Preparation method of atherosclerotic plaque tissue homogenate

Abstract

The invention discloses an atherosclerotic plaque tissue homogenate preparation method, which comprises: S1, obtaining a human plaque biological sample through carotid endarterectomy, and washing blood stains and impurities on the plaque surface with a sodium chloride solution; s2, after cleaning, cutting a part of the biological sample; s3, continuously cutting the sample into blocks; s4, weighing the sample; s5, moving into a 2ml EP tube; s6, adding normal saline in proportion, wherein the weight ratio of the sample to the normal saline is 1: 3; s7, adding two grinding beads; s8, the EP pipe is placed in a homogenizer to be ground; s9, the EP tube is placed on a centrifugal machine for two times of centrifugation, an acetonitrile solvent is added for balancing after the first time of centrifugation, supernate is taken and placed in a new EP tube for standby application after the two times of centrifugation, sample homogenate is obtained, the operation mode of combining low-speed centrifugation and high-speed centrifugation can guarantee that small molecular substances are fully reserved in the supernate, and the detection efficiency of a mass spectrometer is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection and relates to a method for preparing tissue homogenate of atherosclerotic plaque. Background Art

[0002] The existing methods for preparing MALDI sample homogenates of atherosclerotic plaques are different. Currently, the more commonly used method for preparing MALDI sample homogenates is to use a biological sample homogenizer assisted by grinding beads. However, different parameter settings in the instrument program will also affect the homogenization effect. Setting the grinding parameters too fast will cause the EP tube to break during the oscillation process, thereby causing waste of biological samples. Setting the grinding parameters too slowly will result in insufficient grinding and reduce grinding efficiency. At the same time, how to effectively extract the supernatant from the homogenate obtained after sufficient grinding is particularly important for metabolomics mass spectrometry experiments and result analysis. Therefore, it is necessary to design a method for preparing tissue homogenates of atherosclerotic plaques to solve the above problems. Summary of the invention

[0003] In view of the above problems and to overcome the defects of the prior art, the present invention proposes a method for preparing a tissue homogenate of atherosclerotic plaques. The purpose of the present invention is to increase the amount of small molecules in the supernatant, thereby improving the quality of the slurry.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention comprises the following steps:

[0005] S1. Obtain human plaque biological samples through carotid endarterectomy (CEA), and wash the blood and impurities on the surface of the plaque with sodium chloride solution;

[0006] S2. After cleaning, cut out some biological samples;

[0007] S3, continue to cut the sample into pieces;

[0008] S4, weigh the sample;

[0009] S5, transfer into 2 ml EP tube;

[0010] S6. Add normal saline in the ratio of sample weight to normal saline = 1:3;

[0011] S7, add 2 grinding beads;

[0012] S8, placing the EP tube in a homogenizer for grinding;

[0013] S9. Place the EP tube in a centrifuge and centrifuge twice. After the first centrifugation, add acetonitrile solvent to balance. After two centrifugations, take the supernatant and put it in a new EP tube for later use to obtain a sample homogenate.

[0014] Preferably, the mass fraction of the sodium chloride solution is 0.9%.

[0015] Preferably, the two centrifugations are low-speed centrifugation and high-speed centrifugation, respectively, the low-speed centrifugation being performed first and the high-speed centrifugation being performed later.

[0016] Preferably, the high-speed centrifugation speed is 13,000 rpm.

[0017] Preferably, the low-speed centrifugation speed is 3000 rpm.

[0018] Preferably, the low-speed centrifugation speed is 8000 rpm.

[0019] Preferably, the sizes of the two grinding beads are 1 mm and 3 mm respectively.

[0020] Preferably, the sample weight is 500 mg.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention can ensure that the EP tube will not be damaged during the grinding process by adjusting and setting the parameters of the homogenizer, thereby avoiding waste of samples.

[0023] 2. The present invention screens metabolites by setting two different centrifugation speeds. By combining low-speed centrifugation with high-speed centrifugation, it can ensure that small molecules are fully retained in the supernatant.

[0024] 3. In the present invention, after the first centrifugation, the addition of acetonitrile solvent can effectively precipitate the protein, ensure that the small molecules are fully retained in the solution, and improve the detection efficiency of the mass spectrometer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the process of the present invention;

[0026] Figure 2 This is a schematic diagram of mass spectrometry for solution detection in Example 1 of the present invention;

[0027] Figure 3 This is a schematic diagram of mass spectrometry for solution detection in Example 2 of the present invention;

[0028] Figure 4 This is a schematic diagram of mass spectrometry for solution detection in Example 3 of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The following is combined with Figure 1-4 The specific implementation modes of the present invention are described in further detail.

