Adipose tissue cell nucleus extracting solution and application thereof

Abstract

The invention relates to an adipose tissue cell nucleus extracting solution and application thereof. The extracting solution comprises cane sugar, HEPES, inorganic salt, a surfactant, dithiothreitol, an RNA enzyme inhibitor and water. The adipose tissue cell nucleus extracting solution and the cell nucleus extracting method are suitable for adipose of different species and different parts, operation is easy and convenient, the obtained cell nucleus is good in state and relatively high in integrity, and it is hopeful to meet single cell nucleus sequencing and even multi-omics research. The invention lays a sample preparation foundation for transcriptome research of animal fat sample cell nucleuses, and has important significance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an adipose tissue cell nucleus extract and application thereof. Background Art

[0002] Fat is a highly active endocrine organ that is classified as white, brown, or beige depending on its function and plays a key role in metabolism. However, because adipocytes are filled with lipids and have a large morphology, they are not compatible with traditional single-cell methods, resulting in a lack of high-resolution single-cell data.

[0003] The currently reported methods for extracting cell nuclei from adipose tissue have a limited scope of application. It is difficult to obtain high-quality data for fat from different species and different parts, and the experiments lack good repeatability. The method for extracting cell nuclei from animal adipose tissue disclosed in the present invention is applicable to adipose tissue from multiple species and different parts, and can greatly meet the needs of extracting single cell nuclei from adipose tissue from multiple species and different parts. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention aims to provide an adipose tissue cell nucleus extract and its application.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an adipose tissue cell nucleus extract, comprising sucrose, HEPES, an inorganic salt, a surfactant, dithiothreitol, an RNase inhibitor and water.

[0007] The adipose tissue cell nucleus extract obtained by the present invention is suitable for fat from different species and different parts. The extracted cell nuclei are in good condition and have relatively high integrity. The addition of surfactants, inorganic salts and HEPES can improve the extraction effect of cell nuclei, promote nuclear digestion and maintain the integrity of nuclear morphology.

[0008] Preferably, the extract comprises 0.1-0.4M sucrose, 8-12mM HEPES, 8-15mM inorganic salt, 100-300mM dithiothreitol in molar concentration, 0.005-0.02% surfactant in mass percentage, 400-600U / mL RNase inhibitor and water in enzyme activity.

[0009] The molar concentration of the sucrose can be selected from 0.1M, 0.12M, 0.15M, 0.18M, ​​0.2M, 0.22M, 0.25M, 0.28M, 0.3M, 0.32M, 0.35M, 0.38M, 0.4M, etc., the molar concentration of HEPES can be selected from 8mM, 8.5mM, 9mM, 9.5mM, 10mM, 10.5mM, 11mM, 11.5mM, 12mM, etc., the molar concentration of the inorganic salt can be selected from 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, etc., the molar concentration of dithiothreitol can be selected from 100mM, 12 0mM, 150mM, 180mM, 200mM, 220mM, 250mM, 280mM, 300mM, etc., the mass percentage of the surfactant can be selected as 0.005%, 0.008%, 0.01%, 0.012%, 0.015%, 0.018%, 0.02%, etc., the enzyme activity of the RNase inhibitor can be selected as 400U / mL, 420U / mL, 450U / mL, 480U / mL, 500U / mL, 520U / mL, 550U / mL, 580U / mL, 600U / mL, etc., other specific point values ​​within the above numerical range can be selected, so they will not be described one by one here.

[0010] Preferably, the inorganic salt comprises magnesium chloride and potassium chloride.

[0011] The present invention finds that the addition of inorganic salts can promote the digestion of the nucleus, and magnesium chloride and potassium chloride can cooperate with each other, and both are indispensable.

[0012] Preferably, the molar ratio of magnesium chloride to potassium chloride is (0.5-2.5):(8-12).

