Application of human umbilical cord mesenchymal stem cell exosome in promotion of lung development and verification method

Abstract

The invention discloses application of a human umbilical cord mesenchymal stem cell exosome in promotion of lung development and a verification method, and relates to the technical field of biological medicine.The key point of the technical scheme is that the human umbilical cord mesenchymal stem cell exosome remarkably promotes the pulmonary alveolation process and improves the lung structure and function through rich Wnt5a, so that the lung development is promoted, and the lung development is promoted. The morbidity of bronchial lung dysplasia of premature infants is reduced; by activating a Wnt5a / ROCK1 pathway, the exosome enhances differentiation and self-repairing capabilities of alveolar epithelial cells, accelerates synthesis of alveolar surfactants, and improves lung functions and stability; as a biological treatment means, the exosome is high in safety and low in immunogenicity, side effects and complications of drugs are reduced, and clinical transformation potential for treating lung dysplasia of premature infants is shown.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more specifically, to the application and verification method of human umbilical cord mesenchymal stem cell exosomes in promoting lung development. Background Art

[0002] Bronchopulmonary dysplasia (BPD) is the most common and severe chronic lung disease in premature infants, especially in extremely premature infants with a gestational age of less than 28 weeks. With the advancement of medical technology, the pathological characteristics of the new BPD have changed from early lung damage associated with mechanical ventilation and oxygen therapy to a disease characterized by simplified alveolar structure, reduced alveolar number, abnormal capillary development, and impaired lung parenchymal development. Therefore, existing traditional treatments have limited effects in the new BPD, and new treatments urgently need to focus on how to promote lung development and repair.

[0003] In human lungs, alveolar type II epithelial cells (AT2 cells) are key alveolar cell subtypes with the potential for self-renewal and differentiation, and play an important role in the occurrence and repair of BPD. AT2 cells can not only secrete pulmonary surfactant to maintain alveolar tension, but also differentiate into alveolar type I cells after alveolar damage and participate in the regeneration of lung tissue. Therefore, promoting the functional recovery and differentiation of AT2 cells is crucial for improving lung development in children with BPD.

[0004] In the past decade, stem cell therapy, especially human umbilical cord mesenchymal stem cells (hUC-MSCs), has shown significant therapeutic potential in promoting tissue repair and development. Exosomes derived from hUC-MSCs (hUC-MSCs-Exos), as mediators of the paracrine effect of stem cells, can play an important role in the process of tissue repair because they contain a variety of growth factors, cytokines, microRNAs and proteins. Exosomes are small vesicles wrapped by a double lipid membrane, carrying specific active ingredients of donor cells, which can affect the function of target cells by regulating intercellular signaling. However, there are no reports that human umbilical cord mesenchymal stem cell exosomes have the effect of promoting lung development, especially for bronchopulmonary dysplasia caused by premature birth. Summary of the invention

[0005] The purpose of the present invention is to provide an application and verification method of human umbilical cord mesenchymal stem cell exosomes in promoting lung development, so as to solve the problems in the prior art that there is a lack of effective treatment for bronchopulmonary dysplasia caused by premature birth and the mechanism of human umbilical cord mesenchymal stem cell exosomes in promoting lung development has not been fully studied.

[0006] The above technical objective of the present invention is achieved through the following technical solution: application of human umbilical cord mesenchymal stem cell exosomes in promoting lung development, and the preparation process of human umbilical cord mesenchymal stem cell exosomes comprises the following steps:

[0007] Umbilical cord cleaning and tissue cutting: The umbilical cord was cleaned with Dulbecco's phosphate buffered saline, cut longitudinally, the artery and vein were removed, and the soft gel tissue was cut into 1-3 mm 3 small pieces;

[0008] Human umbilical cord mesenchymal stem cell hucMSCs culture: tissue blocks were placed in 6-well plates and incubated with modified Eagle's medium-α containing 10% fetal bovine serum and penicillin / streptomycin at 37°C and 5% CO2 for 10-12 days. The umbilical cord tissue was removed and maintained in culture;

[0009] Serum-free medium treatment: Cultured hucMSCs were maintained in serum-free medium for 48 h;

[0010] Differential centrifugation: Sequential differential centrifugation removes cells, cell debris, and apoptotic remnants;

[0011] Ultracentrifugation: ultracentrifugation was performed at 120,000 × g for 1.5 h and 2 h to collect the exosome pellet;

[0012] Collection and suspension of exosome pellet: The exosome pellet was resuspended in 200 μL of Dulbecco's phosphate buffer for subsequent studies.

[0013] The specific method of maintaining culture is to continue to maintain culture with modified Eagle's medium-α after regularly replacing the culture medium and removing the umbilical cord tissue until the cells are expanded to the 3rd to 5th generation and the human umbilical cord mesenchymal stem cells are expanded to a 10-layer cell culture chamber.

[0014] The specific centrifugation process for removing cells, cell debris and apoptotic residues in the differential centrifugation step is as follows: centrifugation at 750g centrifugal force for 10 minutes, centrifugation at 3000g centrifugal force for 20 minutes, and centrifugation at 12000g centrifugal force for 45 minutes.

