CN116407552A - Application of miR-17-5p in the preparation of products for preventing or treating pulmonary fibrosis - Google Patents

Abstract

The invention relates to an application of an embryonic stem cell exosome in preparing a product for preventing or treating pulmonary fibrosis. The research of the application shows that miR-17-5p can reduce the expression of the fibrosis factor by inhibiting the expression of Thbs2, so that the lung fibrosis and lung inflammation can be effectively relieved.

Description

Application of miR-17-5p in the preparation of products for preventing or treating pulmonary fibrosis

technical field

The invention relates to the field of biotechnology, in particular to an application of miR-17-5p in the preparation of products for preventing or treating pulmonary fibrosis.

Background technique

Interstitial pneumonia is a disease of pulmonary interstitial inflammation and fibrosis caused by a variety of reasons, mainly invading the pulmonary interstitium and alveolar space, including alveolar epithelial cells, capillary endothelial cells, basement membrane, blood vessels and lymph nodes, and periductal tissue eventually leads to pulmonary interstitial fibrosis and loss of alveolar capillary function.

According to different etiologies, interstitial pneumonia is mainly divided into interstitial pneumonia of known cause and idiopathic interstitial pneumonia. Interstitial pneumonia of known cause includes hypersensitivity pneumonitis, drug-induced interstitial pneumonia, connective tissue disease-associated interstitial pneumonia, pneumoconiosis, etc.; idiopathic interstitial pneumonia has 6 subtypes based on tissue type Including usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia, bronchiolitis obliterans with organizing pneumonia, respiratory bronchiolitis with interstitial non-disease (ILD), desquamative interstitial pneumonia and acute interstitial pneumonia.

The main clinical manifestation of interstitial pneumonia is dyspnea, followed by cough, and some patients may be accompanied by systemic symptoms such as fever and fatigue. Disease progression in interstitial pneumonia causes pulmonary fibrosis. In the early stage of lung tissue injury, the recruitment of inflammatory cells occurs, leading to the release of chemokines and pro-inflammatory cytokines, followed by massive recruitment of inflammatory cells, tissue remodeling, and accumulation of collagen fibers in the lung. Subsequently, healthy tissue is replaced by an altered extracellular matrix, and alveolar architecture is disrupted, resulting in reduced lung compliance and function, disrupted gas exchange, and ultimately respiratory failure and death.

The current drugs for the treatment of interstitial pneumonia and pulmonary fibrosis mainly include staying away from predisposing factors, anti-inflammatory therapy, anti-fibrosis therapy, and prevention and treatment of hypoxemia. Anti-inflammatory drugs are mainly glucocorticoids, which have serious side effects. Finding new drugs for the treatment of interstitial pneumonia and pulmonary fibrosis is a difficult and important task to be solved urgently.

Contents of the invention

The research of this application found that miR-17-5p can reduce the expression of fibrosis factors by inhibiting the expression of Thbs2, so that it can effectively alleviate the occurrence of pulmonary fibrosis, and has the effect of preventing and treating pulmonary fibrosis and lung inflammation. effect, and miR-17-5p is a small molecule RNA with high safety and few side effects.

Therefore, the present application provides an application of miR-17-5p in the preparation of products for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation. In addition, a medicament capable of preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation is also provided.

In one of the embodiments, the disease related to pulmonary fibrosis is interstitial pneumonia.

In one embodiment, the disease related to pulmonary fibrosis is idiopathic interstitial pneumonia, common interstitial pneumonia or idiopathic nonspecific interstitial pneumonia.

In one of the embodiments, the miR-17-5p comes from stem cell exosomes.

In one of the embodiments, the miR-17-5p is derived from human embryonic stem cell exosomes.

In one embodiment, the product includes an active ingredient, and the active ingredient includes one or both of miR-17-5p and miR-17-5p mimics.

In one of the embodiments, the nucleotide sequence of the miR-17-5p mimic is shown in SEQ ID NO: 3-4.

In one of the embodiments, the product further includes a carrier for delivering miR-17-5p and/or miR-17-5p mimics into the body.