[0013] Among them, the specific point values ​​in (0.5-2.5) can be selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.5, etc., and the specific point values ​​in (8-12) can be selected from 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be listed here one by one.

[0014] Preferably, the surfactant comprises IGEPAL CA-630.

[0015] The present invention found that the addition of surfactants can improve the digestion effect of cell nuclei, the selection of surfactant types can affect the extraction effect of cell nuclei, and the use of other surfactants can cause damage to the nuclear membrane. The effect of IGEPAL CA-630 is better than other surfactants.

[0016] In a second aspect, the present invention provides a use of the adipose tissue cell nucleus extract according to the first aspect in preparing an adipose tissue cell nucleus extract product.

[0017] Preferably, the product comprises a kit.

[0018] In a third aspect, the present invention provides a method for extracting adipose tissue cell nuclei, the method comprising:

[0019] (1) mixing adipose tissue with the adipose tissue cell nucleus extract described in the first aspect, extracting to obtain a first mixed solution;

[0020] (2) filtering the first mixed solution, centrifuging, removing the upper lipid layer, and resuspending to obtain a resuspended solution;

[0021] (3) The resuspension is centrifuged and the resulting precipitate is resuspended in a resuspension buffer.

[0022] The present invention adopts adipose tissue cell nucleus extract in combination with a specific cell nucleus extraction method, and the extracted cell nucleus has higher quality.

[0023] Preferably, the ratio of adipose tissue to adipose tissue cell nucleus extract is 1 g: (30-50) mL.

[0024] The specific point values ​​in (30-50) can be selected as 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, etc. Other specific point values ​​within the above numerical range can also be selected, and they will not be described one by one here.

[0025] Preferably, the extraction temperature is 0-4°C and the extraction time is 3-5 min.

[0026] The temperature can be selected at 0°C, 0.5°C, 1°C, 1.5°C, 2°C, 2.5°C, 3°C, 3.5°C, 4°C, etc., and the time can be selected at 3min, 3.2min, 3.5min, 3.8min, 4min, 4.2min, 4.5min, 4.8min, 5min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be described one by one here.

[0027] Preferably, the centrifugation speed in step (2) is 800-1200 g and the time is 8-12 min.

[0028] The rotation speed can be selected as 800g, 820g, 850g, 880g, 900g, 920g, 950g, 980g, 1000g, 1020g, 1050g, 1080g, 1100g, 1120g, 1150g, 1180g, 1200g, etc., and the time can be selected as 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min, 12min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0029] The centrifugal speed in step (3) is 400-600g and the time is 8-12min.

[0030] The rotation speed can be selected as 400g, 420g, 450g, 480g, 500g, 520g, 550g, 580g, 600g, etc., and the time can be selected as 8min, 8.5min, 9min, 9.5min, 10min, 10.5min, 11min, 11.5min, 12min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0031] Preferably, the resuspension buffer comprises 0.15-0.35% PBS, 0.5-1.5% PBSA in terms of mass percentage, 30-60 U / mL RNase inhibitor in terms of enzyme activity, and 1.95-2.05 mM magnesium chloride and water in terms of molar concentration.

[0032] The enzyme activity of the RNase inhibitor can be selected from 30U / mL, 32U / mL, 34U / mL, 36U / mL, 38U / mL, 40U / mL, 42U / mL, 44U / mL, 46U / mL, 48U / mL, 50U / mL, 52U / mL, 54U / mL, 56U / mL, 58U / mL, 60U / mL, etc. The mass percentage of PBS can be selected from 0.15%, 0.18%, 0.2%, 0.22%, 0.25%, 0.28%, 0.3%, 0.32%, 0. The mass percentage of PBSA can be selected as 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, etc. The molar concentration of magnesium chloride can be selected as 1.95mM, 1.96mM, 1.97mM, 1.98mM, 1.99mM, 2mM, 2.01mM, 2.02mM, 2.03mM, 2.04mM, 2.05mM, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be described one by one here.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The adipose tissue cell nucleus extract and cell nucleus extraction method obtained by the present invention are applicable to fat of different species and different parts, are easy to operate, and the obtained cell nuclei are in good condition and relatively high integrity, which can meet the needs of later RNA-Seq research, and are expected to meet the needs of single cell nuclear sequencing (snRNA-Seq) and even multi-omics research. The present invention lays a foundation for sample preparation for transcriptome research of cell nuclei of animal fat samples, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 1.

