Application of miR-221-3p-containing adipose-derived stem cell exosome in products for treating / relieving stroke

Abstract

The invention relates to an application of an adipose-derived stem cell exosome containing miR-221-3p in a product for treating / relieving stroke, and belongs to the technical field of biology. According to the present invention, the adipose-derived stem cell exosome is subjected to gene modification, that is, the exosome is extracted after the miR-221-3p is overexpressed on the adipose-derived stem cells, such that the gene-modified miR-221-enriched adipose-derived stem cell exosome (ADSC-EVsmiR-221) is innovatively constructed, such that the adipose-derived stem cell exosome (ADSC-EVsmiR-221) has strong immune regulation ability, and can provide good treatment effect on stroke.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of adipose stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke. Technical Background

[0002] Stroke, commonly known as stroke, is a neurological disease with high mortality and disability rates. It is the third leading cause of death and disability worldwide. Its main treatment goal is to restore blood flow as soon as possible after the onset of symptoms. The current clinical method for restoring patency of stroke blood vessels is mainly intravenous thrombolysis, but the effective window time of this treatment method is very narrow. A high proportion of patients do not recover their neurological function after treatment. Survivors have sequelae of varying degrees, which seriously affect their quality of life. Therefore, finding an effective treatment method to reduce the mortality and disability rate after stroke and improve the sequelae of stroke is of great significance to improving the quality of life of patients and reducing the social burden. In recent years, studies have found that exosomes have become a potential diagnosis and treatment method for stroke because of their neuroprotective and neurorepair effects. In addition, exosomes can cross the blood-brain barrier and circulate in peripheral blood and cerebrospinal fluid, and can be used as excellent non-invasive biomarkers for the diagnosis and prognosis of ischemic stroke. Summary of the invention

[0003] Based on this, the present application provides a method of enriching miR-221-3p in adipose stem cell exosomes, which can inhibit the polarization of microglia M1, reduce neuronal damage after stroke, reduce the area of ​​stroke infarction, reduce neurological damage after stroke, promote the recovery of limb coordination and motor function after stroke, and improve stroke sequelae.

[0004] A use of adipose stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke, wherein the exosomes are extracted from adipose stem cells after overexpressing miR-221-3p.

[0005] In one embodiment, the method for preparing the exosomes is as follows:

[0006] Step 1, constructing and culturing adipose stem cells overexpressing miR-221-3P;

[0007] Step 2: Extract exosomes by ultracentrifugation.

[0008] In one embodiment, step 1 is specifically: using RNA-fit transfection reagent to transfect miR-221-3p mimic on P5 generation adipose stem cells to construct adipose stem cells overexpressing miR-221-3P, replacing the adipose stem cell culture medium 24 hours after transfection with a culture medium prepared with exosome-free serum, and collecting the adipose stem cell supernatant after continuing to culture for 24 hours.

[0009] In one embodiment, the step 2 is specifically as follows: the adipose stem cell supernatant obtained in step 1 is first centrifuged at a low speed of 500g for 10 minutes to remove cell debris and large particles, then centrifuged at a low speed of 2000g for 30 minutes to remove a large amount of cell debris and organelles to obtain a crude exosome precipitate, and then centrifuged at a high speed of 10000g for 60 minutes to remove small fragments and membrane pieces to obtain a medium exosome precipitate; finally, ultracentrifuged at 121000g for 60 minutes to obtain a final exosome precipitate, which is resuspended in sterile PBS to obtain adipose stem cell exosomes containing miR-221-3p.

[0010] In one embodiment, the exosomes can inhibit microglial M1 polarization and improve neurological function damage after stroke in vivo.

[0011] In one embodiment, the product comprises food, medicine or health care product.

[0012] In one embodiment, the drug comprises adipose stem cell exosomes containing miR-221-3p, a drug carrier, and / or a pharmaceutical excipient.

[0013] In one embodiment, the drug carrier includes microcapsules, microspheres or nanoparticles; and the pharmaceutical excipients include excipients and additives.

[0014] In one embodiment, the pharmaceutical excipients include anti-adhesive agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculating agents, filter aids and release retardants.

[0015] In one embodiment, the dosage form of the drug comprises granules, capsules, tablets, pills or oral liquids.

[0016] Beneficial effects:

[0017] This method can be used to genetically modify adipose stem cell exosomes, that is, to extract exosomes after overexpressing miR-221 in adipose stem cells, and to innovatively construct "genetically modified" adipose stem cell exosomes (ADSC-EVs miR-221 ), which makes it have stronger immune regulation ability and can play a better therapeutic effect on stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1:Transcriptome sequencing confirmed that miR-221-3p was highly expressed in adipose stem cell exosomes.

