CN108872187B - Method for detecting drug resistance by using Raman imaging technology and application thereof - Google Patents
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Abstract
The invention discloses a method for detecting drug resistance by using a Raman imaging technology, which comprises the following steps: (1) respectively carrying out drug resistance treatment on the wild type and the drug resistance type of the tumor cell line, and then respectively taking each nucleolar region and each cytoplasmic region as a Raman imaging technology detection sample; (2) preparing a Raman reference spectrum by scanning the detection sample obtained in the step (1), and obtaining a characteristic peak of the drug-resistant cell; (3) detecting an unknown sample to be detected by using a Raman spectrometer to obtain a Raman spectrum of the sample to be detected; (4) and (4) analyzing whether the Raman spectrum of the sample to be detected obtained in the step (3) has a characteristic peak which is the same as that of the Raman reference spectrum, namely, carrying out qualitative identification according to the characteristic peak position and the intensity of the Raman spectrum of the drug-resistant tumor cells. The method for detecting the drug resistance by using the Raman imaging technology has the advantages of high detection speed and high efficiency, and has important practical application value for clinically detecting the drug resistance of cells.
Description
Technical Field
The invention relates to the technical field of medicine detection, in particular to a method for detecting drug resistance by using a Raman imaging technology and application thereof.
Background
The Raman imaging technology is a new generation of rapid, high-precision and surface scanning laser Raman technology, perfectly combines a confocal microscope technology and a laser Raman spectrum technology, is used as a third generation Raman technology, and has the characteristics of high speed and extremely high resolution imaging. Compared with the original traditional Raman application technology, the new generation Raman imaging speed is 600 times of that of the conventional Raman mapping, and high-fraction Raman images of the sample can be obtained within minutes.
Drug resistance (drug resistance) refers to the resistance of microorganisms, parasites and tumor cells to the action of chemotherapeutic drugs, and once drug resistance occurs, the chemotherapeutic action of the drugs is significantly reduced. Drug resistance can be classified into acquired drug resistance and natural drug resistance according to the cause of the occurrence of the drug resistance. Natural resistance may also exist for a pathogen in nature, such as a strain of bacteria. Tumors are a group of malignant diseases caused by tumor-driving genes and are characterized by high morbidity and mortality. The greatest challenge encountered during tumor therapy is the problem of therapeutic resistance. The point that the targeted therapy causes drug resistance is proved by clinical practice, so that the reason why the tumor has drug resistance needs to be known from the source to really 'take the drug to the symptom', thereby delaying the drug resistance as much as possible. However, the tumor is constantly changing dynamically, and new tumor cells are continuously generated, which include some variant progeny, so that it can be said that the drug resistance is the result of "tumor evolution".
At present, drug resistance detection is generally molecular level detection or cell level detection, the procedure is generally complex, and the Raman imaging technology is not applied to drug resistance detection.
In summary, how to quickly and effectively solve the technical problem of drug resistance of a drug is a problem that those skilled in the art are in urgent need to solve.
Disclosure of Invention
In order to rapidly and effectively detect the problem of drug resistance, the invention aims to provide a method for detecting the drug resistance by using a Raman imaging technology.
The invention also aims to provide the application of the method in detecting the drug resistance of the cells.
In order to achieve the above object, the present invention firstly provides a method for detecting drug resistance by using raman imaging technology, the method comprising the following steps:
(1) respectively carrying out drug resistance treatment on the wild type and the drug resistance type of the tumor cell line, and then respectively taking each nucleolar region and each cytoplasmic region as a Raman imaging technology detection sample;
(2) preparing a Raman reference spectrum by scanning the detection sample obtained in the step (1), and obtaining a characteristic peak of the drug-resistant cell;
(3) detecting an unknown sample to be detected by using a Raman spectrometer to obtain a Raman spectrum of the sample to be detected;
(4) and (4) analyzing whether the Raman spectrum of the sample to be detected obtained in the step (3) has a characteristic peak which is the same as that of the Raman reference spectrum, namely, qualitatively identifying according to the peak position and the intensity of the characteristic peak of the Raman spectrum of the drug-resistant tumor cell, and identifying whether the cell generates drug resistance.
Preferably, the tumor cell comprises a lung cancer, liver cancer, ovarian cancer, uterine cancer or breast cancer cell.
