Method for extracting mouse intestinal nerve synapses

Abstract

The invention relates to the technical field of molecular biology, in particular to a method for extracting mouse intestinal nerve synapses, which specifically comprises the steps of pretreatment, centrifugation, gradient centrifugation and the like. According to the method, the mouse intestinal nerve synapses are accurately extracted by utilizing a gradient centrifugation method, the extracted synaptic protein is high in purity and stable in structure, the mechanism of the intestinal nerve synapses can be known, the pathophysiology of many digestive system diseases can be disclosed, and the method has important significance on the pathogenesis of the diseases.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a method for extracting mouse intestinal nerve synapses. Background Art

[0002] Enteric neural synapses are an important part of the enteric nervous system (ENS), which is often called the "second brain". It has relatively independent functions and can autonomously control various physiological activities of the intestine. The ENS is composed of a large number of neurons and glial cells, which are connected by synapses to form a complex neural network. These neural synapses are not only involved in the control of intestinal motility, but also involve multiple functions of the intestine, such as sensation, secretion, and vascular regulation. In recent years, with the in-depth study of ENS, people have gradually realized that ENS plays a key role in the occurrence and development of many digestive system diseases, such as irritable bowel syndrome (IBS), Crohn's disease, and chronic constipation. Therefore, in-depth research on the ENS, especially the mechanism of enteric neural synapses, is of great significance for revealing the pathophysiology of these diseases.

[0003] In order to better understand the function and pathological mechanism of ENS, researchers need to develop effective methods to extract and study mouse enteric synapses. Traditional extraction methods have certain limitations, such as difficulty in maintaining the integrity of synaptic structure or possible damage to nerve cells during the extraction process. In addition, synaptic extraction methods are relatively cumbersome.

[0004] Therefore, it is particularly important to develop a new extraction method that can maintain the integrity of synaptic structure and function and is easy to operate. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and propose a method for extracting mouse intestinal nerve synapses. Through gradient centrifugation technology, intestinal nerve synapses are effectively separated and enriched to improve the purity of mouse intestinal nerve synapses, improve the purity of extracted synaptic proteins and maintain their structural stability.

[0006] To achieve the purpose of the present invention, the technical solution provided by the present invention is as follows:

[0007] A method for extracting mouse intestinal neural synapses comprises the following steps:

[0008] S1. Remove the small intestine tissue of mice under low temperature conditions, cut the intestinal lumen longitudinally, and then fully clean the small intestine to remove the intestinal contents;

[0009] S2. Transfer the small intestinal tissue to a Ca-free medium containing 5 mM EDTA and 1 mM DTT.2+ Mg 2+ The small intestine tissue was obtained by spinning and washing in Hank's equilibrium solution and vortexing, and then using a filter to obtain the small intestine tissue, which was repeated several times.

[0010] S3. Divide the small intestinal tissue into 5 cm pieces on average, add 1 mL of synaptic extraction protection solution to every 5 cm of small intestinal tissue; transfer to a tissue grinder, grind thoroughly, transfer the sample to a 1.5 mL EP tube, and store it in crushed ice;

[0011] S4. Perform the first centrifugation on the sample, take the supernatant and transfer it to a new 1.5mL EP tube; then perform the second centrifugation, remove the supernatant, retain the precipitate, add 1mL of 0.4M sucrose solution to each tube and mix thoroughly;

[0012] S5. Prepare 1.2M sucrose solution, 1M sucrose solution, and 0.8M sucrose solution and store them at 4°C. Tilt the ultracentrifuge tube at 45° and slowly add 1.2M sucrose solution (1mL), 1M sucrose solution (1mL), and 0.85M sucrose solution (1mL) along the tube wall in the order of 1.2M sucrose solution (1mL), 1M sucrose solution (1mL), and 0.85M sucrose solution (1mL) to form a gradient sucrose solution. Finally, slowly add the 0.4M sucrose solution to which the precipitate obtained in S4 is completely mixed to finally form a sucrose gradient solution of 1.2M, 1M, 0.8M, and 0.4M.

