Use of SKP-SC-EVs in the preparation of a product for treating Parkinson's disease

Abstract

The application discloses application of skin precursor cell induced Schwann cell derived vesicles (SKP-SC-EVs) in preparation of a product for treating Parkinson's disease, wherein the SKP-SC-EVs have a protective effect in rotenone or MPP+-induced cell injury, and in a MPTP-induced mouse Parkinson's disease model, the SKP-SC-EVs can be given by a nasal instillation method to improve the motor ability of the mouse and restore the olfactory function, and the product can be used for research and treatment of Parkinson's disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and particularly relates to application of SKP-SC-EVs in preparation of a product for treating Parkinson's disease. BACKGROUND

[0002] Parkinson's disease (PD) is one of the common neurodegenerative diseases, and its key pathological feature is the degeneration of the substantia nigra striatal-dopaminergic pathway. The main clinical manifestations include motor symptoms and non-motor symptoms. The motor symptoms include bradykinesia, resting tremor, rigidity and postural instability, etc. The non-motor symptoms include sensory disorders, sleep disorders, autonomic dysfunction, neuropsychiatric disorders, etc. The pathogenesis of PD is still unclear. The most important treatment is drug therapy. Levodopa preparations are the most effective drugs. However, the current treatment mainly improves symptoms and cannot stop the death of dopaminergic neurons and the progression of the disease.

[0003] With the development of stem cell technology, the research on using stem cell therapy to treat Parkinson's disease has made a major breakthrough, which is expected to bring a complete revolution to the treatment of PD. Stem cells can exert neurorestorative and neuroprotective effects in vivo through mechanisms such as replacing neurons, secreting neurotrophic factors, and regulating neural microenvironment. The stem cells currently used in research mainly include induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, and neural stem cells.

[0004] Skin-derived precursors (SKPs) are derived from neural crest stem cells in the embryonic period and exist in the adult period. They can be obtained from adult skin, are abundant in source, are easy to obtain, and are easy to culture and expand in vitro. Skin-derived precursor cells from rodents and humans can be differentiated into skin-derived precursor cell-induced Schwann cells (SKP-SCs), which can be massively expanded in vitro and have been proven to promote the regeneration of peripheral nerves. It has been found that in a 6-OHDA-induced SH-SY5Y cell PD model, SKP-SCs can reduce 6-OHDA-induced cytotoxicity through the PI3K / AKT / Bcl-2 pathway, regulate autophagy, and exert a neuroprotective effect. In a 6-OHDA-induced mouse PD model, stereotactic injection of SKP-SCs into the brain can alleviate 6-OHDA-induced behavioral impairment and reduce the loss of dopaminergic neurons. However, the identity, biological effects and safety of cells transplanted into the body are not completely clear at present, and the transplanted cells are mostly allogeneic in origin, which will cause immunogenicity after transplantation and is not conducive to clinical promotion.

[0005] In recent years, the use of cell-derived vesicles (EVs) to replace cell therapy is on the rise. Vesicles contain a variety of bioactive substances (proteins, mRNA, microRNA, lncRNA and DNA, etc.), which are taken up by target cells after being released from cells to trigger corresponding biological effects. Recent studies have shown that a variety of cell-derived vesicles are an important way of intercellular interaction in the nervous system, and play an important role in peripheral nerve development and regeneration by regulating the regenerative microenvironment. Stem cell-derived vesicles can cross the blood-brain barrier and have certain protective ability for PD model animals. Vesicles can be obtained in large quantities in vitro, and are easier to control the quality, and have more advantages in safety and regulation. Compared with cells, they are immunologically inert and do not require autologous sources. Vesicles can also be combined with in vitro culture modification to regulate the composition of released components, which is more conducive to the later quantitative use. SUMMARY

[0006] The present application provides the use of SKP-SC-EVs in the preparation of a product for treating Parkinson's disease, to solve the technical problem that there is no special clinical drug for Parkinson's disease.

[0007] To solve the above technical problems, one technical solution adopted by the present application is: the use of SKP-SC-EVs in the preparation of a product for treating Parkinson's disease.

[0008] Another technical solution adopted by the present application is: a product for treating Parkinson's disease, comprising SKP-SC-EVs.

[0009] Preferably, the concentration of SKP-SCs-EVs reaches 4.9×10 10 Particles / ml.

[0010] Preferably, the particle size of SKP-SCs-EVs is greater than or equal to 80nm and less than or equal to 220nm.

[0011] Preferably, the product includes drops, oral liquid, health products.

