Method for treating prosopalgia

Abstract

The invention discloses a trigeminal neuralgia treatment method, and relates to the technical field of trigeminal neuralgia treatment. The direct effect of the M2 on the proinflammatory and pain induction characteristics of the Schwann cells can play an important role in the multi-aspect analgesic effect of the M2; the microenvironment around nerves can be changed by increasing m < 2 > m macrophages after SHED-CM, so that NP is cured; immunohistochemical analysis shows that the number of p near-end side CD206 + F4 / 80 + macrophages of qualified thiocyanate is remarkably increased through sheet-CM treatment, however, TNF-alpha + S100beta + proinflammatory Schwann cells and TNF-alpha + F4 / 80 + M1 macrophages are remarkably reduced after sheet-CM treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of trigeminal neuralgia treatment, and particularly relates to a method for treating trigeminal neuralgia. Background Art

[0002] Neuropathic pain (NP), injuries or diseases of the somatosensory system often lead to highly debilitating chronic pain. Currently, there is no effective drug that can completely and definitively treat NP. We investigated the therapeutic potential of conditioned medium (CM) from human exfoliated deciduous teeth (SHED-CM) stem cells in a partial sciatic nerve ligation (PSL) model of NP using mice. PSL can cause abnormal pain sensations such as allodynia and hyperalgesia. In behavioral tests, intravenous injection of shed-cm treatment improved psl-induced hypersensitivity. We found that treatment with SHED-CM led to the recruitment of M2 macrophages in the injured sciatic nerve and ipsilateral L4 / L5 dorsal root ganglia, and inhibited the activity of microglia in the spinal cord. Notably, specific depletion of mannose-glycosylated fusiforms against intimal m2 macrophages significantly reduced the antinociceptive effect of SHED-CM. Intravenous injection of mm2CM induced by SHED-CM (M2-CM) improved psl-induced hypersensitivity. We found that M2-CM directly inhibited the expression of nociceptive receptors and proaminergic mediators in Schwann cells. Taken together, our data indicate that SHED-CM improves NP by inducing anti-m2 macrophages, and for this purpose, we propose a method for treating trigeminal neuralgia. Summary of the Invention

[0003] The purpose of the present invention is: to solve the problems mentioned in the above background art, the present invention provides a method for treating trigeminal neuralgia.

[0004] The present invention specifically adopts the following technical solutions to achieve the above purpose:

[0005] A method for treating trigeminal neuralgia, comprising:

[0006] Step 1, experimental preparation: Prepare mice under standard laboratory conditions and provide water and food;

[0007] Step 2, partial sciatic nerve ligation and drug administration: All mice were anesthetized by intraperitoneal injection of three mixtures (10 ml / kg). The sciatic nerve (thiocyanate) was exposed at a high position, and the dorsal side of the thiocyanate was carefully released from the surrounding tissues. Its dorsal side was carefully supported by a glass rod without pressing the nerve against the underlying structure. A 50 silk thread bent into a small needle was inserted into the nerve, and 1 / 3 to 1 / 2 of the nerve thickness was tightly ligated. Then, 3 to 4 skin sutures were used to close the incision. In the sham control group, the scn was exposed through a small incision, but no ligation was performed before closing the incision;

[0008] Step 3, Preparation of conditioned medium from human exfoliated deciduous teeth-derived stem cells: After crown and root separation, the dental pulp was digested with 3 mg / ml type I collagenase and 4 mg / ml dispase for 1 h at 37°C. Single cell suspensions (3×104 cells / ml) were placed on culture dishes and then incubated in an atmosphere of 100% relative humidity at 37°C and 5% carbon dioxide. After 48 h of culture, the medium was collected and centrifuged for 3 min at 440 / g. The supernatant was collected and centrifuged for 3 min at 17400 / g. The resulting supernatant was used for differential experiments of SHED-CM or fibroblast-CM. The protein concentration in shed-CM and fibro-CM was measured using a BCA protein assay kit and adjusted to 3 μg / ml using DMEM.

[0009] Step 4, Behavioral tests: All candidate mice were acclimated to the testing environment for 2 h per day for at least 2 days before baseline testing. Mice with stable data were selected for outcome-free testing. They were individually placed on a 5×5 mm metal mesh floor covered with small plastic containers and allowed to acclimate to the environment for approximately 2 - 3 h before testing. Mechanical hypersensitivity was measured using an electronic von Frey apparatus, and von Frey filaments were applied to the middle of the plantar surface of the hind paw. The withdrawal response was measured 5 times for each hind paw. If the mouse showed an active paw withdrawal response after stimulation, it was recorded as a positive response. Motor function was tested using a rotarod. Before the thermal test, they were acclimated to the instrument. The rotarod was accelerated from 4 to 40 revolutions per minute, and the observation period was 1 min. The time when the mouse fell off the rod was recorded.

[0010] Step 5, Real-time quantitative polymerase chain reaction: Fresh sciatic nerves, covering 1 cm long connectors at early day 3, or human Schwann cells were collected, torn in 1.5 ml RNase-free tubes, homogenized with Isogen, and chloroform was added. After centrifugation at iced temperature for 30 min at 4°C, the aqueous phase containing RNA was transferred to a fresh tube. Then RNA was precipitated by centrifugation with isopropanol and 75% ethanol. The purified total RNA was quantified using a spectrophotometer, and RNA integrity was checked on a 1.5% agarose gel. The purified total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase and random primers, and amplified using Thunderbird SYBR qPCR Mix.

