Application of miR-3470b in preparation of medicine for treating Parkinson's disease

Abstract

The invention relates to the technical field of biological pharmacy, in particular to application of miR-3470b in preparation of a medicine for treating Parkinson's disease. The effect and mechanism of the miR-3470b carried by the exosome in DA cell protection are found for the first time through cell and animal experiments. By inhibiting the synthesis of the exosome and observing the information carried by the exosome, the effect of protecting DA cells is achieved; secondly, screening and verifying that the main miRNA carried in the exosome is miR-3470b through miRNA-seq, and exploring that the specific mechanism of the exosome for protecting the DA cells is that the co-cultured DA cells are protected through targeting GREM1; and finally, further verifying that the exosome miR-3470b secreted by the SIX2 overexpressed microglial cells has a protection effect on DA cells through in-vivo experiments, and can improve the motor ability of PD mice. According to the invention, a new target and thought can be provided for a strategy of treating PD by loading miRNA on the exosome.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to the application of miR-3470b in the preparation of drugs for treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease. Its main pathological features are the loss of dopamine (DA) cells and the formation of Lewy bodies in the substantia nigra compacta (SNpc) of the midbrain. Its clinical symptoms mainly include motor dysfunction characterized by resting tremor, rigidity, bradykinesia and postural balance disorders, and non-motor symptoms such as constipation, sleep disorders and depression, which bring great inconvenience to the lives of PD patients. At present, the drug treatment of PD mainly focuses on increasing dopamine in the brain, such as levodopa and dopamine receptor agonists, which cannot slow down or prevent the progressive death of DA cells. Therefore, finding new therapeutic targets and saving damaged DA cells is one of the difficulties faced in current PD research.

[0003] Exosomes (Exos) are a type of nano-scale extracellular vesicles that are widely recognized at present, with a diameter of about 30nm-150nm. Studies have found that exosomes can contain a variety of non-coding RNAs, mRNAs, proteins and lipids. They are produced, packaged and secreted outside the cells by cells, and are taken up by receptor cells to release the contents into the receptor cells. It is reported that in the central nervous system, microglia can secrete exosomes into the extracellular environment. These exosomes act on nerve cells and other glial cells, participate in the regulation of brain structure and function, and play an important role in the occurrence and development of various brain diseases. Because the special membrane structure of exosomes can protect the internal RNA from enzymatic degradation, it has been considered to be a non-coding RNA-based intercellular communication carrier, especially miRNA. MiRNA is a class of small non-coding RNAs with 17-24 nucleotides, which mediate post-transcriptional gene silencing by binding to mRNA. MiRNA can stably exist in the cell fluid and participate in intracellular activities, including cell proliferation, differentiation and migration. In recent years, the role of miRNA in exosomes has received increasing attention, and the mode of information transmission by exosomes is also considered to be the third mode of intercellular communication.

[0004] In summary, it is necessary to explore the application of miRNA in exosomes in Parkinson's disease. Summary of the invention

[0005] To solve the above problems, the present invention provides the use of miR-3470b in the preparation of a drug for treating Parkinson's disease.

[0006] The present invention is achieved through the following technical solutions:

[0007] The use of miR-3470b in the preparation of a drug for treating Parkinson's disease, wherein the nucleotide sequence of miR-3470 is shown in SEQ ID NO.1, which is tcactctgtagaccaggctgg.

[0008] Preferably, the miR-3470b is produced by exosomes of microglia overexpressing SIX2.

[0009] Preferably, the specific method for producing miR-3470b is to overexpress SIX2 by infecting BV-2 microglial cells with a lentivirus carrying the SIX2 gene, and then screen BV2 cells that stably overexpress SIX2 through antibiotics. At this time, the exosomes that can be secreted by BV2 cells are rich in miR-3470b.

[0010] Preferably, the microglial cells are mouse microglial cell line BV-2 cells.

[0011] Preferably, the drug contains miR-3470b as the only active ingredient.

