CD (carbon deuterium) signals are collected at 2912 cm -1 and 2133 cm -1 , respectively, and the CD / CH ratio is calculated by 2633 cm-1 The wave number collection non-resonant data to remove non-resonant signal in the later data processing, the GES-1 cell imaging results of 30% D2O culture condition are shown in Figure 1, and the GES-1 cell imaging results of 50% D2O culture condition are shown in Figure 2.

[0065] The carbon deuterium signal intensity of CD channel reaction cells, that is, the reaction cells are cultured with culture medium containing heavy water, and the newly synthesized cell components containing carbon deuterium chemical bonds related to cell metabolism, the grayish white background in the figure is the carbon deuterium signal, the brighter the signal, the stronger the signal, and the signal intensity can reflect the metabolic intensity of the cells. As can be seen from the figure, the gray part in the figure becomes brighter and brighter with the increase of time, indicating that the deuterium in the heavy water is participating in the metabolism of GES-1 cells over time.

[0066] The signal intensity in the imaging results is analyzed, and the results are shown in Figure 3. Under the same culture time (24 hours), there is almost no difference between 30% D2O culture and 50% D2O culture on cell CH signal (Figure 3A), and 50% D2O culture makes the cell CD signal (Figure 3B) and CD / CH signal ratio (Figure 3C) slightly higher than 30% D2O culture, so both 30% D2O culture and 50% D2O culture can be used as culture conditions. Under the same culture conditions (30% D2O or 50% D2O), the culture time (12 hours or 24 hours) has no obvious effect on the CD signal intensity and the CD / CH signal ratio.

[0067] Example 4: Drug comparison experiment analysis

[0068] The cell slides were cultured with normal culture medium and culture medium with the highest concentration of drug GC4 (gemcitabine 200 μM + cisplatin 100 μM) respectively, and the liquid was changed after 6 hours of culture (37℃, 5% CO2), the waste liquid was absorbed and washed with PBS, then 30% D2O culture medium and 30% D2O culture medium containing the highest concentration of drug were added for 48 hours, and then fixed and imaged after 48 hours. The CH (carbon hydrogen) and CD (carbon deuterium) signals were collected at 2912 cm -1 and 2133 cm -1 The CH (carbon hydrogen) and CD (carbon deuterium) signals were collected at 2912 cm -1The non-resonant data was collected to remove the non-resonant signal in the later data processing, and the imaging results are shown in Figure 4. It can be seen that in the image with the drug added, the CH signal has lost the cell morphology, indicating that the drug has caused great damage to the tumor cells and tumor-related cells; at the same time, the metabolism of GES-1 cells is greatly reduced, as can be seen from the CD signal diagram, the CD signal of the untreated group is much higher than that of the drug-treated group, also indicating that the addition of GC4 drug significantly inhibits the metabolism of cells, causing great damage to tumor and tumor-related cells.

[0069] The signal intensity was analyzed, and the results are shown in Figure 5. Not only the CH and CD signals are greatly weakened, but also the ratio of CD / CH is significantly reduced. Using this method, the influence of the drug on tumor and tumor-related cells can be intuitively and quickly reflected by the changes in CH and CD signal intensity.

[0070] Example 5: Drug gradient experiment analysis

[0071] 30% D2O and 50% D2O were used as control groups, 6 gradients were set in each group, and the cells were first cultured on a climbing board for 24 h. The first two gradients were directly added with culture medium, and the last four gradients were added with culture medium containing corresponding concentrations of drugs for continuous culture for 24 h. After culture, the first gradient was continuously added with ordinary culture medium, the second gradient was added with culture medium containing corresponding concentrations of D2O, and the last four gradients were added with culture medium containing corresponding concentrations of D2O and corresponding concentrations of drugs for continuous culture for 24 h. After culture, fixation was performed, and detection was performed. The specific culture conditions of the six groups are as follows:

[0072] (1) complete culture medium (control group);

[0073] (2) 30% / 50% heavy water complete culture medium;

[0074] (3) 30% / 50% heavy water complete culture medium containing experimental maximum drug concentration x 0.001 (0.2 μM gemcitabine + 0.1 μM cisplatin);

[0075] (4) 30% / 50% heavy water complete culture medium containing experimental maximum drug concentration x 0.01 (2 μM gemcitabine + 1 μM cisplatin);

[0076] (5) 30% / 50% heavy water complete culture medium containing experimental maximum drug concentration x 0.1 (20 μM gemcitabine + 10 μM cisplatin);

[0077] (6) 30% / 50% heavy water complete culture medium containing experimental maximum drug concentration (200 μM gemcitabine + 100 μM cisplatin).

[0078] I. Imaging detection of cells without distinguishing protein signal and lipid signal under 30% D2O culture conditions

[0079] At 2912cm -1 and 2133cm -1 CH and CD signals were acquired separately and transmitted through a 1902cm channel. -1 Wavenumber acquisition of non-resonance data was used to remove non-resonance signals during subsequent data processing. The imaging results are shown in Figure 6. It can be clearly seen that the CH signal becomes worse and worse as the drug concentration increases, indicating that the drug has caused great damage to the tumor and tumor-related cells. From the CD signal map, it can be seen that the signal without drug is much higher than the signal after drug addition, while there is almost no CD signal in the control group without D2O.

