A method for detecting cell nuclear resonance frequencies

Abstract

This invention discloses a method for detecting the intrinsic frequency of cell nuclei. S1: Surgically obtained tumor specimens are placed in a 4°C culture medium under sterile conditions and washed twice with a pre-cooled phosphate-buffered saline solution at 4°C. S2: Then, the specimens are digested using 0.125% trypsin in an incubator, followed by the addition of a suitable amount of culture medium containing fetal bovine serum to terminate the digestion. S3: The digested mixture is filtered, and the filtered cells are seeded at a density of 5000 cells per well in a 6-well plate for culture. This method is a systematic strategy for detecting the intrinsic frequency of tumor cell nuclei in vitro. Tumor cells are obtained from patient tumor tissue obtained during surgery. Lentiviral transfection is used to label the nuclei of the tumor cells, enabling them to emit bright fluorescence. The labeled cells are then excited with ultrasound of different frequencies to identify the intrinsic frequency of the tumor cell nuclei. This more accurately identifies the resonance frequency of tumor cells, providing a guarantee for subsequent tumor cell killing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for detecting cell nuclear resonance frequency. Background Technology

[0002] Malignant tumors are one of the most significant causes of death and harm to human health. Current treatments primarily include surgery, radiotherapy, chemotherapy, and targeted therapy. However, some patients still do not respond well to these treatments. In recent years, physical methods for treating tumors have begun to be used clinically.

[0003] Sound waves are mechanical vibration waves of objects. They have a strong ability to penetrate tissues and have been widely used in clinical diagnosis and treatment, such as imaging and high-intensity focused ultrasound.

[0004] Resonance refers to the phenomenon where a physical system vibrates with a larger amplitude at a specific frequency than at other frequencies. These specific frequencies and wavelengths are called resonant frequencies. At the resonant frequency, a very small periodic vibration can produce a large vibration because the system stores kinetic energy. When the resistance is very small, the resonant frequency is approximately equal to the system's natural frequency (or inherent frequency), which is the frequency of free oscillation.

[0005] Theoretically, the natural frequency of a structural system depends only on its inherent properties (such as mass and stiffness). When the periodic excitation frequency approaches its natural frequency, the system amplitude tends towards infinity, i.e., resonance occurs. Compared to healthy cells, tumor cells differ from normal cells in nucleus size, stiffness, and mass; therefore, their natural frequencies should theoretically differ. This difference can be used to selectively kill tumor cells. When the natural frequency of a tumor cell nucleus is applied, it causes the nucleus to vibrate significantly, leading to tumor cell death. This frequency has no effect on normal cells.

[0006] However, the physical characteristics of tumor cells in different locations (such as lung cancer and liver cancer) are heterogeneous, and even tumors in different individuals from the same location exhibit heterogeneity. Therefore, based on the principles of precision and personalized treatment, the intrinsic frequency of the cell nucleus in the target tumor should be detected before using ultrasound resonance therapy.

[0007] Therefore, this invention proposes a method for detecting the resonance frequency of cell nuclei. Summary of the Invention

[0008] The purpose of this invention is to provide a method for detecting the resonance frequency of cell nuclei, so as to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a method for detecting the resonance frequency of cell nuclei, the specific steps of which include: S1: placing the tumor specimen obtained by surgery into a culture medium at 4°C under sterile conditions, and washing it twice with a phosphate buffer solution pre-cooled at 4°C; S2: Then digest with 0.125% trypsin in an incubator, and then add an appropriate amount of culture medium containing fetal bovine serum to stop the digestion; S3: Filter the digested mixture, and seed the filtered cells into 6-well plates at a density of 5000 cells per well for culture; S4: Tumor cells are transfected with lentiviruses to make the cells express enhanced green fluorescent protein (EGFP) carrying a nuclear localization sequence. The EGFP synthesized in the cytoplasm will be transported to the cell nucleus, making the cells emit bright fluorescence. S5: Place the cells under a fluorescence microscope, observe them through the FITC channel, and take photos for recording. S6: Next, the labeled cells were excited by ultrasound at different frequencies, and the images were taken again; S7: If the frequency is the inherent frequency of the tumor cell nucleus, the nucleus will vibrate at a high frequency. The exposure time is much longer than the vibration period. Under long exposure, the fluorescent area of ​​the nucleus at this time will be larger than that of the nucleus when it is not vibrating, so the inherent frequency of the tumor cell nucleus can be detected.

[0010] Preferably, in step S2, the digestion time with 0.125% trypsin in an incubator is 15-30 minutes, and the temperature is maintained at 37°C.

[0011] Preferably, in step S3, a 40μm cell sieve is used for filtration.

