Umbilical cord mesenchymal stem cell exosome as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to an umbilical cord mesenchymal stem cell exosome and a preparation method and application thereof.The umbilical cord mesenchymal stem cell exosome is derived from umbilical cord mesenchymal stem cells expressing S100g-MTOR binding domain fusion protein and can inhibit excessive autophagy through an S100g / MTOR signal channel, and therefore intestinal mucosal barrier damage caused by antibiotics is relieved. The preparation method comprises the following steps: constructing an expression vector, transfecting and screening a stable expression cell strain, performing serum-free culture, collecting a culture supernatant, purifying the exosome and the like. The exosome disclosed by the invention can be used for preparing a medicine for treating antibiotic-related intestinal mucosal barrier injury, has the advantages of strong targeting property, definite action mechanism, remarkable curative effect and the like, and provides a new choice for preventing and treating antibiotic-related intestinal adverse reactions.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an umbilical cord mesenchymal stem cell exosome, a preparation method thereof, and an application. More precisely, the present invention provides an S100g signal pathway-optimized mesenchymal stem cell exosome that regulates autophagy through the S100g / MTOR signal pathway to treat antibiotic-induced intestinal mucosal barrier damage. Background Art

[0002] Exosomes are extracellular vesicles with a diameter between 40 - 160 nm, secreted by cells and present in various body fluids, carrying various bioactive molecules such as nucleic acids, proteins, lipids, and metabolites. As an important mediator of intercellular communication, exosomes play a key role in physiological and pathological processes. In recent years, exosomes have become an ideal carrier for drug delivery due to their natural biocompatibility, low immunogenicity, and high stability.

[0003] Exosomes derived from mesenchymal stem cells (MSCs) have been widely studied and used in the treatment of various diseases. Li et al. reported in 《Stem Cell Research & Therapy》 (2022 Jul 28; 379) that it was found that extracellular vesicles derived from allogeneic adipose mesenchymal stem cells could more efficiently promote the rapid recovery of indicators such as creatinine and blood urea nitrogen to reach normal physiological homeostasis levels. In addition, this study also found using ultra-high performance liquid chromatography-mass spectrometry that the metabolic levels of 6 molecules such as carnitine, melibiose, D-glucosamine, cytidine, dihydroorotic acid, and stachyose were highly correlated with the occurrence of post-renal acute kidney injury and were potential targets for extracellular vesicles of allogeneic adipose mesenchymal stem cells to treat post-renal acute kidney injury. This study provided a useful reference for exploring and developing new and efficient methods for treating post-renal acute kidney injury. In addition, Wu et al. found in 《Biomaterials》 (2019, 206: 87 - 100) that exosomes derived from mesenchymal stem cells in the infrapatellar fat pad were rich in miR-100-5p, which could enhance chondrocyte autophagy by inhibiting the MTOR signal pathway, thereby protecting articular cartilage and improving gait abnormalities in osteoarthritis.

[0004] Antibiotics are important drugs for treating infectious diseases, but long-term, broad-spectrum, and high-dose use of antibiotics can cause a series of adverse reactions, especially intestinal-related diseases such as antibiotic-associated diarrhea, irritable bowel syndrome, and inflammatory bowel disease. According to relevant research reports, the annual global consumption of antibiotics exceeds 40 billion doses and is still increasing. The adverse effects of antibiotics on the host intestine are mainly through reducing intestinal microbial diversity, direct toxic damage of the drug, and screening of drug-resistant flora, thereby reducing the immunity of host cells and increasing epithelial cell death.

[0005] The intestinal mucosal barrier, as a complex structure that separates the internal environment from the luminal environment, includes a physical barrier, a chemical barrier, a biological barrier, and an immune barrier. Among them, the physical barrier is formed by the tight junctions of intestinal epithelial cells and the mucus layer. Many studies have shown that inhibiting cell proliferation, promoting cellular inflammatory stress, and causing excessive autophagy in cells are the main reasons for antibiotic-induced intestinal mucosal barrier damage. Currently, the treatment methods for antibiotic-related intestinal barrier damage mainly include supplementing probiotics, fecal microbiota transplantation, short-chain fatty acids, and dietary nutritional support, etc., but these treatment methods have limited effects and certain side effects.

[0006] S100g protein (also known as calbindin-D9k, Calbindin-D9k) is a vitamin D-dependent calcium-binding protein and belongs to the S100 family. This protein is highly expressed in intestinal epithelial cells, and its main function is to mediate the transport of calcium ions from the apical side of intestinal epithelial cells (the entrance regulated by TRPV6 calcium channels) across the cytoplasm to the basolateral side (pumping calcium into the blood through calcium pumps such as PMCA1). S100g contains two EF-hand domains, including an N-terminal non-classical EF hand and a C-terminal canonical EF hand, which can efficiently bind and transport calcium ions.

[0007] However, so far, there are few research reports on the role of S100g in regulating autophagy and the treatment of antibiotic-related intestinal barrier damage using umbilical cord mesenchymal stem cell exosomes through the S100g / MTOR signaling pathway. MTOR (mammalian target of rapamycin) is a key metabolic regulator and plays a central role in autophagy regulation. The activation of MTOR can inhibit the autophagy process, while its inhibition promotes autophagy. The current challenge lies in how to precisely regulate the autophagy process in intestinal epithelial cells to protect the integrity of the intestinal mucosal barrier during antibiotic treatment.

[0008] Therefore, there is an urgent need to develop a new treatment method that can effectively regulate the autophagy process and protect the intestinal mucosal barrier from antibiotic damage. Summary of the Invention

[0009] The purpose of the present invention is to provide an umbilical cord mesenchymal stem cell exosome, its preparation method and application, to solve the problem of the lack of effective means to protect antibiotic-related intestinal mucosal barrier damage in the prior art.

[0010] The inventors of the present invention found in long-term research work that antibiotics can cause excessive autophagy in intestinal epithelial cells, thereby leading to cell apoptosis, decreased expression of tight junction proteins, and damage to the intestinal mucosal barrier function. The S100g protein can regulate the phosphorylation state of MTOR by interacting with the MTOR protein, and then precisely regulate the autophagy process. The present invention ingeniously designs an umbilical cord mesenchymal stem cell exosome containing an S100g-MTOR binding domain fusion protein, realizing the precise regulation of the autophagy process and effectively protecting the intestinal mucosal barrier from antibiotic damage.

[0011] The first aspect of the present invention provides a kind of umbilical cord mesenchymal stem cell exosome, which is derived from umbilical cord mesenchymal stem cells expressing S100g-MTOR binding domain fusion protein. The diameter of the exosome is 40-160nm, and it expresses CD9, CD63 and Alix markers, and can inhibit excessive autophagy through the S100g / MTOR signaling pathway, and is used for treating intestinal mucosal barrier injury caused by antibiotics.

[0012] Preferably, the S100g-MTOR binding domain fusion protein comprises the amino acid sequence of human-derived S100g protein, the flexible linker peptide sequence GGSGSGG and the binding domain sequence composed of the amino acids at positions 2115-2150 of MTOR protein. The S100g protein has two EF-hand calcium binding domains.

[0013] In one embodiment of the present invention, the amino acid sequence of the S100g-MTOR binding domain fusion protein is as follows:

[0014] MSAKKSPEEIREKIFDIFDGDKSGKDFHEELKLANTSLLKHVLQSKVAKSMTDSEEEIREAFKVFDKNGDGRISEDEIKE LAQAFSLFDQDGRTITLDEVEFPQGGSGSGG LERVRTLGFQRSFTVLDREQHRENLVAFYSKEDAHE

[0015] Among them, the first 79 amino acids are the human-derived S100g protein sequence, GGSGSGG is the flexible linker peptide, and the last 36 amino acids are the MTOR binding domain sequence.

[0016] The exosome of the present invention inhibits excessive autophagy, reduces apoptosis, and thus alleviates intestinal mucosal barrier injury caused by antibiotics by increasing the p-MTOR / MTOR ratio, decreasing the LC3 II / I ratio, and decreasing the p62 expression in intestinal epithelial cells.

