Cell nuclear fiber layer in-situ separation method
Through an in-situ separation method of the cell nuclear fiber layer including adherent culture, separation fluid treatment, fixation fluid fixation and cleaning fluid cleaning, the problem of difficulty in separating and studying the in-situ structure of the cell nuclear fiber layer in the prior art is solved, and the acquisition of the complete structure of the cell nuclear fiber layer is achieved, and the disclosure of the function of the nuclear fiber layer is promoted.
Patent Information
- Application Number
- CN202510290869.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-05-30
AI Technical Summary
The existing technology is difficult to effectively isolate and study the in situ structure of the cell nuclear fiber layer, which affects the comprehensive disclosure of the function of the nuclear fiber layer.
A method for in situ separation of cell nuclear fiber layers is provided, including adherent culture, treatment of the nuclear skeleton with separation fluid, fixing the nuclear skeleton with fixing fluid, and cleaning the cell nuclear fiber layer. The separation fluid, fixation fluid and cleaning fluid all contain specific ingredients to ensure the integrity and structural retention of the nuclear fiber layer.
This method can simply obtain a complete structure of fiber layer, which is convenient to detect the nuclear fiber layer structure in a physiological state, and helps to fully reveal the functions of the nuclear fiber layer.
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Figure CN120060115A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of bioseparation technology, and particularly relates to a method for in-situ separation of nuclear lamina. Background Art
[0002] The nuclear lamina plays important functions in mammalian cells, mainly including the following aspects: ① supporting the nuclear structure, ② regulating gene expression, ③ regulating DNA repair, and ④ being related to the disassembly of the nuclear membrane. The lamins form a skeletal structure that supports the inner side of the nuclear envelope, enabling the nucleus to maintain its normal shape and size. The structure of the nuclear lamina determines its supporting ability for the nucleus. Studying the in-situ structure of the nuclear lamina is of great significance for studying the functions of the nuclear lamina. Therefore, in-depth research on the in-situ structure of the nuclear lamina is of great significance for comprehensively revealing the functions of the nuclear lamina. Thus, there is an urgent need to provide a solution for the complete separation of the nuclear lamina of the cell. Summary of the Invention
[0003] The purpose of the present invention is to provide a method for in-situ separation of nuclear lamina of the cell, which can more simply obtain a nuclear lamina with a complete structure and is convenient for detecting the structure of the nuclear lamina under physiological conditions.
[0004] A method for in-situ separation of nuclear lamina of the cell provided by the present invention includes: after adherent culture of the cells to be separated, aspirating and discarding the culture medium and washing, treating with a separation solution on ice for 1 h - 3 h and washing to obtain a nuclear skeleton, fixing the nuclear skeleton with a fixing solution, and then washing with a washing solution to obtain an in-situ nuclear lamina of the cell; the separation solution includes 0.1% - 2% of a separating agent and an enzyme inhibitor with a concentration of 15 μL / mL - 25 μL / mL; the fixing solution includes 0.5% - 3% of a fixing agent, and the fixing agent includes glutaraldehyde and osmium tetroxide; the washing solution includes 10% - 100% of a cleaning agent, and the cleaning agent includes ethanol.
[0005] Optionally, the enzyme inhibitor includes serine protease inhibitor, protease inhibitor, and phosphoprotease inhibitor.
[0006] Optionally, the separation solution includes a serine protease inhibitor with a concentration of 18 μL / mL, a protease inhibitor with a concentration of 1 μL / mL, and a phosphoprotease inhibitor with a concentration of 1 μL / mL.
[0007] Optionally, the separating agent includes Nonidet P40 or Triton X-100.
[0008] Optionally, the separation solution includes 0.1% of Triton X-100.
[0009] Optionally, the separation solution includes 1% of Nonidet P40.
[0010] Optionally, when fixing the nuclear skeleton with a fixing solution, the nuclear skeleton is fixed with fixing solution A and then fixing solution B in sequence.
[0011] Optionally, the types of the fixatives in the fixing solution A and the fixing solution B are independent of each other.
[0012] Optionally, the concentrations of the fixatives in the fixing solution A and the fixing solution B are independent of each other.
[0013] Optionally, after fixing the nuclear skeleton with fixing solution A at 20°C - 30°C, the pre-fixed skeleton is separated; after fixing the pre-fixed skeleton with fixing solution B at 20°C - 30°C, the fixed nuclear skeleton is separated; the fixing treatment durations of the fixing solution A and the fixing solution B are independently 0.5 h - 24 h.
[0014] Optionally, the fixing treatment duration of the fixing solution A is 0.5 h - 24 h, and the fixing treatment duration of the fixing solution B is 0.5 h - 2 h.
[0015] Optionally, gradient washing is performed with cleaning solutions of different concentrations, and then drying is carried out to obtain the in-situ nuclear lamina of cells.
