Method for treating neuropathic pain based on stem cells

Abstract

The invention discloses a method for treating neuropathic pain based on stem cells, and relates to the technical field of neuropathic pain treatment. In order to detect the anti-injury feeling activity of M2-CM, PSLmi ges is given, it is found that the M2-CM, instead of notDMEM, can prevent PSL-induced ectopic pain and proinflammatory response in thiocyanate, in addition, the M2-CM also inhibits expression of TRPA1 in Schwann cells in a body, and the result also shows that in an M2-CM group, the I ba1 + positive level on the same side of an fL3 / 4 group is obviously reduced in a DMEM group, in conclusion, in conclusion, the anti-injury feeling activity of the M2-CM is remarkably improved, and the anti-injury feeling activity of the M2-CM is remarkably improved. The SHED-CM part inhibits neuroinflammation and mechanical ectopic pain through the action of M2, and the m2-CM treatment directly inhibits the expression of TRPA1, pro-inflammatory cytokines and ch emotion factors in TNF-alpha activated human Schwann cells, and effectively inhibits the pain induced by ps l.

Description

Technical Field

[0001] The present invention relates to the technical field of neuropathic pain treatment, and in particular to a method for treating neuropathic pain based on stem cells. Background Art

[0002] Lesions or diseases of the sensory nervous system cause NP, characterized by allodynia and hyperalgesia. The diversity and complex pathophysiology of these diseases hinder the development of effective treatments for NP. No single drug can completely and definitively treat NP.

[0003] In recent years, mesenchymal stem cell therapy has shown great therapeutic potential in clinical and preclinical studies. It is worth noting that in most pregnancies, neural function is restored mainly through paracrine / trophic mechanisms. Stem cells secrete a series of trophic and immunomodulatory factors and can be collected through serum-free conditioned medium (CM). Stem cell CM has significant therapeutic effects on central nervous system and PNS damage, neurodegenerative and autoimmune diseases. Dental pulp stem cells (DPSCs) have a powerful analgesic effect on neuropathy induced by osteoarthritis and pain. Therefore, we propose a stem cell-based approach to treat neuropathic pain. Summary of the Invention

[0004] The purpose of the present invention is to solve the problems mentioned in the above background technology. The present invention provides a method for treating neuropathic pain based on stem cells.

[0005] In order to achieve the above-mentioned purpose, the present invention specifically adopts the following technical solutions:

[0006] A stem cell-based method for treating neuropathic pain, comprising the following steps:

[0007] Step 1: Prepare the animal;

[0008] Step 2: nerve ligation and drug administration;

[0009] Step 3, preparing culture medium;

[0010] Step 4: Behavioral testing;

[0011] Step 5: Depletion of M2 macrophages in vivo;

[0012] Step 6: Statistical analysis.

[0013] Furthermore, the step 1 includes: preparing two groups of mice, placing them in a standard laboratory, and feeding them with water and food, one group being the experimental group and the other being the control group.

[0014] Furthermore, the standard laboratory is made of a transparent glass box, placed outdoors with good lighting conditions, and equipped with a thermostat with a room temperature of 25°C.

[0015] Furthermore, the step 2 includes: exposing the hot sciatic nerve of the test group to a high place, injecting anesthetic to anesthetize it, and ligating it. After completion, the incision is sutured with skin sutures.

[0016] Furthermore, the preparation of the culture medium in step 3 comprises the following steps:

[0017] S1. Prepare materials and select deciduous teeth from 6 to 12 years old;

[0018] S2, culture, after the crown and root are separated, they are dissolved and the single cell submembrane is placed in a culture dish for culture;

[0019] S3, incubation, placing the culture medium after the culture is completed in an incubation device for incubation;

[0020] S4. Centrifugation: Place the culture medium after incubation in a centrifuge for centrifugation and collect the supernatant.

[0021] Furthermore, the incubation conditions in S3 are 37° C., 5% carbon dioxide and 100% relative humidity.

[0022] Furthermore, the centrifugation time in S4 is 3 minutes.

[0023] Furthermore, the behavioral test in step 4 includes: placing the mice in the test group and the control group on a metal mesh plate, and performing hypersensitivity measurement using a mechanical hypersensitivity measurement device.

[0024] Furthermore, the in vivo M2 macrophage depletion in step five includes: clearing M2 macrophages from PSL mice.

[0025] Furthermore, the statistical analysis in step six includes: testing the differences between the means of three or more groups.