[0017] The second aspect of the present invention provides a method for preparing the above-mentioned S100g signaling pathway-optimized mesenchymal stem cell exosome, which comprises the following steps:

[0018] (1) Construct an expression vector expressing S100g-MTOR binding domain fusion protein;

[0019] (2) Transfect the expression vector into human umbilical cord mesenchymal stem cells, and screen to obtain a cell line stably expressing S100g-MTOR binding domain fusion protein;

[0020] (3) Culture the cell line in serum-free medium for 48-72 hours;

[0021] (4) Collect the culture supernatant and remove cells and cell debris by centrifugation;

[0022] (5) Isolate and purify exosomes by sucrose-deuterium oxide combined with ultra-high speed centrifugation;

[0023] (6) Characterize and verify the function of the obtained exosomes.

[0024] Preferably, the expression vector in step (1) is the pcDNA3.1(+) vector, which contains the human CMV enhancer / promoter, the coding sequence of the S100g-MTOR binding domain fusion protein, and the SV40 polyA termination signal.

[0025] More preferably, in step (2), the transfection is carried out by liposome transfection method. After transfection, G418 at a concentration of 400 - 800 μg / ml is used for screening for 7 - 14 days to obtain a stable expression cell line; the passage number of the human umbilical cord mesenchymal stem cells is 3 - 6 passages.

[0026] Further preferably, the serum-free medium in step (3) is DMEM-low glucose medium, and 0.5 - 5% human platelet lysate (hPL) or 0.5 - 1% human serum albumin (HSA) can be added; the culture conditions are 37°C, 5% CO2, and 1% O2 hypoxic pretreatment can be selectively carried out for 24 - 48 hours to increase the yield of exosomes.

[0027] In another embodiment of the present invention, the sucrose-deuterium oxide combined with ultra-high speed centrifugation method in step (5) includes: loading the supernatant pretreated by pre-ultracentrifugation (10,000 g, 30 minutes) onto the top of a 10 - 60% sucrose density gradient, centrifuging at 120,000 g for 16 - 18 hours, collecting exosomes in the density range of 1.13 - 1.19 g / ml, diluting with PBS and precipitating by centrifugation at 120,000 g for 70 minutes. The obtained exosomes are resuspended with PBS and filtered through a 0.22 μm filter.

[0028] The third aspect of the present invention provides the application of the above-mentioned S100g signaling pathway optimized mesenchymal stem cell exosomes in the preparation of drugs for treating antibiotic-related intestinal barrier injury.

[0029] Preferably, the exosomes are administered at a dose of 1×10 10 -1×10 11 particles / kg, once every 1 - 3 days, and the treatment course is 2 - 4 weeks.

[0030] More preferably, the administration route is intraperitoneal injection, intravenous injection or oral administration of enteric-coated preparations; the antibiotic-related intestinal barrier injury includes antibiotic-related diarrhea, antibiotic-related intestinal inflammation, increased intestinal permeability caused by antibiotics, and decreased expression of tight junction proteins caused by antibiotics.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] 1. The present invention first proposes an innovative strategy for regulating autophagy through the S100g / MTOR signaling pathway to protect the intestinal mucosal barrier from antibiotic damage, opening up a new direction for the treatment of antibiotic-related intestinal mucosal barrier injury.

[0033] 2. The S100g-MTOR binding domain fusion protein-expressing exosomes designed by the present invention have a dual mechanism of action: on the one hand, through the regulation of calcium signals mediated by S100g, and on the other hand, through the regulation of autophagy balance by the MTOR pathway, achieving a multi-level protective effect on intestinal epithelial cells.

[0034] 3. The exosomes of the present invention have obvious advantages compared with traditional treatment methods: the mechanism of action is more direct and clear, not affected by the intestinal flora status, the curative effect is more stable and predictable, and it is applicable during antibiotic treatment; compared with hormonal drugs, there are no immunosuppressive adverse reactions and no long-term use safety hazards, and the action is more targeted and specific; compared with directly using S100g protein, the bioavailability mediated by exosomes is higher, avoiding rapid protein degradation, and can penetrate the mucus layer to reach epithelial cells; compared with ordinary MSC-Exo, the S100g-MBD function is enhanced, the autophagy regulation ability is improved, the affinity for intestinal epithelial cells is enhanced, and the treatment effect is increased by 50-80%.

[0035] 4. The present invention has established a complete preparation process, including genetic modification technology, serum-free culture technology and advanced exosome isolation and purification technology, laying a foundation for large-scale production of high-quality exosome preparations.

[0036] 5. The therapeutic effect of the present invention has been fully verified by in vitro and in vivo experiments, indicating that the exosomes have a significant therapeutic effect and application prospect in antibiotic-related intestinal mucosal barrier injury. Description of the Drawings

[0037] Figure 1 . Characterization of hucMSCs-Exo.

[0038] (a) TEM image of exosome morphology, scale bar: 200 nm.

[0039] (b) Exosome particle size distribution curve detected by NTA.

[0040] (c) Western blot images of CD9, CD63, and Alix in two exosomes.

[0041] Figure 2 . Effects of hucMSC-Exo on antibiotic (clindamycin hydrochloride)-induced intestinal mucosal barrier injury in mice.

[0042] (a) Schematic diagram of the mouse experiment design.

[0043] (b) Line graph of the percentage of mouse body weight gain from day 0 to day 28.

[0044] (c) In vivo imaging of FD4 in mice and quantitative analysis of the total radiation value of mice.

[0045] (d) Representative colon pictures of antibiotic-induced intestinal injury and quantitative analysis of colon length.

[0046] (e) HE staining of mouse colon, original magnification ×10 (left), ×200 (right), crypts (yellow arrows), capillaries (green arrows), goblet cells (blue arrows).

[0047] (f) Immunohistochemical images of Claudin-1, Occludin, and ZO-1 and quantitative analysis of the mean optical density, original magnification x400.

[0048] (g) RT-qPCR results of Occludin, Claudin-1, and ZO-1 genes, as well as Western Blot images and analysis of relative protein expression levels (h).

[0049] Figure 3 . Analysis of microbial diversity among the blank group, antibiotic group, and high-concentration exosome group.

[0050] (a) Venn diagram for analyzing shared and unique characteristic sequences among groups.

[0051] (b) Column chart of the species abundance at the phylum level of the mouse fecal flora.

[0052] (c) Analysis of differences in α-diversity indices (chao1, observed_features, shannon, simpson box plots).

[0053] (d) Analysis of differences in β-diversity indices, principal coordinate analysis (PCoA) based on Weighted Unifrac distance, and non-metric multi-dimensional scaling analysis (NMDS).

[0054] (e) LEfSe analysis diagram. For species with LDA score ≥ 3.5, on the left is the antibiotic group vs the blank group, and on the right is the control group vs the exosome group.

[0055] Figure 4 . The effect of exosomes on the damage of intestinal cell mucosal barrier caused by antibiotics (clindamycin hydrochloride).

[0056] (a) The cck8 results show the effect of antibiotic concentrations (50μm, 100μm, 200μm) on inhibiting the proliferation of NCM460 cells.

[0057] (b) and (c) Western blot images and relative protein expression levels of ZO-1, Occludin, and Claudin-1 under the damage of antibiotics (100μm, 200μm).

[0058] (d) The results of the cell proliferation experiment of exosome treatment on intestinal cells damaged by antibiotics (200μm).

[0059] (e), (f), and (i) Western blot images, relative protein expression levels, and RT-qPCR results of ZO-1, Occludin, and Claudin-1 proteins in intestinal cells damaged by antibiotics (200μm) treated with exosomes.

[0060] (g) and (h) Transwell images and cell quantification results of intestinal cells damaged by antibiotics treated with exosomes.

[0061] Figure 5 . Transcriptomic analysis of high-concentration MSCs-Exo in improving antibiotic-induced intestinal barrier damage.

[0062] (a) Volcano plot of differentially expressed genes in mouse colon tissues (left: Antibiotic group vs Control group, right: exosome group vs Antibiotic group).

[0063] (b) Display of the number of differential genes in the two groups corresponding to Figure a. Red indicates high gene expression, and green indicates low expression.

[0064] (c) RT-qPCR results of the S100g gene in mouse colon tissues.

[0065] (d) and (e) KEGG and GO analyses of differential genes in the Antibiotic group vs Exosome group.

[0066] (f) Immunofluorescence image of S100g protein in NCM460 cells at a magnification of 40x.

[0067] (g) RT-qPCR results of the S100g gene in NCM460 cells.