[0036] Figure 2 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 2.

[0037] Figure 3 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 3.

[0038] Figure 4 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 4.

[0039] Figure 5 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 5.

[0040] Figure 6 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 6.

[0041] Figure 7 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 7.

[0042] Figure 8 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 8.

[0043] Fig. 9 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 9.

[0044] Fig.10 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 10.

[0045] Fig.11 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 11.

[0046] Fig.12 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 12.

[0047] Fig.13This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Application Example 13.

[0048] Fig.14 This is a trypan blue staining microscopy image of the cell nucleus suspension prepared in Comparative Application Example 1.

[0049] Fig.15 This is a trypan blue staining microscope detection image of the cell nucleus suspension prepared in comparative application example 2. DETAILED DESCRIPTION

[0050] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0051] The sources of the functional ingredients contained in the products involved in the following embodiments and comparative examples are as follows (only the functional ingredients are reflected, and the necessary auxiliary ingredients contained in other commercially available raw materials are not repeated):

[0052] IGEPAL CA-630 is a product purchased from Sigma-Aldrich with the trade name of I8896;

[0053] The RNase inhibitor was purchased from Thermo Fisher Scientific under the trade name EO0381;

[0054] Triton X 100 was purchased from Sigma-Aldrich under the trade name T8787;

[0055] Tween 20 is purchased from Sigma-Aldrich Company with the trade name of P9416.

[0056] Preparation Example 1

[0057] This preparation example provides a resuspension buffer, which includes 0.2% PBS, 30 U / mL RNase inhibitor, 1% PBSA in terms of mass percentage, and 2 mM magnesium chloride and the remainder water in terms of molar concentration.

[0058] The preparation method thereof is as follows: physically mixing the preparation raw materials to obtain the product.

[0059] Preparation Example 2

[0060] This preparation example provides a resuspension buffer, which includes 0.15% PBS, 40 U / mL RNase inhibitor, 1.5% PBSA in terms of mass percentage, and 1.95 mM magnesium chloride and the remainder water in terms of molar concentration.

[0061] The preparation method is similar to that of Preparation Example 1.

[0062] Preparation Example 3

[0063] This preparation example provides a resuspension buffer, which includes 0.3% PBS, 60 U / mL RNase inhibitor, 0.5% PBSA in terms of mass percentage, and 2.05 mM magnesium chloride and the remainder water in terms of molar concentration.

[0064] The preparation method is similar to that of Preparation Example 1.

[0065] Example 1

[0066] The present embodiment provides an adipose tissue cell nucleus extract, which includes 0.3 M sucrose, 10 mM HEPES, 1.5 mM magnesium chloride, 10 mM potassium chloride, and 200 mM dithiothreitol in terms of molar concentration, 0.01% IGEPAL CA-630 in terms of mass percentage, and 500 U / mL of RNase inhibitor and the balance of water in terms of enzyme activity.

[0067] The preparation method thereof is as follows: physically mixing the preparation raw materials to obtain the product.

[0068] Example 2

[0069] The present embodiment provides an adipose tissue cell nucleus extract, which includes 0.1 M sucrose, 12 mM HEPES, 2.5 mM magnesium chloride, 12 mM potassium chloride, and 100 mM dithiothreitol in terms of molar concentration, 0.005% IGEPAL CA-630 in terms of mass percentage, and 600 U / mL of RNase inhibitor and the balance water in terms of enzyme activity.

[0070] The preparation method is similar to that of Example 1.