[0019] Figure 2 :In vitro experiments confirmed that miR-221-3p inhibited the polarization of M1 microglia and the expression of inflammatory genes; (A is the expression of INOS gene; B is the expression of IL1β gene; C is the expression of TNFɑ gene; D is the expression of IL6 gene).

[0020] Figure 3 : In vivo experiments confirmed that nasal drops of agomiR miR-221-3p improved the neurological function scores of stroke mice (A: modified neurological function score (mNSS scale); B: balance beam test results).

[0021] Figure 4 : Adipose stem cell exosomes with overexpression of miR-221 better inhibit the polarization of microglia (A: expression of miR-221 in adipose stem cell exosomes; B: expression of microglia marker CD86; C: expression of inflammatory factor IL6). DETAILED DESCRIPTION

[0022] It is worth noting that the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0023] Example 1: Preparation method of adipose stem cell exosomes enriched with miRNA

[0024] The present invention is based on the screening of transcriptome sequencing of adipose stem cell exosomes to screen for miRNA-221-3p molecules with high expression abundance in adipose stem cell exosomes. In addition, miR-221-3p has been reported as an anti-inflammatory factor. Therefore, the key molecule miR-221-3p ( Figure 1 Based on this, miR-221-3p was enriched in exosomes of adipose stem cells. The specific method was to use RNA-fit transfection reagent to transfect miR-221-3p mimic on P5 adipose stem cells to construct adipose stem cells that overexpress miR-221-3P. 24 hours after transfection, the adipose stem cell culture medium was replaced with a medium prepared with exosome-free serum, and the adipose stem cell supernatant was collected after continuing to culture for 24 hours.

[0025] The supernatant of the adipose stem cells collected above was further subjected to ultracentrifugation to collect exosomes. First, low-speed centrifugation at 500 g was used for 10 minutes to remove cell debris and large particles. Then, low-speed centrifugation at 2000 g was used for 30 minutes to remove a large amount of cell debris and organelles to obtain a crude exosome precipitate. High-speed centrifugation at 10000 g was then used for 60 minutes to remove small fragments and membranes to obtain a medium exosome precipitate. Finally, ultracentrifugation at 121000 g was used for 60 minutes to obtain a final exosome precipitate, which was resuspended in sterile PBS to obtain a usable adipose stem cell exosome precipitate enriched in miR-221-3p.

[0026] Example 2: In vitro experiments

[0027] LPS was first used to induce M1 polarization of BV2 microglia. Six hours after LPS treatment, RNA-fit transfection reagent was used to overexpress miR-221-3p in the BV2 microglia cell line, so that BV2 microglia overexpressed miR-221-3p after polarization. RNA samples of BV2 microglia were extracted 24 hours later, and QPCR experiments were subsequently performed to detect the effect of miR-221-3p overexpression on the expression of microglial M1 polarization markers and inflammatory factors (including INOS, IL1β, TNFɑ and IL6).

[0028] like Figure 2 As shown in the figure (NC: untreated BV2 cells transfected with negative control fragments; M1+NC: BV2 microglia (M1-state BV2 microglia) cells transfected with negative control fragments after 6 hours of LPS treatment; miR-221mimic: untreated BV2 microglia transfected with miR-221mimic fragments; BV2 microglia (M1-state BV2 microglia) cells transfected with miR-221mimic fragments after 6 hours of LPS treatment), miR-221-3p was verified in the BV2 microglia polarization model to inhibit the M1 polarization of microglia. MiR-221-3p inhibited LPS-induced microglia M1 polarization by reducing the expression levels of inflammatory-related genes (INOS, IL1β, TNFɑ and IL6) in BV2 microglia, and finally inhibited the inflammatory response of microglia.

[0029] Example 3: In vivo experiments

[0030] To verify the effect of miR-221-3p on the neuromotor function of stroke mice, we first constructed a miR-221-3p agomiR that can be stably expressed in vivo, and used suture embolization surgery to construct a stroke model in C57BL6 mice. We then administered miR-221-3p agomiR via intranasal drops on D1, D3, and D5 after modeling to construct a miR-221-3p overexpression model in the mouse brain. At D7, the modified neurological score (mNSS) scale was used to evaluate the effect of miR-221-3p overexpression on neurological function damage in stroke mice, and the balance beam experiment was used to evaluate its effect on motor function damage in stroke mice.

[0031] like Figure 3 As shown (SHAM: sham operation control group; MCAO: stroke model group undergoing MCAO surgery; MCAO+agomirNC: mice nasally dripped agomir negative control fragment one hour after stroke modeling; MCAO+agomir 221: mice nasally dripped agomir-221 overexpression fragment one hour after stroke modeling, causing overexpression of miR-221 in the brains of stroke mice), verified on a stroke animal model (MCAO surgery), the results showed that: the mNSS neurological function score and balance beam test confirmed that nasal drip of miR-221-3p agomir can improve the neurological and motor function damage of stroke mice.