[0031] In this embodiment, by Figure 1 As shown, the present invention comprises the following steps:

[0032] S1. Obtain human plaque biological samples through carotid endarterectomy (CEA), and wash the blood and impurities on the surface of the plaque with sodium chloride solution;

[0033] S2. After cleaning, cut out some biological samples;

[0034] S3, continue to cut the sample into pieces;

[0035] S4, weigh the sample;

[0036] S5, transfer into 2 ml EP tube;

[0037] S6. Add normal saline in the ratio of sample weight to normal saline = 1:3;

[0038] S7, add 2 grinding beads;

[0039] S8, placing the EP tube in a homogenizer for grinding;

[0040] S9. Place the EP tube in a centrifuge and centrifuge twice. After centrifugation, take the supernatant and put it in a new EP tube for later use to obtain a sample homogenate.

[0041] In this embodiment 1, by Figure 2 As shown;

[0042] 1. Pre-grinding treatment of atherosclerotic plaque biological samples

[0043] ① Use 0.9% sodium chloride solution to rinse the blood and impurities on the surface of the plaque.

[0044] ② After cleaning, cut out part of the biological sample (just cut out the part that includes the lesion to be studied).

[0045] ③Continue to cut the sample into pieces.

[0046] ④ Weigh the sample (mg) (block sample should be around 500mg).

[0047] ⑤Transfer into 2ml EP tube.

[0048] ⑥ Add normal saline (μl) according to the ratio (sample weight: normal saline = 1:3).

[0049] ⑦ Add 2 grinding beads (1 each of 1 mm and 3 mm).

[0050] 2. Homogenizer

[0051] ① Balance the EP tube in the test tube slot of the instrument and start the instrument after balancing.

[0052] Setting parameters:

[0053] Grinding temperature -20℃

[0054] Operating frequency 33HZ

[0055] Run time 30s

[0056] Pause time 30s

[0057] Running times: 10 times (10 times as a group, after 10 times, choose to continue grinding or stop running according to the grinding situation)

[0058] ② When no large pieces of tissue are visible in the EP tube and the homogenate has a uniform color, it means that the grinding is sufficient. Use a pipette to transfer the homogenate in the EP tube into a new EP tube for later use.

[0059] 3. High-speed centrifugation

[0060] After balancing in the test tube slot of the centrifuge, perform the first centrifugation (low speed).

[0061] Setting parameters:

[0062] Operating speed 13000 rpm

[0063] Running time 10min

[0064] Centrifugation temperature 4℃

[0065] After centrifugation, take the supernatant to a new EP tube for later use.

[0066] 4. Precipitate impurities

[0067] ① After high-speed centrifugation, add acetonitrile solvent in proportion (supernatant: acetonitrile solvent = 1:3).

[0068] ② Mix well (vortex on a vortexer for 1-2 minutes).

[0069] 5. High-speed centrifugation

[0070] After adding acetonitrile, balance it in the test tube slot of the centrifuge and perform a second centrifugation (high speed).

[0071] Setting parameters:

[0072] Operating speed 13000 rpm

[0073] Running time 10min

[0074] Centrifugation temperature 4℃

[0075] After centrifugation, take the supernatant (stock solution) into a new EP tube for later use. Recorded as stock solution A.

[0076] In this embodiment 2, by Figure 3 As shown:

[0077] Pre-grinding treatment of atherosclerotic plaque biological samples

[0078] ① Use 0.9% sodium chloride solution to rinse the blood and impurities on the surface of the plaque.

[0079] ② After cleaning, cut out part of the biological sample (just cut out the part that includes the lesion to be studied).

[0080] ③Continue to cut the sample into pieces.

[0081] ④ Weigh the sample (mg) (block sample should be around 500mg).

[0082] ⑤Transfer into 2ml EP tube.

[0083] ⑥ Add normal saline (μl) according to the ratio (sample weight: normal saline = 1:3).

[0084] ⑦ Add 2 grinding beads (1 each of 1 mm and 3 mm).

[0085] 2. Homogenizer

[0086] ① Balance the EP tube in the test tube slot of the instrument and start the instrument after balancing.