More preferably, the tumor cell is a lung cancer tumor cell and the agent for drug-resistant treatment is a paclitaxel agent.
Preferably, the characteristic peak of the Raman spectrum of the drug-resistant tumor cell is that a vibration peak of 1515cm & lt-1 & gt to 1525cm & lt-1 & gt appears in the cytoplasm region of the cell.
More preferably, the characteristic peak of the Raman spectrum of the drug-resistant tumor cell is 1520cm-1 of the vibration peak of beta-carotene in the cytoplasm region of the cell.
Preferably, the detection condition of the Raman spectrometer is that the wavelength is 532nm, and the excitation power is 235 μ W/μm2 on the sample plane.
Furthermore, the invention also provides application of the method in detecting cell drug resistance.
Preferably, the drug resistance is the drug resistance of taxol which is an anti-tumor drug.
Advantageous effects
The method for detecting the drug resistance by using the Raman imaging technology has the advantages of high detection speed and high efficiency, and has important practical application value for clinically detecting the drug resistance of cells.
Drawings
FIG. 1 shows different spectra of light waves in drug-resistant lung cancer cells.
The figure has 4 peaks from top to bottom, which are respectively marked as 1, 2, 3 and 4, and the schematic diagram is as follows:
the more protein part (basically near the cell nucleus) of the 1-Zhi-1Red paclitaxel sample;
the lipid-rich fraction (essentially near the cytoplasmic domain) of the 2-Zhi-1Green paclitaxel sample;
the lipid-rich fraction of the 3-Ori-3Red non-paclitaxel treated sample;
4-Ori-3Green samples not treated with paclitaxel had a more protein fraction (essentially near the nucleus);
the vertical line shows that the second spectrum shows the vibrational peak of 1520cm-1 beta-carotene, which is the characteristic peak of taxol.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
General grade fetal bovine serum purchased from the biotechnological institute of Beijing Yuan Heng san Ma
Premium fetal bovine serum from Lanzhou Bailing Biotechnology Ltd
RPMI1640 Medium was purchased from Gibco, USA as dry powder
Taxol is purchased from Doderelike Biotech Ltd
The taxol is a natural secondary metabolite separated and purified from the bark of a gymnosperm yew, and has good anti-tumor effect through clinical verification, and particularly has special effects on ovarian cancer, uterine cancer, breast cancer and the like with high incidence rate of cancer. Paclitaxel is the most popular anticancer drug in the international market in recent years, and is considered to be one of the most effective anticancer drugs in human for the next 20 years. In recent years, the global population and the cancer incidence rate have increased explosively, and the demand for paclitaxel has also increased significantly.
Example 1 detection of paclitaxel resistance in Lung cancer tumor cells Using Raman imaging
The A549 lung cancer tumor cell line wild type and drug-resistant type used by the invention are purchased from Shanghai Beinoco Biotechnology Co. The experimental cell lines were all placed at 37 ℃ with 10% fetal calf serum added and 5% CO2Culturing in an incubator.
RPMI1640 cell culture medium preparation (1L): 1640 Dry powder (Gibco) in one bag, NaHCO32.0g/L, dissolving in triple distilled water, slowly stirring at room temperature for 4 hours, adjusting the pH value to 7.2 by using concentrated hydrochloric acid, fixing the volume to 1L, roughly filtering by using filter paper, then filtering and sterilizing by using a 0.22-micron microporous filter membrane (the pH value generally rises by 0.1-0.3 after filtering), subpackaging in 500mL glass bottles, sealing by using a sealing membrane, storing at 4 ℃, and adding penicillin (the final concentration is 100U/mL), streptomycin sulfate (the final concentration is 100U/mL) and fetal calf serum (the total volume is 10%) before use.
A549 lung cancer cell line wild type and drug-resistant type are selected, and paclitaxel (1 micromole/liter) with the death amount more than half of the death amount is added into the drug-resistant cell line. The two were placed in IPMI1640 medium and 10% fetal bovine serum and cultured normally at 37 ℃ and the cells were observed to grow well.