In one of the embodiments, the method of delivering miR-17-5p and/or miR-17-5p mimics by the carrier includes one or more of the following: delivery of transfection reagents, delivery of liposome nanoparticles, coupling co-delivery, multimeric nanoparticle delivery, exosome delivery and nanozyme delivery. .

In one of the embodiments, the dosage form of the product is tablet, capsule, granule, pill, injection or sustained release preparation.

A medicament comprising an active ingredient comprising miR-17-5p and/or a miR-17-5p mimic.

Description of drawings

Fig. 1 is the result of the influence of BLM of different concentrations on the expression of TGFβ1 of Beas-2b cell and cell activity in embodiment 1;

Fig. 2 is the result of the Fn mRNA level of NC mimic group, NC inhibitor group, miR-17-5p mimic group and miR-17-5p inhibitor group before and after BLM treatment in embodiment 2;

Fig. 3 is the result of the mRNA level of type I collagen before and after BLM treatment of NC mimic group, NC inhibitor group, miR-17-5p mimic group and miR-17-5p inhibitor group in embodiment 2;

Fig. 4 is the result of Western blot in embodiment 2;

Fig. 5 is the result of the expression level of Thbs2 in embodiment 3;

6 is the principle of whether the mutation in Example 4 can be combined with the wild-type Thbs2 gene and the activity result of luciferase;

Fig. 7 is the schematic diagram of the dosing regimen and the results of histological section of the 8th day execution in embodiment 6;

Fig. 8 is a schematic diagram of the dosing regimen and the results of tissue sections of the 21st day sacrificed in Example 6.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully below, and the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that the present disclosure will be thorough and complete.

The term "and/or" includes any and all combinations of one or more of the associated listed items.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field of the invention. The terms used herein in the description of the present invention are for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

It should be noted that the "active ingredient" herein refers to any ingredient that provides pharmacological activity or other direct effects or affects the structure or any function of the body of humans and other animals in the diagnosis, cure, mitigation, treatment, or prevention of diseases. The "adjuvant" herein includes, but is not limited to, pharmaceutically acceptable adjuvants. A pharmaceutically acceptable excipient is one that is compatible with other ingredients of a pharmaceutical preparation and is suitable for contact with tissues or organs of a recipient (such as a human or animal). There is no or minimal complication of toxicity, irritation, allergic reaction, immunogenicity or other problems during use.

According to the study of the present application, miR-17-5p reduces the expression of fibrosis-related factors by down-regulating the high expression of Thbs2 induced by bleomycin, thereby achieving the effect of anti-fibrosis and reducing inflammation.

Bleomycin (BLM), also known as bleomycin, is a cytotoxic glycoprotein isolated from Streptomyces verticillium. It is clinically used as a broad-spectrum antitumor/antibacterial drug to treat various cancers. BLM can cause DNA strand breaks in tumor cells and induce apoptosis. However, due to the lack of BLM hydrolase in the lungs, when BLM is taken in excess of the therapeutic dose, it will cause pulmonary fibrosis, so it is often used in the construction of animal models of pulmonary fibrosis . BLM has a wide range of applications, and can induce pulmonary fibrosis in rodents such as mice, rats or rabbits, dogs and various primates. BLM can be administered in a variety of ways, including intraperitoneal injection, tail vein injection, tracheal administration, nasal instillation, and aerosol inhalation, and both single administration and repeated administration can well induce pulmonary fibrosis in animals . At present, the most commonly used route of administration is tracheal administration. The pulmonary fibrosis induced by tracheal administration can better simulate the pathological changes of human pulmonary fibrosis, and has the advantages of short modeling time and small dosage.

Based on the above, one embodiment of the present application provides an application of miR-17-5p in the preparation of products for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation.

In some embodiments, the above-mentioned products for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation (referred to as "products") comprising miR-17-5p can prevent or treat diseases related to pulmonary fibrosis . In other embodiments, the above product comprising miR-17-5p can prevent or treat diseases related to lung inflammation. In other embodiments, the above products comprising miR-17-5p are capable of preventing or treating diseases associated with both pulmonary fibrosis and pulmonary inflammation. Further, the disease associated with pulmonary fibrosis and pulmonary inflammation is interstitial pneumonia. Furthermore, the diseases related to pulmonary fibrosis and pulmonary inflammation are idiopathic interstitial pneumonia, common interstitial pneumonia or idiopathic nonspecific interstitial pneumonia. Specifically, idiopathic interstitial pneumonia is divided into 6 subtypes based on tissue type, including usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia, bronchiolitis obliterans with organizing pneumonia, respiratory Bronchitis with interstitial non-disease (ILD), desquamative interstitial pneumonia, and acute interstitial pneumonia.

