Cell nucleus skeleton in-situ separation method

Abstract

The invention provides a cell nucleus skeleton in-situ separation method, and relates to the technical field of biological separation. The in-situ separation method provided by the invention comprises the following steps: carrying out adherent culture on cells to be separated, then absorbing and discarding a culture solution, cleaning, treating on ice by using a separation solution for 1-3 hours, and cleaning to obtain a cell in-situ nucleus skeleton; the separating liquid comprises a separating agent with the concentration of 0.1-2% and enzyme inhibitors with the concentration of 15-25 [mu] L / mL, and the enzyme inhibitors comprise a serine protease inhibitor, a proteinase inhibitor and a phosphoproteinase inhibitor. According to the method, the complete cell nucleus skeleton can be simply and efficiently separated, the biomechanical property of the cell nucleus can be more accurately detected, and meanwhile, an important tool is provided for in-vitro research on dynamic expression of genes.

Description

Technical Field

[0001] The present invention relates to the technical field of biological separation, and in particular to a method for in-situ separation of nuclear skeleton. Background Art

[0002] The nuclear skeleton is the core support structure in the cell nucleus composed of a protein fiber network, mainly composed of lamins, nuclear matrix proteins and chromosome skeletons. These components form a three-dimensional framework through dynamic interactions, running through the nuclear membrane, chromosomes and nucleoli, and play an irreplaceable role in maintaining the physical shape of the cell nucleus and exercising normal biological functions. Among them, the nuclear skeleton is not only the physical skeleton of the cell nucleus, but also can dynamically regulate genome activities, and its structural integrity is directly related to cell survival, cell differentiation and disease development.

[0003] In recent years, the importance of nuclear skeleton research has been continuously increasing in the exploration of disease mechanisms and clinical transformation. Research shows that the abnormalities of nuclear skeleton components are closely related to the occurrence and development of various diseases such as progeria, muscular dystrophy, neurodegenerative diseases and cancer metastasis. However, due to the complex composition, strong dynamics and high intertwining with chromatin of the nuclear skeleton, the premise for analyzing its functional mechanism lies in how to efficiently and reliably separate the nuclear skeleton, which can provide an important tool for in vitro reconstruction of chromatin higher-order structure and research on gene dynamic expression.

[0004] Currently, the separation of the nuclear skeleton mainly uses a solvent to dissolve the nuclear membrane lipid components in the cell nucleus, then uses nuclease to degrade free DNA / RNA to remove chromatin, and finally washes with a salt solution to remove soluble proteins, and the remaining insoluble components are regarded as the nuclear skeleton. However, the above method has a relatively cumbersome operation process, and the obtained nuclear skeleton morphology is not complete enough. Therefore, it is urgent to provide a solution to improve the above problems. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for in-situ separation of nuclear skeleton, which can simply and efficiently separate the complete nuclear skeleton, can more accurately detect the biomechanical properties of the cell nucleus, and at the same time provide an important tool for in vitro research on gene dynamic expression.

[0006] A method for in-situ separation of nuclear skeleton provided by the present invention includes: after adherent culture of the cells to be separated, aspirate the culture medium and wash, treat with a separation solution on ice for 1 h - 3 h and wash to obtain the in-situ nuclear skeleton of the cells; 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, and the enzyme inhibitor includes serine protease inhibitor, protease inhibitor and phosphoprotease inhibitor.

[0007] Optionally, after the cells to be separated are adherently cultured and spread to 70%-80% of the bottom area of the container, discard the culture medium.

[0008] Optionally, after discarding the culture medium, wash with pre-cooled PBS buffer.

[0009] Optionally, the temperature of the pre-cooled PBS buffer is 3°C - 5°C.

[0010] Optionally, wash 2 - 4 times with the pre-cooled PBS buffer.

[0011] Optionally, when treating with the separation solution on ice, pre-cool the separation solution in advance, and the temperature of the pre-cooled separation solution is 3°C - 5°C.

[0012] Optionally, the separating agent includes Nonidet P40 or Triton X-100.

[0013] Optionally, the separation solution includes 0.1% Triton X-100.

[0014] Optionally, the separation solution includes 1% Nonidet P40.

[0015] Optionally, the separation solution includes serine protease inhibitor at a concentration of 18 μL / mL, protease inhibitor at a concentration of 1 μL / mL, and phosphoprotease inhibitor at a concentration of 1 μL / mL.