[0016] Optionally, when performing gradient washing, the concentration gradient of the cleaning solution increases.
[0017] Optionally, the concentration difference between adjacent gradients of the cleaning solution is independently 5% - 30%.
[0018] Optionally, when performing gradient washing with cleaning solutions of different concentrations, the washing duration of each gradient of the cleaning solution is independently 10 min - 60 min.
[0019] Optionally, the cells to be separated are cultured adherently and spread to 70% - 80% of the bottom area of the container, and then the culture medium is aspirated.
[0020] Optionally, when treating with the separation solution on ice, the separation solution is pre-cooled in advance, and the temperature of the pre-cooled separation solution is 3°C - 5°C.
[0021] Optionally, after aspirating the culture medium, pre-cooled PBS buffer is used for washing.
[0022] Optionally, the temperature of the pre-cooled PBS buffer is 3°C - 5°C.
[0023] Optionally, the pre-cooled PBS buffer is used for washing 2 - 4 times.
[0024] Optionally, after treatment on ice, washing is performed 2 - 4 times with PBS buffer at 3°C - 5°C.
[0025] Optionally, the separation solution is used to treat on ice for 2 h. Description of the Drawings
[0026] Figure 1 It is a morphological characterization and protein fluorescence map of intact cells and in-situ nuclear lamina of cells obtained in Example 1 under a laser confocal microscope;
[0027] Figure 2 It is a graph of height, roughness and morphological characterization of the in-situ nuclear lamina of cells obtained in Example 1 under an atomic force microscope;
[0028] Figure 3 It is a structural characterization map of the in-situ nuclear lamina of cells obtained in Example 1 under an atomic force microscope;
[0029] Figure 4 It is a flow chart of a method for in-situ separation of nuclear lamina provided by the present invention. Detailed Embodiments
[0030] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein shall have the ordinary meanings understood by those of ordinary skill in the art in the field to which the present invention belongs.
[0031] See Figure 4 , the present invention provides a method for in-situ separation of nuclear lamina, including the following steps:
[0032] S1. After adherent culture of the cells to be separated, aspirate the culture medium and wash;
[0033] S2. Treat with a separation solution on ice for 1 h - 3 h and wash to obtain a nuclear skeleton;
[0034] S3. Fix the nuclear skeleton with a fixing solution;
[0035] S4. Wash with a washing solution to obtain an in-situ nuclear lamina of cells.
[0036] Actually, when performing step S1, the cells to be separated are adherently cultured and spread to 70% - 80% of the bottom area of the container, then the culture medium is aspirated, and after aspirating the culture medium, the cells are washed with pre-cooled PBS buffer. Specifically, the temperature of the pre-cooled PBS buffer is 3°C - 5°C, and the cells are washed with the pre-cooled PBS buffer 2 - 4 times.
[0037] Actually, the separation solution used in step S2 includes 0.1%-2% of a separating agent and enzyme inhibitors with a concentration of 15 μL / mL - 25 μL / mL. Specifically, the used ones include serine protease inhibitor (PMSF), protease inhibitor, and phosphoprotease inhibitor.
[0038] In some embodiments, when using the separation solution to process on ice in step S2, the separation solution is pre-cooled in advance, and the temperature of the pre-cooled separation solution is 3°C - 5°C. In addition, the separating agent includes Nonidet P40 or Triton X-100. Specifically, the separation solution includes 0.1% of Triton X-100, or includes 1% of Nonidet P40.
[0039] In some embodiments, the separation solution includes serine protease inhibitor with a concentration of 18 μL / mL, protease inhibitor with a concentration of 1 μL / mL, and phosphoprotease inhibitor with a concentration of 1 μL / mL in step S2. In some embodiments, after processing on ice in step S2, it is washed 2 - 4 times with PBS buffer solution at 3°C - 5°C, and the separation solution is used to process on ice for 2 h.
[0040] Actually, the fixing solution used in step S3 includes 0.5%-3% of a fixing agent, and the fixing agent includes glutaraldehyde and osmium tetroxide. Specifically, when performing step S2, the nuclear skeleton is fixed successively with fixing solution A and fixing solution B, and the types and concentrations of the fixing agents in fixing solution A and fixing solution B are independent of each other.
[0041] In some embodiments, when performing step S3, the nuclear skeleton is fixed with fixing solution A at 20°C - 30°C and then the pre-fixed skeleton is separated, and the pre-fixed skeleton is fixed with fixing solution B at 20°C - 30°C and then the fixed nuclear skeleton is separated. In addition, the fixing treatment durations of fixing solution A and fixing solution B are independently 0.5 h - 24 h. Preferably, the fixing treatment duration of fixing solution A is 0.5 h - 24 h, and the fixing treatment duration of fixing solution B is 0.5 h - 2 h.