[0011] Step 6, Immunohistochemistry: Mice in each group were deeply anesthetized before intracardiac perfusion, then injected with 4% paraformaldehyde. Then, the fixed SCN, L4 / L5 DRG, and L3-L4 spinal cord were collected from the mice. After fixation with 4% paraformaldehyde, they were dehydrated overnight in 25% sucrose, and the frozen tissues were embedded in OCT compound at 4°C. The thalamus and DRG were longitudinally sectioned into 10-μm-thick slices, and the spinal cord was sectioned into 30-μm-thick slices. The slices were mounted on glass slides, permeabilized with 0.3% Triton X-100 in PBS for 15 min, blocked with 5% normal donkey serum at 25-27°C for 1 h, and incubated overnight with the following primary antibodies: anti-f4 / 80, rabbit anti-cd206, rabbit anti-antibody 1, rabbit GFAP, anti-S100β, and anti-TNF-α. The next day, they were washed with 0.3% Triton X-100 PBS, incubated with fluorescent-conjugated secondary antibodies at -25°C for 1 h, washed with PBS with 0.3% Triton X-100, and incubated with DAPI at room temperature for 5 min. Finally, the slices were mounted on glass slides with mounting medium and covered with coverslips. Images of the tissues were captured using a conventional fluorescence microscope and a confocal microscope. The proximal CD206 / F4 / 80 and TNF-α / S100β positive cells and CD206 / F4 / 80 positive cells in DRG were counted at 400-fold magnification, and iba1 positive cells in the dorsal and ventral horns of the spinal cord were counted at 200-fold magnification. All positive cells were counted from at least three overlapping slices of 8 groups of animals;

[0012] Step 7, In vivo depletion of M2 macrophages: The poly-aminated liposome macrophage D depletion kit was used to deplete M2 macrophages from PSL mice. After PSL, SHED-CM was injected daily for 7 days. Starting from the 4th day, or 6.2 mg / kg of mannosylated liposome was injected. Control group mice received mannosylated liposomes without dichloromethane disodium salt in the same volume of m-Clodrosome-M2 macrophages, which express mannose receptors. 10 animals in each group were sacrificed for tissue collection 7 days after PSL;

[0013] Step 8, Preparation of M2-CM: Place the femoral bone marrow cells of 8-week-old male mice in 6 culture dishes, differentiate them into macrophages in DMEM with 20 ng / ml macrophage colony-stimulating factor, at 37 °C, in an atmosphere of 5% CO2 for 7 days. Then incubate the macrophages with serum-free DMEM and SHED-CM for 24 h. Capture the phase-contrast images of the induced macrophages using a digital microscope camera. Detect the mRNA expression of M2-type cell markers or trophic factors by qPCR. The macrophages induced by shed-cm are named M2 macrophages. Wash the induced macrophages twice with PBS, replace the medium with serum-free DMEM, after incubating for 24 h, collect the cell pellets, centrifuge at 440 g for 5 min, then collect the supernatant, take 400 g, centrifuge for 5 min, and use the obtained ultra-clear supernatant as M2-CM in subsequent in vivo and vitreous experiments;

[0014] Step 9, In vitro activation of human Schwann cells: Seed the cells in 6-cm culture dishes, incubate them with 10 ng / ml TNF-α in serum-free DMEM or rM2-CM for 24 h, and then collect the cells for gene analysis;

[0015] Step 10, Flow cytometry: Scrape the macrophages derived from the bone marrow of mice treated with shed-cm using a cell scraper, centrifuge at 400 g for 5 min, wash the cells with FACS incubation buffer and 1% bovine serum albumin, and incubate them with cd206-PE and F4 / 80-FITC antibodies in FACS incubation buffer. Subsequently, analyze them using a flow cytometer;

[0016] Step 11, Statistical analysis: The means of the two groups were statistically analyzed using Student's t-test.

[0017] Further, in Step 1, male ICR mice aged 7 - 11 weeks are used.

[0018] Further, the laboratory in Step 1 is a plastic cage with a 12-h light / dark cycle and a temperature between 23 and 24 °C.

[0019] Further, in Step 2, the mice are deeply anesthetized with a mixture of 5.0 mg / ml, 1.0 mg / ml, and 5.0 mg / ml midazolam, diluted with distilled water for injection (79% of the total volume).

[0020] Further, the type of skin suture used in Step 2 is 4-0.

[0021] Furthermore, the human skin fibroblast cell line in step three is derived from individuals over 36 years old, obtained from the Health Science Research Resource Bank at the 12th passage. The SHEDs and fibroblasts are 1×105. After several passages, the SHEDs or fibroblasts with a confluence of 70%-80% are washed twice, and the medium is replaced with serum-free DMEM.

[0022] Furthermore, the primers used in step five measure the fluorescence intensity of the inserted SYBR Green and are normalized to the fluorescence intensity of glyceraldehyde-3-phosphate dehydrogenase.

[0023] Furthermore, the density of each culture dish in step nine is 3×10, with a total of 5 cells.

[0024] Furthermore, in step eleven, Tukey's multiple comparison test is used to test the differences between the means of three or more groups. All statistical analyses are performed using the statistical analysis software R, and a p-value of <0.05 is used to declare statistical significance.