[0015] Another object of the present invention is to provide an application of the above-mentioned human umbilical cord mesenchymal stem cell exosomes in promoting lung development, wherein the identification method of the human umbilical cord mesenchymal stem cell exosomes is through transmission electron microscopy, nanoparticle tracking analysis and nanoflow cytometry, wherein the nanoflow cytometry analyzes the expression of exosome protein markers, and the specific expression includes the following: positive expression of CD9, CD63, and CD81; the transmission electron microscope is used to observe the morphological characteristics of the exosomes of human umbilical cord mesenchymal stem cells.

[0016] The specific steps of the transmission electron microscope observation are as follows:

[0017] Fixation: Samples were pre-fixed with 2.5% glutaraldehyde and then post-fixed with 1% osmium tetroxide;

[0018] Dehydration: The samples were gradually dehydrated with acetone, and the concentration gradient of the dehydrating agent was 30% → 50% → 70% → 80% → 90% → 95% → 100%;

[0019] Infiltration: Mix the dehydrating agent and Epon-812 embedding medium in a ratio of 3:1, 1:1, and 1:3, and infiltrate sequentially;

[0020] Embedding: Samples were embedded in pure Epon-812 embedding medium;

[0021] Semi-thin positioning and ultra-thin sectioning: The alveolar region of mouse lung tissue was selected under a light microscope, and ultra-thin sections of 60 to 90 nm were cut using an ultra-thin slicer and collected on a copper grid;

[0022] Staining: Sections were first stained with methenamine for 10 to 15 minutes and then with lead citrate for 1 to 2 minutes.

[0023] The present invention is further configured as follows: the human umbilical cord mesenchymal stem cell exosomes promote lung development by promoting the expression of related proteins, wherein the proteins include mature alveolar proteins SPB, SPC and ATP-binding cassette transporter A3.

[0024] The present invention is further configured as follows: compared with premature infants, full-term infants' umbilical cord mesenchymal stem cell exosomes can accelerate the lung development process by regulating the Wnt5a / ROCK1 pathway to promote the expression of RhoA.

[0025] Among them, the Wnt5a / ROCK1 pathway specifically refers to that the exosomes of umbilical cord mesenchymal stem cells of full-term infants directly regulate the phosphorylation of ROCK1 in lung tissue through naturally enriched Wnt5a, promote the autophagy of alveolar type II cells and the development of lamellar bodies, promote alveolarization and reduce the incidence of BPD in premature infants.

[0026] By adopting the above technical scheme, the present invention finds that the exosomes of umbilical cord mesenchymal stem cells of full-term infants are rich in Wnt5a, activating the atypical Wnt signaling pathway, especially the Wnt5a / ROCK1 pathway, promoting the differentiation, migration and self-repair ability of alveolar type II cells. Wnt5a directly interacts with ROCK1, accelerates the reorganization of the actin cytoskeleton by phosphorylating ROCK1, and further enhances the process of alveolar formation and maturation.

[0027] Exosomes from umbilical cord mesenchymal stem cells of full-term infants accelerate the synthesis of alveolar surfactant by promoting the formation of LBs and the expression of related proteins (SPC, SPB, ABCA3). The increase in surfactant can effectively maintain the surface tension of the alveoli, prevent alveolar collapse, and maintain the normal structure and function of the alveoli, which is particularly important for the lung development of premature infants.

[0028] Exosomes from umbilical cord mesenchymal stem cells of full-term infants improved the expression of RhoA, enhanced the expression of autophagy-related protein LC3B, promoted the development of LBs during the autophagy process of AT2 cells, contributed to the stability of alveolar cells and the production of pulmonary surfactant, and enhanced autophagy was closely related to the formation of LBs, indicating that full-term Exos further promoted the development and maturation of the lung through RhoA and autophagy mechanisms.

[0029] Exosomes from umbilical cord mesenchymal stem cells of full-term infants provide new ideas and potential therapies for the treatment of respiratory problems in premature infants, especially bronchopulmonary dysplasia (BPD), by improving alveolarization, accelerating alveolar maturation, and promoting the synthesis of surfactant. This mechanism reduces drug side effects and enhances the self-repair ability of lung tissue, allowing premature infants to recover normal lung function faster.

[0030] In summary, the present invention has the following beneficial effects:

[0031] 1. The human umbilical cord mesenchymal stem cell exosomes of the present invention significantly promote the process of alveolarization through their rich Wnt5a, accelerate the development and maturation of alveoli, and improve alveolar structure and lung function. This effect provides an effective intervention for lung development and respiratory problems in premature infants; human umbilical cord mesenchymal stem cell exosomes activate the Wnt5a / ROCK1 pathway, enhance the differentiation and self-repair ability of alveolar epithelial cells, thereby effectively promoting the repair of alveolar epithelium and improving the recovery effect after lung tissue damage;

[0032] 2. Human umbilical cord mesenchymal stem cell exosomes significantly accelerate the synthesis of alveolar surfactant and promote the expression of proteins related to alveolar surfactant, thereby reducing the surface tension of the alveolar wall, preventing alveolar collapse, and improving lung stability and functionality; human umbilical cord mesenchymal stem cell exosomes promote lung development, enhance lung repair, increase surfactant production and other mechanisms. Human umbilical cord mesenchymal stem cell exosomes have a significant effect on reducing respiratory complications in premature infants and bronchopulmonary dysplasia, reducing dependence on mechanical ventilation and oxygen therapy, and improving the quality of life of premature infants;