[0012] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0013] Preferably, the pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

[0014] Preferably, the pharmaceutically acceptable dosage forms include tablets, capsules, granules, injections, pills, powders or pastes.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention provides the use of miR-3470b in the preparation of drugs for the treatment of Parkinson's disease. The present invention intends to discover for the first time the role and mechanism of miR-3470b carried by exosomes in protecting DA cells through cell and animal experiments. The present invention inhibits exosome synthesis and observes that the information carried by exosomes has the effect of protecting DA cells; secondly, the main miRNA carried in the exosomes is screened and verified by miRNA-seq as miR-3470b, and the specific mechanism of its protection of DA cells is to protect co-cultured DA cells by targeting GREM1; finally, in vivo experiments further verify that exosomal miR-3470b secreted by microglia overexpressing SIX2 has a protective effect on DA cells, and can improve the motor ability of PD mice. The present invention can provide new targets and ideas for the strategy of exosomes loaded with miRNA to treat PD. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is a diagram showing that the exosomes in the supernatant of SIX2-BV2 cells of the present invention protect damaged DA neurons.

[0019] Figure 1 In the figure, A is a bar graph of TH expression in DA cells after WB detection using the exosome inhibitor GW4689; B is a statistical graph of TH expression in DA cells after WB detection using the exosome inhibitor GW4689; C is a graph of apoptosis of DA cells after TUNEL detection using the exosome inhibitor GW4689; D is a graph of DA cell viability after CCK8 detection using the exosome inhibitor GW4689; E is a statistical graph of DA cell viability after CCK8 detection using the exosome inhibitor GW4689, (n=3, *P<0.05, **P<0.01, ***P<0.001).

[0020] Figure 2 Figure 2 shows the purification and identification of exosomes in the present invention.

[0021] Figure 2 In the figure, A is a transmission electron microscopy image of exosomes, Scale bar: 100 nm; B is the number and size distribution of microglial exosomes detected by NTA; C is WB detection of exosome marker proteins CD9, CD63, TSG101 and Golgi marker protein Grasp65.

[0022] Figure 3 This is a diagram showing that the exosomes derived from SIX2-BV2 of the present invention are engulfed by DA cells and protect DA cells.

[0023] Figure 3 In the figure, A is a picture of DA cells engulfing co-cultured exosomes; B is a picture of CCK8 exploring the effect of different concentrations of exosomes on DA cell viability; C is a picture of WB detecting TH expression in DA cells after the addition of exosomes; D is a statistical picture of WB detecting TH expression in DA cells after the addition of exosomes; E is a picture of TUNEL detecting DA cell apoptosis after the addition of exosomes; F is a statistical picture of TUNEL detecting DA cell apoptosis after the addition of exosomes; G is a picture of CCK8 detecting DA cell viability, (n=3, **P<0.01, ***P<0.001).

[0024] Figure 4 This is a diagram of the miRNA in the SIX2-BV2-derived exosomes protecting DA neurons of the present invention.

[0025] Figure 4 In the figure, A is qPCR verification of the knockdown effect of the exosome synthase Drosha; B is a band diagram of TH expression in DA cells after WB detection of Drosha knockdown; C is a statistical diagram of TH expression in DA cells after WB detection of Drosha knockdown; D is a TUNEL detection of DA cell apoptosis after Drosha knockdown; E is a statistical diagram of DA cell apoptosis after TUNEL detection of Drosha knockdown, (n=3, **P<0.01, ***P<0.001).

[0026] Figure 5 This is the exosome miRNA-seq and verification diagram of the present invention.

[0027] Figure 5 A is a heat map of the difference in exosome miRNA expression from BV2 in different groups; B is a volcano map of the difference in exosome miRNA expression from BV2 in different groups; C is a statistical diagram of the difference in exosome miR-3470b verified by qPCR; D is a statistical diagram of the difference in MPP verified by qPCR + Statistical graph of miR-3470b after treatment of DA cells; E is the statistical graph of miR-3470b after DA cells phagocytized exosomes, verified by qPCR (n=3, **P<0.01, ***P<0.001).

[0028] Figure 6 This is a diagram showing that miR-3470b in the exosomes of the present invention protects damaged DA cells.