[0087] Setting parameters:

[0088] Grinding temperature -20℃

[0089] Operating frequency 33HZ

[0090] Run time 30s

[0091] Pause time 30s

[0092] Running times: 10 times (10 times as a group, after 10 times, choose to continue grinding or stop running according to the grinding situation)

[0093] ② When no large pieces of tissue are visible in the EP tube and the homogenate has a uniform color, it means that the grinding is sufficient. Use a pipette to transfer the homogenate in the EP tube into a new EP tube for later use.

[0094] 3. Low speed centrifugation

[0095] After balancing in the test tube slot of the centrifuge, perform the first centrifugation (low speed).

[0096] Setting parameters:

[0097] Operating speed 3000 rpm

[0098] Running time 10min

[0099] Centrifugation temperature 4℃

[0100] After centrifugation, take the supernatant to a new EP tube for later use.

[0101] 4. Precipitate impurities

[0102] ① After low-speed centrifugation, add acetonitrile solvent in proportion (supernatant: acetonitrile solvent = 1:3).

[0103] ② Mix well (vortex on a vortexer for 1-2 minutes).

[0104] 5. High-speed centrifugation

[0105] After adding acetonitrile, balance it in the test tube slot of the centrifuge and perform a second centrifugation (high speed).

[0106] Setting parameters:

[0107] Operating speed 13000 rpm

[0108] Running time 10min

[0109] Centrifugation temperature 4℃

[0110] After centrifugation, take the supernatant (stock solution) into a new EP tube for later use. Recorded as stock solution B.

[0111] In this embodiment three, by Figure 4 As shown:

[0112] 3. Pre-grinding treatment of atherosclerotic plaque biological samples

[0113] ① Use 0.9% sodium chloride solution to rinse the blood and impurities on the surface of the plaque.

[0114] ② After cleaning, cut out part of the biological sample (just cut out the part that includes the lesion to be studied).

[0115] ③Continue to cut the sample into pieces.

[0116] ④ Weigh the sample (mg) (block sample should be around 500mg).

[0117] ⑤Transfer into 2ml EP tube.

[0118] ⑥ Add normal saline (μl) according to the ratio (sample weight: normal saline = 1:3).

[0119] ⑦ Add 2 grinding beads (1 each of 1 mm and 3 mm).

[0120] 2. Homogenizer

[0121] ① Balance the EP tube in the test tube slot of the instrument and start the instrument after balancing.

[0122] Setting parameters:

[0123] Grinding temperature -20℃

[0124] Operating frequency 33HZ

[0125] Run time 30s

[0126] Pause time 30s

[0127] Running times: 10 times (10 times as a group, after 10 times, choose to continue grinding or stop running according to the grinding situation)

[0128] ② When no large pieces of tissue are visible in the EP tube and the homogenate has a uniform color, it means that the grinding is sufficient. Use a pipette to transfer the homogenate in the EP tube into a new EP tube for later use.

[0129] 3. Low speed centrifugation

[0130] After balancing in the test tube slot of the centrifuge, perform the first centrifugation (low speed).

[0131] Setting parameters:

[0132] Operating speed 8000 rpm

[0133] Running time 10min

[0134] Centrifugation temperature 4℃

[0135] After centrifugation, take the supernatant to a new EP tube for later use.

[0136] 4. Precipitate impurities

[0137] ① After low-speed centrifugation, add acetonitrile solvent in proportion (supernatant: acetonitrile solvent = 1:3).

[0138] ② Mix well (vortex on a vortexer for 1-2 minutes).

[0139] 5. High-speed centrifugation

[0140] After adding acetonitrile, balance it in the test tube slot of the centrifuge and perform a second centrifugation (high speed).

[0141] Setting parameters:

[0142] Operating speed 13000 rpm

[0143] Running time 10min

[0144] Centrifugation temperature 4℃

[0145] After centrifugation, take the supernatant (stock solution) into a new EP tube for later use. Recorded as stock solution C.

[0146] In this embodiment, by Figure 2-4 As shown:

[0147] 1. Dilute the stock solution

[0148] ① Dilute the obtained stock solutions A, B and C by 5 times in proportion (stock solution: physiological saline = 1:5), and prepare the diluted solutions of the stock solutions for later use.