[0068] Figure 6 . Related research on the mechanism of exosomes in antibiotic-induced intestinal barrier injury.

[0069] (a) Western blot images and relative protein expression levels of proteins related to autophagy (p-MTOR / MTOR, P62, LC3II / I) and apoptosis (Bax, Bak) induced by antibiotics (100, 200 μM).

[0070] (b) Western blot images and relative protein expression levels of proteins related to autophagy and apoptosis induced by antibiotics (200 μM) treated with low and high concentrations of exosomes.

[0071] (c) Flow cytometry diagrams and quantitative results of cell apoptosis.

[0072] (d) Transmission electron microscopy observation of cell structure, red represents autophagosomes, blue represents mitochondria, scale bar, 1 μm.

[0073] Figure 7 . Effect of down-regulation of the S100g gene on antibiotic-induced intestinal epithelial cell barrier injury.

[0074] (a) Immunofluorescence images of S100G protein (magnification 40x) and RT-qpcr results (b) after transfection of NCM460 cells with si-NC and si-S100g for 48 h.

[0075] (c) Results of cell proliferation experiments in the si-NC group, si-NC + antibiotic group, and si-S100g + antibiotic group after adding antibiotics (200 μM).

[0076] (d) Cell migration images and quantitative results.

[0077] (e) Flow cytometry images and quantitative results of cell apoptosis.

[0078] (f) Western blot images and relative protein expression levels of intestinal epithelial cell mucosal barrier proteins (ZO-1, Occludin, Claudin-1), autophagy proteins (p-MTOR / MTOR, P62, LC3II / I), and apoptosis proteins (Bax, Bak). Detailed implementation methods

[0079] The present invention will be further described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0080] Example 1: Construction of an expression vector for S100g-MTOR binding domain fusion protein

[0081] This example aims to design and construct an expression vector capable of highly expressing the S100g-MTOR binding domain fusion protein in human umbilical cord mesenchymal stem cells.

[0082] First, based on the sequence information of the human S100g gene (NM_004057.4) in the GenBank database, the full-length S100g coding sequence was designed and synthesized. At the same time, according to the information of the human MTOR protein (P42345) in the UniProt database, the amino acid fragment at positions 2115-2150 of the MTOR protein was selected as the MTOR binding domain. To ensure the independent folding of the two functional domains, a flexible linker peptide GGSGSGG was designed to connect them. The entire fusion protein coding sequence was codon-optimized to adapt to human cell expression.

[0083] During gene synthesis, an NheI restriction site and a Kozak sequence (GCCACC) were added to the 5' end to enhance translation efficiency, and a BamHI restriction site was added to the 3' end. The purity of the synthesized gene was ≥98%, and the sequence was verified to be completely correct by sequencing. The complete amino acid sequence of the S100g-MTOR binding domain fusion protein is as follows:

[0084] MSAKKSPEEIREKIFDIFDGDKSGKDFHEELKLANTSLLKHVLQSKVAKSMTDSEEEIREAFKVFDKNGDGRISEDEIKE LAQAFSLFDQDGRTITLDEVEFPQGGSGSGG LERVRTLGFQRSFTVLDREQHRENLVAFYSKEDAHE

[0085] Among them, the first 79 amino acids (MSAKKS...EFPQ) are the human S100g protein sequence, GGSGSGG is the flexible linker peptide, and the last 36 amino acids (LERVRT...DAHE) are the MTOR binding domain sequence. The S100g protein contains two EF-hand calcium-binding domains, located at amino acids 13-48 (N-terminal non-canonical EF hand) and 45-79 (C-terminal canonical EF hand), respectively.

[0086] Next, we used NheI and BamHI restriction endonucleases to double-digest the pcDNA3.1(+) vector (Invitrogen, V79020) and the synthesized S100g-MTOR binding domain fusion gene. The digestion reaction was carried out at 37 °C for 2 hours, and then the digested products were purified by 0.8% agarose gel electrophoresis. The purified vector and the insert fragment were ligated using T4 DNA ligase (NEB, M0202S) at a molar ratio of 1:3 at 16 °C for 16 hours.

[0087] The ligation products were transformed into competent Escherichia coli DH5α and spread on LB plates containing 100 μg / ml ampicillin, and cultured overnight at 37 °C. 10 - 15 monoclonal colonies were picked for colony PCR screening using the T7 forward primer and the BGH reverse primer. The positive clones were sent for sequencing to confirm the sequence integrity. The sequencing verification results showed that the constructed expression vector pcDNA3.1(+)-S100g-MBD contained the complete coding sequence of the S100g-MTOR binding domain fusion protein, and the sequence accuracy was 100%. In addition, through NheI / BamHI double-digestion verification, it was confirmed that the size of the inserted fragment was correct (about 360 bp).

[0088] Example 2: Isolation and culture of human umbilical cord mesenchymal stem cells (hucMSCs)

[0089] This example aims to establish a stable culture system for human umbilical cord mesenchymal stem cells (hucMSCs) to lay the foundation for subsequent exosome production.

[0090] Under the condition of obtaining informed consent, fresh umbilical cord tissue was collected from the placenta of healthy women who gave birth vaginally. The umbilical cord tissue was rinsed 3 times with cold PBS containing penicillin-streptomycin under sterile conditions to remove residual blood. After carefully removing the umbilical cord blood vessels (umbilical vein and umbilical artery), the Wharton's jelly part was cut into small pieces about 1 - 2 mm 3 and placed into DMEM medium containing 1 mg / ml type I collagenase (Sigma-Aldrich, C0130), and digested at 37 °C for 5 hours, gently shaking to promote tissue digestion.

[0091] The digested tissue fluid was filtered through a 100 μm cell sieve, and the filtrate was collected. Subsequently, the cells were collected by centrifugation at 300 g for 10 minutes. The cell pellet was resuspended in complete medium (DMEM-low glucose, 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin) and seeded into culture flasks at a density of 5,000 cells / cm 2 The cells were cultured at 37 °C in a 5% CO2 condition. After 48 hours, the medium was changed to remove non-adherent cells and tissue debris, and then the medium was changed every 3 days.

[0092] After about 7 - 10 days of primary cell culture, when the cells reached 80 - 90% confluence, they were digested with 0.25% trypsin - EDTA (Gibco, 25200056) for 3 - 5 minutes. An equal volume of DMEM containing 10% FBS was added to terminate the digestion, and the cells were collected by centrifugation at 300g for 5 minutes. Subsequently, the cells were passaged at a ratio of 1:3 to establish a stable hucMSCs cell line.

[0093] To identify the obtained cells, we performed the following tests:

[0094] 1. Morphological observation: The obtained cells showed a typical spindle - shaped morphology, grew adherently, and were arranged in a swirling pattern.

[0095] 2. Surface marker analysis: By flow cytometry, the positive rates of cell surface markers CD73, CD90, and CD105 were all ≥95%, while the positive rates of hematopoietic stem cell markers CD34, CD45, and HLA - DR were all ≤2%, meeting the identification criteria of the International Society for Cellular Therapy (ISCT).

[0096] 3. Multidirectional differentiation potential: The cells were cultured in osteogenic, adipogenic, and chondrogenic induction media for 21 days respectively. Through Alizarin Red, Oil Red O, and Alcian Blue staining, it was confirmed that hucMSCs had the ability to differentiate into bone, fat, and cartilage.

[0097] After identification, it was confirmed that the isolated cells were human umbilical cord mesenchymal stem cells with typical characteristics. To meet the requirements of subsequent exosome production, a master cell bank (MCB) and a working cell bank (WCB) system were established. After the primary hucMSCs were amplified for 2 - 3 passages and passed the identification, they were cryopreserved in liquid nitrogen at a density of 1×10 6 cells / ml using a cryopreservation solution containing 10% DMSO, 40% FBS, and 50% DMEM by programmed cooling (-1°C / min) to establish the MCB. The cells were thawed from the MCB, amplified for 1 passage, and cryopreserved in the same way to establish the WCB. For each exosome production, the cells were thawed from the WCB and amplified no more than 6 passages to ensure the stability of cell characteristics.

[0098] Example 3: Construction of hucMSCs stably expressing the S100g - MTOR binding domain fusion protein

[0099] This example aims to establish a hucMSCs cell line stably expressing the S100g - MTOR binding domain fusion protein.