[0016] Optionally, after treatment on ice, wash 2 - 4 times with PBS buffer at 3°C - 5°C.

[0017] Optionally, treat with the separation solution on ice for 2 h. Description of the Drawings

[0018] Figure 1 It is a morphological characterization diagram of the cell nucleus and the in-situ nuclear skeleton of the cell observed by the laser confocal microscope in the present invention;

[0019] Figure 2 It is a morphological characterization diagram of the cell nucleus and the in-situ nuclear skeleton of the cell observed by the scanning electron microscope in the present invention;

[0020] Figure 3 It is a morphological characterization diagram of the cell nucleus and the in-situ nuclear skeleton of the cell observed by the atomic force microscope in the present invention;

[0021] Figure 4 It is a biomechanical property characterization diagram of the cell nucleus and the in-situ nuclear skeleton of the cell observed by the atomic force microscope in the present invention;

[0022] Figure 5This is a detection graph showing the effect of different treatment durations on the content of in-situ nuclear skeleton surface proteins in cells analyzed by Western blotting according to Example 1 of the present invention;

[0023] Figure 6 This is a flowchart of a method for in-situ isolation of nuclear skeleton provided by the present invention. Detailed implementation manners

[0024] 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 to which the present invention belongs.

[0025] Refer to Figure 6 , the present invention provides a method for in-situ isolation of nuclear skeleton, including the following steps:

[0026] S1. After adherent culture of the cells to be separated, aspirate the culture medium and wash;

[0027] S2. Treat with the separation solution on ice for 1 h - 3 h and wash to obtain the in-situ nuclear skeleton of the cells.

[0028] Actually, the separation solution used in step S2 includes 0.1% - 2% of a separating agent and an enzyme inhibitor with a concentration of 15 μL / mL - 25 μL / mL. Specifically, the used ones include serine protease inhibitor (PMSF), protease inhibitor, and phosphatase inhibitor.

[0029] In some embodiments, when performing step S1, after adherent culture of the cells to be separated until they spread to 70% - 80% of the bottom area of the container, aspirate the culture medium, and after aspirating the culture medium, wash with pre-cooled PBS buffer solution. Specifically, the temperature of the pre-cooled PBS buffer solution is 3°C - 5°C, and wash with the pre-cooled PBS buffer solution 2 - 4 times.

[0030] In some embodiments, when using the separation solution to treat on ice in step S2, pre-cool the separation solution 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.

[0031] In some embodiments, in step S2, the separation solution includes serine protease inhibitor at a concentration of 18 μL / mL, protease inhibitor at a concentration of 1 μL / mL, and phosphoprotease inhibitor at a concentration of 1 μL / mL. In some embodiments, when performing step S2, after treatment on ice, the cells are washed 2 to 4 times with PBS buffer at 3°C - 5°C, and treated with the separation solution on ice for 2 h.

[0032] Example 1

[0033] This Example 1 provides a method for in-situ isolation of nuclear skeleton, including the following steps:

[0034] S1. Inoculate human umbilical vein endothelial cells (HUVECs) into a culture dish (with a bottom area of 10 cm 2 ), use DMEM as the culture medium, and culture 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;

[0035] S2. Add 200 μL of the separation 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 the in-situ nuclear skeleton of the cells. The separation solution includes 1% Nonidet P40, 18 μL / mL serine protease inhibitor, 1 μL / mL protease inhibitor, and 1 μL / mL phosphoprotease inhibitor.

[0036] Example 2

[0037] This Example 2 provides a method for in-situ isolation of nuclear skeleton. The difference from Example 1 is that the separation solution used in step S2 includes 0.1% Triton X-100, 18 μL / mL serine protease inhibitor, 1 μL / mL protease inhibitor, and 1 μL / mL phosphoprotease inhibitor.

[0038] Comparative Example 1

[0039] This Comparative Example 1 provides a method for in-situ isolation of nucleus. The difference from Example 1 is that in step S2, it is left standing on ice for 10 min.

[0040] Comparative Example 2

[0041] This Comparative Example 2 provides a method for in-situ isolation of nucleus. The difference from Example 2 is that in step S2, it is left standing on ice for 10 min.