[0012] A method for verifying that SKP-SC-EVs reduce damage to SH-SY5Y cells, comprising the following steps: +

[0013] S11. Culturing SH-SY5Y cells;

[0014] S12. Adding SKP-SC-EVs solution and Rot or MPP + solution in the cell suspension containing SH-SY5Y cells in sequence;

[0015] S13. Detecting the activity of SH-SY5Y cells based on CCK8 reagent.​

[0016] A method for verifying SKP-SC-EVs on a MPTP-induced PD model, comprising the following steps:

[0017] S21. Construct a mouse PD model based on a MPTP-induced solution;

[0018] S22. Perform nasal feeding on the mouse PD model based on a SKP-SC-EVs solution;

[0019] S23. Perform Nissl staining or TH immunofluorescence staining on the mouse PD model.

[0020] The beneficial effects of the present application are: the present application provides skin precursor cell-induced Schwann cell-derived vesicles (SKP-SC-EVs), which can increase cell viability in rotenone or 1-methyl-4-phenylpyridinium ion (MPP + ) induced cell damage, in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced mouse Parkinson's disease model, the method of intranasal instillation is used to give SKP-SC-EVs, and the recovery of mouse motor ability and olfactory function is detected by behavior, which can be used for research and treatment of Parkinson's disease. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings, wherein:

[0022] Figure 1 Figures for identifying SKP and induced differentiated SKP-SC in the present application by light microscope and immunocytochemical staining; (A) SKP is cultured to the third generation, and the cells grow in spherical colonies; (b) SKP immunocytochemical staining identification figure; (C) SKP-SC culture light microscope figure; (D) SKP-SC immunocytochemical staining identification figure;

[0023] Figure 2 Figures for identifying SKP-SC-derived EVs in the present application; (A) NTA detects EVs particle size; (B) Western Blot detects EVs markers CD9, CD81 and TSG101.

[0024] Figure 3Effects of SKP-SC-EVs on the viability of damaged SH-SY5Y cells in the present application; (A) CCK-8 assay of the effects of different concentrations of SKP-SC-EVs on the viability of 50 μM rotenone (Rot) damaged SH-SY5Y cells; (B) CCK-8 assay of the effects of SKP-SC-EVs on the viability of 20 mM 1-methyl-4-phenylpyridinium ion (MPP+) damaged SH-SY5Y cells;

[0025] Figure 4 Construction of MPTP-induced mouse PD model in the present application; (A) Rotarod time of MPTP-induced mouse model; (B) olfactory function detection of MPTP-induced mouse model;

[0026] Figure 5 Effects of SKP-SC-EVs nasogastric feeding on the movement and olfactory function of MPTP-induced mouse PD model in the present application; (A) effects of SKP-SC-EVs on the rotarod time of PD model mice; (B) effects of SKP-SC-EVs on the olfactory function of PD model mice;

[0027] Figure 6 Comparison chart of SKP-SC-EVs nasogastric feeding on the loss of midbrain neurons in MPTP-induced mouse PD model in the present application, detected by Nissl staining;

[0028] Figure 7 Comparison chart of SKP-SC-EVs nasogastric feeding on the loss of brain tyrosine hydroxylase (TH) positive neurons in MPTP-induced mouse PD model in the present application, detected by TH immunofluorescence staining. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0030] The present application provides skin precursor cell-induced Schwann cell-derived vesicles (SKP-SC-EVs). Application in the preparation of products for treating Parkinson's disease.

[0031] The present application also provides a product for treating Parkinson's disease, comprising SKP-SC-EVs.

[0032] In the present application, the concentration of SKP-SC-EVs reaches 4.9×10 10Particles / ml, SKP-SCs-EVs have a particle size greater than or equal to 80 nm and less than or equal to 220 nm.

[0033] Optionally, the product includes drops, oral liquids, and health supplements. In different embodiments, products containing SKP-SC-EVs can be made into different forms such as drops, oral liquids, or health supplements, which can be selected according to the actual situation.

[0034] Example 1

[0035] A method for preparing SKP-SC-EVs includes the following steps:

[0036] Step 1. Culture and differentiation of SKP-SCs: A 1 cm × 2 cm piece of dorsal skin from a newborn SD rat was isolated and placed in dissection solution. Subcutaneous tissue was removed on ice. After rinsing twice with dissection solution, the tissue was minced and transferred to a 15 mL centrifuge tube. 2 mL of type XI collagenase (1 mg / mL) and DNase were added, and the tube was incubated at 37 ℃ in a 5% CO2 incubator. The tissue was pipetted every 15 min until it became cloudy. 10 mL of basal culture medium was added to stop the digestion. The tube was centrifuged at 4 ℃ and 1200 rpm for 5 min, the supernatant was discarded, and the tissue was resuspended in 10 mL of culture medium. The resuspended tissue was filtered through a 400-mesh sieve, centrifuged at 4 ℃ and 1200 rpm for 7 min, the supernatant was discarded, and the cells were resuspended in 1 mL of culture medium. Cells were counted at 5.0 × 10⁴ cells / mL and seeded in large dishes. SKPs were cultured in serum-free medium supplemented with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The cells exhibited spherical growth and were identified after passage to the second generation. Figure 1 As shown in A and C. SKPs were digested into single cells with 2 mL of type XI collagenase (0.5 mg / mL) and then cultured adherently. Differentiation of SKPs into SCs was induced using serum-free medium containing adenylate cyclase activator (Forskolin) and exogenous growth factor (HRG). Cell morphology changes during induction were observed. Colonies were picked and expanded for culture. Immunofluorescence staining revealed expression of SC surface markers S100β, GFAP, and p75NTR, as shown in Figure 1. Figure 1 As shown in B and D in the diagram.