[0042] Actually, the cleaning solution used in step S4 includes 10%-100% of a cleaning agent, and the cleaning agent includes ethanol. Specifically, after gradient cleaning with cleaning solutions of different concentrations and then drying, the in-situ nuclear lamina of cells is obtained; among them, when performing gradient cleaning, the concentration of the cleaning solution increases in a gradient manner. Preferably, the concentration difference between adjacent gradients of the cleaning solution is independently 5%-30%. In some embodiments, when performing gradient cleaning with cleaning solutions of different concentrations, the cleaning duration of each gradient cleaning solution is independently 10 min - 60 min.
[0043] Example 1
[0044] Example 1 provides a method for in-situ isolation of nuclear lamina, including:
[0045] S1. Inoculate hepatocytes (LO2) into a culture dish (with a bottom area of 10 cm 2 ), and culture them in a carbon dioxide incubator at 37 °C until the cells spread to approximately 75% of the bottom area of the culture dish. Then, aspirate the culture medium and wash the cells 3 times with PBS buffer pre-cooled at 4 °C.
[0046] S2. Add 200 μL of isolation solution to the culture dish, let it stand on ice for 2 h, then aspirate the residual solution, and wash the cells 3 times with PBS buffer pre-cooled at 4 °C to obtain in-situ nuclear skeleton of cells. The isolation solution includes 1% Nonidet P40, 18 μL / mL serine protease inhibitor, 1 μL / mL protease inhibitor, and 1 μL / mL phosphatase inhibitor.
[0047] S3. Treat the in-situ nuclear skeleton of cells with Fixative A (2% glutaraldehyde aqueous solution) at room temperature for 2 h, then aspirate Fixative A, wash the cells with pre-cooled PBS buffer, add Fixative B (1% osmium tetroxide aqueous solution), continue to treat the cells at room temperature for 2 h, then aspirate Fixative B and wash the cells with pre-cooled PBS buffer to obtain fixed nuclear skeleton.
[0048] S4. Use ethanol aqueous solutions with concentrations of 30%, 50%, 70%, 80%, 90% and absolute ethanol to perform gradient washing on the fixed nuclear skeleton. The washing time for each gradient is 30 min, and the washing solution of the current gradient is replaced every 15 min. After washing, dry the cells to obtain in-situ nuclear lamina of cells.
[0049] Structural Characterization
[0050] 1. Observe the morphology and protein fluorescence intensity of intact cells and in-situ nuclear lamina of cells by laser confocal microscopy: Treat hepatocytes (LO2) and the in-situ nuclear lamina of cells obtained in Example 1 with 4% paraformaldehyde for 20 min respectively, then add 0.1% Triton X-100 and treat the cells at room temperature for 10 min, then add 0.5% BSA and block the cells at 37 °C for 1 h, then add 1 μg / mL Lamin A / C antibody and incubate the cells overnight at 4 °C, then add Alexa Fluor 488 secondary antibody and incubate the cells at 37 °C for 1 h, finally add DAPI staining solution and incubate the cells at 37 °C for 10 min, and take CLSM photos as Figure 1 shown. It can be seen from Figure 1 that the fluorescence intensity of Lamin A / C in the in-situ nuclear lamina of cells has no change compared with that of intact cells, indicating that the lamin protein on the nuclear lamina is not damaged.
[0051] 2. Detect the height, roughness, and morphology of the in-situ nuclear skeleton and nuclear lamina of cells using an atomic force microscope: Place the in-situ nuclear skeleton and nuclear lamina of cells obtained in Example 1 on the stage of the atomic force microscope. Scan in tapping mode and in AC mode. Determine the tip and sample stage, turn on the laser and adjust its position to the tip. Lower the tip position to 50 μm from the sample, find the peak and adjust the amplitude and setpoint, then lower the tip to start scanning and draw an image as Figure 2 shown, where Figure 2 The upper left figure in A is the morphology map of the in-situ nuclear lamina and the height curve graph of the in-situ nuclear lamina, Figure 2 The lower left figure in A is the morphology map of the in-situ nuclear skeleton and the height curve graph of the in-situ nuclear skeleton, Figure 2 The left figure in B is the height quantification map of the in-situ nuclear lamina and the in-situ nuclear skeleton, Figure 2 The right figure in B is the roughness quantification map of the in-situ nuclear lamina and the in-situ nuclear skeleton; at the same time, obtain the structure of the in-situ nuclear lamina as Figure 3 shown, where Figure 3 A is the surface morphology and magnified morphology map of the in-situ nuclear lamina, Figure 3 Ba is the fiber diameter quantification map of the in-situ nuclear lamina, Figure 3 Bb is the network pore diameter quantification map of the in-situ nuclear lamina, Figure 3 Bc is the network pore area quantification map of the in-situ nuclear lamina.