[0149] ② Vortex the diluted solution in a vortex machine for 1 minute

[0150] 2. Prepare a mixture of matrix solution and diluent

[0151] ① Mix the diluent and matrix solution in an EP tube according to the ratio (diluent: matrix solution = 1:2), where the matrix solution is DHB 20 mg / ml (DHB: acetonitrile is 7:3);

[0152] ② Vortex the mixture in a vortex machine for 1 minute;

[0153] 3. Spot plate and air dry

[0154] ①Pour the mixed liquid into the target plate groove

[0155] ② Air dry and set aside

[0156] 4. On-machine testing

[0157] The mass spectrum was obtained by detection in MALDI-TOF;

[0158] 5. Experimental Results

[0159] Comparative experimental results:

[0160] Example 1: Extract the supernatant by high-speed centrifugation ( Figure 2 , speed: 13000r / 13000r).

[0161] Example 2: The supernatant was extracted by combining low-speed centrifugation and high-speed centrifugation ( Figure 3 , speed: 3000r / 13000r).

[0162] Example 3: The supernatant was extracted by combining low-speed centrifugation and high-speed centrifugation ( Figure 4, speed: 8000r / 13000r).

[0163] Based on the experimental results obtained, we determined that the metabolites required for annotation in the experiment were those with m / z peaks between 250 and 1200. Based on this experimental result, we paid special attention to the m / z interval between 270 and 850 (marked by the red line). The number of peaks represents the number of substances detected. The higher the peak, the better the response. Figure 2 and Figure 3 By comparison, Figure 3 The number of m / z peaks in the spectrum increased significantly, and the response level was improved. Figure 3 and Figure 2 The contrast is obvious at around m / z values ​​of 400, 450, and 650. The number of peaks near the m / z values ​​increases significantly and the response degree increases. Figure 3 The increase in the number of peaks and the improvement in responsiveness indicate that the combination of low-speed centrifugation and high-speed centrifugation can better extract metabolites in the tissue and retain them in the supernatant, thus avoiding the precipitation of a large amount of substances to be detected by centrifugation. Figure 4 The experimental results are obtained by using the third low-speed centrifugation and high-speed centrifugation combined speed (8000r / 13000r), which is consistent with Figure 2 In comparison, it is once again shown that the combination of low-speed centrifugation and high-speed centrifugation can better extract metabolites in tissues, and can significantly increase the number of detection peaks and the response degree of substances. It can be concluded that the slurry obtained by combining high-speed centrifugation and low-speed centrifugation is more effective.

[0164] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a tissue homogenate of atherosclerotic plaque, characterized in that: The preparation method comprises the following steps: S1. Obtain human plaque biological samples through carotid endarterectomy and wash the blood and impurities on the surface of the plaque with sodium chloride solution; S2. After cleaning, cut out some biological samples; S3, continue to cut the sample into pieces; S4, weigh the sample; S5, transfer to 2 ml EP tube; S6. Add normal saline in the ratio of sample weight to normal saline = 1:3; S7, add 2 grinding beads; S8, placing the EP tube in a homogenizer for grinding; S9. Place the EP tube in a centrifuge and centrifuge twice. After the first centrifugation, add acetonitrile solvent to balance. After two centrifugations, take the supernatant and put it in a new EP tube for later use to obtain a sample homogenate.

2. The method for preparing a tissue homogenate of atherosclerotic plaque according to claim 1, characterized in that: The mass fraction of the sodium chloride solution is 0.9%.

3. The method for preparing a tissue homogenate of atherosclerotic plaque according to claim 2, characterized in that: The two centrifugations are low-speed centrifugation and high-speed centrifugation, respectively, wherein the low-speed centrifugation is performed first and the high-speed centrifugation is performed later.

4. The method for preparing a tissue homogenate of atherosclerotic plaque according to claim 3, characterized in that: The high-speed centrifugation speed is 13,000 rpm.

5. The method for preparing a tissue homogenate of atherosclerotic plaque according to claim 4, characterized in that: The low-speed centrifugation speed is 3000 rpm.

6. The method for preparing a tissue homogenate of atherosclerotic plaque according to claim 4, characterized in that: The low-speed centrifugation speed is 8000 rpm.

7. The method for preparing a tissue homogenate of atherosclerotic plaque according to claim 1, characterized in that: The sizes of the two grinding beads were 1 mm and 3 mm respectively.

8. The method for preparing a tissue homogenate of atherosclerotic plaque according to claim 1, characterized in that: The sample weight was 500 mg.

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