[0013] S6. Perform ultracentrifugation, then take out the ultracentrifuge tube, carefully collect the synaptic proteins between 1M-1.2M sucrose solution, and transfer to a 1.5mL EP tube to obtain the synaptic solution;

[0014] S7. Add the same volume of ice-cold 1% Triton-X to the obtained synaptic solution, incubate on ice for 15 minutes, centrifuge at 30,000 g for 30 minutes at 4°C, remove the supernatant, and take the precipitate as the intestinal synapse.

[0015] As an improvement, the low temperature condition in step S1 is in physiological saline at 4°C.

[0016] As an improvement, the conditions for the spin washing and vortex oscillation in step S2 are: spin washing at 20 rpm for 30 minutes at 37° C., and then vortex oscillation for 20 seconds.

[0017] As an improvement, the conditions for the initial centrifugation treatment in step S4 are: centrifugation at 1000g for 5 minutes at 4°C; the conditions for the secondary centrifugation treatment are: centrifugation at 15000g for 20 minutes at 4°C.

[0018] As an improvement, the ultracentrifugation treatment conditions in step S6 are: centrifugation at 85,000 g for 2 hours at 4°C using an ultracentrifuge.

[0019] The present invention has the advantages that:

[0020] 1. The present invention proposes a scheme for preparing mouse intestinal nerve synapses, and uses a gradient centrifugation method to accurately extract mouse intestinal nerve synapses. The extracted synaptic proteins have high purity and stable structure, which can help understand the mechanism of intestinal nerve synapses and help reveal the pathophysiology of many digestive system diseases, and are of great significance to the pathogenesis of the disease.

[0021] 2. The present invention can effectively separate and enrich intestinal nerve synapses through gradient centrifugation technology, avoiding contamination from other tissues and cells, thereby improving the purity of synaptic proteins. At the same time, the method minimizes damage to the synaptic structure during the extraction process and maintains the integrity of the synapse.

[0022] 3. The method provided by the present invention has clear steps, is easy to operate, and is easy for laboratory personnel to learn and master, which is conducive to promotion and application. Compared with traditional extraction methods, the method of the present invention can complete the extraction of synapses more quickly and improve experimental efficiency. DETAILED DESCRIPTION

[0023] In order to make the purpose, 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 in combination with 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.

[0024] Example 1

[0025] This embodiment discloses a method for extracting mouse intestinal neural synapses, comprising the following steps:

[0026] S1. Remove the small intestine tissue of mice in 4°C normal saline, cut the intestinal lumen longitudinally, and thoroughly clean the small intestine to remove the intestinal contents;

[0027] S2. Transfer the small intestinal tissue to a Ca-free medium containing 5 mM EDTA and 1 mM DTT. 2+ Mg 2+ The intestinal tissue was collected by washing in Hank's balanced solution at 37°C at 20 rpm for 30 min, vortexing for 20 s, and then using a filter to obtain the small intestinal tissue, which was repeated several times.

[0028] S3. The small intestine tissue was divided into 5 cm pieces on average. 1 mL of synaptic extraction and protection solution (Syn-PER from Thermo Fisher Scientific) was added to every 5 cm of small intestine tissue. TM Synaptic protein extraction reagent); transfer to a tissue grinder, grind thoroughly, transfer the sample to a 1.5 mL EP tube, and store in crushed ice;

[0029] S4. Perform the initial centrifugation on the sample at 1000g for 5 minutes at 4°C. Transfer the supernatant to a new 1.5mL EP tube; then perform a secondary centrifugation at 15000g for 20 minutes at 4°C, remove the supernatant, retain the precipitate, and add 1mL of 0.4M sucrose solution to each tube and mix thoroughly;

[0030] S5. Prepare 1.2M sucrose solution, 1M sucrose solution, and 0.8M sucrose solution and store them at 4°C in a Beckman ultracentrifuge tube, tilt it 45°, and slowly add 1.2M sucrose solution (1mL), 1M sucrose solution (1mL), and 0.85M sucrose solution (1mL) along the tube wall in the order of 1.2M sucrose solution (1mL), 1M sucrose solution (1mL), and 0.85M sucrose solution (1mL) to form a gradient sucrose solution; finally, slowly add the 0.4M sucrose solution that is completely mixed with the precipitate obtained in S4 to finally form a sucrose gradient solution of 1.2M, 1M, 0.8M, and 0.4M;