[0029] Figure 6In the figure, A is a graph for qPCR verification of the knockdown effect of miR-3470b; B is a band graph for WB detection of TH expression in DA cells after knockdown of miR-3470b; C is a statistical graph for WB detection of TH expression in DA cells after knockdown of miR-3470b; D is a graph for TUNEL detection of DA cell apoptosis after knockdown of miR-3470b; E is a statistical graph for TUNEL detection of DA cell apoptosis after knockdown of miR-3470b, (n=3, **P<0.01, ***P<0.001).

[0030] Figure 7 This is a diagram showing that miR-3470b of the present invention protects damaged DA cells by inhibiting GREM1.

[0031] Figure 7 In the figure, A is the Venn diagram of miR-3470b targets predicted by Target scan and miR-Walk; B is the network diagram of miR-3470b targets; C is the statistical diagram of miR-3470b targets verified by qPCR; D is the binding site of miR-3470b and GREM1 predicted by Tragetscan; E is the statistical diagram of miR-3470b acting on GREM1 verified by dual luciferase; F is the GREM1 knockdown effect verified by qPCR.

[0032] Figure 8 This is a diagram showing that miR-3470b of the present invention protects damaged DA cells by inhibiting GREM1.

[0033] Figure 8 In the figure, A is the band diagram of WB detection of TH, GREM1 and TGF-β in DA cells; B is the result diagram of TH expression detection; C is the result diagram of GREM1 expression detection; D is the result diagram of TGF-β expression detection; E is the result diagram of TUNEL detection of DA cell apoptosis; F is the statistical diagram of TUNEL detection of DA cell apoptosis, (n=3, **P<0.01, ***P<0.001).

[0034] Fig. 9 This is a diagram of the construction of the PD mouse model and exosome injection of the present invention.

[0035] Fig. 9 In the figure, A is a diagram of PD modeling and tail vein injection; B is a band diagram of TH expression after WB detection of PD modeling; C is a statistical diagram of TH expression after WB detection of PD modeling; D is a diagram of phagocytosis of exosomes by substantia nigra DA cells in vivo, (n=3, **P<0.01).

[0036] Fig.10 The sh-GREM1 model of the mouse brain SNC region of the present invention was successfully constructed.

[0037] Fig.10In the figure, A is a schematic diagram of stereotaxic positioning of mouse brain; B is a diagram of GREM1 knockdown effect detected by WB; C is a statistical diagram of GREM1 knockdown detected by WB (n=3, **P<0.01).

[0038] Fig.11 This is a diagram showing the effects of different groups of exosomes of the present invention on the behavior of PD mice.

[0039] Fig.11 In the figure, A is the pattern diagram of mouse rotarod test; B is the statistical diagram of mouse rotarod test; C is the pattern diagram of mouse climbing pole test; D is the statistical diagram of mouse climbing pole test, (n=5, **P<0.01).

[0040] Fig.12 This figure shows the effects of different groups of exosomes of the present invention on the activation of DA cells and microglia in the SNC region of PD mice.

[0041] Fig.12 In the middle, A is immunofluorescence detection of TH in substantia nigra + and Iba-1 + Cell number chart, B is immunofluorescence detection of TH in substantia nigra + Statistical chart of cell number; C is immunofluorescence detection of Iba-1 in substantia nigra + Cell number statistics; D is the WB detection of substantia nigra TH, GREM1 and TGF-β strip diagram; E is the WB detection of substantia nigra TH statistics; F is the WB detection of GREM1 statistics; G is the WB detection of TGF-β statistics, (n = 3, *P < 0.05, **P < 0.01). DETAILED DESCRIPTION

[0042] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in the present invention. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0043] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0044] The beneficial effects of the present invention are described below by means of specific embodiments:

[0045] Example 1. Effects of exosomes on damaged DA cells

[0046] 1. Exosomes in the supernatant of BV2 cells protect damaged DA cells

[0047] Since the previous experimental results showed that BV2 cells overexpressing SIX2, referred to as SIX2-BV2 cells, have a protective effect on DA cells, and SIX2-BV2 cells are rich in exosomes, the present invention adds GW4689, a sphingomyelinase inhibitor, while culturing SIX2-BV2 cells to inhibit the synthesis of exosomes in microglia, extracts the supernatant, and co-cultures with DA cells. Compared with the normal supernatant co-culture group, WB results show that the TH expression of DA cells in the co-culture group with the addition of GW4689 supernatant is significantly reduced, such as Figure 1 As shown in A to B; TUNEL and CCK8 results showed that after adding GW4689, the apoptosis of co-cultured DA cells increased significantly, as shown in Figure 1 As shown in D to E in Figure , the cell viability decreased significantly. Figure 1 As shown in C, it shows that SIX2-BV2 cells secrete exosomes to protect DA cells.