[0100] First, thaw hucMSCs from WCB and expand and culture them in complete medium for 3 - 4 passages. When the cells reach 70 - 80% confluence, transfection is carried out. 24 hours before transfection, seed the cells in six - well plates at a density of 5×10 4 cells / cm 2 . On the day of transfection, change the medium to DMEM + 10% FBS without antibiotics.

[0101] To optimize the transfection conditions, the following transfection experiments were designed in this study:

[0102] 1. Transfection reagent screening: Test three transfection reagents, Lipofectamine 3000 (Thermo Fisher Scientific, L3000015), FuGENE HD (Promega, E2311), and jetPRIME (Polyplus, 114 - 15) respectively.

[0103] 2. Optimization of DNA:transfection reagent ratio: Test three ratios of 1:2, 1:3, and 1:4 respectively.

[0104] 3. Optimization of DNA concentration: Test three concentrations of 0.5 μg / ml, 1 μg / ml, and 2 μg / ml respectively.

[0105] The transfection process is carried out according to the instructions of each transfection reagent. After incubating the transfection complex at room temperature for 15 - 20 minutes, add it to the cell culture wells. Replace the medium with complete medium 6 hours after transfection and continue culturing for 48 hours.

[0106] Detect the expression level of S100g - MTOR binding domain fusion protein by RT - qPCR and Western blot. The optimal transfection conditions are determined as follows: Lipofectamine 3000 as the transfection reagent, DNA:reagent ratio of 1:3, and DNA concentration of 1 μg / ml. Under these conditions, the transfection efficiency is about 30 - 40%, and the expression level of the fusion protein is the highest.

[0107] To establish a stable cell line expressing the target protein, first determine the optimal screening concentration of G418 through a killing curve experiment. Expose untransfected hucMSCs to different concentrations (200 - 1000 μg / ml) of G418 for 7 days, and determine that the lowest concentration that can kill all cells is 600 μg / ml. Subsequently, screen the transfected cells in a medium containing 400 μg / ml G418 for 7 days, and then increase the concentration to 600 μg / ml and continue screening for 7 days until stable clones are formed.

[0108] Single cell clones were isolated by the limiting dilution method. The screened cells were seeded into 96-well plates at a density of 0.5 cells per well on average and cultured for 2 - 3 weeks until single cell clones formed. Monoclonal cells with good growth were selected for amplification culture. The expression level of the S100g-MTOR binding domain fusion protein was verified by RT-qPCR and Western blot, and its intracellular localization was observed by immunofluorescence. At the same time, the monoclonal cells were continuously passaged for 10 generations to verify the expression stability.

[0109] After screening and verification, multiple hucMSCs clones stably expressing the S100g-MTOR binding domain fusion protein were obtained. Clone 6 with the highest and most stable expression level was selected for subsequent exosome production. It was found by immunofluorescence observation that the S100g-MTOR binding domain fusion protein was mainly distributed in the cytoplasm, and part of it could be transferred to the cell membrane following the change of calcium signal, which was consistent with the typical distribution pattern of S100g protein.

[0110] Example 4: Production and purification of S100g signaling pathway optimized MSC-Exo

[0111] This example aims to establish a stable production and purification process for S100g signaling pathway optimized MSC-Exo.

[0112] As Figure 1 shown, clone 6 of hucMSCs stably expressing the S100g-MTOR binding domain fusion protein obtained in Example 3 was amplified and cultured. When the cells reached 70 - 80% confluence, they were washed 3 times with PBS to remove serum components, and then changed to serum-free medium (DMEM-low glucose basal medium supplemented with 1% ITS and 0.5% HSA). To further optimize exosome production, a hypoxic pretreatment strategy was adopted: the cells were cultured under the conditions of 1% O2, 5% CO2, and 37 °C for 24 hours, and then transferred to serum-free medium for culture.

[0113] After 48 - 72 hours of serum-free culture, the cell culture supernatant was collected. The collected supernatant was first centrifuged at 300g for 10 minutes to remove cells, then centrifuged at 2,000g for 20 minutes to remove cell debris, and the supernatant was filtered through a 0.45 μm filter. Protease inhibitor mixture (1X) was added to the pretreated supernatant, and then subsequent purification was carried out immediately or stored at 4 °C for a short time (not exceeding 24 hours).

[0114] Exosomes were purified by the combined sucrose and deuterium oxide ultra-high speed centrifugation method. The specific steps are as follows:

[0115] 1. Pre-ultracentrifugation: The pretreated supernatant was centrifuged at 10,000g for 30 minutes to remove large particles and microvesicles. The supernatant was carefully collected, avoiding disturbing the precipitate.

[0116] 2. Density gradient preparation: Add PBS solutions containing 60%, 50%, 40%, 30%, 20%, and 10% sucrose into the ultracentrifuge tube from bottom to top, with 0.5 ml for each layer. Let it stand at 4 °C for 30 minutes to form a continuous gradient.

[0117] 3. Sample loading and centrifugation: Carefully layer the supernatant after pre-ultracentrifugation on top of the sucrose gradient, and centrifuge at 120,000 g and 4 °C for 16 hours using a swing-out rotor (SW41).

[0118] 4. Exosome collection: Collect one fraction every 0.5 ml in the order from top to bottom, and detect the CD9, CD63, and Alix markers by Western blot to determine the fraction enriched with exosomes (usually in the fraction with a density of 1.13 - 1.19 g / ml).

[0119] 5. Purification and concentration: Combine the fractions containing exosomes, dilute them 5-fold with PBS, and precipitate the exosomes by centrifugation at 120,000 g and 4 °C for 70 minutes. Discard the supernatant and resuspend the exosome precipitate with an appropriate amount of PBS.

[0120] 6. Sterilization filtration: Filter the resuspended exosomes through a 0.22 μm filter to ensure sterility.

[0121] 7. Sub-packaging and storage: Sub-package the purified exosomes into single-use doses and store them at -80 °C or perform lyophilization.

[0122] Characterize the purified exosomes:

[0123] 1. Morphological analysis: Observe the morphology of exosomes by transmission electron microscopy (Talos L120C, FEI). The purified exosomes show a typical cup-shaped morphology with a diameter between 40 - 160 nm ( Figure 1 a).

[0124] 2. Particle size distribution: Determine the particle size distribution and concentration of exosomes by nanoparticle tracking analysis (ZetaView, Particle Metrix). The results show that the average diameter of exosomes is 105 ± 15 nm and the concentration is 5×10 11 -1×10 12 particles / ml ( Figure 1 b).

[0125] 3. Marker detection: Detect the expression of exosome markers CD9, CD63, and Alix by Western blot, and the results are all positive; while the organelle marker calnexin is negative, confirming that the isolated substance is indeed purified exosomes ( Figure 1 c).

[0126] 4. Detection of S100g-MTOR binding domain fusion protein: The presence of S100g-MTOR binding domain fusion protein in exosomes was confirmed by Western blot and mass spectrometry analysis. The results showed that compared with ordinary MSC-Exo, the S100g-related signals were significantly enhanced in S100g signaling pathway-optimized MSC-Exo.

[0127] Example 5: Functional verification of S100g signaling pathway-optimized MSC-Exo (in vitro experiments)

[0128] This example aims to evaluate the functions of S100g signaling pathway-optimized MSC-Exo in regulating autophagy and protecting intestinal epithelial cells in vitro.

[0129] First, a NCM460 intestinal epithelial cell injury model was established using clindamycin hydrochloride (200 μM, 48 h) Figure 4 a-c). The efficient uptake of exosomes by cells was verified by PKH26 fluorescence labeling. Exosome treatment could significantly increase the viability of antibiotic-damaged cells Figure 4 d), improve cell migration ability Figure 4 g-h), and restore the expression of intestinal barrier proteins (ZO-1, Occludin, Claudin-1) Figure 4 e-f, i).

[0130] 5.1 Establishment of cell model

[0131] The human colon epithelial cell line NCM460 was selected as the in vitro model. First, the optimal conditions for antibiotic (clindamycin hydrochloride) damage were determined. NCM460 cells were seeded in 96-well plates at a density of 4×10 3 cells / well. After 24 hours of culture, different concentrations (0, 50, 100, 200 μM) of clindamycin hydrochloride were added and treated for 24 h, 48 h, and 72 h. The cell viability was detected by CCK-8. The results showed that treatment with 200 μM clindamycin for 48 h could significantly reduce the cell viability by about 40% (p<0.001). Therefore, this condition was selected as the antibiotic damage model for subsequent experiments.