[0033] 3. As a biological treatment, human umbilical cord mesenchymal stem cell exosomes can replace some traditional drug treatments, thereby reducing drug-induced side effects and complications. Because they are derived from human umbilical cord mesenchymal stem cells, they are highly safe and have low immunogenicity. The application of human umbilical cord mesenchymal stem cell exosomes provides new ideas and strategies for the treatment of premature pulmonary dysplasia, demonstrates great potential for clinical transformation, and may become an emerging means of treating pulmonary dysplasia and other related diseases in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0035] Figure 1 This is a comparative analysis result of the phenotypes of full-term exosomes and premature exosomes in Example 2 of the present invention;

[0036] Among them, Figure 1 A: Comparison of transmission electron microscopy images of full-term exosomes and premature exosomes; Appendix Figure 1 B: Nanoparticle tracking analysis results of full-term exosomes and premature exosomes; Appendix Figure 1 C: Nanoflow technology identification of exosome surface markers; Appendix Figure 1 D, E: Schematic diagram of the effects of different storage conditions on the diameter and concentration of exosomes; Appendix Figure 1 F: Western blot showing the expression of exosome-related markers; Appendix Figure 1 G, H: Exosome uptake experiment shows that exosomes are captured by A549 cells; Appendix Figure 1 I: Confocal microscopy results;

[0037] Figure 2 This is a diagram showing the analysis results of the functional and structural changes of full-term exosomes in promoting fetal lung development in Example 3 of the present invention;

[0038] Among them, Figure 2 A: Schematic diagram of the effects of full-term exosomes and premature exosomes on cell growth; Figure 2 B: Schematic diagram of the experimental design of exosome injection during mouse pregnancy; Figure 2 C: Immunofluorescence detection results of α-SMA in E18.5 lung tissue; Appendix Figure 2 D: Hematoxylin-eosin staining results of lung tissues at E13.5 and E18.5; Appendix Figure 2E: Transmission electron microscopy observation results of E18.5 alveolar epithelial cells;

[0039] Figure 3 This is a result diagram of the full-term exosomes promoting the expression of proteins and genes related to lung development in Example 4 of the present invention;

[0040] Among them, Figure 3 A: Western blot detection of SPC and RhoA proteins in E13.5 lung tissue; Figure 3 B: Western blot detection of SPC and RhoA proteins in E18.5 lung tissue; Figure 3 C: Western blot detection of ABCA3 and SPB proteins in E13.5 and E18.5 lung tissues; Appendix Figure 3 D: Real-time PCR detection of SFTPC, RhoA and ABCA3 mRNA in lung tissue at E13.5; Appendix Figure 3 E: Real-time PCR detection of SFTPC, RhoA and ABCA3 mRNA in lung tissue at E18.5; Appendix Figure 3 F: Effects of full-term exosomes and premature exosomes on RhoA mRNA expression;

[0041] Figure 4 This is a proteomic analysis diagram of full-term exosomes and premature exosomes in Example 4 of the present invention;

[0042] Among them, Figure 4 A: Hierarchical clustering analysis of the proteome of full-term exosomes and premature exosomes; Appendix Figure 4 B: GO enrichment analysis of differentially expressed proteins; Appendix Figure 4 C: Bubble diagram of enriched pathways in KEGG enrichment analysis; Figure 4 D: Volcano plot of the screening results of differentially expressed proteins; Figure 4 E: Expression of the top 9 upregulated proteins in the full-term exosome group; Appendix Figure 4 F, G: Experimental results of WB experiment;

[0043] Figure 5 is the analysis of the interaction between Wnt5a and ROCK1 and its potential mechanism in lung development in Example 5 of the present invention;

[0044] Among them, Figure 5 Figure 1. Experimental results of ACo-IP experiment; Attachment Figure 5 B: Wen's analysis and identification results; Attachment Figure 5 C: AlphaFold’s experimental results; Attachment Figure 5 D: Validation results of Co-IP experiment;

[0045] Figure 6This is a diagram for analyzing the mechanism of 5-month-old exosomes promoting phosphorylation of ROCK1 and accelerating autophagy through Wnt5a in an embodiment of the present invention;

[0046] Among them, Figure 6 A: Effects of full-term and premature exosomes on the expression of ROCK1, p-ROCK1, Wnt5a and LC3B in lung tissue; Appendix Figure 6 B: Comparison of LC3B expression in full-term and premature exosome groups at E13.5 and E18.5 stages; Appendix Figure 6 C: Comparison of Wnt5a expression in full-term and premature exosome groups at E13.5 and E18.5; Appendix Figure 6 D: Comparison of ROCK1 expression in full-term and premature exosome groups at E13.5 and E18.5; Appendix Figure 6 E: Comparison of p-ROCK1 / ROCK1 ratios between term and preterm exosome groups at E13.5 and E18.5;

[0047] Figure 7 Schematic diagram of the mechanism of action of exosome Wnt5a in lung development in the embodiment of the present invention;

[0048] Figure 8 The results of the culture and identification of hucMSCs and the expression of their exosome markers in Examples 1 and 2 of the present invention are as follows;

[0049] Among them, Figure 8 A: Growth status of primary hucMSCs at different culture stages; Figure 8 B: Micrograph of hucMSCs progeny; Attachment Figure 8 C: Distribution diagram of exosomes in the cytoplasm; Appendix Figure 8 D, E: positive and negative expression of hucMSCs surface markers;

[0050] Fig. 9 is a schematic diagram of the experimental process of the interaction between Wnt5a and RhoA and the extraction and verification of exosomes in Example 5 of the present invention;