[0080] Analysis of the carbon-hydrogen (CH) bond signal intensity under 30% D2O culture conditions is shown in Figure 7. It can be seen that with a 10-fold increase in drug concentration, the CH bond signal intensity significantly decreased, indicating significant disruption of cell morphology. Analysis of the carbon-deuterium (CD) bond signal intensity under 30% D2O culture conditions is shown in Figure 8. It can be seen that with a 10-fold increase in drug concentration, the CD bond signal intensity significantly decreased, indicating that cell water metabolism was significantly inhibited with increasing drug concentration.

[0081] II. Imaging and Detection of Cells by Differentiating Protein (P) and Lipid (L) Signals under 30% D2O Culture Conditions

[0082] At 2930cm -1 and 2850cm -1 Wavenumbers were collected from CH P and CH L The signal is at 2177cm. -1 and 2135cm -1 Test CD separately P and CD L The signal, and through 1902cm -1 Wavenumber acquisition was used to obtain non-resonant data for removal during subsequent data processing. The imaging results are shown in Figure 9. With increasing drug concentration, CH (including CH4) P and CH L ) and CD (including CD) P and CD L The signals are all weakening, and when the drug concentration reaches a certain level, the changes are greatly reduced.

[0083] The signal strength was analyzed, and the results are shown in Figures 10-13. As can be seen from the figures, regardless of CH... P CH L CD P Still CD LAll are changed with the change of concentration gradient, and consistent with the gradient trend, there is no CH signal strong and CD signal weak, indicating that stimulated Raman scattering imaging can be through the different imaging wave number in different channels to the specificity of tumor and tumor-related cell chemical composition (lipid and protein) for separate quantitative imaging detection, both can measure CH and CD signal in protein, but also can measure its signal in lipid molecules.

[0084] Three, 50% D2O culture conditions without distinguishing protein signal and lipid signal to image detection of cells

[0085] In 2912cm -1 and 2133cm -1 Collect CH and CD signal, and through 1902cm -1 Wave number to collect non-resonant data, to remove non-resonant signal in later data processing, detection imaging results as shown in Figure 14, can be seen that with the increase of drug concentration, CH signal is getting worse and worse, indicating that the drug has caused great harm to the tumor and tumor-related cells, from the CD signal graph, can be seen that the signal without drug is far higher than that after the drug, such as without D2O (control group) then almost no CD signal, the results consistent with the results of 30% D2O culture conditions.

[0086] Four, 50% D2O culture conditions without distinguishing protein (P) and lipid (L) signal to image detection of cells

[0087] In 2930cm -1 and 2850cm -1 Wave number to collect CH P and CH L Signal, in 2177cm -1 and 2135cm -1 Detection of CD P and CD L Signal, and through 1902cm -1 Wave number to collect non-resonant data, to remove non-resonant signal in later data processing, detection imaging results as shown in Figure 15, with the increase of drug concentration, CH and CD signal are weakening, when the drug concentration to a certain concentration, the change greatly weakened, whether CH P , CH L , CD P or CD LThe signal intensity of CH and CD both changed with the concentration gradient, and was consistent with the gradient trend, and there was no case of strong CH signal and weak CD signal, which was consistent with the result obtained by 30% D2O culture. It showed that coherent Raman imaging could be used to quantitatively image the specific chemical composition (lipid and protein) of tumor and tumor-related cells in different channels by different imaging wave numbers, which could measure the signal of CH and CD in protein and lipid molecules.

[0088] Obviously, the above embodiments are only examples for clearly illustrating, and are not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for evaluating the effect of a drug on tumors and tumor-associated cells based on cell metabolic imaging, characterized in that: Culturing tumor and tumor-related cells in a culture medium containing a drug to be tested and a deuterium-containing metabolic marker, imaging the tumor and tumor-related cells by coherent Raman scattering microscopic imaging technology, and quantifying the metabolic product signal of the metabolic marker, and detecting the drug effect of the cell sample based on metabolic inhibition difference by the same detection scheme, for evaluating the effect of the drug to be tested on the tumor and tumor-related cells.

2. The evaluation method according to claim 1, characterized in that: Culturing the tumor and tumor-related cells in a first culture medium containing a drug for a first duration, adding the deuterium-containing metabolic marker, and culturing the tumor and tumor-related cells in a second culture medium containing the drug to be tested and the metabolic marker for a second duration.

3. The evaluation method according to claim 2, characterized in that: The first duration and the second duration are 0-48 hours, and the sum of the first duration and the second duration is 24-96 hours.

4. The evaluation method according to claim 1, characterized in that: The coherent Raman scattering microscopic imaging technology includes one or more of stimulated Raman scattering microscopic imaging and coherent anti-Stokes microscopic imaging technology.

5. The evaluation method of claim 1, wherein: The deuterium-containing metabolic marker includes one or more of heavy water, deuterated palmitic acid, deuterated oleic acid, deuterated amino acid, deuterated glucose, and deuterated cholesterol.

6. The evaluation method according to claim 5, characterized in that: The final concentration of the heavy water in the culture medium is 30-50%.

7. The evaluation method of claim 1, wherein: The culture inoculation density of the tumor to be detected and tumor-related cells is (0.05-1)×10 5 / cm 2 when the coherent Raman optical microscopic imaging technology is detected.

8. The evaluation method of claim 1, wherein: The amount of the culture medium to be added is (100-500) μL / cm 2 .

9. A kit for detecting the effect of a drug on tumors and tumor-associated cells, characterized by: The kit includes reagents used in the evaluation method of any one of claims 1-8.

10. Use of the kit of claim 9 in evaluating the effect of a drug on tumor and tumor-related cells.

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