In some embodiments, miR-17-5p is from stem cell exosomes. Further, miR-17-5p comes from embryonic stem cell exosomes. In an optional specific example, miR-17-5p is derived from human embryonic stem cell exosomes. It can be understood that, in other embodiments, the source of miR-17-5p is not limited to the above, and can also be in other ways, for example, by artificially synthesizing the nucleotide sequence of miR-17-5p.

In some embodiments, the above-mentioned products for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation (referred to as "products") are medicines or foods.

In some embodiments, the above-mentioned product is a drug, and the product includes an active ingredient, and the active ingredient includes one or both of miR-17-5p and miR-17-5p mimics.

microRNA mimic (miRNA mimic) is a chemically synthesized miRNA mimic that can mimic the high-level expression of mature miRNA in cells to enhance the regulation of endogenous miRNA and conduct gain-of-function research. miRNA mimic is a simple and efficient tool for miRNA research. It can be transfected into cells just by wrapping it with a transfection reagent, without the cumbersome operation of constructing a vector, and its transfection efficiency can be observed with a transfection control. miR-17-5p mimic is a microRNA mimic designed for miR-17-5p. In some embodiments, the double-stranded RNA nucleic acid molecules designed by Dharmocon* and chemically modified are endogenous mature miRNA functional activity enhancers and highly efficient mimics; * chemical modification ensures that the antisense strand of miRNA is preferred Binds to a RISC-like complex while preventing off-target reactions involving the sense strand.

In some embodiments, the nucleotide sequence of the miR-17-5p mimic is shown in SEQ ID NO: 3-4. It can be understood that, in other embodiments, the nucleotide sequence of the miR-17-5p mimic is not limited to the above, and can also be other nucleotide sequences designed according to miR-17-5p.

In some embodiments, the above-mentioned product is a medicament, which, in addition to the active ingredient, also includes a carrier for delivering miR-17-5p and/or miR-17-5p mimics into the body. Optionally, the carrier delivery method for miR-17-5p and/or miR-17-5p mimics into the body includes one of the following or Various: transfection reagent delivery, liposomal nanoparticle delivery, conjugation delivery, multimeric nanoparticle delivery, exosome delivery and nanozyme delivery.

It can be understood that the carrier used to deliver miR-17-5p and/or miR-17-5p mimics into the body is not limited to the above, and other vectors capable of delivering miR-17-5p and/or miR-17-5p Mimetic A substance that is delivered to and released in the body.

It can be understood that when the above-mentioned product is a medicine, the dosage form is not particularly limited. Optionally, the dosage forms of the above-mentioned products are tablets, capsules, granules, pills, injections or sustained-release preparations.

In some embodiments, the dosage form of the product is a tablet. In order to make the above products into tablets, various excipients known in the art can be used as auxiliary materials. Optionally, the auxiliary materials include at least one of diluents, binders, wetting agents, disintegrants and lubricants. Further, the diluent is selected from at least one of starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate and calcium carbonate. The binder is selected from starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia mucilage, gelatin paste, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, At least one of ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone and polyethylene glycol. The disintegrating agent is selected from starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, polyoxyethylene, sorbitol, fat At least one of acid esters and sodium dodecylsulfonate. The lubricant is selected from at least one of talcum powder, silicon dioxide, stearate, tartaric acid, liquid paraffin and polyethylene glycol. The wetting agent is selected from at least one of water, ethanol and isopropanol. Of course, the tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

In some embodiments, the above-mentioned product is an injection. In order to prepare the above products into injections, known injection excipients can be added. Optionally, the excipients for injection include at least one of solubilizers, pH regulators and osmotic pressure regulators. Further, the solubilizer is at least one selected from ethanol, isopropanol, propylene glycol, polyethylene glycol, poloxamer, lecithin and hydroxypropyl-β-cyclodextrin. The pH regulator is at least one selected from citrate, phosphate, carbonate, acetate, hydrochloric acid and hydroxide. The osmotic pressure regulator is at least one selected from sodium chloride, mannitol, glucose, phosphate, citrate and acetate.