[0025] The beneficial effects of the present invention are as follows:

[0026] The direct effect of M2 of the present invention on the pro-inflammatory and pain-inducing properties of Schwann cells may play an important role in the multi-faceted analgesic effect of M2; increasing m2m macrophages after SHED-CM can change the perineural microenvironment, thereby curing NP; immunohistochemical analysis shows that shed-CM treatment significantly increases the number of CD206+F4 / 80+ macrophages in the proximal side of the qualified thiocyanate, however, TNF-α+S100β+ pro-inflammatory Schwann cells and TNF-α+F4 / 80+ M1 macrophages are significantly reduced after shed-CM treatment. Description of the Drawings

[0027] Figure 1 It is a schematic diagram showing that intravenous administration of SHED-CM of the present invention can relieve neuropathic pain caused by partial sciatic nerve ligation;

[0028] Figure 2 It is a schematic diagram of the primer sequences used in the qrt-pcr of the present invention;

[0029] Figure 3 It is a schematic diagram showing that SHED-CM treatment of the present invention transforms the pro-inflammatory microenvironment into an anti-inflammatory and tissue-protective microenvironment;

[0030] Figure 4 It is a schematic diagram showing that SHED-CM treatment of the present invention induces M2 macrophages;

[0031] Figure 5 It is a schematic diagram showing that shed-cm treatment of the present invention inhibits the activation of microglia;

[0032] Figure 6 It is a schematic diagram of the effect of M2 deletion on shed-cm-mediated behavior in the present invention;

[0033] Figure 7 It is a schematic diagram of the effect of shed-cm-induced m2 depletion on Schwann cell activators in the present invention;

[0034] Figure 8 It is a schematic diagram of the effect of consuming shed-cm-induced m2 on microglial communication in the present invention;

[0035] Figure 9 It is a schematic diagram of the pain-promoting sensory characteristics of M2-CM inhibiting tnf-α-activated Schwann cells in vitro in the present invention;

[0036] Figure 10 It is a schematic diagram of the analgesic effect of M2-CM in the present invention;

[0037] Figure 11 It is a schematic diagram of the analgesic effect of MCP-1 / sSiglec-9 in the present invention;

[0038] Figure 12 It is a flowchart of the present invention.

[0039] Reference numerals:

[0040] Figure 1 Among them: A, Time course; B, Early ipsilateral paw withdrawal threshold; C, Rotarod confirmed the early stage model; D, Time course of mid-term SHED-CM treatment; E, Mid-term ipsilateral paw withdrawal threshold; F, Time course of late-stage SHED-CM treatment; G, Homologous paw withdrawal threshold;

[0041] Figure 4 Among them: A, B, Representative images of immunofluorescence staining of CD206, F4 / 80 and TNF-α, S100β; C, D, Quantitative detection of the number of CD206+F4 / 80+ cells and the area of TNF-α+S100β+; E, Immunofluorescence staining images of CD206, F4 / 80 in L4 / 5 DRG; F, Quantitative analysis of the number of CD206+F4 / 80+ cells in L4 / 5 DRG;

[0042] Figure 5 Among them: A, B, Representative images of contralateral Iba1 immunofluorescence staining; C, D, Number of Iba1+ cells in the dorsal and ventral horns of L3 / 4 SP;

[0043] Figure 6 Among them: A, Time course of m2 depletion in the early stage model; B, Withdrawal of the ipsilateral paw;

[0044] Figure 7 Among them: A, Representative images of immunofluorescence staining; B, TNF-α, S100β in SCN; C, CD206, F4 / 80; D, Number of CD206+F4 / 80+ cells; E, Quantitative cation analysis of TNF-α+S100β+ area in SCN; F, Number of CD206+F4 / 80+ cells in L4 / 5 DRG;

[0045] Figure 8 Among them: A, Representative images of Iba1 immunofluorescence staining; B, C, Quantitative analysis of the number of Iba1+ cells;

[0046] Figure 9 Among them: A, Flow cytometry analysis of shed-cm-induced M2; B, qPCR analysis of the expression of human Schwann cells treated with M2-CM;

[0047] Figure 10 Among them: A, Time course in the early stage of M2-CM treatment; B, Ipsilateral paw withdrawal threshold in the early stage; C, qPCR analysis of the sciatic nerve 3 days after surgery; D, Immunofluorescence staining of TRPA1, S100β for SCN; E, Quantitative analysis of the TRPA+S100β+ area; F, Immunofluorescence staining of Iba1 in the ipsilateral horn of L 3 / 4 SP; G, H, Quantitative analysis of the number of Iba1+ cells;

[0048] Figure 11 Among them: A, Time course;

[0049] B, Ipsilateral paw withdrawal threshold of the midfoot. Detailed implementation manners

[0050] To make the objectives, 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 with reference to the accompanying drawings in the embodiments of the present invention.