[0132] 5.2 Exosome uptake study

[0133] To verify the uptake of S100g signaling pathway-optimized MSC-Exo by NCM460 cells, the exosomes were labeled with PKH26 fluorescent dye and co-cultured with NCM460 cells for 0.5 h, 2 h, 6 h, and 12 h. The uptake of exosomes was observed by laser confocal microscopy. The results showed that exosomes began to be taken up by cells at 2 h, reached the peak at 6 h, and almost all cells contained exosomes labeled with red fluorescence at 12 h. Flow cytometry analysis showed that the uptake efficiency at 6 h was approximately 85%. At the same time, we also found that S100g signaling pathway-optimized MSC-Exo had a higher cell uptake efficiency compared with ordinary MSC-Exo (85% vs 70%, p<0.05).

[0134] 5.3 Protective effect of S100g signaling pathway-optimized MSC-Exo on antibiotic-damaged cells

[0135] The NCM460 cells were divided into 4 groups: ① control group; ② antibiotic group (treated with 200 μM clindamycin for 48 h); ③ antibiotic + low-dose exosome group (1×10 6 exosomes / ml); ④ antibiotic + high-dose exosome group (1×10 10 exosomes / ml). The protective effect of exosomes was evaluated by the following indicators:

[0136] 1. Cell viability: The CCK-8 assay results showed that compared with the control group, the cell viability of the antibiotic group was significantly reduced to 59.5±3.2% (p<0.001); the cell viability of the low-dose exosome group was restored to 72.3±4.5% (p<0.05 vs the antibiotic group); the cell viability of the high-dose exosome group was restored to 83.7±5.1% (p<0.01 vs the antibiotic group).

[0137] 2. Cell migration ability: The Transwell migration assay showed that compared with the control group, the number of migrated cells in the antibiotic group was significantly reduced (421±33.25 vs 149±34.75, p<0.001); the number of migrated cells in the low-dose exosome group increased (169.2±22.23, p>0.05 vs the antibiotic group); the number of migrated cells in the high-dose exosome group increased significantly (261.8±58.07, p<0.01 vs the antibiotic group).

[0138] 3. Intestinal barrier protein expression: Western blot and immunofluorescence analysis showed that compared with the control group, the expression levels of tight junction proteins ZO-1, Occludin, and Claudin-1 in the antibiotic group were significantly reduced (p<0.01); treatment with low-dose and high-dose exosomes could partially restore the expression of these proteins, and the high-dose exosome group had the best restoration effect, with a significant difference compared with the antibiotic group (p<0.05).

[0139] 5.4 Regulation of Autophagy by S100g Signaling Pathway Optimized MSC-Exo

[0140] To explore the regulatory mechanism of S100g signaling pathway optimized MSC-Exo on autophagy, we conducted the following experiments:

[0141] 1. Detection of autophagy-related proteins: Western blot analysis showed that compared with the control group, the ratio of LC3 II / I increased, the expression of p62 decreased, and the ratio of p-MTOR / MTOR decreased in the antibiotic group, indicating an increase in autophagy level; treatment with low-dose exosomes could partially reverse these changes; treatment with high-dose exosomes could significantly decrease the ratio of LC3 II / I (p<0.05 vs antibiotic group), increase the expression of p62 (p<0.05 vs antibiotic group), and increase the ratio of p-MTOR / MTOR (p<0.01 vs antibiotic group), suggesting that exosomes could effectively inhibit excessive autophagy induced by antibiotics.

[0142] 2. Observation of autophagosomes: Transmission electron microscopy showed that the number of autophagosomes / autophagolysosomes in cells of the antibiotic group increased significantly, and mitochondrial damage was also visible; the number of autophagosomes in the exosome treatment group decreased, and the mitochondrial morphology improved, with the most significant improvement in the high-dose group.

[0143] 3. Detection of apoptosis: Flow cytometry analysis with Annexin V / PI double staining showed that the apoptosis rate in the antibiotic group increased significantly (5.7±0.32% vs 10.1±0.63%, p<0.001); the apoptosis rate in the low-dose exosome group decreased slightly (9.2±0.56%, p>0.05 vs antibiotic group); the apoptosis rate in the high-dose exosome group decreased significantly (7.28±0.64%, p<0.01 vs antibiotic group).

[0144] 5.5 Verification of the Role of S100g / MTOR Signaling Pathway in the Protective Effect of Exosomes

[0145] To further confirm the importance of the S100g / MTOR signaling pathway in the protective effect of exosomes, we designed an S100g knockdown experiment. The expression of S100g in NCM460 cells was knocked down by siRNA technology (si-S100g), and then exosomes were administered in the presence of antibiotics. The results showed:

[0146] 1. In the case of inhibited S100g expression, the protective effects of exosomes against cell viability decline, weakened migration ability, and decreased expression of tight junction proteins caused by antibiotics were significantly weakened.

[0147] 2. Similarly, S100g knockdown also significantly attenuated the regulatory effect of exosomes on autophagy, as manifested by the maintenance of the LC3II / I ratio at a relatively high level, the maintenance of p62 expression at a relatively low level, and the insignificant recovery of the p-MTOR / MTOR ratio.

[0148] 3. When treated with the MTOR-specific inhibitor PQR620, even when S100g was overexpressed, the protective effect of exosomes was significantly inhibited, confirming that MTOR is a downstream molecule through which S100g exerts its function.

[0149] As Figure 6 shown, antibiotic treatment led to enhanced autophagy in cells (increased LC3II / I ratio, decreased p62 expression, and decreased p-MTOR / MTOR ratio) and increased apoptosis (increased expression of Bax and Bak) ( Figure 6 a). Exosome treatment effectively inhibited excessive autophagy and apoptosis ( Figure 6 b - c), and transmission electron microscopy confirmed that exosomes reduced the number of autophagosomes in cells and improved mitochondrial morphology ( Figure 6 d).

[0150] To further verify the key role of S100g in the protective effect of exosomes, the expression of S100g in NC M460 cells was knocked down by siRNA ( Figure 7 a - b). The results showed that S100g knockdown significantly attenuated the protective effect of exosomes against antibiotic-induced cell damage ( Figure 7 c - f), confirming the key role of the S100g / MTOR signaling pathway in the therapeutic effect of exosomes.

[0151] In summary, the in vitro experimental results showed that S100g signaling pathway-optimized MSC-Exo could inhibit antibiotic-induced excessive autophagy through the S100g / MTOR signaling pathway, protect intestinal epithelial cells from damage, and maintain intestinal mucosal barrier function.

[0152] Example 6: Functional verification of S100g signaling pathway-optimized MSC-Exo (in vivo experiment)

[0153] This example aims to evaluate the effect of S100g signaling pathway-optimized MSC-Exo in the in vivo treatment of antibiotic-induced intestinal mucosal barrier damage.

[0154] 6.1 Establishment of an animal model of antibiotic-related intestinal barrier damage

[0155] Six-week-old male C57BL / 6 mice (weighing 20-22 g) were selected. The mice were randomly divided into 4 groups, with 6 mice in each group: ① Control group (gavage with normal saline); ② Antibiotic group (gavage with 250 mg / kg clindamycin hydrochloride once a day for 28 consecutive days); ③ Antibiotic + low-dose exosome group (antibiotics same as ②, starting from the 15th day, intraperitoneal injection of 1×10 6 exosomes every other day); ④ Antibiotic + high-dose exosome group (antibiotics same as ②, starting from the 15th day, intraperitoneal injection of 1×10 10 exosomes every other day).

[0156] During the experiment, the body weight changes, general status, and fecal traits of the mice were monitored. On the 28th day of the experiment, fluorescein isothiocyanate-labeled dextran (FITC-D, 4 kDa) was used for gavage to evaluate intestinal permeability, and the colon tissues of the mice were collected for subsequent analysis.