[0071] Example 3

[0072] This embodiment provides an adipose tissue cell nucleus extract, which includes 0.4 M sucrose, 8 mM HEPES, 0.5 mM magnesium chloride, 8 mM potassium chloride, and 300 mM dithiothreitol in terms of molar concentration, 0.02% IGEPAL CA-630 in terms of mass percentage, and 400 U / mL of RNase inhibitor and the balance of water in terms of enzyme activity.

[0073] The preparation method is similar to that of Example 1.

[0074] Example 4

[0075] This embodiment provides an adipose tissue cell nucleus extract, which differs from Embodiment 1 only in that it does not contain potassium chloride, and its reduced mass is distributed to the mass of magnesium chloride, while other components and contents remain unchanged.

[0076] The preparation method is similar to that of Example 1.

[0077] Example 5

[0078] This embodiment provides an adipose tissue cell nucleus extract, which differs from Embodiment 1 only in that it does not contain magnesium chloride, and its reduced mass is distributed to the mass of potassium chloride, while other components and contents remain unchanged.

[0079] The preparation method is similar to that of Example 1.

[0080] Example 6

[0081] This embodiment provides an adipose tissue cell nucleus extract, which is different from the embodiment 1 only in that the mass percentage of IGEPALCA-630 is changed from 0.02% to 0.002%, and the other components and contents remain unchanged.

[0082] The preparation method is similar to that of Example 1.

[0083] Example 7

[0084] This embodiment provides an adipose tissue cell nucleus extract, which is different from the embodiment 1 only in that the mass percentage of IGEPALCA-630 is changed from 0.02% to 0.1%, and the other components and contents remain unchanged.

[0085] The preparation method is similar to that of Example 1.

[0086] Example 8

[0087] This example provides an adipose tissue cell nucleus extract, which differs from Example 1 only in that IGEPALCA-630 is replaced by Triton X 100 in equal amounts, and other components and contents remain unchanged.

[0088] The preparation method is similar to that of Example 1.

[0089] Example 9

[0090] This example provides an adipose tissue cell nucleus extract, which differs from Example 1 only in that IGEPALCA-630 is replaced by Tween 20 in equal amounts, and other components and contents remain unchanged.

[0091] The preparation method is similar to that of Example 1.

[0092] Comparative Example 1

[0093] This comparative example provides an adipose tissue cell nucleus extract, which differs from Example 1 only in that it does not contain HEPEs, and other components and contents remain unchanged.

[0094] The preparation method is similar to that of Example 1.

[0095] Comparative Example 2

[0096] This comparative example provides an adipose tissue cell nucleus extract, which differs from Example 1 only in that it does not contain IGEPAL CA-630, and other components and contents remain unchanged.

[0097] The preparation method is similar to that of Example 1.

[0098] Application Example 1

[0099] This application example provides a method for extracting animal adipose tissue cell nuclei, the method comprising:

[0100] (1) Take 100 mg of mouse subcutaneous white adipose tissue, put it into a 1.5 mL low-adsorption centrifuge tube, add 50 μL of adipose tissue nuclear extract, and cut the adipose tissue into 3 mm pieces. 3 Next, transfer the tissue homogenate to a 5 mL centrifuge tube, add 2950 μL of adipose tissue nuclear dissociation solution, rinse the scissors and the original centrifuge tube, and extract on ice for 4 minutes. During the extraction, invert and mix 3 times / minute to obtain the extract.

[0101] (2) Filter the extract obtained in step (1) through a 40 μm filter, rinse the original 50 mL centrifuge tube with adipose tissue cell nuclear extract, and adjust the volume to 5 mL.

[0102] (3) The extract obtained in step (2) was centrifuged at 4°C, 1000 g for 10 min, the upper lipid layer was removed and the precipitate was resuspended, and the extract was centrifuged again at 4°C, 500 g for 10 min. The precipitate was resuspended in 0.2 mL of resuspension buffer.