[0051] Please refer to Figure 1 - Figure 11 , the present invention provides a method for treating trigeminal neuralgia, including:

[0052] Step 1, Experimental preparation: Prepare mice under standard laboratory conditions and provide water and food;

[0053] Step 2, partial sciatic nerve ligation and drug administration: All mice were anesthetized by intraperitoneal injection of three mixtures (10 ml / kg). The thermal sciatic nerve (thiocyanate) was exposed at a high position, and the dorsal side of the thiocyanate was carefully released from the surrounding tissues. Its dorsal side was carefully supported by a glass rod without pressing the nerve onto the underlying structure. A 3 / 8 50 silk thread bent into a small needle was inserted into the nerve, and 1 / 3 - 1 / 2 of the nerve thickness was tightly ligated. Then, 3 - 4 skin sutures were used to close the incision. In the sham control group, the scn was exposed through a small incision, but no ligation was performed before closing the incision;

[0054] Step 3, preparation of conditioned medium from human deciduous teeth-derived stem cells shed: After crown and root separation, the dental pulp was digested with 3 mg / ml type I collagenase and 4 mg / ml dispase for 1 h at 37 °C. The single-cell suspension (3×104 cells / ml) was placed on a culture dish and then incubated in an atmosphere of 100% relative humidity at 37 °C and 5% carbon dioxide. After 48 h of culture, the medium was collected and centrifuged for 3 min at 440 / g. The supernatant was collected and centrifuged for 3 min at 17400 / g. The resulting supernatant was used for differential experiments of SHED-CM or fibroblast-CM. The protein concentration in shed-CM and fibro-CM was measured using a BCA protein assay kit and adjusted to 3 μg / ml using DMEM;

[0055] Step 4, behavioral tests: All candidate mice were accustomed to the test environment for 2 h per day for at least 2 days before the baseline test. Mice with stable data were selected for the outcome-free test. They were individually placed on a 5×5 mm metal grid floor covered with small plastic containers and allowed to adapt to the environment for approximately 2 - 3 h before the test. Mechanical hypersensitivity was measured using an electronic von Frey apparatus. The von Frey filament was applied to the middle of the plantar surface of the hind paw, and the withdrawal response was measured 5 times for each hind paw. If the mouse showed an active paw withdrawal response after stimulation, it was recorded as a positive response. Motor function was tested using a rotarod. Before the thermal test, they were accustomed to the instrument. The expansion cylinder accelerated from 4 to 40 revolutions per minute, and they were observed for 1 min. The time when the mouse fell off the rod was recorded;

[0056] Step 5, real-time quantitative polymerase chain reaction: Fresh sciatic nerves, covering a 1-cm-long connective body at an early stage on the 3rd day, or human Schwann cells were collected and torn in a 1.5-ml RNase-free tube. Isogen was added for homogenization, followed by chloroform addition. After centrifugation at 4 °C for 30 min, the aqueous phase containing RNA was transferred to a fresh tube. Then, RNA was precipitated by centrifugation with isopropanol and 75% ethanol. The purified total RNA was quantified using a spectrophotometer, and the RNA integrity was checked on a 1.5% agarose gel. The purified total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase and random primers, and amplification was performed using Thunderbird SYBR qPCR Mix;

[0057] Step 6, Immunohistochemistry: Mice in each group were deeply anesthetized before intracardiac perfusion, then injected with 4% paraformaldehyde. Then, the fixed SCN, L4 / L5 DRG, and L3-L4 spinal cord were collected from the mice. After fixation with 4% paraformaldehyde, they were dehydrated overnight in 25% sucrose, frozen at 4 °C, and the tissues were embedded in OCT compound. The cherry thiocyanate and DRG were longitudinally sectioned into 10-μm-thick slices, and the spinal cord was sectioned into 30-μm-thick slices, which were mounted on glass slides. The slices were permeabilized with 0.3% Triton X-100 in PBS for 15 min, blocked with 5% normal donkey serum at 25-27 °C for 1 h, and incubated overnight with the following primary antibodies: anti-f4 / 80, rabbit anti-cd206, rabbit anti-antibody 1, rabbit GFAP, anti-S100β, and anti-TNF-α. The next day, they were washed with 0.3% Triton X-100 PBS, incubated with fluorescently conjugated secondary antibodies at -25 °C for 1 h, the slices were washed in PBS with 0.3% Triton X-100, and incubated with DAPI at room temperature for 5 min. Finally, the slices were mounted on glass slides with mounting medium and covered with coverslips. Images of the tissues were captured using a conventional fluorescence microscope and a continuous-focus microscope. Proximal CD206 / F4 / 80 and TNF-α / S100β positive cells and CD206 / F4 / 80 positive cells in DRG were counted at 400× magnification, and iba1 positive cells in the dorsal and ventral horns of the spinal cord were counted at 200× magnification. All positive cells were counted from at least three overlapping slices of 8 groups of animals;

[0058] Step 7, In vivo depletion of M2 macrophages: The polyaminochlorosome macrophage D depletion kit was used to deplete M2 macrophages from PSL mice. After PSL, SHED-CM was injected daily for 7 days. Starting from the 4th day, or 6.2 mg / kg mannosylated chlorosome was injected. Control group mice received mannosylated liposomes without disodium clodrosome in the same volume of m-Clodrosome-M2 macrophages, which express mannose receptors. 10 animals in each group were sacrificed for tissue collection 7 days after PSL;