And detecting the spectrums of the wild type and the drug-resistant type of the treated A549 lung cancer tumor cell line by using a Raman-11 type machine (a slit scanning confocal Raman microscope). The method comprises the following specific steps:
scanning a drug-resistant A549 lung cancer tumor cell line wild type and drug-resistant type, randomly selecting 4 drug-resistant and wild type lung cancer cells respectively, and taking each nucleolar region and each cytoplasmic region respectively, and then performing linear scanning under a mirror, wherein the time for scanning each cell is 15 minutes, so as to obtain Raman spectra of the drug-resistant and wild type lung cancer cells. The wavelength of the Raman spectrometer is 532nm, and the excitation laser power on the sample plane is 235 mu W/mu m2。
As shown in FIG. 1, the light wave of lung cancer cells showed different spectra, the vertical line shows that the second spectrum showed a vibrational peak at 1520cm-1 beta-carotene, and the vibrational peak at 1520cm of beta-carotene was repeatedly observed in the Raman spectrum-1. The characteristic peak of the drug resistance of the taxol medicaments is determined through multiple verification, comparison and analysis.
Example 2 detection of drug resistance of Lung cancer tumor cells to paclitaxel Using Raman imaging
The lung cancer tumor cell line H1299 was cultured and treated for drug resistance in the same manner as in example 1.
The spectra of the treated H1299 lung cancer cell line wild-type and drug-resistant were examined using a Raman-11 machine (slit scanning confocal Raman microscope). The method comprises the following specific steps:
scanning drug-resistant H1299 lung cancer tumor cell lines of wild type and drug-resistant type, randomly selecting 4 drug-resistant and wild type lung cancer cells respectively, and taking each nucleolar region and each cytoplasmic region respectively, and then performing linear scanning under a mirror, wherein the time for scanning each cell is 15 minutes, and obtaining Raman spectra of the two cells. The wavelength of the Raman spectrometer is 532nm, and the excitation laser power on the sample plane is 235 mu W/mu m2。
As a result, a vibrational peak of beta-carotene 1520. + -.5 cm was repeatedly observed in the Raman spectrum-1. The lung cancer tumor cell line H1299 was again shown to be resistant to paclitaxel.
The method for detecting the drug resistance by using the Raman imaging technology has the advantages of high detection speed and high efficiency, and has important practical application value for clinically detecting the drug resistance of cells.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (3)
1. A method for detecting drug resistance using raman imaging, said method comprising the steps of:
(1) respectively carrying out taxol reagent drug resistance treatment on the wild type and the drug resistance type of the lung cancer cell line, and then respectively taking each nucleolus region and each cytoplasm region as Raman imaging technology detection samples; the detection condition of the Raman spectrometer is that the wavelength is 532nm, and the excitation power on the sample plane is 235 MuW/Mum 2;
(2) preparing a Raman reference spectrum by scanning the detection sample obtained in the step (1), and obtaining a characteristic peak of the drug-resistant cell;
(3) detecting an unknown sample to be detected by using a Raman spectrometer to obtain a Raman spectrum of the sample to be detected;
(4) analyzing whether the Raman spectrum of the sample to be detected obtained in the step (3) has a characteristic peak which is the same as that of a Raman reference spectrum, namely, carrying out qualitative identification according to the peak position and the intensity of the characteristic peak of the Raman spectrum of the drug-resistant lung cancer cell to realize the identification of whether the cell generates drug resistance; the characteristic peak of the Raman spectrum of the drug-resistant lung cancer cell is 1515cm appeared in the cytoplasm region of the cell-1～1525cm-1A vibration peak.
2. The method of claim 1, wherein the Raman spectrum of the drug-resistant lung cancer cell has a characteristic peak of 1520cm in the cytoplasmic domain of the cell at which a peak of β -carotene is present-1。
3. Use of the method of claim 1 or 2 for detecting drug resistance in a cell.
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Inventor after: Zhan Xiaokai Inventor after: Liu Jie Inventor after: Zhang Sen Inventor after: Lin Lin Inventor after: Hao Xuezhi Inventor before: Zhan Xiaokai Inventor before: Liu Jie Inventor before: Zhang Sen |
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Effective date of registration: 20231218 Address after: 8 worker's Stadium South Road, Chaoyang District, Beijing 100020 Patentee after: BEIJING CHAO-YANG HOSPITAL, CAPITAL MEDICAL University Address before: No. 712, 110 / F, nanxinyuan Middle Road, Chaoyang District, Beijing, 100122 Patentee before: Zhan Xiaokai |