[0032] Example 4: MiR-221 overexpressed adipose stem cell exosomes further inhibit microglial polarization and inflammatory factor expression

[0033] According to the method of Example 1, adipose stem cell exosomes (ADSC-EVs) with enhanced function of miR-221 overexpression were constructed. miR-221mimic ), and the negative control fragment was transfected into adipose-derived stem cells using the same method, and exosomes were extracted using the same method as the control group (ADSC-EVs NC ).

[0034] like Figure 4 As shown (Control: normal BV2 microglia without any treatment; M1: LPS-treated BV2 microglia induced them to become M1 polarized state; M1+ADSC-EVs: ADSC-EVs exosomes of adipose stem cells were given to the control group after LPS induction for 1 hour NC Treatment of BV2 microglia; M1+ADSC-EVs miR-221mimic : One hour after LPS induction, ADSC-EVs with miR-221 overexpression were administered at the same concentration as that of the control group. miR-221mimicTreatment of BV2 microglia), miR-221 overexpressed fat stem cell exosomes better inhibited the polarization of microglia. QPCR was used to detect the concentration of exosomes in the two groups of fat stem cells. The expression of miR-221 in the fat stem cell exosomes obtained by the method of the present invention increased by nearly 10 times compared with the control group. The results are as follows Figure 4 As shown in A. The exosomes obtained were used to treat polarized microglia. Compared with the single adipose stem cell exosome treatment group, the QPCR results showed that the microglial exosomes with miR-221 overexpression better inhibited the expression of microglial M1 polarization marker CD86 than the normal exosomes; QPCR results showed that the microglial exosomes with miR-221 overexpression better inhibited the expression of microglial M1 polarization marker and inflammatory factor IL6 than the normal exosomes. The adipose stem cell exosomes with miR-221 overexpression constructed by us had a more obvious therapeutic effect. The results are shown in Figure 4 As shown in B and C.

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Application of adipose stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke, characterized in that: The exosomes are extracted from adipose stem cells after overexpressing miR-221-3p.

2. The use of adipose stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke according to claim 1, characterized in that: The preparation method of the exosomes is as follows: Step 1, constructing and culturing adipose stem cells overexpressing miR-221-3P; Step 2: Extract exosomes by ultracentrifugation.

3. The use of adipose stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke according to claim 2, characterized in that: The step 1 specifically includes: using RNA-fit transfection reagent to transfect miR-221-3p mimic on P5 adipose-derived stem cells to construct adipose-derived stem cells that overexpress miR-221-3P, replacing the adipose-derived stem cell culture medium 24 hours after transfection with a culture medium prepared with exosome-free serum, and collecting the adipose-derived stem cell supernatant after continuing to culture for 24 hours.

4. The use of adipose stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke according to claim 2, characterized in that: The step 2 is specifically as follows: the adipose stem cell supernatant obtained in step 1 is first centrifuged at a low speed of 500g for 10 minutes to remove cell debris and large particles, then centrifuged at a low speed of 2000g for 30 minutes to remove a large amount of cell debris and organelles to obtain a crude exosome precipitate, and then centrifuged at a high speed of 10000g for 60 minutes to remove small fragments and membrane pieces to obtain a medium exosome precipitate; finally, ultracentrifuged at 121000g for 60 minutes to obtain a final exosome precipitate, which is resuspended in sterile PBS to obtain adipose stem cell exosomes containing miR-221-3p.

5. The use of adipose stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke according to claim 1, characterized in that: The exosomes can inhibit microglial M1 polarization and improve neurological function damage after stroke in vivo.

6. Use of adipose stem cell exosomes containing miR-221-3p according to claim 1 in a product for treating / alleviating stroke, wherein the product comprises food, medicine or health care product.

7. The use of adipose-derived stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke according to claim 6, wherein the drug comprises adipose-derived stem cell exosomes containing miR-221-3p, a drug carrier, and / or a pharmaceutical excipient.

8. The use of adipose stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke according to claim 7, characterized in that: The drug carrier includes microcapsules, microspheres or nanoparticles; the pharmaceutical excipients include excipients and additives.

9. The use of adipose stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke according to claim 7, characterized in that: The pharmaceutical excipients include anti-adhesive agents, penetration enhancers, buffers, plasticizers, surfactants, defoamers, thickeners, inclusion agents, absorbents, humectants, solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, pH regulators, adhesives, disintegrants, fillers, lubricants, wetting agents, integrators, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, foaming agents, suspending agents, coating materials, fragrances, diluents, flocculants and deflocculating agents, filter aids and release retardants.

10. The use of adipose stem cell exosomes containing miR-221-3p in a product for treating / alleviating stroke according to claim 6, characterized in that: The dosage form of the drug includes granules, capsules, tablets, pills or oral liquids.

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