[0051] Among them, Fig. 9 A: Simplified flow chart of hucMSCs exosome extraction; Appendix Fig. 9 B: Transfection results of 293T cells; Fig. 9 C: h-AT2 cell transfection experiment results; Appendix Fig. 9 D: IP experimental results; Attachment Fig. 9 E: Proteomic analysis results. DETAILED DESCRIPTION

[0052] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0053] The human umbilical cords obtained for this study were from full-term and preterm infants at birth (all individuals and their parents provided informed consent to participate in this study, which was approved by the Medical Research Ethics Committee of West China Second Hospital, Sichuan University, Medical Research Ethics Approval No. 2022). All animal experiments were approved by the Chinese Ethics Committee and the Institutional Animal Care of West China Second Hospital and were performed in accordance with ethical standards and in animal care.

[0054] Experimental materials and equipment: phosphate-buffered saline (DPBS, Invitrogen, USA), modified Eagle's medium-α (MEM-α, Gibco, USA), 10% fetal bovine serum (FBS, Invitrogen, USA), carbon-coated grids (ElectronMicroscopy Sciences, USA), transmission electron microscope (JEM-1400FLASH, JP), sodium pentobarbital (IP), ethanol (EtOH), tissue dehydrator (Leica-ASP 6025, GER), phenylmethylsulfonyl fluoride (PMSF);

[0055] Example 1: Obtaining human umbilical cord mesenchymal stem cell exosomes (hucMSC-Exos)

[0056] Primary cell culture: The umbilical cord was cleaned with Dulbecco's phosphate buffered saline and then cut longitudinally to remove the artery and vein. The soft gel tissue was cut into 1-3 mm 3 Small pieces of cells were placed individually in 6-well plates, and modified Eagle's medium-α was supplemented with 10% fetal bovine serum and penicillin / streptomycin. The plates were incubated at 37°C in a humidified atmosphere of 5% CO2 for 10 to 12 days. Primary cells crawled out of the umbilical cord tissue on approximately the 10th day and the first day of the second generation (scale bar = 200 μm). Figure 8 As shown in A;

[0057] During the incubation period, supplemented MEM-α was added regularly until the umbilical cord tissue was finally removed, and after washing the plates three times, the cultures were maintained with MEM-α supplemented with 10% FBS and penicillin / streptomycin. The cells were expanded in the cell culture chamber, and the cells were passaged to the 3rd to 5th passages by digestion and successfully induced to form osteoblasts, adipocytes, and chondrocytes (scale bar = 100 μm). Figure 8 As shown in B. The present invention uses multiple umbilical cords, each umbilical cord produces an independent MSC population, and each independent in vivo experiment uses a different Exos preparation.

[0058] Exosome harvesting and isolation: The cultured hucMSCs were transferred to serum-free medium for 48 hours. The use of serum-free medium is intended to reduce the influence of exogenous exosomes or other contaminants in the serum and ensure that the isolated exosomes are from hucMSCs. The following differential centrifugation steps were used to gradually remove cells, cell debris and apoptotic residues: 750×g, 10 minutes, to remove cells; 3000×g, 20 minutes, to remove cell debris; 12000×g, 45 minutes, to remove apoptotic residues;

[0059] After completing the differential centrifugation to remove impurities, the exosomes are further separated and enriched by ultracentrifugation. The specific operation steps of ultracentrifugation are as follows:

[0060] The supernatant was placed in a Type 50.2 Ti rotor and centrifuged at 120,000 × g for 1.5 hours at 4°C. The collected exosome pellet was gently transferred to a SW41 Ti rotor and centrifuged at 120,000 × g and 4°C for 2 hours.

[0061] Gently resuspend the final exosome pellet in 200 μL of DPBS to ensure that the exosomes are evenly dispersed.

[0062] The present invention successfully extracted and cultured hucMSCs from the umbilical cord of healthy full-term and premature infants by the above method. In order to observe whether hucMSCs contain exosomes, the specific markers CD9 and CD81 of the exosome membrane were detected by IF and observed under a confocal microscope, indicating that exosomes can be distributed in the cytoplasm (scale bar = 50 μm), as shown in the attached figure. Figure 8 C; by detecting their osteogenic, adipogenic and chondrogenic differentiation identification, and by detecting their positive and negative markers by flow cytometry, it was shown that the markers CD105, CD73, CD44, and CD90 were positive, and the markers CD11b and CD31 were negative, as shown in the attached Figure 8 D, and exosomes were extracted from full-term infant umbilical cord mesenchymal stem cells tiUCMSCs and early-term infant umbilical cord mesenchymal stem cells piUCMCSs by ultracentrifugation, as shown in the attached Fig. 9 As shown in A.

[0063] Example 2: Identification of human umbilical cord mesenchymal stem cell exosomes (hucMSC-Exos)

[0064] The identification method of human umbilical cord mesenchymal stem cell exosomes is through transmission electron microscopy, nanoparticle tracking analysis and nanoflow cytometry. Nanoflow cytometry analyzes the expression of exosome protein markers, and the specific expressions include the following: CD9, CS63, and CD81 are positively expressed; transmission electron microscopy is used to observe the morphological characteristics of exosomes of human umbilical cord mesenchymal stem cells.