[0103] The adipose tissue cell nucleus extract is the extract prepared in Example 1, and the resuspension buffer is the resuspension buffer prepared in Preparation Example 1.

[0104] Application Example 2

[0105] This application example provides a method for extracting animal adipose tissue cell nuclei, the method comprising:

[0106] (1) Take 100 mg of mouse gonadal adipose tissue, put it into a 1.5 mL low-adsorption centrifuge tube, add 50 μL of adipose tissue nuclear extract, and cut the adipose tissue into 3 mm pieces. 3 Next, transfer the tissue homogenate to a 5 mL centrifuge tube, add 3950 μL of adipose tissue nuclear dissociation solution, rinse the scissors and the original centrifuge tube, and extract on ice for 5 minutes. During the extraction, invert and mix 3 times / minute to obtain the extract.

[0107] (2) The extract obtained in step (1) was filtered through a 40 μm filter, and the original 50 mL centrifuge tube was rinsed with adipose tissue cell nuclear extract and fixed to 5 mL;

[0108] (3) The extract obtained in step (2) was centrifuged at 4°C, 800g for 12 min, the upper lipid layer was removed, and the precipitate was resuspended, and the extract was centrifuged again at 4°C, 400g for 12 min. The precipitate was resuspended in 0.2 mL of resuspension buffer.

[0109] The adipose tissue cell nucleus extract is the extract prepared in Example 2, and the resuspension buffer is the resuspension buffer prepared in Preparation Example 2.

[0110] Application Example 3

[0111] This application example provides a method for extracting animal adipose tissue cell nuclei, the method comprising:

[0112] (1) Take 100 mg of mouse brown adipose tissue, put it into a 1.5 mL low-adsorption centrifuge tube, add 50 μL of adipose tissue nuclear extract, and cut the adipose tissue into 3 mm pieces. 3 Next, transfer the tissue homogenate to a 5 mL centrifuge tube, add 4950 μL of adipose tissue nuclear dissociation solution, rinse the scissors and the original centrifuge tube, and extract on ice for 3 minutes. During the extraction, invert and mix 3 times / minute to obtain the extract.

[0113] (2) Filter the extract obtained in step (1) through a 40 μm filter, rinse the original 50 mL centrifuge tube with adipose tissue cell nuclear extract, and adjust the volume to 5 mL.

[0114] (3) The extract obtained in step (2) is centrifuged at 4°C, 1200g for 8 min, the upper lipid layer is removed, the precipitate is resuspended, and the extract is centrifuged again at 4°C, 600g for 8 min. The precipitate is resuspended in a resuspension buffer.

[0115] The adipose tissue cell nucleus extract is the extract prepared in Example 1, and the resuspension buffer is the resuspension buffer prepared in Preparation Example 1.

[0116] Application Example 4

[0117] This application example provides a method for extracting animal adipose tissue cell nuclei, the method comprising:

[0118] (1) Take 100 mg of pig visceral adipose tissue, put it into a 1.5 mL low-adsorption centrifuge tube, add 50 μL of adipose tissue nuclear extract, and cut the adipose tissue into 3 mm pieces. 3Next, transfer the tissue homogenate to a 5 mL centrifuge tube, add 2950 μL of adipose tissue nuclear dissociation solution, rinse the scissors and the original centrifuge tube, and extract on ice for 4 minutes. During the extraction, invert and mix 3 times / minute to obtain the extract.

[0119] (2) Filter the extract obtained in step (1) through a 40 μm filter, rinse the original 50 mL centrifuge tube with adipose tissue cell nuclear extract, and adjust the volume to 5 mL.

[0120] (3) The extract obtained in step (2) is centrifuged at 4°C, 900g for 10 min, the upper lipid layer is removed, and the precipitate is resuspended, and the extract is centrifuged again at 4°C, 450g for 11 min. The precipitate is resuspended in a resuspension buffer.