[0052] It can be seen from Figure 2 that in the in-situ isolated nuclear lamina, the nucleolus protrudes significantly (green arrow), the cytoskeleton is intact (red arrow), the network surface grid of the nuclear lamina is clear (black arrow and the nuclear lamina height curve graph in the upper left figure of 2A), and the surface of the in-situ isolated nuclear skeleton does not show a clear grid structure ( Figure 2 black arrow in the lower left figure of A). The height of the nuclear lamina is significantly lower than that of the nuclear skeleton, and the roughness is significantly greater than that of the nuclear skeleton. The lower height of the nuclear lamina compared to the nuclear skeleton is due to the reduction in the height of the nuclear lamina caused by the removal of the surface proteins of the nuclear skeleton during the process of isolating the in-situ nuclear lamina. The higher roughness of the nuclear lamina compared to the nuclear skeleton is due to the increase in the roughness of the nuclear lamina caused by the complete exposure of the network structure of the nuclear lamina during the process of isolating the in-situ nuclear lamina. It can be seen from Figure 3 that through the method for in-situ isolating the nuclear lamina provided by the present invention, a complete grid-like nuclear lamina can be isolated, removing substances such as surface proteins and lipids that wrap the nuclear lamina network.
[0053] Although the embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are all within the scope and spirit of the present invention as described in the claims. Moreover, the present invention as described herein may have other embodiments and can be implemented or realized in various ways.
Claims
1. A method for in situ separation of cell nuclear lamina, characterized in that: include: After the cells to be separated are cultured on the wall, the culture medium is discarded and washed, and the cells are treated with a separation solution on ice for 1h-3h and washed to obtain a nuclear skeleton, and the nuclear skeleton is fixed with a fixative, and then washed with a washing solution to obtain an in situ nuclear lamina of the cells; the separation solution includes 0.1%-2% of a separation agent and an enzyme inhibitor with a concentration of 15μL / mL-25μL / mL; the fixative includes 0.5%-3% of a fixative, and the fixative includes glutaraldehyde and osmic acid; the washing solution includes 10%-100% of a cleaning agent, and the cleaning agent includes ethanol.
2. The in-situ separation method according to claim 1, characterized in that: The enzyme inhibitors include serine protease inhibitors, proteinase inhibitors, and phosphatase inhibitors; preferably, the separation solution includes serine protease inhibitors at a concentration of 18 μL / mL, proteinase inhibitors at 1 μL / mL, and phosphatase inhibitors at 1 μL / mL.
3. The in-situ separation method according to claim 1, characterized in that: The separation agent includes Nonidet P40 or Triton X-100; preferably, the separation solution includes 0.1% Triton X-100, or the separation solution includes 1% Nonidet P40.
4. The in-situ separation method according to claim 1, characterized in that: When using a fixative to fix the nuclear skeleton, fixative A and fixative B are used sequentially to fix the nuclear skeleton; the types of the fixatives in the fixative A and the fixative B are independent of each other, and / or the concentrations of the fixatives in the fixative A and the fixative B are independent of each other.
5. The in-situ separation method according to claim 4, characterized in that: Fix the nuclear skeleton with fixative A at 20°C-30°C and then separate the pre-fixed skeleton, and fix the pre-fixed skeleton with fixative B at 20°C-30°C and then separate the fixed nuclear skeleton; the fixation treatment time of the fixative A and the fixative B are independently 0.5h-24h; preferably, the fixation treatment time of the fixative A is 0.5h-24h, and the fixation treatment time of the fixative B is 0.5h-2h.
6. The in-situ separation method according to claim 1, characterized in that: The cell in situ nuclear lamina is obtained by gradient washing with washing solutions of different concentrations and then drying; wherein the concentration gradient of the washing solution increases during gradient washing, and preferably, the concentration difference of the washing solutions of adjacent gradients is independently 5%-30%.
7. The in-situ separation method according to claim 6, characterized in that: When gradient cleaning is performed using cleaning solutions of different concentrations, the cleaning time of each gradient of the cleaning solution is independently 10 min-60 min.
8. The in-situ separation method according to claim 1, characterized in that: After the cells to be separated are cultured and spread to 70%-80% of the bottom area of the container, the culture medium is discarded; and / or, when the separation solution is used on ice, the separation solution is pre-cooled in advance, and the temperature of the pre-cooled separation solution is 3°C-5°C.
9. The in-situ separation method according to claim 1, characterized in that: After the culture medium is discarded, the cells are washed with pre-cooled PBS buffer; wherein the temperature of the pre-cooled PBS buffer is 3° C.-5° C.; and / or, the cells are washed with pre-cooled PBS buffer 2-4 times.
10. The in-situ separation method according to claim 1, characterized in that: After treatment on ice, wash with 3°C-5°C PBS buffer 2-4 times; and / or, treat with separation solution on ice for 2 hours.