[0026] The beneficial effects of the present invention are as follows:

[0027] The attenuation of M2-CM in vivo of the present invention. In order to detect the anti-nociceptive activity of M2-CM, PSLmiges were administered, and it was found that M2-CM, but not DMEM, could prevent PSL-induced allodynia and pro-inflammatory response in thiocyanate. In addition, M2-CM also inhibited the expression of TRPA1 in Schwann cells in vivo. The results also showed that in the M2-CM group, the Iba1+ positive level on the ipsilateral side of the fL3 / 4 group was significantly reduced in the DMEM group. In summary, these results indicate that SHED-CM partially inhibits neuroinflammation and mechanical allodynia through the action of M2. M2-CM treatment directly inhibits the expression of TRPA1 as well as pro-inflammatory cytokines and ch emotional factors in TNF-α-activated human Schwann cells, and effectively inhibits PSL-induced pain. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a workflow diagram of the present invention;

[0029] Figure 2 It is a workflow diagram for preparing culture medium in the present invention;

[0030] Figure 3 Schematic diagram of the analgesic effect of M2-CM in the present invention.

[0031] Reference numerals: Figure 3 Middle: A, Time course of the early phase of M2-CM treatment; B, Early ipsilateral paw withdrawal threshold; C, qPCR analysis of the sciatic nerve 3 days after surgery; D, Immunofluorescence staining of the SCN for TRPA1 and S100β; E, Quantitative analysis of the TRPA+S100β+ area; F, Immunofluorescence staining of Iba1 in the ipsilateral horn of the l 3 / 4SP; G, H, Quantitative analysis of the number of Iba1+ cells. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0033] See also Figure 1 - Figure 3 The present invention provides a method for treating neuropathic pain based on stem cells, comprising the following steps:

[0034] Step 1: Prepare the animal;

[0035] Step 2: nerve ligation and drug administration;

[0036] Step 3, preparing culture medium;

[0037] Step 4: Behavioral testing;

[0038] Step 5: Depletion of M2 macrophages in vivo;

[0039] Step 6: Statistical analysis.

[0040] In this embodiment, preferably, step one includes: preparing two groups of mice, placing them in a standard laboratory, and feeding them with water and food, one group being the experimental group and the other being the control group; the number of mice in each group is selected according to actual needs, and feeding them with water and food can ensure the maturity of the mice, and with the cooperation of the experimental group and the control group, the subsequent test results can be directly understood.

[0041] In this embodiment, preferably, the standard laboratory uses a transparent glass box, which is placed outdoors with good lighting conditions and is equipped with a thermostat with a room temperature of 25°C; the transparent box is convenient for observation, the lighting conditions are good, and the observation effect can be guaranteed, while the constant temperature conditions can ensure the survival rate of the mice.

[0042] In this embodiment, preferably, step 2 includes: exposing the hot sciatic nerve of the test group to a high place, anesthetizing it by injecting anesthetics, and ligating it. After completion, the incision is sutured with skin sutures.

[0043] In this embodiment, preferably, the preparation of the culture medium in step 3 includes the following steps:

[0044] S1. Prepare materials and select deciduous teeth from 6 to 12 years old;

[0045] S2, culture, after the crown and root are separated, they are dissolved and the single cell submembrane is placed in a culture dish for culture;

[0046] S3, incubation, placing the culture medium after the culture is completed in an incubation device for incubation;

[0047] S4. Centrifugation: Place the culture medium after incubation in a centrifuge for centrifugation and collect the supernatant.

[0048] In this embodiment, preferably, the incubation condition in S3 is 37° C., 5% carbon dioxide, and 100% relative humidity.

[0049] In this embodiment, preferably, the centrifugation time in S4 is 3 minutes.

[0050] In this embodiment, preferably, the behavioral test in step 4 includes: placing the mice in the test group and the control group on a metal mesh plate, and performing hypersensitivity measurement using a mechanical hypersensitivity measurement device.

[0051] In this embodiment, preferably, the depletion of M2 macrophages in vivo in step 5 includes: clearing M2 macrophages from PSL mice.

[0052] In this embodiment, preferably, the statistical analysis in step six includes: testing the differences between the means of three or more groups.

[0053] The working principle and use process of the present invention:

[0054] Step 1. Prepare animals. Prepare two groups of mice, place them in a standard laboratory, and feed them with water and food. One group is the experimental group and the other is the control group. The number of mice in each group is selected according to actual needs. Feeding water and food can ensure the maturity of the mice. With the cooperation of the experimental group and the control group, the subsequent test results can be directly understood. The standard laboratory uses a transparent glass box, placed outdoors with good lighting conditions, and equipped with a thermostat. The room temperature is 25°C; the transparent box is easy to observe, the lighting conditions are good, which can ensure the observation effect, and the constant temperature conditions can ensure the survival rate of the mice.

[0055] Step 2: Nerve ligation and drug administration: The hot sciatic nerve of the experimental group was exposed at a high place, anesthetized by injection of anesthetic, and ligated. After completion, the incision was sutured with skin sutures.