In addition, an embodiment of the present application also provides a medicament, which includes an active ingredient, and the active ingredient includes miR-17-5p and/or miR-17-5p mimics.

In addition, an embodiment of the present application also provides a method for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation, the method adopts miR-17-5p and/or miR-17-5p mimics to achieve Prevention or treatment of pulmonary fibrosis and/or inflammation of the lungs.

More specifically, one embodiment of the present application provides a method for preventing or treating diseases related to pulmonary fibrosis, which uses miR-17-5p and/or miR-17-5p mimics to prevent pulmonary fibrosis or treatment.

In addition, an embodiment of the present application provides a method for preventing or treating diseases related to pulmonary fibrosis and pulmonary inflammation, the method uses miR-17-5p and/or miR-17-5p mimics to achieve pulmonary fibrosis and prevention or treatment of lung inflammation.

In addition, an embodiment of the present application also provides a method for preventing or treating diseases related to lung inflammation, which uses miR-17-5p and/or miR-17-5p mimics to prevent or treat lung inflammation treat.

Further, the above-mentioned methods for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation are aimed at interstitial pneumonia. Furthermore, the above-mentioned methods for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation are aimed at idiopathic interstitial pneumonia, common interstitial pneumonia or idiopathic non-specific interstitial pneumonia.

In an optional specific example, the above-mentioned methods for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation can be used for COVID-19 patients. Pneumonia caused by novel coronavirus infection (Coronavirus disease2019, COVID-19) is pneumonia caused by severe acute respiratory syndrome coronavirus 2 (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) infection. During the course of COVID-19 lesions, the formation of pulmonary fibrosis can be predicted early through the irregular interface and parenchymal bands, and clinical intervention can be performed on patients with COVID-19 as early as possible, which may reverse the development of lung diseases to a certain extent and improve the prognosis.

The following will be described in detail in conjunction with specific embodiments. The following examples do not include other components except unavoidable impurities unless otherwise specified. The reagents and instruments used in the examples are all conventional choices in the art unless otherwise specified. The experimental methods for which specific conditions are not indicated in the examples are implemented according to conventional conditions, such as the conditions described in literature, books or the method recommended by the manufacturer. In the following examples, *p<0.05; **p<0.01; ***p<0.001. Unless otherwise specified, in the following examples, unless otherwise specified, "exo" hereinafter is the abbreviation of exosomes.

Example 1

Cellular fibrosis model construction

In order to study the molecular mechanism of embryonic stem cell exosomes in alleviating pulmonary fibrosis, a cell fibrosis model was constructed at the cellular level using BLM. TGFβ1 is considered as the "molecular switch" of fibrosis, which plays a key role in promoting the occurrence and development of fibrosis. Specific operations include:

1. Beas-2b cells were cultured with DMEM/HD complete medium and placed in a constant temperature incubator at 37°C and 5% CO ₂ saturated humidity. During normal culture, fresh complete medium was replaced every 2 days, and when the confluence of cells reached 90%, passage was carried out according to the ratio of 1:3.

2. When Beas-2b cells were treated with BLM, the cells were seeded in 6-well plates, and when the cells adhered to the wall and the confluence reached 70%, an intervention experiment was performed (the most effective expression of BLM-induced cell fibrosis was screened by the expression of TGFβ1). optimal concentration). in:

1) The reverse transcription experiment used PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa), kit. SYBR Green qPCR mix system (Abgent Company) was used for qPCR, and the operation was performed according to the instructions of the kit. Primer sequence:

hTGFβ1-F: TACCTGAACCCGTGTTGCTCTC (SEQ ID NO: 1);

hTGFβ1-R: GTTGCTGAGGTATCGCCAGGAA (SEQ ID NO: 2).

2) Determination of cell viability

(1) Beas-2b cells were seeded in a 96-well plate, and BLM was added to make the final concentration of 0, 0.5, 1, and 2 μg/mL the next day, and the cells were treated for 24 hours.