[0059] Step 8, Preparation of M2-CM: Place the femoral bone marrow cells of 8-week-old male mice in 6 culture dishes, differentiate them into macrophages in DMEM with 20 ng / ml macrophage colony-stimulating factor, at 37 °C, in an atmosphere of 5% CO2 for 7 days. Then, incubate the macrophages with serum-free DMEM and SHED-CM for 24 h. Capture the phase-contrast images of the induced macrophages using a digital microscope camera. Detect the mRNA expression of M2-type cell markers or trophic factors by qPCR. The macrophages induced by shed-cm are named M2 macrophages. Wash the induced macrophages twice with PBS, replace the medium with serum-free DMEM, and after incubating for 24 h, collect the iron ingots, centrifuge at 440 g for 5 min, then collect the supernatant. Take 400 g and centrifuge for 5 min. The resulting ultra-clear liquid is used as M2-CM in subsequent in vivo and vitreous experiments;

[0060] Step 9, In vitro activation of human Schwann cells: Seed the cells in 6-cm culture dishes, incubate them with 10 ng / ml TNF-α in serum-free DMEM or rM2-CM for 24 h, and then collect the cells for gene analysis;

[0061] Step 10, Flow cytometry: Scrape the macrophages derived from the bone marrow of mice treated with shed-cm using a cell scraper, centrifuge at 400 g for 5 min, wash the cells with FACS incubation buffer and 1% bovine serum, and incubate them with cd206-PE and F4 / 80-FITC antibodies in FACS incubation buffer. Subsequently, analyze them using a flow cytometer;

[0062] Step 11, Statistical analysis: The means of the two groups were statistically analyzed using the Student's t-test.

[0063] In this example, preferably, male ICR mice aged 7 - 11 weeks are used in Step 1.

[0064] In this example, preferably, the laboratory in Step 1 has a 12 h dark / light cycle and a temperature between 23 and 24 °C in a plastic cage.

[0065] In this example, preferably, in Step 2, the mice are deeply anesthetized with a mixture of 5.0 mg / ml, 1.0 mg / ml, and 5.0 mg / ml midazolam, diluted with distilled water for injection (79% of the total volume).

[0066] In this example, preferably, the skin suture used in Step 2 is size 4-0.

[0067] In this embodiment, preferably, in step three, the human skin fibroblast cell line is derived from individuals over 36 years old, obtained from the Health Science Research Resource Bank at the 12th passage. The SHEDs and fibroblasts are 1×105. After several generations, the SHEDs or fibroblasts with a confluence of 70%-80% are washed twice, and the medium is replaced with serum-free DMEM.

[0068] In this embodiment, preferably, in step five, the primers used measure the fluorescence intensity of the inserted SYBR Green and are normalized to the fluorescence intensity of glyceraldehyde-3-phosphate dehydrogenase.

[0069] In this embodiment, preferably, in step nine, the density of each culture dish is 3×10, with a total of 5 cells.

[0070] In this embodiment, preferably, in step eleven, Tukey's multiple comparison test is used to test the differences between the means of three or more groups. All statistical analyses are performed using the statistical analysis software R, and a p-value of <0.05 is used to declare statistical significance.

[0071] The working principle and usage process of the present invention:

[0072] Step one, experimental preparation: Prepare mice under standard laboratory conditions and provide water and food. Among them, the mice used are 7-11-week-old male ICR mice, and the laboratory is a plastic cage with a 12h dark / light cycle and a temperature between 23 and 24 °C.

[0073] Step two, partial sciatic nerve ligation and drug administration: The mice are deeply anesthetized with a mixture of 5.0 mg / ml, 1.0 mg / ml, and 5.0 mg / ml midazolam, diluted with distilled water for injection (79% of the total volume). All mice are anesthetized by intraperitoneal injection of the three mixtures (10 ml / kg). The thermal sciatic nerve (sulphate) is exposed at a high position, and the dorsal side of the sulphate is carefully released from the surrounding tissues, and its dorsal side is carefully supported by a glass rod without pressing the nerve against the underlying structure. A 3 / 8 50 silk thread bent into a small needle is inserted into the nerve, and 1 / 3 to 1 / 2 of the nerve thickness is tightly ligated. Then, 3 to 4 skin sutures are used to close the incision, and the skin suture model selected is 4-0. In the sham operation control group, the scn is exposed through a small incision, but no ligation is performed before the incision is closed. The right 1 / 3 to 1 / 2 of the sulphate is tightened to induce NP. After nerve ligation, a decrease in the threshold of tactile stimulation is observed 3 days after PSL and remains at this level for at least 2 weeks. SHED-CM is injected intravenously daily, but Fibro-CM is administered immediately after PSL (early) and is not inhibited.

[0074] Step 3, Preparation of conditioned medium from human exfoliated deciduous teeth stem cells: After the crown and root were separated, the dental pulp was digested with 3 mg / ml type I collagenase and 4 mg / ml dispase for 1 h at 37°C. The single-cell suspension (3×104 cells / ml) was placed on a culture dish and then incubated in an atmosphere of 100% relative humidity at 37°C and 5% carbon dioxide. The human skin fibroblast cell line was derived from individuals over 36 years old and obtained from the Health Science Research Resource Bank at passage 12. The SHEDs and fibroblasts were seeded at 1×105. After several passages, the SHEDs or fibroblasts with 70%-80% confluence were washed twice, and the medium was replaced with serum-free DMEM. After culturing for 48 h, the medium was collected and centrifuged for 3 min at 440 / g. The supernatant was collected and centrifuged for 3 min at 17400 / g. The resulting supernatant was used for the differential experiment of SHED-CM or fibroblast-CM. The protein concentration in shed-CM and fibro-CM was measured using a bicinchoninic acid protein assay kit and adjusted to 3 μg / ml using DMEM.