[0121] The adipose tissue cell nucleus extract is the extract prepared in Example 1, and the resuspension buffer is the resuspension buffer prepared in Preparation Example 1.

[0122] Application Example 5

[0123] This application example provides a method for extracting animal adipose tissue cell nuclei, the method comprising:

[0124] (1) Take 100 mg of mouse brown adipose tissue, put it into a 1.5 mL low-adsorption centrifuge tube, add 50 μL of adipose tissue nuclear extract, and cut the adipose tissue into 3 mm pieces. 3 Next, transfer the tissue homogenate to a 5 mL centrifuge tube, add 2950 μL of adipose tissue nuclear dissociation solution, rinse the scissors and the original centrifuge tube, and extract on ice for 4 minutes. During the extraction, invert and mix 3 times / minute to obtain the extract.

[0125] (2) Filter the extract obtained in step (1) through a 40 μm filter, rinse the original 50 mL centrifuge tube with adipose tissue cell nuclear extract, and adjust the volume to 5 mL.

[0126] (3) The extract obtained in step (2) is centrifuged at 4°C, 1100 g for 11 min, the upper lipid layer is removed, and the precipitate is resuspended, and the extract is centrifuged again at 4°C, 550 g for 10 min. The precipitate is resuspended in a resuspension buffer.

[0127] The adipose tissue cell nucleus extract is the extract prepared in Example 2, and the resuspension buffer is the resuspension buffer prepared in Preparation Example 2.

[0128] Application Example 6

[0129] This application example provides a method for extracting animal adipose tissue cell nuclei, the method comprising:

[0130] (1) Take 100 mg of mouse brown adipose tissue, put it into a 1.5 mL low-adsorption centrifuge tube, add 50 μL of adipose tissue nuclear extract, and cut the adipose tissue into 3 mm pieces. 3 Next, transfer the tissue homogenate to a 5 mL centrifuge tube, add 2950 μL of adipose tissue nuclear dissociation solution, rinse the scissors and the original centrifuge tube, and extract on ice for 5 minutes. During the extraction, invert and mix 3 times / minute to obtain the extract.

[0131] (2) The extract obtained in step (1) was filtered through a 40 μm filter, and the original 50 mL centrifuge tube was rinsed with adipose tissue cell nuclear extract and fixed to 5 mL;

[0132] (3) The extract obtained in step (2) was centrifuged at 4°C, 950g for 12 min, the upper lipid layer was removed and the precipitate was resuspended, and the extract was centrifuged again at 4°C, 500g for 11 min. The precipitate was resuspended in 0.2 mL of resuspension buffer.

[0133] The adipose tissue cell nucleus extract is the extract prepared in Example 3, and the resuspension buffer is the resuspension buffer prepared in Preparation Example 3.

[0134] Application Example 7

[0135] This application example provides a method for extracting animal adipose tissue cell nuclei, which differs from application example 1 only in that the adipose tissue nuclear dissociation solution prepared in example 1 is replaced with the adipose tissue nuclear dissociation solution prepared in example 4, and other operations remain unchanged.

[0136] Application Example 8

[0137] This application example provides a method for extracting animal adipose tissue cell nuclei, which differs from application example 1 only in that the adipose tissue nuclear dissociation solution prepared in example 1 is replaced with the adipose tissue nuclear dissociation solution prepared in example 5, and other operations remain unchanged.

[0138] Application Example 9

[0139] This application example provides a method for extracting animal adipose tissue cell nuclei, which differs from application example 1 only in that the adipose tissue nuclear dissociation solution prepared in example 1 is replaced with the adipose tissue nuclear dissociation solution prepared in example 6, and other operations remain unchanged.

[0140] Application Example 10

[0141] This application example provides a method for extracting animal adipose tissue cell nuclei, which differs from application example 1 only in that the adipose tissue nuclear dissociation solution prepared in example 1 is replaced with the adipose tissue nuclear dissociation solution prepared in example 7, and other operations remain unchanged.