[0065] To assess the morphology of Exos, 5 μL of sample was adsorbed on carbon-coated grids for one minute. The grids were made hydrophilic by glow discharge for 30 seconds and any excess liquid was then carefully eliminated. The grids were subsequently washed with 1% hexamethylenetetramine for 15 seconds. After removing any remaining excess liquid, the morphology of the adsorbed Exos was examined using transmission electron microscopy.

[0066] The specific steps of transmission electron microscope observation are as follows:

[0067] Fixation: Samples were pre-fixed with 2.5% glutaraldehyde and then post-fixed with 1% osmium tetroxide;

[0068] Dehydration: The samples were gradually dehydrated with acetone, and the concentration gradient of the dehydrating agent was 30% → 50% → 70% → 80% → 90% → 95% → 100%;

[0069] Infiltration: Mix the dehydrating agent and Epon-812 embedding medium in a ratio of 3:1, 1:1, and 1:3, and infiltrate sequentially;

[0070] Embedding: Samples were embedded in pure Epon-812 embedding medium;

[0071] Semi-thin positioning and ultra-thin sectioning: The alveolar region of mouse lung tissue was selected under a light microscope, and ultra-thin sections of 60 to 90 nm were cut using an ultra-thin slicer and collected on a copper grid;

[0072] Staining: Sections were first stained with methenamine for 10 to 15 minutes and then with lead citrate for 1 to 2 minutes.

[0073] Next, images of the copper grids were collected using a JEM-1400FLASH transmission electron microscope (TOK, JP). Each copper grid was first observed under a low-power microscope to view the entire tissue, and then images of the observed area were collected to examine specific lesions.

[0074] Nanoparticle Tracking Analysis: The size and concentration distribution of Exos were carefully evaluated on a state-of-the-art Nanoparticle Tracking Analysis system using NTA (ZetaView, Particle Metrix, GER), following the methodology outlined previously. In the procedure, the sample was introduced into the pump and the Brownian motion of the nanoparticles was carefully monitored. Simultaneously, the particle size was determined according to the Stokes-Einstein equation, while the particle concentration in the solution was accurately calculated based on the number of tracked particles. Prior to analysis, the Exos samples were carefully diluted in vesicle-free ultrapure water to ensure optimal measurement conditions.

[0075] The results of the identification of fresh exosomes in both groups by the above-mentioned nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) showed a heterogeneous exosome population with an average diameter of 150 nm and demonstrated typical exosome morphological characteristics, with almost no difference between the two groups, as shown in the attached Figure 1 A, attached Figure 1 To further verify that exosomes are membrane structures rather than protein aggregates, we used nanoflow technology to detect typical exosome markers CD9, CD63, and CD81, as shown in the attached figure. Figure 1 C. In order to conduct more rigorous follow-up experiments, the present invention compared the exosome particles and diameters stored at three different temperatures, 4°C, -80°C and stored in liquid nitrogen for 7 days.

[0076] The present invention found that in the short term, there was no significant difference in the diameter of exosomes between the three temperatures. However, the particles in the 4°C group showed the most, as shown in the attached Figure 1 D, attached Figure 1 E, which provides evidence to support the stability of exosomes in subsequent experiments of the present invention. The present invention also uses Western blotting to show that Exos-related markers CD9, CD63 and TSG101 are expressed, while calnexin is negative, as shown in the attached Figure 1 As shown in F, there was almost no difference between the two groups.

[0077] In order to prove that both groups of exosomes can be taken up by cells, the present invention simultaneously used two membrane dyes and successfully proved that the exosomes themselves, rather than the membrane dyes, can be taken up by cells. The present invention observed no significant difference in cell uptake between the two groups of exosomes, as shown in the attached Figure 1 G, attached Figure 1 H. In addition, with the help of phalloidin, the present invention captures that exosomes can not only be taken up by cells, but also break through the nuclear membrane, as shown in the attached Figure 1 As shown in I.

[0078] Example 3: Construction of human umbilical cord mesenchymal stem cell exosomes (hucMSC-Exos) animal model

[0079] The experimental mice used in this study were SPF-grade C57BL / 6J mice, 8-12 weeks old female mice and 10-12 weeks old male mice, weighing 18-22 g, purchased from Jiangsu Zhidikang Biotechnology Co., Ltd. This animal experiment has been approved by the Experimental Animal Ethics Committee of West China Second Hospital, Sichuan University, and all operations were carried out in accordance with relevant animal management regulations. All experimental animals were raised in the SPF Experimental Animal Center of West China Second Hospital, Sichuan University.

[0080] Mice are more active at night and rest during the day, so they were chosen to be caged at 6-7 pm. At 8-9 am the next day, a 200 μl pipette tip was used to check for vaginal plugs. If a white plug was seen at the vaginal opening or the 200 μl pipette tip was obviously blocked, it was considered pregnant and marked as E0.5 day. E0.5-day-old female and male mice were divided into a control group, a premature exosome group, and a term exosome group (n=3).

[0081] The premature exosome group and the full-term exosome group were injected with 100 μL hucMSCs through the tail vein at E10.5 and E15.5, respectively. The exosome concentration was accurately measured before injection and injected at a concentration of 2×10^10 particles / kg. The control group was injected with an equal volume of DPBS for two consecutive days, and the fetal rat lungs were obtained by cesarean section at E13.5 and E18.5.