[0056] Step 3: preparing the culture medium, comprising the following steps:

[0057] S1. Prepare materials and select deciduous teeth from 6 to 12 years old;

[0058] S2, culture, after the crown and root are separated, they are dissolved and the single cell submembrane is placed in a culture dish for culture;

[0059] S3, incubation, placing the culture medium after the culture is completed in an incubation device for incubation, and the incubation conditions are 37° C., 5% carbon dioxide, and 100% relative humidity;

[0060] S4. Centrifugation: Place the culture medium after incubation in a centrifuge and centrifuge for 3 minutes to collect the supernatant.

[0061] Conditioned medium from human exfoliated deciduous teeth with stem cells prevents NP and maintains low-growth function after PSL. We ligated the right 1 / 3 to 1 / 2 of the microfascicle with thiocyanate to induce NP. After nerve ligation, a decrease in the threshold to tactile stimulation was observed. This threshold was reached at least 3 days after PSL and remained at this level for at least 2 weeks. Daily intravenous administration of SHED-CM, but not Fibro-CM, was given immediately after PSL (early stage).

[0062] Step 4: Behavioral test: Place the mice in the experimental group and the control group on a metal mesh plate, and perform hypersensitivity measurement using a mechanical hypersensitivity measurement device.

[0063] Step 5: Depletion of M2 macrophages in vivo, eliminating M2 macrophages from PSL mice.

[0064] To examine the antinociceptive activity of M2-CM in vivo, PSLmiges were administered and M2-CM, but not DMEM, was found to prevent PSL-induced allodynia and proinflammatory responses in thiocyanate ( Figure 3 AC), in addition, M2-CM also inhibited the expression of TRPA1 in Schwann cells in vivo. The results also showed that in the M2-CM group, the Iba1+ positive level in the ipsilateral fL3 / 4 group was significantly reduced compared with that in the DMEM group ( Figure 3 FH), Taken together, these results indicate that SHED-CM inhibits neuroinflammation and mechanical allodynia in part through the action of M2, and that M2-CM treatment directly suppresses the expression of TRPA1 as well as proinflammatory cytokines and CH mood factors in TNF-α-activated human Schwann cells and effectively inhibits PSL-induced pain.

[0065] Step 6: Statistical analysis to test the differences between the means of three or more groups.

[0066] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for treating neuropathic pain based on stem cells, characterized in that: The steps include: Step 1: Prepare the animal; Step 2: nerve ligation and drug administration; Step 3, preparing culture medium; Step 4: Behavioral testing; Step 5: Depletion of M2 macrophages in vivo; Step 6: Statistical analysis.

2. The method for treating neuropathic pain based on stem cells according to claim 1, characterized in that: The step 1 includes: preparing two groups of mice, placing them in a standard laboratory, and feeding them with water and food, one of which is a test group and the other is a control group.

3. The method for treating neuropathic pain based on stem cells according to claim 2, characterized in that: The standard laboratory is made of a transparent glass box, placed outdoors with good lighting conditions, and equipped with a thermostat, with the room temperature at 25°C.

4. The method for treating neuropathic pain based on stem cells according to claim 1, characterized in that: The second step includes: exposing the hot sciatic nerve of the test group to a high place, injecting anesthetic to anesthetize it, and ligating it. After completion, the incision is sutured with skin sutures.

5. The method for treating neuropathic pain based on stem cells according to claim 1, characterized in that: The preparation of culture medium in step 3 comprises the following steps: S1. Prepare materials and select deciduous teeth from 6 to 12 years old; S2, culture, after the crown and root are separated, they are dissolved and the single cell submembrane is placed in a culture dish for culture; S3, incubation, placing the culture medium after the culture is completed in an incubation device for incubation; S4. Centrifugation: Place the culture medium after incubation in a centrifuge for centrifugation and collect the supernatant.

6. The method for treating neuropathic pain based on stem cells according to claim 5, characterized in that: The incubation conditions in S3 were 37° C., 5% carbon dioxide, and 100% relative humidity.

7. The method for treating neuropathic pain based on stem cells according to claim 1, characterized in that: The centrifugation time in S4 is 3 minutes.

8. The method for treating neuropathic pain based on stem cells according to claim 1, characterized in that: The behavioral test in step 4 includes: placing the mice in the test group and the control group on a metal mesh plate, and performing hypersensitivity measurement using a mechanical hypersensitivity measurement device.

9. The method for treating neuropathic pain based on stem cells according to claim 1, characterized in that: The in vivo M2 macrophage depletion in step 5 includes: clearing M2 macrophages from PSL mice.

10. The method for treating neuropathic pain based on stem cells according to claim 1, characterized in that: The statistical analysis in step six includes: testing the differences between the means of three or more groups.

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