[0142] Application Example 11

[0143] This application example provides a method for extracting animal adipose tissue cell nuclei, which differs from application example 1 only in that the adipose tissue nuclear dissociation solution prepared in example 1 is replaced with the adipose tissue nuclear dissociation solution prepared in example 8, and other operations remain unchanged.

[0144] Application Example 12

[0145] This application example provides a method for extracting animal adipose tissue cell nuclei, which differs from application example 1 only in that the adipose tissue nuclear dissociation solution prepared in example 1 is replaced with the adipose tissue nuclear dissociation solution prepared in example 9, and other operations remain unchanged.

[0146] Application Example 13

[0147] This application example provides a method for extracting nuclei from animal adipose tissue cells, which differs from application example 1 only in that step (3) is "centrifuging the extract obtained in step (2) at 4°C, 500g for 10 min, resuspending the precipitate with a resuspension buffer, centrifuging again at 4°C, 500g for 10 min, and resuspending the precipitate with 0.2 mL of resuspension buffer to obtain a nuclear suspension, and other operations remain unchanged.

[0148] Comparative application example 1

[0149] This comparative application example provides a method for extracting animal adipose tissue cell nuclei, which differs from Application Example 1 only in that the adipose tissue nuclear dissociation solution prepared in Example 1 is replaced by the adipose tissue nuclear dissociation solution prepared in Comparative Example 1, and other operations remain unchanged.

[0150] Comparative Application Example 2

[0151] This comparative application example provides a method for extracting animal adipose tissue cell nuclei, which differs from Application Example 1 only in that the adipose tissue nuclear dissociation solution prepared in Example 1 is replaced by the adipose tissue nuclear dissociation solution prepared in Comparative Example 2, and other operations remain unchanged.

[0152] Test Example 1

[0153] The adipose tissue cell nuclear suspension prepared in the application example and the comparative application example was stained with trypan blue, and the cell nuclear morphology was observed under a microscope. For example, the trypan blue staining microscopic examination image of the adipose tissue cell nuclear suspension prepared in application example 1 is as follows: Figure 1 The results of the nuclear yield per unit mass and the percentage of nuclear impurities are shown in Table 1.

[0154] Table 1

[0155] Group Unit mass yield (pieces / mg) Nuclear membrane integrity (%) Application Example 1 4832 Nuclear morphology intact (>95%) Application Example 2 3074 Nuclear morphology intact (>95%) Application Example 3 7222 Nuclear morphology intact (>95%) Application Example 4 1648 Nuclear morphology intact (>95%) Application Example 5 5772 Nuclear morphology intact (>95%) Application Example 6 6818 Nuclear morphology intact (>90%) Application Example 7 618 Nuclear morphology intact (>50%) Application Example 8 1217 Nuclear morphology intact (>90%) Application Example 9 1326 Nuclear membrane damage (<50%) Application Example 10 7538 Diffuse nuclear rim (<50%) Application Example 11 8607 Nuclear membrane damage (<50%) Application Example 12 1672 Nuclear membrane damage (<50%) Application Example 13 1322 Nuclear morphology intact (>80%) Comparative application example 1 1322 Nuclear membrane damage (<80%) Comparative Application Example 2 473 Nuclear morphology intact (>90%)

[0156] According to the data in the table, although there are differences in the yield of nuclei in the nucleus suspensions obtained by the method for extracting nuclei from adipose tissue of different parts of animals provided in Application Examples 1-4 of the present invention, the nuclei are relatively complete and have fewer impurities, indicating that the method can be applied to adipose tissue of different parts and species; in Application Examples 7-8 and Comparative Application Examples 1-2, after removing potassium chloride, magnesium chloride, HEPES or IGEPAL CA-630 from the adipose tissue nuclear dissociation solution, the nuclear yield is reduced; this shows that the above components can improve the extraction effect of nuclei, promote nuclear digestion, and maintain the integrity of nuclear morphology, and removal will affect the yield of the nucleus suspension.