(2) Add 10 μL CCK-8 solution to each well, and continue to incubate in the cell culture incubator for 0.5-4h.

(3) Measure the absorbance at 450 nm.

It was found through experiments that BLM could induce high expression of TGFβ1 at both 1 μg/mL and 2 μg/mL, but at 2 μg/mL, the cytotoxicity was greater and the cell viability was significantly reduced (Figure 1A and Figure 1B). Therefore, a BLM concentration of 1 μg/mL was chosen to induce fibrosis in Beas-2b cells in subsequent experiments.

Example 2

miR-17-5p in the treatment of interstitial pneumonia and pulmonary fibrosis

1. mRNA level

NC mimics (mimics-NC or NC-mimics, as a blank control), NC inhibitors (inhibitor-NC or NC-inhibitor, as a blank control for inhibitors), miR-17-5p were added to Beas-2b cells, respectively. Mimics (mimics-17-5p), miR-17-5p inhibitor (inhibitor-17-5p or 17-5p-inhibitor), and 1 μg/mL bleomycin was added on the second day to induce pneumonia and pulmonary fibrosis Bleomycin was added or not as a control. Wherein: the sequences of NC mimics, NC inhibitors, miR-17-5p mimics and miR-17-5p inhibitors are as follows:

miR-17-5p mimic positive strand (mimic-17-5p sense):

CAAAGUGCUUACAGUGCAGGUAG (SEQ ID NO: 3);

miR-17-5p mimic antisense (mimic-17-5p antisense):

ACCUGCACUGUAAGCACUUUGUU (SEQ ID NO: 4);

miR-17-5p inhibitor (17-5p-inhibitor):

CUACCUGCACUGUAAGCACUUUG (SEQ ID NO: 5);

NC-mimics sense (NC-mimics sense): UUCUCCGAACGUGUCACGUTT (SEQ ID NO: 6);

NC-mimics antisense: ACGUGACACGUUCGGAGAATT (SEQ ID NO: 7);

NC inhibitor (NC-inhibitor): CAGUACUUUUGUGUGUAGUACAA (SEQ ID NO: 8).

Note: In the above sequence, the mimics are double-stranded, and the inhibitor is single-stranded.

The primer sequences used to detect the mRNA levels of Fn and type I collagen are as follows:

hFn-F: ACAACACCGAGGTGACTGAGAC (SEQ ID NO: 9);

hFn-R: GGACACAACGATGCTTCCTGAG (SEQ ID NO: 10);

hCollagen I-F: GATTCCCTGGACCTAAAGGTGC (SEQ ID NO: 11);

hCollagen I-R: AGCCTCTCCATCTTTGCCAGCA (SEQ ID NO: 12).

The results are shown in Figures 2 to 3. From Figure 2 to Figure 3, it can be seen that the mRNA levels of Fn and type I collagen in cells transfected with NC mimic (mimic-NC) and NC inhibitor and added with BLM were significantly higher than those in other groups, while miR-17-5p mimic Phytotransfection significantly decreased Fn and type I collagen mRNA levels. It can be seen that miR-17-5p has the effect of preventing/treating interstitial pneumonia and pulmonary fibrosis.

2. Protein level

The protein expression levels related to fibrosis were verified by Western blot, and the results are shown in Figure 4.

It can be seen from the figure that in the Beas-2b cell fibrosis induced by bleomycin, in addition to the significant up-regulation of the protein levels of FN and α-SMA proteins related to pneumonia and pulmonary fibrosis, the protein level of Thbs2 was also significantly up-regulated. It showed that Thbs2 was positively correlated with pneumonia and pulmonary fibrosis.

Example 3

miR-17-5p regulates Thbs2 expression

To study whether miR-17-5p regulates the expression of Thbs2, we added NC mimics, NC inhibitors, miR-17-5p mimics, and miR-17-5p inhibitors to Beas-2b cells (the specific sequences of each substance As shown in Example 2, the specific operations include: when the cells in the 6-well plate are 80% confluent, each group transfects Beas-2b cells with 2.5 μg of nucleic acid and 3.5 μL of transfection reagent Lipo3000 (Invitrogen), respectively), and On the second day, add or not add bleomycin to induce pneumonia and pulmonary fibrosis or control, and detect with qPCR (the results are shown in Figure 5), wherein the primer sequences used for qPCR:

hThbs2-F: CAGTCTGAGCAAGTGTGACACC (SEQ ID NO: 13);

hThbs2-R: TTGCAGAGACGGATGCGTGTGA (SEQ ID NO: 14).