[0075] Step 4, Behavioral tests: All candidate mice were acclimated to the testing environment for 2 h per day for at least 2 days before the baseline test. Mice with stable data were selected for the outcome-free test. They were individually placed on a 5×5 mm wire mesh floor covered with small plastic containers and allowed to acclimate to the environment for approximately 2-3 h before the test. Mechanical hypersensitivity was measured using an electronic von Frey apparatus. The von Frey filament was applied to the middle of the plantar surface of the hind paw, and the withdrawal response was measured 5 times for each hind paw. If the mouse showed an active paw withdrawal response after stimulation, it was recorded as a positive response. Motor function was tested using a rotarod. Before the thermal test, they were acclimated to the instrument. The rotarod cylinder accelerated from 4 to 40 revolutions per minute, and the observation lasted for 1 min. The time when the mouse fell off the rod was recorded. In the von Frey test, the threshold on the 3rd day in the shed-cm group was 6.73±1.50 and 4.29±1.49 in the group added with DMEM. On the 7th day, the threshold in the shed-CM group was 8.39±0.99 g, which was significantly higher than that in the DMEM group (4.30±1.78 g). In addition, the analgesic effect of SHED-CM disappeared 7 days after the last injection. The analysis of motor function by rotarod rest showed that the motor function of PSL mice treated with DMEM decreased, while that of SHED-CM mice was comparable to that of uninjured wild-type mice during the treatment period (days 1-7) and decreased immediately after the last injection of SHED-CM ( Figure 1 C).

[0076] Step 5, Real-time quantitative e polymerase chain reaction: Fresh sciatic nerves, covering a 1-cm long connector at the early 3rd day, or collecting human Schwann cells, tearing them in a 1.5-ml RNase-free tube, adding chloroform after homogenizing with Isogen, centrifuging at 4 °C for 30 min, transferring the aqueous phase containing RNA to a fresh tube, then precipitating RNA by centrifugation with isopropanol and 75% ethanol, purifying the total RNA and quantifying it with a spectrophotometer, checking the RNA integrity with 1.5% agar segments, converting the purified total RNA into cDNA using M-MLV reverse transcriptase and random primers, and amplifying on Thunderbird SYBR qPCR Mix; the primers used measured the fluorescence intensity of the inserted SYBR Green and normalized it to the fluorescence intensity of glyceraldehyde-3-phosphate dehydrogenase.

[0077] Step 6, Immunohistochemistry: Each group of mice was deeply anesthetized before intracardiac perfusion, then injected with 4% paraformaldehyde. Then, the fixed SCN, L4 / L5 DRG, and L3-L4 spinal cord were collected from the mice. After fixation with 4% paraformaldehyde, they were dehydrated overnight in 25% sucrose and cryo-embedded in OCT compound at 4 °C (Sakura thiocyanate and DRG were longitudinally divided into 10-μm thick sections, and the spinal cord was cut into 30-μm thick sections, mounted on glass slides. The sections were permeabilized with 0.3% Triton X-100 in PBS for 15 min, blocked with 5% normal donkey serum at 25-27 °C for 1 h, and incubated overnight with the following primary antibodies: anti-F4 / 80, rabbit anti-CD206, rabbit anti-antibody 1, rabbit anti-GFAP, anti-S100β, and anti-TNF-α. The next day, they were washed with 0.3% Triton X-100 PBS, incubated with fluorescent-conjugated secondary antibodies at -25 °C for 1 h, the sections were washed in PBS with 0.3% Triton X-100, and incubated with DAPI at room temperature for 5 min. Finally, the sections were mounted on glass slides with mounting medium and covered with coverslips. Images of the tissues were captured using an ordinary fluorescence microscope and a continuous focus microscope. The proximal CD206 / F4 / 80 and TNF-α / S100β positive cells and CD206 / F4 / 80 positive cells in DRG were counted at 400-fold magnification, and iba1 positive cells in the dorsal and ventral horns of the spinal cord were counted at 200-fold magnification. All positive cells were counted from at least three overlapping sections of 8 groups of animals;

[0078] Step 7, Depletion of M2 macrophages in vivo: The polyaminated clodrosome macrophage D depletion kit was used to deplete M2 macrophages from PSL mice. After PSL, SHED-CM was injected daily for 7 consecutive days. Starting from the 4th day, or 6.2 mg / kg of mannosylated clodrosome was injected. Control group mice received mannosylated liposomes without disodium clodronate. In the same volume of m-Clodrosome-M2 macrophages, mannose receptors were expressed. 10 animals were sacrificed in each group for tissue collection 7 days after PSL;

[0079] Step 8, Preparation of M2-CM: Bone marrow cells from 8-week-old male mice were placed in 6 culture dishes and differentiated into macrophages in DMEM with 20 ng / ml macrophage colony-stimulating factor at 37 °C in an atmosphere of 5% CO2 for 7 days. Then the macrophages were incubated with serum-free DMEM and SHED-CM for 24 h. Phase contrast images of the induced macrophages were captured using a digital microscope camera. The mRNA expression of m2-type cell markers or trophic factors was detected by qPCR. The macrophages induced by shed-cm were named M2 macrophages. The induced macrophages were washed twice with PBS, the medium was replaced with serum-free DMEM, and after incubation for 24 h, the supernatant was collected by centrifugation at 440 g for 5 min, and then centrifuged at 400 g for 5 min. The resulting ultra-clear supernatant was used as M2-CM in subsequent in vivo and vitreous experiments; M2-CM treatment directly inhibited the expression of TRPA1, pro-inflammatory cytokines and chemokines in TNF-α-activated human Schwann cells, and effectively inhibited PSL-induced pain. The direct effect of M2 on the pro-inflammatory / pain-inducing properties of Schwann cells may play an important role in the multi-faceted analgesic effect of M2.