[0082] Lung parenchyma morphometry and immunofluorescence: The general morphology of lung tissue was observed by hematoxylin-eosin (H&E) staining. 5 μm thick lung sections were selected and imaged at 200× and 400× magnification using an Olympus BX53 microscope and calibrated by standard micrometer images. Immunofluorescence (IF) was used to study the expression and localization of specific markers. Fresh lung tissue sections were first deparaffinized and rehydrated, followed by antigen retrieval with 1× EDTA at high temperature and blocked with 5% donkey serum. Sections were incubated with the primary antibody α-SMA at 4°C overnight, then washed with 3% PBST and incubated with fluorescent dye-labeled secondary antibodies and DAPI. Finally, the sections were examined and images were recorded using a FV3000 confocal microscope.

[0083] After anesthesia by intraperitoneal injection of 60 mg / kg sodium pentobarbital, the lungs were flushed with phosphate buffer at a constant pressure of 20-25 cmH2O through the right ventricle. The left lung was carefully removed and stored at -80°C. The right lung was then inflated in situ with 4% paraformaldehyde, the pressure was stabilized at 15-20 cmH2O, and then immersed in 4% PFA overnight. After fixation, the lung tissue was transferred to 75% ethanol and dehydrated in a tissue dehydrator overnight. Subsequently, the tissue was embedded in paraffin, and the four parts of the right lung were embedded separately.

[0084] In order to verify the different effects of the two groups of Exos in lung development, the present invention first observed A549 cells by CCK-8 method and found that both groups of exosomes could promote proliferation, among which the full-term Exos group was more effective. Figure 2 A. In the present invention, two groups of exosomes were injected into mice through the tail vein at 10.5 days and 15.5 days of pregnancy, and fetal lung tissue was harvested 48 hours after the two injections, as shown in the attached Figure 2 As shown in B.

[0085] It is well known that the expression level of α-SMA increases as lung development progresses during lung tissue maturation. In the model of the present invention, it was found that in E18.5 lung tissue, after two full-term Exos treatments, the expression of α-SMA was significantly overexpressed, and the degree of alveolarization was higher, as shown in the attached Figure 2 C, attached Figure 2 As shown in D, it is inferred that full-term Exos have a potential role in promoting fetal lung development.

[0086] Subsequently, the present invention examined the ultrastructure of alveolar epithelial cells under a transmission electron microscope. Compared with the control group, the glycogen of AT2 cells in the fetal lung tissue of the full-term Exos group was significantly reduced, while the number of lamellar bodies increased and the structure was more complete, as shown in the attached Figure 2 As shown in E, the effect of promoting lung development was further confirmed.

[0087] Example 4: Human umbilical cord mesenchymal stem cell exosomes (hucMSC-Exos) promote the expression of lung development-related proteins

[0088] Characterization of exosome markers: The isolated exosomes were characterized by conventional methods. First, we used Western Blot (WB) analysis and selected CD9, CD63, and TSG101 as positive markers and Calnexin as a negative control to verify the presence of specific proteins in exosomes.

[0089] Western Blot (WB) analysis: In each tissue block (0.3 cm × 0.3 cm), lysis buffer containing PMSF, phostop and phosphatase inhibitors was used for treatment, and 500 μL of lysis buffer was added to each tissue. After the resulting mixture was homogenized, the lysate was collected and the precipitate was removed by centrifugation. Then, 30 μg of extracted protein was separated by electrophoresis on a 10-12% SDS-PAGE gel. The protein was transferred to a 0.45 μm PVDF membrane by electrophoresis and then blocked in a PBS solution containing 0.5‰ Triton-100 and 5% skim milk powder for 1 hour. The membrane was incubated with the primary antibody overnight at 4°C, and then treated with a secondary antibody conjugated to horseradish peroxidase. Finally, protein expression was detected and visualized by enhanced chemiluminescence.

[0090] To further distinguish exosomes from protein aggregates, nano-flow cytometry was then used to analyze exosome surface markers. After the concentration of the sample was determined by nanoparticle tracking analysis, it was adjusted to 1×10 7 The particles / mL were incubated with 5 μL of PE-conjugated CD9, PE-conjugated CD63, and APC-conjugated CD81 antibodies at room temperature in the dark for 45 minutes. Intermittent shaking was used during the incubation to ensure adequate mixing. For analysis, a Cytoflex flow cytometer was used, equipped with a 405 / 10 filter, high event rate, VSSC-H parameters to optimize the scattered signal, and the optical path was calibrated by particle beads. The instrument was cleaned with 1% sodium hypochlorite solution and rinsed with ultrapure water filtered by a 0.1 μm Millex-VV filter before detection. After the positive signal was detected, the relevant data were recorded. To verify the membrane structure of the exosomes, 0.1% Triton X-100 solution was introduced and analyzed again after incubation at room temperature for 1-2 minutes. The disappearance of the positive signal was accompanied by an increase in the background signal, indicating that the detected particles were exosomes rather than protein aggregates.

[0091] As the most important marker of AT2 cells, the expression level of SPC is highly correlated with the degree of alveolar development. In order to further determine whether full-term Exos can promote lung development, we detected the expression of SPC. The present invention found that in the full-term Exos group of Example 3, the protein expression of SPC increased at both E13.5 and E18.5, while the gene expression was first inhibited at E13.5, but accelerated at E18.5, as shown in the attached Figure 3 A, attached Figure 3 B and attached Figure 3 As shown in D, RhoA and its family are responsible for regulating lung endothelial and smooth muscle cells and are closely related to lung development.