[0037] Step 2. Isolation and Identification of EVs Derived from SKP-SCs: SKP-SCs were seeded in 10 cm culture dishes. When the cell density reached approximately 80-90%, the culture medium was replaced with serum-free medium and cultured for another 48 h. The supernatant was collected, centrifuged at 500 g for 5 min at 4 ℃ to remove cell debris, and then filtered through a 0.22 μm filter. Extracellular vesicles were extracted using the exoEasy Maxi Kit (QIAGEN). Figure 2As shown in Figure A, SKP-SC-EVs exhibit a typical cup-shaped morphology as identified by transmission electron microscopy. Nanoparticle tracer analysis (NTA) was used to detect particle motion, concentration, particle size, and distribution of the EVs. The results showed that the concentration of SKP-SCs-EVs reached 4.9 × 10⁻⁶. 10 Particles / ml, with particle sizes concentrated in the range of 80-220 nm, such as Figure 2 As shown in B in the figure. Western blot results show that EVs express their surface markers CD9, CD81, and TSG101, as shown in Figure B. Figure 2 As shown in C.

[0038] Example 2

[0039] This application provides an SKP-SC-EVs for Rot / MPP + The validation method for reducing damage to SH-SY5Y cells includes the following steps:

[0040] S11. Culture SH-SY5Y cells.

[0041] Specifically, SH-SY5Y cells were cultured using a medium containing DMEM, 10% FBS, 100 U / mL penicillin, and 100 U / mL streptomycin, and were maintained in an incubator at 37°C with saturated humidity containing 95% air and 5% CO2.

[0042] S12. Add SKP-SC-EVs solution and Rot or MPP sequentially to the cell suspension containing SH-SY5Y cells. + Solution.

[0043] Specifically, cell damage and SKP-SC-EVs treatment: SH-SY5Y cell suspension was treated with 4×10⁻⁶ cells / mL. 4 Cells were seeded at a density of 100 μL per well in 96-well plates. After cell attachment, the culture medium was aspirated, and the SKP-SC-EVs concentration was diluted to different concentrations (102 ppm) and added to each well. 6 10 7 10 8 10 9 Particles / mL (diluted with SH-SY5Y cell culture medium) were cultured in SKP-SC-EVs for 24 h; the culture medium was removed, and cultured in medium with a final concentration of 50 μM Rot or 20 mM MPP+ for another 24 h.

[0044] S13. Detect the viability of SH-SY5Y cells using CCK8 reagent.

[0045] Specifically, CCK8 detects cell viability: according to the kit instructions, CCK8 reaction solution is prepared in advance at a ratio of 1:9 (CCK8 reagent: culture medium); remove the culture medium, add 100 μL / well CCK8 reaction solution, wrap the culture plate with tin foil, and place it in the incubator for 1 h; use the microplate reader to measure the absorbance (OD450) at 450 nm wavelength. The results are shown in Figure 3 As shown in the results, SKP-SC-EVs can prevent Rot / MPP+ induced cell viability decline and show concentration dependence.

[0046] The present application also provides a method for verifying the SKP-SC-EVs on the MPTP induced PD model, comprising the following steps:

[0047] S21. Based on the MPTP induction solution, a mouse PD model is constructed;

[0048] S22. Based on the SKP-SC-EVs solution, the mouse PD model is nasally fed;

[0049] S23. The mouse PD model is subjected to Nissl staining or TH immunofluorescence staining.

[0050] The following is the experimental process of the present application:

[0051] Example 3 Construction of MPTP induced mouse PD model.

[0052] Construction of MPTP induced mouse PD model: 28, 8W C57BL / 6J male mice were randomly divided into 2 groups, control group (Control) and model group (MPTP), 14 in each group, and placed in the animal room for 3 days to get used to the environment. MPTP was diluted to 100 μg / μL with normal saline, and stored at -20℃ after aliquoting. When used, it was diluted with normal saline to a final concentration of 1 μg / μL. The MPTP group was injected intraperitoneally with MPTP, 20 mg / kg, once a day, for 7 consecutive days; the Control group was injected intraperitoneally with normal saline at the same time, for 7 consecutive days.