[0042] Structural Characterization

[0043] 1. Observe the morphology of cell nuclei and in-situ nuclear skeletons of cells using a laser confocal microscope: Take CLSM photos of the in-situ nuclear skeletons of cells obtained in Examples 1 to 2 and the cell nuclei obtained in Comparative Examples 1 to 2 as shown in Figure 1 . It can be seen from Figure 1 that by treating cell nuclei with the separation solution for a long time in Examples 1 to 2, clear and complete nuclear skeletons (within the green circles) can be obtained, while the outlines of the cell nuclei obtained in Comparative Examples 1 to 2 are relatively blurred and the nucleoli are not obvious.

[0044] 2. Observe the morphology of cell nuclei and in-situ nuclear skeletons of cells using a scanning electron microscope: Fix the complete cells after completing step S1 in Example 1, the in-situ nuclear skeletons of cells obtained in Examples 1 to 2, and the cell nuclei obtained in Comparative Examples 1 to 2 with 4% paraformaldehyde for 30 min, then wash them 3 times with double-distilled water, and perform scanning electron microscopy characterization under a 10-μm scale bar as shown in Figure 2 .

[0045] It can be seen from Figure 2 that the morphology of the complete cells (red arrows) (HUVEC NR) can clearly show the protrusions and pseudopodia on the cell surface (black arrows). In the morphologies of the cell nuclei obtained in Comparative Example 1 (HUVEC BN Nonidet P40) and Comparative Example 2 (HUVEC BNTriton X-100), it can be seen that the cytoplasm and cell pseudopodia disappear, leaving the cell nucleus and nuclear skeleton (green arrows). While in the morphologies of the in-situ nuclear skeletons of cells obtained in Example 1 (HUVEC NS Nonidet P40) and Example 2 (HUVEC NS Triton X-100), it can be seen that the cell nuclei are rougher, which indicates that the nuclear membrane proteins and proteins inside the cell nuclei are damaged.

[0046] 3. Observe the morphology of cell nuclei and in-situ nuclear skeletons of cells using an atomic force microscope: Fix the complete cells after completing step S1 in Example 1, the in-situ nuclear skeletons of cells obtained in Examples 1 to 2, and the cell nuclei obtained in Comparative Examples 1 to 2 with 4% paraformaldehyde for 30 min, then wash them 3 times with double-distilled water, and perform atomic force microscopy characterization under a 6-μm scale bar and in gas phase using an atomic force microscope as shown in Figure 3 , where Figure 3 A is the AFM morphology diagram and the corresponding height curve diagram of the complete cells, cell nuclei, and in-situ nuclear skeletons of cells, Figure 3 B is the height quantitative diagram of the complete cells, cell nuclei, and in-situ nuclear skeletons of cells.

[0047] It can be seen from Figure 3As can be seen from Figure A, treating cells with Triton X–100 and Nonidet P40 can both remove the cytoplasm and retain the cell nucleus. From the height curves, it can be found that after removing the cytoplasm, the heights of the obtained cell nuclei and the in-situ nuclear scaffolds are both lower than the cell nucleus region of the intact cells, and the height of the in-situ nuclear scaffold is lower than that of the cell nucleus. This is because the nuclear membrane of the in-situ nuclear scaffold is damaged compared to the cell nucleus, resulting in a decrease in height.

[0048] As can be seen from Figure 3 Figure B, the height of the intact cells (HUVEC NR) is 699.3 nm ± 21.80 nm, the height of Comparative Example 2 (HUVEC BN Triton X-100) is 414.70 nm ± 10.74 nm, the height of Example 2 (HUVEC NS Triton X-100) is 385.34 nm ± 10.84 nm, the height of Comparative Example 1 (HUVEC BN Nonidet P40) is 405.67 nm ± 11.84 nm, and the height of Example 1 (HUVEC NS Nonidet P40) is 272.29 nm ± 9.82 nm. Therefore, it can be shown that the nuclear membrane and nuclear contents in the in-situ nuclear scaffolds of the cells obtained by treating the cell nuclei with Nonidet P40 are removed more thoroughly.

[0049] 4. Observation of the biomechanical properties of cell nuclei and in-situ nuclear scaffolds using an atomic force microscope: For the intact cells after completing step S1 in Example 1, as well as the in-situ nuclear scaffolds of the cells obtained in Examples 1 to 2 and the cell nuclei obtained in Comparative Examples 1 to 2, after fixing with 4% paraformaldehyde for 30 min and washing 3 times with double-distilled water, the atomic force microscope mechanical property detection was carried out using an atomic force microscope under a 6-μm scale and liquid-phase testing as Figure 4 shown, where Figure 4 Figure A is the AFM Young's modulus diagram of intact cells, cell nuclei, and in-situ nuclear scaffolds, Figure 4 Figure B is the height quantification diagram of intact cells, cell nuclei, and in-situ nuclear scaffolds, Figure 4 Figure C is Figure 4 the Young's modulus quantification diagram of Figure A.