[0048] 2. Purification and identification of exosomes

[0049] SIX2-BV2 cells were cultured in 15 cm culture dishes. After LPS treatment, the culture medium was collected and exosomes were extracted by differential centrifugation + ultracentrifugation. TEM detected that the exosomes were cup-shaped vesicles, such as Figure 2 As shown in A in the figure; the particle size of the extracted vesicles detected by NTA technology is concentrated in the range of 30 nm, as shown in Figure 2 As shown in B; WB results showed that the exosome marker proteins CD9, CD63, and TSG101 were significantly increased compared with the normal cell group, and the Golgi marker protein Grasp65 was significantly decreased compared with the normal BV2 group, as shown in Figure 2 As shown in C. The above results show that the vesicles purified by the present invention are exosomes.

[0050] 3. BV2-derived exosomes are engulfed by DA cells and protect DA cells

[0051] The purified exosomes were dyed red using the lipophilic dye PKH26 and added to the DA cell culture medium. After 24 hours, the DA cell skeleton was dyed green using phalloidin. The fluorescence results showed that the DA cells engulfed the exosomes of the BV2 cells, such as Figure 3 As shown in A. SIX2-BV2 cell exosomes were added to 1-methyl-4-phenylpyridinium, i.e., MPP, at concentrations of 0 μg / ml, 2 μg / ml, 4 μg / ml, 8 μg / ml, 10 μg / ml, 30 μg / ml, and 50 μg / ml. +In the DA neurons treated with CCK8, the results showed that when the exosome concentration reached 10 μg / ml, the activity of DA cells was close to the normal level, and the upward trend was gentle, so the concentration of 10 μg / ml was selected for subsequent experiments. Figure 3 As shown in B. After the addition of exosomes, relative to MPP + The WB results showed that the expression of TH in DA cells in the exosome treatment group was significantly increased. Figure 3 As shown in Figures C to D, TUNEL and CCK8 results showed that cell apoptosis in the exosome treatment group was significantly reduced. Figure 3 As shown in E to F in Figure , cell viability increased significantly. Figure 3 As shown in G, this indicates that the exosomes of SIX2-BV2 cells are engulfed by DA cells and protect DA cells.

[0052] Example 2: miRNA in SIX2-BV2 exosomes protects damaged DA cells

[0053] 1. miRNA in SIX2-BV2-derived exosomes protects DA cells

[0054] Exosomes are vesicles that encapsulate complex RNA and proteins. In order to verify whether the miRNA in the exosomes plays a protective role, the present invention uses a small interfering reagent of the miRNA synthetase Drosha to knock down the expression of Drosha. The qPCR results show that the expression of Drosha in BV2 cells transfected with si-Drosha-2686 is significantly reduced. Figure 4 As shown in A. The exosomes of Drosha knockdown BV2 cells were purified and added to MES23.5 cells. WB results showed that after knockdown of miRNA synthetase, the expression of TH in DA cells decreased significantly, as shown in Figure 4 As shown in B to C; TUNEL staining results show that after knocking down miRNA synthetase, DA cell apoptosis increased, as shown in Figure 4 As shown in D to E, this indicates that miRNAs in exosomes protect DA cells.