[0012] Preferably, in step S5, the fluorescence microscope used is a confocal microscope or a super-resolution microscope.

[0013] Preferably, in steps S5 and S6, the settings parameters for the two photos need to be consistent.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This method is a systematic strategy for detecting the intrinsic frequency of tumor cell nuclei in vitro. Tumor cells are obtained from tumor tissue obtained from patients during surgery. The nuclei of the tumor cells are labeled with lentivirus transfection, so that the nuclei can emit bright fluorescence. The labeled cells are excited by ultrasound of different frequencies to find the intrinsic frequency of the tumor cell nuclei. The resonant frequency of tumor cells can be identified more accurately, which provides a guarantee for subsequent killing of tumor cells. Attached Figure Description

[0015] Figure 1This is a schematic diagram of the process of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Please see Figure 1 The present invention provides a technical solution: a method for detecting the resonance frequency of cell nuclei, the specific steps of which include: S1: placing the tumor specimen obtained by surgery into a culture medium at 4°C under sterile conditions, and washing it twice with a phosphate buffer solution pre-cooled at 4°C; S2: Then digest with 0.125% trypsin in an incubator, and then add an appropriate amount of culture medium containing fetal bovine serum to stop the digestion; S3: Filter the digested mixture, and seed the filtered cells into 6-well plates at a density of 5000 cells per well for culture; S4: Tumor cells are transfected with lentiviruses to make the cells express enhanced green fluorescent protein (EGFP) carrying a nuclear localization sequence. The EGFP synthesized in the cytoplasm will be transported to the cell nucleus, making the cells emit bright fluorescence. S5: Place the cells under a fluorescence microscope, observe them through the FITC channel, and take photos for recording. S6: Next, the labeled cells were excited by ultrasound at different frequencies, and the images were taken again; S7: If the frequency is the intrinsic frequency of the tumor cell nucleus, the nucleus will vibrate at a high frequency. Since the exposure time is much longer than the vibration period, the fluorescent area of ​​the nucleus under long exposure will be larger than that of the nucleus when not vibrating, thus allowing the intrinsic frequency of the tumor cell nucleus to be detected. In this embodiment, preferably, in step S2, the digestion time with 0.125% trypsin in the incubator is 15-30 minutes, and the temperature is maintained at 37°C. In this embodiment, preferably, in step S3, a 40μm cell sieve is used for filtration. In this embodiment, preferably, in step S5, a confocal microscope or a super-resolution microscope is used for fluorescence microscopy. In this embodiment, preferably, in steps S5 and S6, the settings for the two photographs should be consistent for better comparison and a more intuitive comparison of the results.

[0018] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting the resonance frequency of cell nuclei, characterized in that: The specific steps include: S1: Place the tumor specimen obtained by surgery into a culture medium at 4°C under sterile conditions and wash it twice with a phosphate buffer solution pre-cooled at 4°C. S2: Then, digestion was performed using 0.125% trypsin in an incubator, followed by the addition of an appropriate amount of culture medium containing fetal bovine serum to terminate the digestion. S3: Filter the digested mixture, and seed the filtered cells into 6-well plates at a density of 5000 cells per well for culture; S4: Tumor cells are transfected with lentiviruses to make the cells express enhanced green fluorescent protein (EGFP) carrying a nuclear localization sequence. The EGFP synthesized in the cytoplasm will be transported to the cell nucleus, making the cells emit bright fluorescence. S5: Place the cells under a fluorescence microscope, observe them through the FITC channel, and take photos for recording. S6: Next, the labeled cells were excited by ultrasound at different frequencies, and the images were taken again; S7: If the frequency is the inherent frequency of the tumor cell nucleus, the nucleus will vibrate at a high frequency. The exposure time is much longer than the vibration period. Under long exposure, the fluorescent area of ​​the nucleus at this time will be larger than that of the nucleus when it is not vibrating, so the inherent frequency of the tumor cell nucleus can be detected.

2. The method for detecting cell nuclear resonance frequency according to claim 1, characterized in that: In step S2, the digestion time with 0.125% trypsin in an incubator is 15-30 minutes, and the temperature is maintained at 37°C.

3. The method for detecting cell nuclear resonance frequency according to claim 1, characterized in that: In step S3, a 40μm cell sieve is used for filtration.

4. The method for detecting cell nuclear resonance frequency according to claim 1, characterized in that: In step S5, the fluorescence microscope used is a confocal microscope or a super-resolution microscope.

5. The method for detecting cell nuclear resonance frequency according to claim 1, characterized in that: In steps S5 and S6, the settings parameters for taking photos must be consistent for both photos.

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