[0050] As can be seen from Figure 4 Figure A and Figure 4As can be seen from Figure B, the Young's modulus of the intact cells (HUVEC NR) is 5.15 kPa ± 0.15 kPa, the Young's modulus of Comparative Example 2 (HUVEC BN Triton X-100) is 20.46 kPa ± 2.35 kPa, the Young's modulus of Comparative Example 1 (HUVEC BN Nonidet P40) is 18 kPa ± 1.86 kPa, the Young's modulus of Example 2 (HUVEC NS Triton X-100) is 26.12 kPa ± 0.98 kPa, and the Young's modulus of Example 1 (HUVEC NS Nonidet P40) is 22.02 kPa ± 0.88 kPa. As can be seen from Figure 4 Figure Figure 4 , the Young's modulus of the in-situ nuclear skeleton and the cell nucleus obtained from Example 1 to Example 2 and Comparative Example 1 to Comparative Example 2 is significantly higher than that of the intact cells, and the Young's modulus of the cell nucleus is higher than that of the nuclear region, and the Young's modulus of the in-situ nuclear skeleton is higher than that of the cell nucleus.

[0051] 5. Based on Example 1, Western blot analysis was used to analyze the effect of different treatment durations on the surface protein content of the in-situ nuclear skeleton of cells: After treating with the same separation solution as in Example 1 for 0 min, 10 min, 30 min, 60 min, and 90 min in step S2, PBS with a final concentration of 1 mmol / L PMSF and a protein phosphatase inhibitor was added respectively, and the nuclear skeleton was suspended by pipetting and centrifuged at 4°C and 1000 rpm for 10 min to collect the precipitate. After adding 0.1% SDS to the precipitate, it was pipetted evenly and left standing on ice for 40 min (vortexed for 15 s - 20 s every 10 min); after centrifuging at 4°C and 13000 g for 10 min, the nuclear skeleton protein was obtained; according to the ratio of loading buffer: nuclear skeleton protein = 1:5, loading buffer was added to the protein sample and placed in boiling water for 5 min - 10 min, and then polyacrylamide gel electrophoresis was performed to obtain a Western blot image as Figure 5 shown, where Figure 5 A is the WB protein band, Figure 5 B is the quantitative curve of the protein content changing with time after different treatment durations.

[0052] As can be seen from Figure 5It can be seen that as the processing duration increases, the protein content and types on the nuclear skeleton are continuously decreasing. When the action duration of 1% Nonidet P40 is 1 h, GAPDH in the cytoplasm, Hif-1α in the nucleus, and gp210 protein on the nuclear membrane are completely removed, while the nuclear lamina protein LaminA / C, histone H3, and cytoskeletal protein β-actin are still on the substrate. When the action duration is 2 h, only LaminA / C, H3, and β-actin remain on the substrate.

[0053] Although the embodiments of the present invention have been described in detail above, it is obvious to those skilled in the art that various modifications and changes can be made to these embodiments. However, it should be understood that such modifications and changes are all within the scope and spirit of the present invention described in the claims. Moreover, the present invention described herein can have other embodiments and can be implemented or realized in various ways.

Claims

1. A method for in situ separation of a cell nuclear skeleton, 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 separation solution on ice for 1h-3h and washed to obtain the in situ nuclear skeleton of the cells; the separation solution includes 0.1%-2% of the separation agent and 15μL / mL-25μL / mL of enzyme inhibitors, and the enzyme inhibitors include serine protease inhibitors, proteinase inhibitors, and phosphatase inhibitors.

2. The in-situ separation method according to claim 1, characterized in that: The cells to be separated are cultured and spread to 70%-80% of the bottom area of ​​the container, and then the culture medium is discarded.

3. 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.

4. The in-situ separation method according to claim 1, characterized in that: When the separation solution is used for treatment on ice, the separation solution is precooled in advance, and the temperature of the precooled separation solution is 3°C-5°C.

5. 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.

6. The in-situ separation method according to claim 1, characterized in that: The separation solution includes 18 μL / mL of serine protease inhibitor, 1 μL / mL of proteinase inhibitor, and 1 μL / mL of phosphatase inhibitor.

7. 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.

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