Rat intestinal nerve cell separation method

Abstract

The invention discloses a rat intestinal nerve cell separation method, which comprises the following steps: taking a rat intestinal muscle layer, digesting the rat intestinal muscle layer into single cells, carrying out flow antibody staining and flow cytometry sorting on cell surface molecules such as CD9 and CD45, and purifying the rat intestinal nerve cells, and comprises the following steps: firstly, preparing the single cells, secondly, staining the single cells, and finally, separating the rat intestinal nerve cells. And finally, separating by flow cytometry. The method is short in time consumption, and real-time separation can be realized; the cell surface molecular tags aiming at the rat intestinal nerve cells are determined to be CD45 negative and CD9 positive, and the rat intestinal nerve cells can be rapidly separated in real time for scientific research and clinical application.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for separating rat intestinal nerve cells. Background Art

[0002] The enteric nervous system is a complex system composed of nerve cells and glial cells in the intestinal wall. It can autonomously regulate intestinal motility, transmit nutritional information, sense bacterial flora disorders, and work synergistically with the intestinal immune system, intestinal flora, and other intestinal cell tissues to maintain intestinal function and homeostasis. The enteric nervous system is associated with the occurrence and development of a variety of local or systemic diseases, including congenital megacolon, achalasia, inflammatory bowel disease, cancer, diabetes, neurodevelopmental disorders, and neurodegenerative diseases.

[0003] Enteric nerve cells are the core components of the enteric nervous system. Establishing methods to separate and purify enteric nerve cells can be used in academic research to study the special structural functions of enteric nerve cells that are different from those of central nervous system nerve cells; to explore their interactions with other types of intestinal cells; and to explore their important role in the occurrence and development of related diseases. In clinical practice, it can be used to discover early biomarkers of related diseases; and to predict and evaluate patients' responses and prognosis to specific treatment options.

[0004] Existing neural cell separation technologies, including culture separation, density gradient centrifugation, magnetic cell sorting, microfluidics, etc., are mainly used to separate neural cells in the central nervous system, but are not suitable for real-time separation of intestinal neural cells. This technology will effectively fill this gap. Summary of the invention

[0005] The purpose of the invention is to provide a method for isolating rat intestinal nerve cells, which solves the problems existing in the prior art.

[0006] The technical solution adopted by the present invention is to take the intestinal muscularis of rats and digest it into single cells, and then purify rat intestinal nerve cells by flow antibody staining and flow cytometry sorting of cell surface molecules such as CD9 and CD45. The existing nerve cell separation technology, cell culture separation method takes several days and cannot guarantee real-time effect; density gradient centrifugation separation method is suitable for central nervous cells with large cell density differences, and is not suitable for intestines with small cell density differences; magnetic cell sorting method and microfluidics technology both require clear cell surface molecules as separation labels, and there are currently no molecular labels for rat intestinal nerve cells.

[0007] Single cell preparation:

[0008] 1. Take a SD rat weighing 200-300 g and euthanize it with carbon dioxide.

[0009] 2. Take the rat colon, peel off the intermuscular nerve layer, transfer it to 0.5-2 mg / ml Librase enzyme, and digest it in a 37°C water bath for 20 minutes to 1 hour.

[0010] 3. Transfer to trypsin and incubate in 37°C water bath for 10 to 20 minutes.

[0011] 4. Centrifuge and discard the supernatant.

[0012] Flow cytometry sorting:

[0013] 1. Add antibody mixture, including anti-cell surface label CD9 antibody (using volume concentration 1:200-1:1000), CD45 (1:200-1:1000), and death cell dye DAPI, and stain at 4°C or room temperature in the dark for 20 minutes to 1 hour.

[0014] 2. Centrifuge, remove the supernatant, add Neurobasal medium, and perform flow cytometry sorting. The cell population that meets the requirements of DAPI-negative for the death cell dye, CD45-negative for the cell surface molecule label, and CD9-positive for the cell surface molecule label is the high-purity enteric neural cell.

[0015] The present invention takes a short time and can achieve real-time separation; it is determined that the cell surface molecular labels for rat intestinal nerve cells are CD45 negative and CD9 positive. Through the present invention, rat intestinal nerve cells can be separated quickly and in real time for scientific research and clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a flowchart of rat enteric neuronal cell isolation;

[0017] Figure 2 This is a diagram showing the in vitro culture of isolated intestinal neurons. DETAILED DESCRIPTION

[0018] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 The figure shows a flowchart of rat intestinal nerve cell isolation; the rat intestinal muscularis is obtained and digested into single cells, and then the rat intestinal nerve cells are purified by flow antibody staining and flow cytometric sorting of cell surface molecules such as CD9 and CD45.

[0020] Figure 2 As shown, the in vitro culture display of isolated intestinal nerve cells: the isolated rat intestinal nerve cells were cultured in vitro, and after adhesion, they were fluorescently stained with the nerve cell label Tuj1 and observed under a fluorescence microscope, confirming that the isolated cells were all Tuj1 positive, that is, they were all intestinal nerve cells.

[0021] 1. Single cell preparation:

[0022] 1. Take a SD rat weighing 200-300 g and euthanize it with carbon dioxide.

[0023] 2. Take the rat colon, peel off the intermuscular nerve layer, transfer it to 0.5-2 mg / ml Librase enzyme, and digest it in a 37°C water bath for 20 minutes to 1 hour.

[0024] 3. Transfer to trypsin and incubate in 37°C water bath for 10 to 20 minutes.

[0025] 4. Centrifuge and discard the supernatant.

[0026] 2. Flow cytometry sorting:

[0027] 1. Add antibody mixture, including anti-cell surface label CD9 antibody (using volume concentration 1:200-1:1000), CD45 (1:200-1:1000), and death cell dye DAPI, and stain at 4°C or room temperature in the dark for 20 minutes to 1 hour.