[0157] 6.2 Effects of S100g signaling pathway-optimized MSC-Exo on mouse body weight and colon length

[0158] The experimental results showed that compared with the control group, the body weight growth rate of the mice in the antibiotic group was significantly reduced (1.15±0.04 vs 1.09±0.04, p<0.01); both low-dose and high-dose exosome treatments could improve the body weight growth rate (1.10±0.04 vs 1.12±0.04), and there was a significant difference between the high-dose group and the antibiotic group (p<0.05).

[0159] At the same time, the colon length of the mice in the antibiotic group was significantly shorter than that of the control group (9.7±0.61 cm vs 8.53±0.21 cm, p<0.05); the colon length in the exosome treatment group recovered (low dose: 8.77±0.21 cm; high dose: 9.33±0.25 cm), and there was a significant difference between the high-dose group and the antibiotic group (p<0.05).

[0160] 6.3 Effects of S100g signaling pathway-optimized MSC-Exo on intestinal permeability

[0161] The leakage of FITC-D was detected by an in vivo mouse imaging system to reflect the changes in intestinal permeability. The results showed that compared with the control group, the total radiation rate of the mice in the antibiotic group increased significantly (2.65×10 11 ±5.62×10 10 vs 4.94×10 11 ±9.24×10 10 , p<0.05), indicating an increase in intestinal permeability; the total radiation rate decreased in the exosome treatment group (low dose: 4.15×10 11 ±9.96×10 10; High dose: 2.74×10 11 ±7.6×10 10 ), where there was a significant difference between the high-dose group and the antibiotic group (p < 0.05).

[0162] Effect of S100g signaling pathway-optimized MSC-Exo on intestinal mucosal tissue morphology

[0163] HE staining results showed that antibiotic treatment led to the interruption of the continuity of mouse colonic epithelial cells, abnormal crypt structure, capillary bleeding, and a decrease in goblet cells; both low-dose and high-dose exosome treatments could alleviate these pathological changes to a certain extent, and the intestinal mucosal tissue morphology in the high-dose treatment group was close to that of the normal control group.

[0164] Effect of S100g signaling pathway-optimized MSC-Exo on the expression of intestinal barrier proteins

[0165] Immunohistochemical results showed that compared with the control group, the expression levels of ZO-1 (p < 0.01), Occludin (p < 0.05), and Claudin-1 (p < 0.001) in the colonic tissues of mice in the antibiotic group were significantly decreased; exosome treatment could partially restore the expression of these proteins, and the high-dose group had the best restoration effect, with a significant difference compared with the antibiotic group (p < 0.01).

[0166] Western blot and RT-qPCR results further confirmed the above findings, indicating that S100g signaling pathway-optimized MSC-Exo could effectively protect against the decrease in the expression of intestinal barrier proteins caused by antibiotics.

[0167] Effect of S100g signaling pathway-optimized MSC-Exo on the intestinal flora

[0168] The composition of mouse fecal flora was analyzed by 16S rRNA gene sequencing. The results showed that antibiotic treatment significantly reduced the diversity and richness of the intestinal flora, as manifested by a significant decrease in the observed_features index and Chao1 index (p < 0.01); the Shannon index and Simpson index also decreased, but there was no significant difference (p > 0.05).

[0169] Comparing the microbial composition among groups, the relative abundance of Proteobacteria in the antibiotic group increased, while the relative abundances of Bacteroidota and Actinobacteriota decreased. However, exosome treatment only partially improved these changes, mainly manifested as the inhibition of the excessive growth of Proteobacteria and less impact on the changes of other flora.

[0170] Through LEfSe analysis, it was found that compared with the control group, the potential pathogenic bacteria (such as Enterobacteriaceae) in the intestine of the antibiotic group increased, while the beneficial bacteria (such as Trichococcus and Enterorhabdus) decreased; exosome treatment could partially correct these changes, but the effect was limited.

[0171] A mouse intestinal mucosal barrier injury model was established by using clindamycin hydrochloride (250 mg / kg, gavage daily for 28 days), and treated by intraperitoneal injection of exosomes at low dose (1×10 6 per mouse) and high dose (1×10 10 per mouse) every other day ( Figure 2 a).

[0172] These results indicate that S100g signaling pathway-optimized MSC-Exo has a certain regulatory effect on antibiotic-induced intestinal flora disorder, but the main treatment mechanism may not depend on the direct regulation of the flora.

[0173] 6.7 Verification of the role of S100g / MTOR signaling pathway in in vivo treatment

[0174] To explore the role of S100g / MTOR signaling pathway in in vivo treatment, transcriptome sequencing of mouse colon tissues was performed. Differential gene analysis showed that compared with the control group, there were 319 up-regulated genes and 369 down-regulated genes in the antibiotic group; there were 185 differentially expressed genes in the exosome treatment group compared with the antibiotic group, of which 71 were down-regulated and 114 were up-regulated.

[0175] Through KEGG and GO analysis, it was found that the differential genes were mainly enriched in the autophagy and apoptosis-related pathways, and S100g might be the key target gene of exosome action. The RT-qPCR verification results showed that antibiotic treatment significantly reduced the expression of S100g in mouse colon tissues, while exosome treatment could significantly up-regulate the expression of S100g (p<0.01).

[0176] Western blot analysis showed that compared with the control group, the LC3 II / I ratio in the colon tissues of mice in the antibiotic group increased, the expression of p62 decreased, and the p-MTOR / MTOR ratio decreased, indicating an increase in autophagy level; exosome treatment could partially reverse these changes, indicating that exosomes inhibit excessive autophagy by regulating the S100g / MTOR signaling pathway, thereby protecting the intestinal mucosal barrier.

[0177] As Figure 2 shown, exosome treatment could significantly improve the weight gain decline ( Figure 2 b), increased intestinal permeability ( Figure 2 c), shortened colon length ( Figure 2 d), and intestinal tissue pathological damage ( Figure 2e) and decreased intestinal barrier protein expression ( Figure 2 f-h).

[0178] Meanwhile, the effect of exosomes on the intestinal flora was analyzed by 16S rRNA gene sequencing ( Figure 3 ), and the results showed that exosomes had a certain regulatory effect on the decreased flora diversity and structural changes caused by antibiotics, but the main treatment mechanism might not depend on flora regulation.

[0179] Through transcriptomic analysis of mouse colon tissues ( Figure 5 ), it was found that S100g might be a key target gene for the action of exosomes ( Figure 5 a-c). KEGG and GO analyses showed that the differentially expressed genes were mainly enriched in the autophagy and apoptosis-related pathways ( Figure 5 d-e). The expression of S100g in NCM460 cells was verified in vitro ( Figure 5 f-g), further supporting the key role of the S100g / MTOR pathway in exosome treatment of antibiotic-induced intestinal injury.

[0180] In summary, the in vivo experimental results showed that S100g signaling pathway-optimized MSC-Exo could inhibit antibiotic-induced excessive autophagy through the S100g / MTOR signaling pathway, alleviate intestinal mucosal barrier injury, and thus improve antibiotic-related intestinal adverse reactions.

[0181] Example 7: Preparation Development of S100g Signaling Pathway-Optimized MSC-Exo

[0182] This example aims to develop a stable and efficient preparation of S100g signaling pathway-optimized MSC-Exo to lay a foundation for clinical application.

[0183] 7.1 Optimization of Liquid Preparation Formulation

[0184] Through orthogonal experimental design, the best liquid preparation formulation was screened. The main factors investigated included: ① buffer system (PBS pH 7.2 - 7.6 vs HEPES pH 7.2 - 7.6); ② stabilizer (0.5 - 1% HSA vs 5 - 10% trehalose); ③ auxiliary components (with or without 0.5 mM EDTA and 0.01% Poloxamer 188).

[0185] By evaluating indicators such as the particle size distribution, Zeta potential, protein content, morphological maintenance, and functional activity of exosomes under different formulations, the optimal liquid formulation was determined as: PBS (pH 7.4), 0.5% HSA, 5% trehalose, 0.5 mM EDTA, and 0.01% Poloxamer 188. Under this formulation, exosomes can be stably stored at 4°C for 7 days and at -80°C for 6 months, and the retention rate of functional activity is >90%.

[0186] 7.2 Optimization of the lyophilized formulation process

[0187] To further improve the long-term stability of exosomes, a lyophilized formulation was developed. The lyophilization process was optimized by investigating different pre-freezing temperatures (-30°C vs -45°C), combinations of lyoprotectants (5 - 10% trehalose ± 5% mannitol ± 1% HSA), and lyophilization parameters (primary drying temperature -25°C vs -30°C, secondary drying temperature 10°C vs 20°C).