The qPCR results showed that the expression of Thbs2 in cells transfected with miR-17-5p mimic alone decreased by about 50% (Fig. 5, the third bar graph from left to right). Consistent with Example 2, the high expression of Thbs2 can be induced after BLM stimulation (FIG. 5, the second histogram from left to right).

Example 4

miR-17-5p directly binds Thbs2

In order to verify whether miR-17-5p interacts with Thbs2, we performed a dual luciferase reporter gene experiment, and cloned the 3'-UTR-WT and 3'-UTR-MUT of the Thbs2 gene into the gene containing firefly luciferase, respectively. On the pcs2 plasmid, the plasmid was then co-transfected into 293T cells with the pRLTK plasmid containing Renilla luciferase and the miR-17-5p mimic/control miRNA. After 48 hours, the bioluminescence was detected by a microplate reader to characterize the fluorescence Sulfase activity, the results are shown in Figure 6. Wherein, the dual-luciferase reporter gene detection method is carried out using a dual-luciferase reporter gene detection kit (from TAKARA), and the specific steps include:

(1) After the plasmids were co-transformed for 48 hours, the culture medium was aspirated and washed twice with PBS gently.

(2) Dilute 5×PLB into 1×PLB with sterilized ultrapure water, and place it on ice.

(3) Add 80-100 μL of 5×PLB to each well, place on a shaker at room temperature for 15 minutes to lyse.

(4) Centrifuge at 12000 rpm for 10 min at room temperature.

(5) Add 20 μL of supernatant to the flow tube, and then add 100 μL of LAR II solution, vortex gently and mix well, then test on the machine, delay for 2 seconds, and test for 10 seconds.

(6) Take out the flow tube, add 100 μL Stop&Glo regent, vortex gently and mix well, then test on the machine, delay for 2 seconds, and test for 10 seconds.

The results of microplate reader detection are shown in Figure 6. It can be seen from Figure 6 that the luciferase activity of cells transfected with Thbs2 3'-UTR-WT was reduced, which proved that miR-17-5p could directly interact with Thbs2, thereby reducing the expression of luciferase reporter gene.

Example 5

MiR-17-5p electroporation of 293 exosomes

1. Mouse miR-17-5p is synthesized by Sangon Biotech, and its sequence is as follows:

mouse miR-17-5p mimics-forward:CAAAGUGCUUACAGUGCAGGUAG

(SEQ ID NO: 15);

mouse miR-17-5p mimics-reverse: ACCUGCACUGUAAGCACUUUGUU (SEQ ID NO: 16).

2. 293T cells are anchorage-dependent epithelioid cells, and the growth medium is DMEM (containing 10% FBS). The 293T cells are grown and proliferated to form a monolayer of cells.

3. Extraction of exosomes from 293T cells: the supernatant of 293T cells forming a monolayer of cells in step 2 was collected. Centrifuge at 500 g for 5 min and collect the supernatant. Centrifuge at 2000g for 10min and collect the supernatant. Centrifuge at 10000g for 20min and collect the supernatant. Then centrifuge at 100,000 g for 1 h 40 min to collect the precipitate, wash the precipitate once with PBS, centrifuge again at 100,000 g for 1 h 40 min, collect the precipitate, and resuspend with an appropriate amount of PBS to obtain exosomes. Exosomes (10 mg, 1 mL) and miR-17-5p (100 μm, 100 μL) were electroporated in an electroporation cuvette to obtain miR-17-5p-exo, which was stored at -80°C for later use.