[0028] 2. Centrifuge, remove the supernatant, add Neurobasal medium, and perform flow cytometry sorting. The cell population that meets the requirements of DAPI negative, CD45 negative, and CD9 positive is the high-purity enteric neural cells.

[0029] Embodiment 1:

[0030] 1. Single cell preparation:

[0031] 1. Take a 200 g SD rat and euthanize it with carbon dioxide.

[0032] 2. Take the rat colon, peel off the intermuscular nerve layer, transfer it to 1 mg / ml Librase enzyme, and digest it in a 37°C water bath for 40 minutes.

[0033] 3. Transfer to trypsin and incubate in 37°C water bath for 15 minutes.

[0034] 4. Centrifuge and discard the supernatant.

[0035] 2. Flow cytometry sorting:

[0036] 1. Add antibody mixture containing antibodies against cell surface label CD9 (using volume concentration 1:400), CD45 (1:400), and death cell dye DAPI, and stain at 4℃ in the dark for 40 minutes.

[0037] 2. Centrifuge, remove the supernatant, add Neurobasal medium, and perform flow cytometry sorting. The sorting method is as follows Figure 1As shown, the sorted cells are high-purity enteric neural cells.

[0038] Embodiment 2:

[0039] 1. Single cell preparation:

[0040] 1. Take a 250 g SD rat and euthanize it with carbon dioxide.

[0041] 2. Take the rat colon, peel off the intermuscular nerve layer, transfer it to 0.5 mg / ml Librase enzyme, and digest it in a 37°C water bath for 60 minutes.

[0042] 3. Transfer to trypsin and incubate in 37°C water bath for 10 minutes.

[0043] 4. Centrifuge and discard the supernatant.

[0044] Secondary flow cytometry sorting:

[0045] 1. Add antibody mixture containing antibodies against cell surface label CD9 (using volume concentration 1:600), CD45 (1:800) and death cell dye DAPI, and stain at 4℃ in the dark for 60 minutes.

[0046] 2. Centrifuge, remove the supernatant, add Neurobasal medium, and perform flow cytometry sorting. The cell population that meets the requirements of DAPI negative, CD45 negative, and CD9 positive is the high-purity enteric neural cells.

[0047] Embodiment three:

[0048] 1. Single cell preparation:

[0049] 1. Take a 300 g SD rat and euthanize it with carbon dioxide.

[0050] 2. Take the rat colon, peel off the intermuscular nerve layer, transfer it to 1.5 mg / ml Librase enzyme, and digest it in a 37°C water bath for 30 minutes.

[0051] 3. Transfer to trypsin and incubate in 37°C water bath for 20 minutes.

[0052] 4. Centrifuge and discard the supernatant.

[0053] Secondary flow cytometry sorting:

[0054] 1. Add antibody mixture containing antibodies against cell surface label CD9 (using volume concentration 1:200), CD45 (1:1000) and death cell dye DAPI, and stain for 20 minutes at room temperature away from light.

[0055] 2. Centrifuge, remove the supernatant, add Neurobasal medium, and perform flow cytometry sorting. The cell population that meets the requirements of DAPI negative, CD45 negative, and CD9 positive is the high-purity enteric neural cells.

Claims

1. A method for isolating rat intestinal nerve cells, characterized in that: The rat intestinal muscularis was obtained and digested into single cells, and then the rat enteric neural cells were purified by flow antibody staining of cell surface molecules such as CD9 and CD45 and flow cytometric sorting, which included the following summarized steps: first single cell preparation, second single cell staining, and finally flow cytometric separation.

2. The method for isolating rat enteric nerve cells according to claim 1, characterized in that: Single cell preparation 1) Take a SD rat and euthanize it with carbon dioxide; 2) Take the rat colon, peel off the myenteric nerve layer, transfer to 0.5-2 mg / ml Librase enzyme, and digest in a water bath for 20 minutes to 1 hour; 3) Transfer to trypsin and place in a 37°C water bath for 10-20 minutes; 4) Centrifuge at 1000-2000 rpm, discard the supernatant, and the resulting precipitate is the single cell.

3. The method for isolating rat enteric nerve cells according to claim 1, characterized in that: Single cell staining includes: adding antibody mixture to the obtained cell pellet, and staining at 4°C or room temperature in the dark for 20 minutes to 1 hour.

4. The method for isolating rat enteric nerve cells according to claim 1 or 3, characterized in that: Flow cytometry sorting includes: centrifuging the treated cell precipitate mixture at a speed of 1000-2000 rpm, removing the supernatant, adding Neurobasal culture medium to resuspend the precipitate, and performing flow cytometry sorting. The cell population that meets the requirements of death cell dye DAPI negative, cell surface molecule label CD45 negative, and cell surface molecule label CD9 positive is the high-purity enteric nerve cell.

5. The method for isolating rat enteric nerve cells according to claim 1, characterized in that: SD rats weighing 200-300 g.

6. The method for isolating rat enteric nerve cells according to claim 2, characterized in that: Water bath temperature 37°C.

7. The method for isolating rat enteric nerve cells according to claim 1, characterized in that: The antibody mixture refers to a solution containing three dye components: an antibody labeled with a fluorescent group for the cell surface molecule label CD9; an antibody labeled with a fluorescent group for the cell surface molecule label CD45; and a dead cell dye DAPI.

8. The method for isolating rat enteric nerve cells according to claim 7, characterized in that: The volume concentration of the antibody labeled with fluorescent group for cell surface molecule label CD9 is 1:200~1:1000.

9. The method for isolating rat enteric nerve cells according to claim 7, characterized in that: The volume concentration of the fluorescent group antibody labeled with the cell surface molecule CD45 is 1:200 to 1:1000.

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