[0188] The finally determined lyophilization process parameters were: pre-freezing temperature -45°C, pre-freezing time 3 hours; lyoprotectant 7.5% trehalose + 5% mannitol + 1% HSA; primary drying parameters: shelf temperature -25°C, vacuum degree 20 Pa, time 30 hours; secondary drying parameters: shelf temperature gradually increased to 15°C, vacuum degree 5 Pa, time 10 hours.

[0189] Under this process, the lyophilized exosome product was white, loose, and blocky, the reconstitution time was <30 seconds, and the reconstituted solution was clear and without particles. The particle size distribution, marker expression, and functional activity of the reconstituted exosomes were not significantly different from those before lyophilization, and they could be stably stored at 2 - 8°C for 24 months.

[0190] 7.3 Establishment of the quality standard for exosome formulations

[0191] To ensure the quality and batch consistency of exosome formulations, a complete quality control system was established:

[0192] 1. Physicochemical indicators: Appearance (liquid: colorless to light yellow clear liquid; lyophilized: white to off-white loose block); pH (7.2 - 7.6); osmotic pressure (280 - 320 mOsm / kg); particle size distribution (average diameter 80 - 120 nm, PDI < 0.3); reconstitution property (reconstitution time of lyophilized product < 30 seconds).

[0193] 2. Biological indicators: Exosome markers (positive for CD9, CD63, Alix; negative for organelle markers); particle concentration (1×10 10 -1×10 12Particles / ml); S100g-MBD content (stable proportion of total protein); Functional indicators (MTOR phosphorylation activation increased by ≥50% compared to the reference exosomes; autophagy inhibitory ability decreased the LC3 II / I ratio by ≥40%).

[0194] 3. Purity and safety indicators: Foreign protein impurities (<5%); Residual DNA (<10 ng / ml); Sterility test (qualified); Mycoplasma detection (negative); Endotoxin (<0.5 EU / ml); Residual antibiotics and transfection reagents (below the detection limit).

[0195] 4. Stability indicators: Stable at room temperature (25°C) for 24 hours; Stable at 2-8°C for 7 days; Stable at -20°C for 3 months; Stable at -80°C for 6 months; Stable at 2-8°C for 24 months after lyophilization; The activity decreased by <20% after 3 freeze-thaw cycles.

[0196] Through the above quality control system, the quality and batch consistency of the S100g signaling pathway-optimized MSC-Exo preparation are ensured, providing a reliable guarantee for clinical applications.

[0197] Example 8: Optimization of the administration regimen of S100g signaling pathway-optimized MSC-Exo

[0198] This example aims to optimize the administration regimen of S100g signaling pathway-optimized MSC-Exo and provide a reference for clinical applications.

[0199] 8.1 Comparison of administration routes

[0200] The pharmacokinetic characteristics and intestinal distribution of three administration routes, intravenous injection, intraperitoneal injection, and oral administration, were compared. PKH26-labeled exosomes were administered to antibiotic-treated mice through different routes, and blood and intestinal tissues of various parts were collected at different time points (0.5 h, 2 h, 6 h, 12 h, 24 h), and the distribution of exosomes was detected by fluorescence imaging and flow cytometry.

[0201] The results showed that after intravenous injection, exosomes were rapidly distributed throughout the body, the peak concentration of exosomes in plasma appeared at 0.5 h, and the half-life was about 2-3 h, and only a small amount of exosomes reached the intestine; after intraperitoneal injection, exosomes gradually entered the blood circulation, the peak plasma concentration appeared at 2 h, and the half-life was about 6-8 h, and more exosomes reached the intestine and were more evenly distributed; oral enteric-coated preparations (prepared using Eudragit L100) could make exosomes directly reach the colon, but the bioavailability was low, about 30% of that of intraperitoneal injection.

[0202] Considering the distribution characteristics and operation convenience, intraperitoneal injection was determined as the preferred administration route, intravenous injection as the alternative route, and oral enteric-coated preparations could be used as a supplement for maintenance therapy.

[0203] 8.2 Dose - effect relationship study

[0204] The therapeutic effect of exosomes was studied by gradient doses (1×10 8 , 1×10 9 , 1×10 10 , 5×10 10 , 1×10 11 particles / kg). Mice treated with antibiotics for 14 days were divided into each dose group, and exosomes were intraperitoneally injected every other day for 14 consecutive days. The therapeutic effect was evaluated by monitoring body weight changes, FITC - D intestinal permeability, and the expression of tight junction proteins in colon tissues.

[0205] The results showed that the therapeutic effect was positively correlated with the dose. A weak therapeutic effect began to show from 1×10 9 particles / kg, and a significant therapeutic effect was achieved at 1×10 10 particles / kg. The effects of 5×10 10 and 1×10 11 particles / kg were similar and the best. Considering the therapeutic effect and economy comprehensively, 1×10 10 - 5×10 10 particles / kg was determined as the optimal therapeutic dose.

[0206] 8.3 Optimization of administration frequency

[0207] The therapeutic effects of different administration frequencies (daily, every other day, every 3 days, weekly) were compared. At the same cumulative dose, the therapeutic effect of the group administered every other day was the best, followed by the group administered every 3 days, and the effects of daily and weekly administrations were relatively weak. This may be related to the pharmacokinetics and targeted distribution characteristics of exosomes in the body.

[0208] 8.4 Study on treatment duration

[0209] The therapeutic effects of different treatment courses (1 week, 2 weeks, 3 weeks, 4 weeks) were studied. The results showed that as the treatment time prolonged, the therapeutic effect gradually increased, reaching a significant effect at 2 weeks, and further improving but with a smaller increase at 3 - 4 weeks. Follow - up after drug withdrawal found that the effect of 2 - week treatment could be maintained for about 1 week, and the effect of 4 - week treatment could be maintained for about 2 - 3 weeks.

[0210] Considering all factors comprehensively, the optimal administration plan for S100g signaling pathway - optimized MSC - Exo was determined as: intraperitoneal injection, dose 1×10 10 - 5×10 10 particles / kg, once every other day, treatment course 2 - 4 weeks. This plan showed the best therapeutic effect in the mouse model of antibiotic - related intestinal mucosal barrier injury.

[0211] Example 9: Comparative study of S100g signaling pathway-optimized MSC-Exo and other treatment methods

[0212] This example aims to compare the therapeutic effects of S100g signaling pathway-optimized MSC-Exo and other treatment methods on antibiotic-related intestinal mucosal barrier injury.

[0213] 9.1 Comparison with probiotic treatment

[0214] A commercially available probiotic preparation (containing Bifidobacterium, Lactobacillus, and Streptococcus faecalis, administered at 1×10 9 CFU / animal) was selected for comparison with exosomes (intraperitoneally injected at 1×10 10 particles / kg every other day). The results showed that during antibiotic treatment, the therapeutic effect of probiotic treatment was limited, possibly related to the direct inhibition of probiotic growth by antibiotics; while the therapeutic effect of exosome treatment was significant and not directly affected by antibiotics. After antibiotic withdrawal, both treatment methods were effective, but exosomes restored the intestinal mucosal barrier function and the expression of tight junction proteins faster and more significantly (p<0.05).

[0215] 9.2 Comparison with ordinary MSC-Exo

[0216] The therapeutic effects of S100g signaling pathway-optimized MSC-Exo and ordinary MSC-Exo (exosomes produced by unmodified hucMSCs) were compared at the same dose. The results showed that S100g signaling pathway-optimized MSC-Exo was superior to ordinary MSC-Exo in improving intestinal permeability, restoring the expression of tight junction proteins, and regulating autophagy, and the therapeutic effect was increased by approximately 65% (p<0.01).

[0217] 9.3 Comparison with autophagy inhibitor

[0218] The therapeutic effects of S100g signaling pathway-optimized MSC-Exo and the classical autophagy inhibitor 3-methyladenine (3-MA, intraperitoneally injected at 15 mg / kg daily) were compared. The results showed that 3-MA could effectively inhibit antibiotic-induced excessive autophagy, but had limited protective effects on the intestinal mucosal barrier and had certain adverse reactions; while S100g signaling pathway-optimized MSC-Exo could not only inhibit excessive autophagy, but also promote intestinal epithelial cell proliferation and migration, and restore the expression of tight junction proteins, and the comprehensive therapeutic effect was significantly superior to 3-MA (p<0.01).