[0157] According to Examples 9-10, when the concentration of IGEPAL CA-630 in adipose tissue nuclear dissociation was reduced, the cell nucleus yield was significantly reduced and the nuclear membrane was damaged. When the concentration was increased, the edges of the cell nuclei were more diffuse and the nuclei were swollen and broken. At the same time, in order to verify the importance of the surfactant in the present invention, we replaced IGEPAL CA-630 with Triton X100 and Tween 20 and found that in the application example where Triton X100 was added, the cell nuclei were severely damaged, and the amount of cell nuclei replaced with Tween 20 was significantly reduced and the impurities in the nuclear suspension increased. From the above, it can be seen that the effect of the surfactant IGEPAL CA-630 in the present invention is better than other surfactants. In addition, according to Application Example 13, in the present invention, after two centrifugal digestions, the nuclear yield of the cell nuclear suspension after removing the oil layer in the middle will be improved and the oil droplet residue will be reduced.

[0158] In summary, the dissociation components and process provided by the present invention can be well applied to adipose tissue to dissociate high-quality cell nucleus suspension, and have strong practicality.

[0159] The applicant declares that the present invention illustrates an adipose tissue cell nucleus extract and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0160] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0161] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A fat tissue cell nucleus extract, characterized in that: The extracting solution comprises sucrose, HEPES, inorganic salt, surfactant, dithiothreitol, RNase inhibitor and water.

2. The adipose tissue nuclear extract according to claim 1, characterized in that: The extract comprises 0.1-0.4M sucrose, 8-12mM HEPES, 8-15mM inorganic salt, 100-300mM dithiothreitol in terms of molar concentration, 0.005-0.02% surfactant in terms of mass percentage, 400-600U / mL RNAse inhibitor and water in terms of enzyme activity.

3. The adipose tissue cell nuclear extract according to claim 1 or 2, characterized in that: The inorganic salts include magnesium chloride and potassium chloride; Preferably, the molar ratio of magnesium chloride to potassium chloride is (0.5-2.5):(8-12); Preferably, the surfactant comprises IGEPAL CA-630.

4. Use of the adipose tissue nuclear extract according to any one of claims 1 to 3 in the preparation of an adipose tissue nuclear extract product.

5. A method for extracting adipose tissue cell nuclei, characterized in that: The method comprises: (1) mixing adipose tissue with the adipose tissue cell nucleus extract according to any one of claims 1 to 3, extracting to obtain a first mixed solution; (2) filtering the first mixed solution, centrifuging, removing the upper lipid layer, and resuspending to obtain a resuspended solution; (3) The resuspension is centrifuged and the resulting precipitate is resuspended in a resuspension buffer.

6. The method for extracting adipose tissue nuclei according to claim 5, characterized in that: The ratio of the adipose tissue to the adipose tissue cell nucleus extract is 1 g: (30-50) mL.

7. The method for extracting adipose tissue nuclei according to claim 5 or 6, characterized in that: The extraction temperature is 0-4°C and the extraction time is 3-5 minutes.

8. The method for extracting adipose tissue nuclei according to any one of claims 5 to 7, characterized in that: The centrifugal speed in step (2) is 800-1200g and the time is 8-12min.

9. The method for extracting adipose tissue nuclei according to any one of claims 5 to 8, characterized in that: The centrifugal speed in step (3) is 400-600g and the time is 8-12min.

10. The method for extracting adipose tissue nuclei according to any one of claims 5 to 9, characterized in that: The resuspension buffer comprises PBS 0.15-0.35%, PBSA 0.5-1.5% in terms of mass percentage, RNA inhibitor 30-60 U / mL in terms of enzyme activity, and magnesium chloride 1.95-2.05 mM and water in terms of molar concentration.

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