[0092] In the results of the present invention, full-term Exos promoted the expression of RhoA in the early fetus, while premature Exos reduced the mRNA expression of RhoA. Figure 3 As shown in A, B and E. At the same time, as markers of AT2 cell development, the expression of ABCA3 and mature SPB increased in the full-term Exos group, as shown in the attached Figure 3 C, attached Figure 3 As shown in F.

[0093] In order to explore the possible mechanism of the difference between full-term Exos and premature Exos in promoting lung development, the present invention performed proteomic sequencing on these two groups of Exos and found differentially expressed proteins, such as the attached Figure 4As shown in A. By performing Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis on the differentially expressed proteins in the proteome sequencing results, it was found that the differentially expressed proteins were mainly involved in regulating the differentiation potential of stem cells and enriched the signaling pathways that regulate stem cell pluripotency, as shown in the attached figure. Figure 4 B, Attachment Figure 4 C. By further analyzing the data of differentially expressed proteins in this pathway, it was found that the expression of Wnt5a was significantly upregulated in the full-term Exos group, as shown in the attached Figure 4 As shown in D~G.

[0094] It is well known that the Wnt pathway plays a key role in maintaining cell stemness and fate determination, including canonical (Wnt / β-catenin dependent) and non-canonical (β-catenin independent) pathways. As a representative of the Wnt family that activates non-canonical pathways, Wnt5a can directly regulate stem cell differentiation fate through the Wnt5a / PCP and Wnt5a / Ca2+ pathways, and can also interact with the canonical Wnt pathway represented by Wnt3a. However, confirmation of this phenotype will require additional direct experimental evidence.

[0095] Example 5: Analysis of the interaction between Wnt5a and ROCK1 and its potential mechanism in lung development

[0096] The data in Example 4 show that Wnt5a may play a key role in the promotion of lung development by full-term exosomes (Exos). The specific mechanism of action of Wnt5a is shown in the attached Figure 7 As shown in the attached figure, the present invention first overexpressed Wnt5a-Flag in primary human alveolar type II epithelial cells (h-AT2). Fig. 9 B, Attachment Fig. 9 C, and protein co-immunoprecipitation (Co-IP) experiments were performed using Flag magnetic beads. Mass spectrometry analysis was performed as shown in the attached Figure 5 A and attached Fig. 9 As shown in Figure 2, compared with the IgG control group, the study found 900 protein peptides, and the top ten proteins were ranked and analyzed, as shown in the attached figure. Figure 5 B, to further explore its biological significance. After proteomic analysis, we found RhoA peptides in the IP group, indicating that Wnt5a can interact with RhoA. However, in the results shown in WB, there was no expression of RhoA in the IP group, as shown in the attached Fig. 9 As shown in E.

[0097] The present invention discovered the protein ROCK1, which is a protein serine / threonine kinase, also known as Rho-associated, coiled-coil containing protein kinase 1. The prediction by the Alphafold server showed that there may be an interaction between Wnt5a and ROCK1, such as the attached Figure 5 C. Co-IP and pull-down experiments further confirmed that Wnt5a can directly interact with ROCK1. Figure 5 As shown in D.

[0098] The immunoprecipitation and pull-down assay steps are as follows:

[0099] First, h-AT2 cells were transfected with MSC-EGFP-Wnt5a-flag plasmid. After 72 hours, proteins were extracted using NP40 lysis buffer supplemented with 1× phenylmethylsulfonyl fluoride (PMSF), PhosStop, and phosphatase inhibitors. At the same time, Flag beads and IgG beads were washed separately using NP40 lysis buffer. Protein samples were divided into three groups: Input, IgG, and IP groups. The corresponding beads were added to the IgG and IP groups, incubated overnight at 4°C, and the beads were retained and washed three times. After equal volumes and concentrations of recombinant human Wnt5a-GST protein and recombinant human ROCK1-His protein were mixed, they were divided into GST-Wnt5a+ROCK1-His (Input) group, IgG group, and GST pull-down group. IgG beads and GST beads were added to the IgG and GST pull-down groups, respectively, incubated overnight at 4°C, and then the beads were retained and washed three times for subsequent experiments.

[0100] ROCK1 is ubiquitously expressed in multiple rat tissues, including lung, liver, spleen, kidney, and testis. This is consistent with its function as a key regulator of cell motility, tumor cell invasion, and actin cytoskeletal organization. Therefore, the direct interaction between Wnt5a and ROCK1 is considered to be the core mechanism for the role of Wnt5a in alveolar development.

[0101] The above research results show that the expression level of RhoA in the full-term exosome (Exos) group is increased. ROCK1 has been shown to be able to interact with proteins such as RhoA. As a member of the serine / threonine kinase family, ROCK1 usually exercises its regulatory function through the phosphorylation pathway.

[0102] In order to further evaluate whether full-term Exos can mediate the phosphorylation of ROCK1, the present invention detected the expression of ROCK1, phosphorylated ROCK1 (p-ROCK1), Wnt5a and autophagy-related protein LC3B in fetal lung tissue. The results showed that the expression of p-ROCK1 and Wnt5a in the full-term Exos group was significantly increased. Figure 6 As shown, this suggests that term Exos may regulate lung development by promoting the phosphorylation of ROCK1.