[0080] Step 9, In vitro activation of human Schwann cells: The cells were seeded in 6-cm culture dishes at a density of 3×10 per dish, a total of 5 dishes. After incubation with 10 ng / ml TNF-α in 3 serum-free DMEM or rM2-CM for 24 h, the cells were collected for gene analysis.

[0081] Step 10, Flow cytometry: Macrophages derived from the bone marrow of shed-cm-treated mice were scraped with a cell scraper, centrifuged at 400 g for 5 min, washed with FACS incubation buffer and 1% bovine serum, and incubated with cd206-PE and F4 / 80-FITC antibodies in FACS incubation buffer. Subsequently, they were analyzed using a flow cytometer;

[0082] Step Eleven, Statistical Analysis: The means of the two groups were statistically analyzed using Student's t-test, and Tukey's multiple comparison test was used to test for differences between the means of three or more groups. All statistical analyses were performed using the statistical analysis software R, and a p-value of <0.05 was used to declare statistical significance.

[0083] The foregoing description of the disclosed embodiments enables those skilled in the art to make 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 invention. Thus, the invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A treatment method for trigeminal neuralgia, characterized in that, Including: Step 1, Experimental preparation: Prepare mice under standard laboratory conditions and provide water and food; Step 2, Partial sciatic nerve ligation and drug administration: All mice were anesthetized by intraperitoneal injection of three mixtures (10 ml / kg). The thermal sciatic nerve (thiocyanate) was exposed at a high position, and the dorsal side of the thiocyanate was carefully released from the surrounding tissues. Its dorsal side was carefully supported by a glass rod without pressing the nerve against the underlying structure. A 3 / 8 50 silk thread bent into a small needle was inserted into the nerve, and 1 / 3 - 1 / 2 of the nerve thickness was tightly ligated. Then, 3 to 4 skin sutures were used to close the incision. In the sham operation control group, the sciatic nerve was exposed through a small incision, but no ligation was performed before closing the incision; Step 3, Preparation of conditioned medium from human deciduous teeth-derived stem cells shed: After crown and root separation, the dental pulp was digested with 3 mg / ml type I collagenase and 4 mg / ml dispase at 37 °C for 1 h. The single-cell suspension (3×104 cells / ml) was placed on a culture dish and then incubated in an atmosphere of 100% relative humidity at 37 °C and 5% carbon dioxide. After 48 h of culture, the medium was collected and centrifuged for 3 min at 440 / g. The supernatant was collected and centrifuged for 3 min at 17400 / g. The resulting supernatant was used for differential experiments of SHED-CM or fibroblast-CM. The protein concentration in shed-CM and fibro-CM was measured using a bicinchoninic acid protein assay kit and adjusted to 3 μg / ml using DMEM; Step 4, Behavioral tests: All candidate mice were accustomed to the test environment for 2 h per day for at least 2 days before the baseline test. Mice with stable data were selected for the outcome-free test. They were individually placed on a 5×5 mm metal grid floor covered with small plastic containers and allowed to adapt to the environment for about 2 - Ⅲ h before the test. Mechanical hypersensitivity was measured using an electronic von Frey device. The von Frey filament was applied to the middle of the plantar surface of the hind paw, and the withdrawal response was measured 5 times for each hind paw. If the mouse showed an active paw withdrawal response after stimulation, it was recorded as a positive response. Motor function was tested using a rotarod. Before the thermal test, they were accustomed to the instrument. The rotarod was accelerated from 4 to 40 revolutions per minute, and the observation was carried out for 1 min. The time when the mouse fell off the rod was recorded; Step 5, Real-time quantitative polymerase chain reaction: Fresh sciatic nerve, covering a 1-cm-long segment at an early stage of day 3, or human Schwann cells were collected and torn in a 1.5-ml RNase-free tube. Isogen was added for homogenization, followed by chloroform addition. After centrifugation at 4 °C for 30 min, the aqueous phase containing RNA was transferred to a fresh tube. Then, RNA was precipitated by centrifugation with isopropanol and 75% ethanol. The purified total RNA was quantified using a spectrophotometer, and RNA integrity was checked on a 1.5% agarose gel. The purified total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase and random primers, and amplification was performed on Thunderbird SYBR qPCR Mix; Step 6, Immunohistochemistry: Mice in each group were deeply anesthetized before intracardiac perfusion, then injected with 4% paraformaldehyde. Then, the fixed SCN, L4 / L5 DRG, and L3-L4 spinal cord were collected from the mice. After fixation with 4% paraformaldehyde, they were dehydrated overnight in 25% sucrose and cryo-embedded in OCT compound at 4°C (Sakura thiocyanate and DRG were longitudinally sectioned into 10-μm-thick slices, and the spinal cord was sectioned into 30-μm-thick slices and mounted on glass slides. The sections were permeabilized with 0.3% Triton X-100 in PBS for 15 min, blocked with 5% normal donkey serum at 25-27°C for 1 h, and incubated overnight with the following primary antibodies: anti-f4 / 80, rabbit anti-cd206, rabbit anti-antibody 1, rabbit GFAP, anti-S100β, and anti-TNF-α. The next day, they were washed with 0.3% Triton X-100 PBS, incubated with a fluorescence-conjugated secondary antibody at -25°C for 1 h, the sections were washed in PBS with 0.3% Triton X-100, and incubated with DAPI at room temperature for 5 min. Finally, the sections were mounted on glass slides with mounting medium and covered with coverslips. Images of the tissues were captured using a conventional fluorescence microscope and a continuous-focus microscope. Proximal CD206 / F4 / 80 and TNF-α / S100β positive cells and CD206 / F4 / 80 positive cells in DRG were counted at 400× magnification, and iba1 positive cells in the dorsal and ventral horns of the spinal cord were counted at 200× magnification. All positive cells were counted from at least three overlapping sections of 8 groups of animals; Step 7, In vivo depletion of M2 macrophages: The polyaminated liposome macrophage D clearance kit was used to deplete M2 macrophages from PSL mice. After PSL, SHED-CM was injected daily for 7 days. Starting from the 4th day, or 6.2 mg / kg of mannosylated liposome was injected. Control group mice received mannosylated liposomes without disodium clodronate in the same volume of m-Clodrosome-M2 macrophages, which express mannose receptors. 10 animals in each group were sacrificed for tissue collection 7 days after PSL; Step 8, Preparation of M2-CM: Femoral bone marrow cells from 8-week-old male mice were placed in 6 culture dishes and differentiated into macrophages in DMEM with 20 ng / ml macrophage colony-stimulating factor at 37°C in an atmosphere of 5% CO2 for 7 days. Then, the macrophages were incubated with serum-free DMEM and SHED-CM for 24 h. Phase-contrast images of the induced macrophages were captured using a digital microscope camera. The mRNA expression of m2-type cell markers or trophic factors was detected by qPCR. The macrophages induced by shed-cm were named M2 macrophages. The induced macrophages were washed twice with PBS, the medium was replaced with serum-free DMEM, and after incubation for 24 h, the supernatant was collected by centrifugation at 440 g for 5 min, and then centrifuged at 400 g for 5 min. The resulting ultra-clear supernatant was used as M2-CM in subsequent in vivo and vitreous experiments; Step 9, in vitro activation of human Schwann cells: Seed the cells in a 6-cm culture dish, incubate with 10 ng / ml TNF-α in serum-free DMEM or rM2-CM for 24 h, and then collect the cells for gene analysis. Step 10, flow cytometry: Scrape the shed-cm-treated mouse bone marrow-derived macrophages with a cell scraper, centrifuge at 400 g for 5 min, wash the cells with FACS incubation buffer and 1% bovine serum albumin, and incubate with cd206-PE and F4 / 80-FITC antibodies in FACS incubation buffer. Subsequently, analyze them using a flow cytometer. Step 11, statistical analysis: The means of the two groups were statistically analyzed using Student's t-test.