[0055] 2. Exosome miRNA-seq

[0056] In order to determine which miRNA plays a major role, normal BV2 cells and SIX2-BV2 cells were treated with LPS for 24 h, and the exosomes of the two groups of cells were purified for miRNA-seq. The results showed that compared with the NC group, the expression of miR-3470b and miR-1843b in the exosomes of the SIX2 overexpression group was significantly increased, such as Figure 5As shown in A and B in Figure 2, after prediction of target genes, the present invention found that only the target gene of miR-3470b was related to the survival of DA cells, so the subsequent experiments chose to treat miR-3470b. The qPCR results showed that the expression of miR-3470b in the exosomes of the SIX2 overexpression group was significantly increased, as shown in Figure 2. Figure 5 As shown in C, compared with normal DA cells, MPP + There was no significant change in miR-3470b in DA cells in the treatment group. Figure 5 As shown in D, after the intake of SIX2-BV2 exosomes, the expression of miR-3470b in DA cells increased significantly, as shown in Figure 5 As shown in E, this indicates that the change in miR-3470b expression in DA cells is due to the uptake of exogenous miR-3470b.

[0057] Example 3: miR-3470b in exosomes protects damaged DA cells

[0058] In order to determine whether miR-3470b in exosomes protects DA cells, the present invention uses sh-miR-3470b lentivirus to transfect SIX2-BV2 cells, and qPCR results show that miR-3470b is significantly knocked down, such as Figure 6 As shown in A. After 24 h of LPS treatment, the exosomes were purified and added to the DA cell culture medium. WB results showed that the protective effect of the exosomes with knockdown of miR-3470b was significantly weakened, and the expression of TH in DA cells was significantly reduced, as shown in Figure 6 As shown in B to C in Figure 1, TUNE results showed that the apoptosis of DA cells in the miR-3470b knockdown exosome group increased. Figure 6 As shown in D to E. This indicates that miR-3470b in the exosomes of BV2 cells protects DA cells.

[0059] Example 4: miR-3470b protects damaged DA cells by inhibiting GREM1

[0060] After MES23.5 cells ingested exosomes from BV2 cells overexpressing SIX2, the expression of miR-3470b increased. It is unclear which gene miR-3470b binds to to exert its effect. Therefore, the present invention used miR-Walk and Targetscan to jointly predict the target genes of miR-3470b. The results showed that there were six common target genes, such as Figure 7 As shown in A and B in Figure 4. qPCR verification showed that the expression of FXYD5, GREM1 and SPCS3 genes was significantly decreased, as shown in Figure 7As shown in C in the figure, only GREM1 among these three target genes is related to cell survival, so the present invention selects GREM1 for subsequent treatment. The results of dual luciferase analysis show that after the binding site mutation, the fluorescence intensity decreases, indicating that GREM1 is the target gene of miR-3470b. Figure 7 As shown in D and E in Figure 2. MES23.5 cells were transfected with sh-GREM1 lentivirus, and qPCR results showed that the expression of GREM1 in the sh-GREM1-7 transfection group was significantly decreased, as shown in Figure 2. Figure 7 As shown in F. Compared with the exosome group with miR-3470b knockdown, WB results showed that when miR-3470b knockdown BV2 exosomes were added and GREM1 was knocked down in MES23.5 cells, the expression of TH in MES23.5 cells was significantly increased, and the expression of TGF-β was significantly increased, as shown in Figure 8 As shown in A to D; TUNEL results showed that the apoptosis of MES23.5 cells was significantly reduced. Figure 8 As shown in Figures E to F. The above results indicate that exosome-encapsulated miR-3470b inhibits GREM1 expression and promotes TGF-β expression in MES23.5 cells, thereby protecting DA cells.

[0061] Example 5: Verification of the effects of SIX2-BV2 exosomes on the behavior and DA cells of PD mice

[0062] 1. Construction of PD mouse model and exosome injection

[0063] To construct the PD mouse model, mice were intraperitoneally injected with 30 mg / kg of MPTP for five consecutive days, and the control group was treated with the same volume of saline. Fig. 9 As shown in A. Five days after injection, WB was used to observe the expression of TH in the substantia nigra of the midbrain. The results showed that compared with the Sham group, the expression of TH in the substantia nigra of the midbrain of the MPTP group mice was significantly reduced, as shown in Fig. 9 B and C in Figure 1. The PD mouse model was successfully established. The purified exosomes were dyed red using PKH-26 and injected into the tail vein of the mouse. Fig. 9 As shown in A, brain slices were taken three days later for observation. Fluorescence results showed that DA cells in the substantia nigra engulfed the exosomes injected through the tail vein. Fig. 9 As shown in D.