[0103] It is well known that in alveolar type II epithelial cells (AT2 cells), lamellar bodies (LBs) are not only their characteristic structures, but also the surfactant they secrete plays a key role in alveolar expansion and morphological maintenance. Therefore, AT2 cells are considered to be the most important cell population in the occurrence and prognosis of bronchopulmonary dysplasia (BPD).

[0104] In recent years, many studies have shown that the development of lamellar bodies is closely related to the autophagy process of AT2 cells. Some studies even suggest that lamellar bodies may be a special type of autophagosome. In this study, we also observed that the expression of the autophagy-related biomarker LC3B was significantly upregulated in the lung tissue of the full-term exosome (Exos) group. Figure 6 This further supports the hypothesis that term Exos may regulate lung development by promoting the autophagic process.

[0105] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. The application of human umbilical cord mesenchymal stem cell exosomes in promoting lung development is characterized by: The preparation process of human umbilical cord mesenchymal stem cell exosomes comprises the following steps: Umbilical cord cleaning and tissue cutting: The umbilical cord was cleaned with Dulbecco's phosphate buffered saline, cut longitudinally, the artery and vein were removed, and the soft gel tissue was cut into 1-3 mm 3 small pieces; Human umbilical cord mesenchymal stem cell hucMSCs culture: tissue blocks were placed in 6-well plates and incubated with modified Eagle's medium-α containing 10% fetal bovine serum and penicillin / streptomycin at 37°C and 5% CO2 for 10-12 days. The umbilical cord tissue was removed and maintained in culture; Serum-free medium treatment: Cultured hucMSCs were maintained in serum-free medium for 48 h; Differential centrifugation: Sequential differential centrifugation removes cells, cell debris, and apoptotic remnants; Ultracentrifugation: ultracentrifugation was performed at 120,000 × g for 1.5 h and 2 h to collect the exosome pellet; Collection and suspension of exosome pellet: The exosome pellet was resuspended in 200 μL of Dulbecco's phosphate buffer for subsequent studies.

2. The use of human umbilical cord mesenchymal stem cell exosomes in promoting lung development according to claim 1, characterized in that: The specific method of maintaining culture is to continue to maintain culture with modified Eagle's medium-α after regularly replacing the culture medium and removing the umbilical cord tissue until the cells are expanded to the 3rd to 5th generation and the human umbilical cord mesenchymal stem cells are expanded to a 10-layer cell culture chamber.

3. The use of human umbilical cord mesenchymal stem cell exosomes in promoting lung development according to claim 1, characterized in that: The specific centrifugation process for removing cells, cell debris and apoptotic residues in the differential centrifugation step is as follows: centrifugation at 750g centrifugal force for 10 minutes, centrifugation at 3000g centrifugal force for 20 minutes, and centrifugation at 12000g centrifugal force for 45 minutes.

4. A use of human umbilical cord mesenchymal stem cell exosomes as claimed in any one of claims 1 to 3 in promoting lung development, characterized in that: The method for identifying human umbilical cord mesenchymal stem cell exosomes is to identify them through transmission electron microscopy, nanoparticle tracking analysis and nanoflow cytometry. The nanoflow cytometry analyzes the expression of exosome protein markers, and the specific expressions include the following: positive expression of CD9, CD63, and CD81; the transmission electron microscope is used to observe the morphological characteristics of exosomes of human umbilical cord mesenchymal stem cells.

5. The use of human umbilical cord mesenchymal stem cell exosomes in promoting lung development according to claim 4, characterized in that: The specific steps of the transmission electron microscope observation are as follows: Fixation: Samples were pre-fixed with 2.5% glutaraldehyde and then post-fixed with 1% osmium tetroxide; Dehydration: The samples were gradually dehydrated with acetone, and the concentration gradient of the dehydrating agent was 30% → 50% → 70% → 80% → 90% → 95% → 100%; Infiltration: Mix the dehydrating agent and Epon-812 embedding medium in a ratio of 3:1, 1:1, and 1:3, and infiltrate sequentially; Embedding: Samples were embedded in pure Epon-812 embedding medium; Semi-thin positioning and ultra-thin sectioning: The alveolar region of mouse lung tissue was selected under a light microscope, and ultra-thin sections of 60 to 90 nm were cut using an ultra-thin slicer and collected on a copper grid; Staining: Sections were first stained with methenamine for 10 to 15 minutes and then with lead citrate for 1 to 2 minutes.

6. The use of human umbilical cord mesenchymal stem cell exosomes in promoting lung development according to claim 1, characterized in that: The human umbilical cord mesenchymal stem cell exosomes promote lung development by promoting the expression of related proteins, including mature alveolar proteins SPB, SPC and ATP-binding cassette transporter A3.

7. The use of human umbilical cord mesenchymal stem cell exosomes in promoting lung development according to claim 1, characterized in that: Compared with the exosomes from premature infants, the exosomes from umbilical cord mesenchymal stem cells of full-term infants can accelerate the lung development process by regulating the Wnt5a / ROCK1 axis and promoting the expression of RhoA.

8. The use and verification method of human umbilical cord mesenchymal stem cell exosomes in promoting lung development according to claim 7, characterized in that: The Wnt5a / ROCK1 pathway is specifically that the exosomes of umbilical cord mesenchymal stem cells of full-term infants directly regulate the phosphorylation of ROCK1 in lung tissue through naturally enriched Wnt5a, promote the autophagy of alveolar type II cells and the development of lamellar bodies, promote alveolarization and reduce the incidence of BPD in premature infants.

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