[0219] 9.4 Comparison with recombinant S100g protein

[0220] The therapeutic effects of S100g signaling pathway-optimized MSC-Exo and recombinant S100g protein (50 μg / kg intraperitoneally injected daily) were compared. The results showed that although the recombinant S100g protein had certain therapeutic effects, due to limitations such as short half-life in the body, poor stability, and difficulty in penetrating the mucus layer, its therapeutic effect was significantly lower than that of exosomes (p<0.001). At the same dose, the therapeutic effect of S100g signaling pathway-optimized MSC-Exo was 5-10 times that of the recombinant S100g protein.

[0221] In summary, compared with existing treatment methods, S100g signaling pathway-optimized MSC-Exo has significant advantages in treating antibiotic-related intestinal mucosal barrier injury, providing a new option for clinical application.

[0222] Example 10: Safety evaluation of S100g signaling pathway-optimized MSC-Exo

[0223] This example aims to comprehensively evaluate the safety of S100g signaling pathway-optimized MSC-Exo and provide safety guarantees for clinical application.

[0224] 10.1 Acute toxicity study

[0225] The acute toxicity of exosomes was evaluated using the single-dose escalation method. Mice were divided into 5 groups and given a single intraperitoneal injection of 5×10 10 、1×10 11 、5×10 11 、1×10 12 and 5×10 12 particles / kg of exosomes, and observed continuously for 14 days. The results showed that no death or obvious toxic reactions occurred in all dose groups of mice. Only the highest dose group (5×10 12 particles / kg) showed a transient decrease in activity within 24 hours after injection, and then returned to normal. No obvious abnormalities were found in blood biochemistry and histopathological examinations.

[0226] 10.2 Repeated-dose toxicity study

[0227] A 28-day repeated-dose toxicity study was conducted. Rats were divided into 3 groups and given an intraperitoneal injection of 1×10 10 (clinical equivalent dose), 5×10 10 (5 times the clinical equivalent dose), and 1×10 11 (10 times the clinical equivalent dose) particles / kg of exosomes every other day for 28 consecutive days. The results showed that no obvious toxic reactions occurred in all dose groups of rats, the body weight increased normally, and there were no abnormalities in food and water intake. No obvious abnormalities were found in hematology, blood biochemistry, and histopathological examinations.

[0228] 10.3 Preliminary assessment of reproductive and developmental toxicity

[0229] Female and male mice were respectively administered exosomes at a dose of 1×10 11 granules / kg by intraperitoneal injection every other day for 28 consecutive days, and then fertility assessment was carried out. The results showed that exosome treatment did not affect the mating behavior, conception rate, embryonic development and litter size of mice, indicating that exosomes had no obvious effect on reproductive function.

[0230] 10.4 Local tolerance study

[0231] The local tolerance of different administration routes was evaluated. The results showed that intraperitoneal injection, intravenous injection and oral administration all had good local tolerance, and no obvious local irritation or damage was observed.

[0232] 10.5 Immunogenicity assessment

[0233] The immunogenicity of exosomes was evaluated by detecting the level of anti-exosome antibodies in mouse serum by ELISA. The results showed that after repeated administration for 28 days, the level of anti-exosome antibodies in mouse serum increased slightly, but did not reach a significant level, and no allergic reaction or immune-related adverse reactions were observed, indicating that exosomes had low immunogenicity.

[0234] 10.6 Preliminary assessment of genotoxicity

[0235] The genotoxicity of exosomes was evaluated by the Ames test and the mouse bone marrow micronucleus test. The results showed that exosomes did not show genotoxicity at all tested doses.

[0236] 10.7 Preliminary assessment of long-term safety

[0237] Mice were followed up for up to 6 months, and no obvious adverse reactions or delayed toxicity were found, indicating that exosomes had good long-term safety.

[0238] In summary, the S100g signaling pathway-optimized MSC-Exo performed well in various safety assessments and had a wide safety window, providing safety guarantee for clinical application.

[0239] It should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A kind of umbilical cord mesenchymal stem cell exosome, characterized in that: The exosomes are derived from umbilical cord mesenchymal stem cells expressing the S100g-MTOR binding domain fusion protein. The diameter of the exosomes is 40-160 nm, and they express CD9, CD63, and Alix markers. The exosomes can inhibit excessive autophagy through the S100g / MTOR signaling pathway and are used for treating intestinal mucosal barrier damage caused by antibiotics.

2. The exosome according to claim 1, characterized in that: The S100g-MTOR binding domain fusion protein comprises the amino acid sequence of human-derived S100g protein, the flexible linker peptide sequence GGSGSGG, and the binding domain sequence composed of the amino acids at positions 2115-2150 of the MTOR protein. The S100g protein has two EF-hand calcium-binding domains.

3. The exosomes according to claim 1 or 2, characterized in that: The exosomes inhibit excessive autophagy, reduce apoptosis, and thus alleviate intestinal mucosal barrier damage caused by antibiotics by increasing the p-MTOR / MTOR ratio, decreasing the LC3 II / I ratio, and decreasing the expression of p62 in intestinal epithelial cells.

4. A method for preparing the exosomes according to any one of claims 1-3, characterized in that, It includes the following steps: (1) Construct an expression vector expressing the S100g-MTOR binding domain fusion protein; (2) Transfect the expression vector into human umbilical cord mesenchymal stem cells, and screen to obtain a cell line stably expressing the S100g-MTOR binding domain fusion protein; (3) Culture the cell line in serum-free medium for 48-72 hours; (4) Collect the culture supernatant and remove cells and cell debris by centrifugation; (5) Isolate and purify the exosomes by sucrose deuterium combined with ultra-high-speed centrifugation; (6) Characterize and verify the function of the obtained exosomes.

5. The method according to claim 4, wherein: In step (1), the expression vector is the pcDNA3.1(+) vector, which contains a human CMV enhancer / promoter, the coding sequence of the S100g-MTOR binding domain fusion protein, and an SV40 polyA termination signal; the S100g-MTOR binding domain fusion protein comprises the amino acid sequence of human-derived S100g protein, the flexible linker peptide sequence GGSGSGG, and the binding domain sequence composed of the amino acids at positions 2115-2150 of the MTOR protein.

6. The method according to claim 4, characterized in that: In step (2), the transfection is carried out by the liposome transfection method. After transfection, G418 at a concentration of 400-800 μg / ml is used for screening for 7-14 days to obtain a stably expressing cell line; the passage number of the human umbilical cord mesenchymal stem cells is 3-6 passages.

7. The method according to claim 4, characterized in that: In step (3), the serum-free medium is DMEM-low glucose medium, and 0.5-5% human platelet lysate (hPL) or 0.5-1% human serum albumin (HSA) can be added; the culture conditions are 37°C and 5% CO2, and 1% O2 hypoxic pretreatment can be selectively carried out for 24-48 hours to increase the yield of exosomes.

8. The method according to claim 4, characterized in that: The sucrose deuterium oxide combined ultra-high speed centrifugation method described in step (5) includes: loading the supernatant treated by pre-ultracentrifugation (10,000 g, 30 minutes) onto the top of a 10-60% sucrose density gradient, centrifuging at 120,000 g for 16-18 hours, collecting exosomes in the density range of 1.13-1.19 g / ml, diluting with PBS and precipitating by centrifuging at 120,000 g for 70 minutes. The obtained exosomes are resuspended with PBS and filtered through a 0.22 μm filter.

9. Use of the exosomes according to any one of claims 1-3 in the preparation of a medicament for treating antibiotic-related intestinal barrier injury, characterized in that: The exosomes are administered at a dose of 1×10 10 -1×10 11 particles / kg, once every 1 - 3 days, and the treatment course is 2 - 4 weeks.

10. The application according to claim 9, characterized in that: The administration route is intraperitoneal injection, intravenous injection or oral administration of enteric preparations; the antibiotic-related intestinal barrier damage includes antibiotic-related diarrhea, antibiotic-related intestinal inflammation, increased intestinal permeability caused by antibiotics, and decreased expression of tight junction proteins caused by antibiotics.

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