[0031] S6. Centrifuge at 85,000 g for 2 hours at 4°C in a Beckman ultracentrifuge, then remove the ultracentrifuge tube, carefully collect the synaptic proteins between 1M and 1.2M sucrose solution, and transfer to a 1.5 mL EP tube to obtain a synaptic solution;

[0032] S7. Add the same volume of ice-cold 1% Triton-X to the obtained synaptic solution, incubate on ice for 15 minutes, centrifuge at 30,000 g for 30 minutes at 4°C, remove the supernatant, and take the precipitate as the intestinal synapse.

[0033] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for extracting mouse intestinal synapses, characterized in that: The steps include: S1. Remove the small intestine tissue of mice under low temperature conditions, cut the intestinal lumen longitudinally, and then fully clean the small intestine to remove the intestinal contents; S2. Transfer the small intestinal tissue to a Ca-free medium containing 5 mM EDTA and 1 mM DTT. 2+ Mg 2+ The small intestine tissue was obtained by spinning and washing in Hank's equilibrium solution and vortexing, and then using a filter to obtain the small intestine tissue, which was repeated several times. S3. Divide the small intestinal tissue into 5 cm pieces and add 1 mL of synaptic extraction and protection solution to every 5 cm of small intestinal tissue; Transfer to a tissue grinder, grind thoroughly, transfer the sample to a 1.5 mL EP tube, and store in crushed ice; S4. Perform the first centrifugation on the sample, take the supernatant and transfer it to a new 1.5mL EP tube; then perform the second centrifugation, remove the supernatant, retain the precipitate, add 1mL of 0.4M sucrose solution to each tube and mix thoroughly; S5. Prepare 1.2M sucrose solution, 1M sucrose solution, and 0.8M sucrose solution and store them at 4°C. Tilt the ultracentrifuge tube at 45° and slowly add 1.2M sucrose solution (1mL), 1M sucrose solution (1mL), and 0.85M sucrose solution (1mL) along the tube wall in the order of 1.2M sucrose solution (1mL), 1M sucrose solution (1mL), and 0.85M sucrose solution (1mL) to form a gradient sucrose solution. Finally, slowly add the 0.4M sucrose solution to which the precipitate obtained in S4 is completely mixed to finally form a sucrose gradient solution of 1.2M, 1M, 0.8M, and 0.4M. S6. Perform ultracentrifugation, then take out the ultracentrifuge tube, carefully collect the synaptic proteins between 1M-1.2M sucrose solution, and transfer to a 1.5mL EP tube to obtain the synaptic solution; S7. Add the same volume of ice-cold 1% Triton-X to the obtained synaptic solution, incubate on ice for 15 minutes, centrifuge at 30,000 g for 30 minutes at 4°C, remove the supernatant, and take the precipitate as the intestinal synapse.

2. A method for extracting mouse intestinal neural synapses according to claim 1, characterized in that: The low temperature condition in step S1 is in physiological saline at 4°C.

3. A method for extracting mouse intestinal neural synapses according to claim 1, characterized in that: The conditions for the spin washing and vortex oscillation in step S2 are: spin washing at 20 rpm for 30 minutes at 37° C., and then vortex oscillation for 20 seconds.

4. The method for extracting mouse intestinal neural synapses according to claim 1, characterized in that: The conditions for the initial centrifugation in step S4 are: centrifugation at 1000 g for 5 minutes at 4°C; the conditions for the secondary centrifugation are: centrifugation at 15000 g for 20 minutes at 4°C.

5. The method for extracting mouse intestinal neural synapses according to claim 1, characterized in that: The ultracentrifugation treatment conditions in step S6 are: centrifugation at 85,000 g for 2 hours at 4°C using an ultracentrifuge.