2. The method for treating trigeminal neuralgia according to claim 1, wherein: In Step 1, male ICR mice aged 7 - 11 weeks were used.

3. A method for treating trigeminal neuralgia according to claim 1, characterized in that: In Step 1, the laboratory conditions were a 12-h light / dark cycle and a plastic cage with a temperature between 23 and 24 °C.

4. A method for treating trigeminal neuralgia according to claim 1, characterized in that: In Step 2, the mice were deeply anesthetized with a mixture of 5.0 mg / ml, 1.0 mg / ml, and 5.0 mg / ml midazolam and diluted with distilled water for injection (79% of the total volume).

5. A method for treating trigeminal neuralgia according to claim 1, characterized in that: In Step 2, the skin suture used was size 4-0.

6. A method for treating trigeminal neuralgia according to claim 1, characterized in that: In Step 3, the human skin fibroblast cell line was derived from individuals over 36 years old, obtained from the Health Science Research Resources Bank at passage 12. The SHEDs and fibroblasts were at a density of 1×10⁵. After several passages, the SHEDs or fibroblasts at 70% - 80% confluence were washed twice, and the medium was replaced with serum-free DMEM.

7. A treatment method for trigeminal neuralgia according to claim 1, characterized in that: In Step 5, the primers used measured the fluorescence intensity of the inserted SYBR Green and were normalized to the fluorescence intensity of glyceraldehyde-3-phosphate dehydrogenase.

8. A method for treating trigeminal neuralgia according to claim 1, characterized in that: In Step 9, the density of each culture dish was 3×10⁵ cells, with a total of 5 dishes.

9. A method for treating trigeminal neuralgia according to claim 1, characterized in that: In Step 11, Tukey's multiple comparison test was used to test for differences between the means of three or more groups. All statistical analyses were performed using the statistical analysis software R, and a p-value of <0.05 was used to declare statistical significance.

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