Example 6

miR-17-5p encapsulated exosomes in the treatment of bleomycin-induced interstitial pneumonia

We used bleomycin (BLM), a broad-spectrum antitumor drug, to construct a mouse model of interstitial pneumonia and pulmonary fibrosis. BLM injection dose and method: intratracheal injection 3mg/kg. They were divided into 6 groups, with 10 C57BL male mice in each group. On day 0, they were given BLM or the same amount of PBS as a control, and miR-17-5p was given to the tail vein after 1 day, 3 days, 5 days, and 7 days after BLM injection. -exo (inject 100 μL of 100 μg/100 μL per 20 g mouse) or the same amount of exo or the same amount of PBS, and sacrifice the mice on the 8th day and the 21st day for histological detection, the results are shown in the figure 7 to 8. Figure 7 is a schematic diagram of the dosing regimen and the results of tissue sections for killing on the 8th day; Figure 8 is a schematic diagram of the dosing scheme and the results of tissue sections for killing on the 21st day.

Figure 7 is the 7-day interstitial pneumonia stage. It can be seen that the HE stained lung structure of the BLM group was destroyed, the alveoli disappeared, and inflammatory cell infiltration and interstitial fiber deposition appeared. Relatively normal, inflammatory cell infiltration is not obvious. Therefore, miR-17-5p-exo treatment significantly alleviated interstitial pneumonia. BLM-induced lung injury in mice began to show fibrosis at 14 days, and the degree of fibrosis was the most severe at 21 days to 28 days. Figure 8 is the stage of pulmonary fibrosis at 21 days. We found by H&E staining that after 21 days of BLM induction in mice, it caused changes in the lung structure, resulting in collapsed alveoli and thickened airway epithelium, suggesting the emergence of interstitial pulmonary fibrosis, while miR-17-5p- These symptoms were significantly improved in exo-treated mice.

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

The above-mentioned embodiments only express several implementation modes of the present invention, which are convenient for a specific and detailed understanding of the technical solution of the present invention, but should not be construed as limiting the protection scope of the invention patent. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. It should be understood that technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are within the protection scope of the appended claims of the present invention. Therefore, the protection scope of the patent for the present invention shall be determined by the content of the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims (10)

1. Application of miR-17-5p in the preparation of products for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation. 2. The application of miR-17-5p according to claim 1 in the preparation of products for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation, characterized in that, the The disease was interstitial pneumonia. 3. The application of miR-17-5p according to claim 1 or 2 in the preparation of products for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation, characterized in that, the miR-17- 5p comes from stem cell exosomes. 4. The application of miR-17-5p according to claim 3 in the preparation of products for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation, characterized in that, said miR-17-5p is from Human embryonic stem cell exosomes. 5. The application of miR-17-5p according to claim 1, 2 or 4 in the preparation of products for the prevention or treatment of diseases associated with pulmonary fibrosis and/or pulmonary inflammation, characterized in that the products include An active ingredient, the active ingredient comprising one or both of miR-17-5p and miR-17-5p mimics. 6. The application of miR-17-5p according to claim 5 in the preparation of products for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation, characterized in that, the miR-17-5p simulates The nucleotide sequence of the compound is shown in SEQ ID NO: 3-4. 7. The application of miR-17-5p according to claim 5 in the preparation of products for the prevention or treatment of diseases associated with pulmonary fibrosis and/or pulmonary inflammation, characterized in that the product also includes a carrier, wherein The vectors described above are used to deliver miR-17-5p and/or miR-17-5p mimics in vivo. 8. The use of miR-17-5p according to claim 7 in the preparation of products for the prevention or treatment of diseases associated with pulmonary fibrosis and/or pulmonary inflammation, characterized in that the carrier delivers miR-17- 5p and/or miR-17-5p mimics include one or more of: transfection reagent delivery, liposomal nanoparticle delivery, conjugation delivery, multimeric nanoparticle delivery, exosome delivery, and Nanozyme delivery. 9. The application of miR-17-5p according to any one of claims 1, 2, 4 and 6 to 8 in the preparation of products for preventing or treating diseases related to pulmonary fibrosis and/or pulmonary inflammation, wherein It is characterized in that the dosage form of the product is tablet, capsule, granule, pill, injection or sustained-release preparation. 10. A medicament, characterized in that it comprises an active ingredient, and the active ingredient includes miR-17-5p and/or miR-17-5p mimics.

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