[0053] Mouse behavioral test: after the administration is completed, the rotarod and olfactory behavioral training is performed for 3 consecutive days, and the behavioral test is performed on the 4th day to compare the differences in motor and sensory functions between the Control group and the MPTP group. Rotarod test: place the mice on a rotarod instrument with an initial speed of 4 rpm and a maximum speed of 40 rpm (5 min to reach the maximum speed), record the time at which the mouse falls to the ground, each mouse is tested 5 times, and the average value is taken, as shown in Figure 4A, MPTP group mice on the time of the rotating rod significantly reduced. Olfactory experiment: in the mouse cage, 3 cm thick litter, placed in the four corners and the center of the cheese bar at 1 cm below the litter, from the corner down the mouse, start timing, record the time of mice to find cheese bar, 5 times test, the results take the average as Figure 4 B, MPTP group mice to find cheese bar time significantly increased.

[0054] Example 4: Investigation of the effect of SKP-SCs-EVs on the motor and sensory function of PD model mice.

[0055] SKP-SC-EVs nasal feeding: after the successful construction of MPTP-induced mouse PD model, Control and MPTP groups were divided into 2 groups, namely Control+Buffer group, Control+EVs group, MPTP+Buffer group, MPTP+EVs group, 7 in each group, respectively, by nasal feeding of SKP-SC-EVs or solvent (Buffer) control. Each mouse was dropped into hyaluronidase in the same side of the nostril, and after 30 min of treatment, SKP-SC-EVs (1×10 9 Particle / mouse) was slowly dropped to make it fully absorbed, and SKP-SC-EVs was given by nasal feeding every 3 days for a total of 4 times. After the last instillation, the behavior training was performed for 3 consecutive days, and the rotating rod and olfactory behavior test was performed on the 4th day, the method was the same as above, and the results were as follows: Figure 5 A and B, SKP-SC-EVs nasal feeding can increase the time of PD model mice on the rotating rod and shorten the time to find cheese bar, suggesting that SKP-SC-EVs can improve the motor and olfactory function of MPTP-induced PD mice.

[0056] Nissl staining: after the behavior experiment, the mouse was perfused, the midbrain tissue was taken out and placed in 4% paraformaldehyde for overnight fixation at 4°C; the brain tissue was placed in 10%, 20%, 30% sucrose solution for gradient dehydration, until the brain tissue sank to the bottom, the brain tissue in 30% sucrose solution was taken out and embedded with 5% sucrose, and frozen section was performed; Nissl staining solution was used for staining for 20 min, distilled water was washed for 2 times, each for 1 min, 95% ethanol was dehydrated for 2 min, and xylene was transparent for 2 times, each for 5 min; neutral gum was used for mounting, and the results were observed and photographed under a microscope as Figure 6 SKP-SC-EVs can reduce the loss of midbrain neurons in MPTP-induced mouse PD model.

[0057] TH immunofluorescence staining: 1 mL blocking solution was added on the above-mentioned frozen sections, and blocked at room temperature for 2 h; the primary antibody TH was diluted with blocking solution (1:500), and an appropriate amount of diluted primary antibody was added, and placed in a dark box (water was added in the dark box for moisturizing), and incubated at 4°C overnight; washed with PBS for 3 times, each for 5 min; the secondary antibody was diluted with blocking solution (1:500), and an appropriate amount of diluted secondary antibody was added, and placed in a dark box, and incubated at room temperature for 2 h; washed with PBS for 3 times, each for 5 min; anti-fluorescence quenching mounting agent was added, and mounted, and bubbles were avoided, and a Zeiss fluorescence microscope was used for observation and photographing, and the results were as shown in Figure 7 FIG. 6 shows that SKP-SC-EVs can prevent the reduction of TH positive neurons in a MPTP-induced mouse PD model.

[0058] The above only describes the embodiments of the present application, and does not limit the patent scope of the present application, and any equivalent structure or equivalent process transformation using the content of the specification and drawings of the present application, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. The application of SKP-SC-EVs in the preparation of drugs for treating Parkinson's disease, characterized in that, The SKP-SC-EVs are vesicles derived from Schwann cells induced by skin progenitor cells; the concentration of the SKP-SCs-EVs reaches 4.9 × 10⁻⁶. 10 Particles / ml; the particle size of the SKP-SCs-EVs is greater than or equal to 80 nm and less than or equal to 220 nm; wherein the SKP-SCs-EVs express their surface markers CD9, CD81 and TSG101.

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