[0064] 2. The sh-GREM1 model in the SNC region of the mouse brain was successfully constructed

[0065] In order to reduce the expression of GREM1 in the substantia nigra of mice, 1 μL of GREM1 knockdown virus was injected into the substantia nigra of mice, and MPTP was intraperitoneally injected seven days later as well as SIX2-exo and miR-3470b. KD-exo tail vein injection model, behavioral testing was performed 12 days later, and brain tissue was taken for WB and immunofluorescence 17 days later. Fig.10 As shown in A. After the midbrain of mice was collected, WB results showed that the expression of GREM1 in the sh-GREM1 group was significantly reduced compared with that in the sham group. Fig.10 As shown in B to C.

[0066] 3. Effects of exosomes from different groups on the behavior of PD mice

[0067] In order to clarify the protective effect and mechanism of miR-3470b in SIX2-BV2 cell exosomes on DA cells of PD mice, different groups of exosomes were injected at the same time as MPTP modeling. After the modeling was completed, the rotating rod test and climbing pole test were performed to observe the changes in the motor ability of mice. The rotating rod test showed that compared with the sham group, the falling latency of mice in the MPTP group and the miR-3470b exosome knockdown group was significantly reduced. The falling latency of mice in the exosome group and the miR-3470b knockdown and GREM1 knockdown group was significantly increased compared with the MPTP group, as shown in Figure 2. Fig.11 As shown in A and B in Figure 2. The results of the pole climbing experiment showed that compared with the sham group, the time taken by mice in the MPTP group and the miR-3470b knockdown exosome group to climb down from the top was significantly increased. The time taken by mice in the exosome group and the miR-3470b knockdown and GREM1 knockdown groups to climb down from the top was significantly reduced compared with the MPTP group. Fig.11 As shown in C and D.

[0068] 4. Effects of exosomes from different groups on the activation of DA cells and microglia in the SNC region of PD mice

[0069] After the model was successfully established, immunofluorescence and immunoblotting were used to observe the changes in the number of TH and microglia in the midbrain. The immunofluorescence results showed that compared with the sham group, the TH in the substantia nigra of the midbrain in the MPTP group and the miR-3470b knockdown exosome group was significantly higher than that in the sham group. + The number of cells decreased significantly, and Iba-1 + Compared with the MPTP group, the number of TH cells in the exosome group and the miR-3470b knockdown and GREM1 knockdown groups was significantly higher than that in the MPTP group. + The number of cells increased, Iba-1 + Decreased cell number, such as Fig.12As shown in A, B and C in Figure 2. WB results showed that compared with the sham group, the expression of TH and TGF-β proteins in the midbrain of the MPTP group and the miR-3470b knockdown exosome group was significantly decreased, and the expression of GREM1 protein was significantly increased. Compared with the MPTP group, the expression of TH and TGF-β proteins in the midbrain of the mice in the exosome group and the miR-3470b knockdown and GREM1 knockdown groups was significantly increased, and the expression of GREM1 protein was significantly decreased. Fig.12 As shown in D, E, F and G in FIG.

[0070] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0071] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. The use of miR-3470b in the preparation of a drug for treating Parkinson's disease, characterized in that: The nucleotide sequence of miR-3470b is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The miR-3470b was produced by exosomes of microglia overexpressing SIX2.

3. The use according to claim 2, characterized in that: The specific method for producing miR-3470b is to overexpress SIX2 by infecting BV-2 microglial cells with a lentivirus carrying the SIX2 gene, and then screen BV2 cells that stably overexpress SIX2 through antibiotics. At this time, the exosomes that can be secreted by BV2 cells are rich in miR-3470b.

4. The use according to claim 2, characterized in that: The microglial cells are mouse microglial cell line BV-2 cells.

5. The use according to claim 1, characterized in that: The drug contains miR-3470b as the only active ingredient.

6. The use according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that: The pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

8. The use according to claim 1, characterized in that: The pharmaceutically acceptable dosage forms include tablets, capsules, granules, injections, pills, powders or pastes.

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