Stable production of mesenchymal stromal cell exosome containing anti-inflammatory miRNA effective components, and preparation method and application thereof
The exosome production method constructed using hTERT immortalized human placental mesenchymal matrix cells has solved the problems of low production efficiency and low purity of exosomes, realizing large-scale production and high-quality stability of exosomes, reducing costs, and providing feasible technical support for their clinical application.
Patent Information
- Application Number
- CN202511113763.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-11
- Publication Date
- 2025-11-28
AI Technical Summary
In existing technologies, the production efficiency of mesenchymal matrix cell exosomes is low, the purity is low, and the cost is high, making it impossible to translate laboratory research into clinical applications. Furthermore, the heterogeneity of primary cell donors and the passage process lead to large batch-to-batch quality differences, making it difficult to meet the needs of large-scale production.
hTERT immortalized human placental mesenchymal stromal cells (iMSC022) were used as the source of exosome production. Immortalized cell lines were constructed through lentivirus transduction and cultured in serum-free medium. Exosomes were extracted by centrifugation, PEG precipitation and ultrafiltration concentration to ensure cell stability and high quality of exosomes.
This technology enables large-scale production of exosomes, reduces batch-to-batch quality variations, improves the stability and therapeutic activity of anti-inflammatory miRNAs, simplifies the production process, reduces costs, and provides a stable and sustainable cell source for the industrialization of exosomes.
Smart Images
Figure CN121022751A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, specifically to a method for the stable production of mesenchymal matrix cell exosomes containing effective anti-inflammatory miRNA components, and its preparation and application. Background Technology
[0002] Mesenchymal stromal cell (MSC)-derived exosomes (Exosomes) have shown significant potential in the treatment of inflammation-related diseases (such as pulmonary fibrosis and liver fibrosis) due to their rich content of bioactive components such as anti-inflammatory miRNAs. However, their clinical translation and industrial application still face core bottlenecks related to cell source and stability. MSCs from different donors exhibit significant heterogeneity. Even from the same tissue source (such as bone marrow, adipose tissue, or placenta), their cell composition and functional characteristics (such as pro-angiogenic capacity and immunomodulatory strength) vary due to differences in donor age and health status, directly leading to fluctuations in the types and amounts of anti-inflammatory miRNAs in the secreted Exosomes. Furthermore, primary MSCs have limited in vitro proliferation capacity, and increased passage times can easily lead to cell senescence or genetic instability, not only reducing Exo yield but also potentially altering the expression patterns of their anti-inflammatory components, resulting in batch-to-batch quality variations. To ensure the safety and efficacy of clinical applications, repeated testing and validation of multiple batches of Exo are required, significantly increasing the complexity and cost of the production process and severely hindering its large-scale application.
[0003] The large-scale preparation and purification of exosomes is another key obstacle to their industrialization. Primary MSCs typically rely on 2D adherent culture, resulting in low cell density and limited growth space, leading to extremely low Exosome yields that are insufficient to meet clinical dosage requirements. Serum components added to traditional culture systems to maintain cell viability can contaminate Exo secreted by MSCs, increasing the difficulty of subsequent purification and potentially introducing exogenous contaminants (such as foreign proteins and viruses), affecting the purity and safety of Exo. Furthermore, existing laboratory-scale purification techniques (such as ultracentrifugation and density gradient centrifugation) are cumbersome, time-consuming, and have small single-batch processing volumes, making industrial scale-up impossible. These process defects result in low Exo production efficiency and high costs, severely hindering its translation from laboratory research to clinical application.
[0004] Therefore, how to obtain high-quality, stable, and sustainable single-cell sources of exosomes, break through the cell proliferation limit, and achieve large-scale production of exosomes is an urgent problem to be solved. Summary of the Invention
[0005] The main objective of this invention is to propose a stable method for producing mesenchymal matrix cell exosomes containing anti-inflammatory miRNA active components, as well as their preparation and application. This aims to solve the problems of low production efficiency, low purity, and high cost of existing exosomes, which prevent the transformation from laboratory research to clinical application.
[0006] To achieve the above objectives, this invention proposes a method for the stable production of mesenchymal matrix cell exosomes containing effective anti-inflammatory miRNA components, comprising the following steps:
[0007] S1. Obtain placental tissue and isolate and culture primary placental mesenchymal matrix cells;
[0008] S2. Immortalized placental mesenchymal matrix cell line was constructed by transducing the hTERT gene with lentivirus;
[0009] S3. The immortalized placental mesenchymal matrix cell line is cultured in serum-free medium, and the cell supernatant is collected.
[0010] S4. The obtained supernatant was successively subjected to centrifugation, PEG precipitation and ultrafiltration concentration to extract exosomes.
[0011] Preferably, in step S1, the isolation and culture of primary placental mesenchymal matrix cells includes: cutting placental villus tissue into fragments of 1-2 mm in size, digesting with trypsin, and then inoculating and culturing until the cell confluence reaches 75-85%.
[0012] Preferably, in step S3, the serum-free culture medium is MSCBM culture medium containing 4% human platelet lysate.
[0013] The present invention also proposes a method for preparing mesenchymal matrix cell exosomes as described above.
[0014] Preferably, the mesenchymal matrix cell exosomes comprise miR-146a-5p, miR-29a-3p, miR-29b-3p, let-7b-3p, and miR-21-5p.
[0015] Preferably, the exosomes containing miR-146a-5p, miR-29a-3p, miR-29b-3p, let-7b-3p, and miR-21-5p have a particle size distribution between 50 and 200 nm.
[0016] The present invention also proposes a pharmaceutical composition in which the active ingredient is mesenchymal matrix cell exosomes as described above.
[0017] The present invention also proposes the application of mesenchymal matrix cell exosomes as described above in the preparation of antifibrotic drugs.
[0018] Preferably, the fibrosis includes pulmonary fibrosis, liver fibrosis, or skin fibrosis.
[0019] Preferably, the fibrosis is pulmonary fibrosis, and the application includes the following steps: injecting exosomes as described above into the trachea of rats, wherein the dose of exosomes is 5 × 10⁻⁶.6 ~5×10 8 Item / time.
[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0021] (1) This invention significantly overcomes the limitations of traditional primary cell lines by using hTERT immortalized human placental mesenchymal stromal cells (iMSC022) from a single donor as the source for exosome (Exo) production. Compared to primary mesenchymal stromal cells (MSCs), which require multiple donors to construct cell banks due to their limited proliferation capacity, iMSC022, after lentiviral transfection and antibiotic selection, exhibits high homogeneity and can be stably passaged for more than 60 generations while maintaining phenotypic and functional stability. This eliminates the impact of donor heterogeneity and tissue origin differences on Exo quality from the source, greatly reducing batch-to-batch variability. Simultaneously, the immortalization characteristic endows the cells with unlimited proliferation capacity, enabling large-scale Exo production by directly scaling up upstream cell culture. This solves the problem of low primary cell yields and difficulty in meeting clinical dosage requirements, providing a stable and sustainable cell source guarantee for the industrialization of Exo.
[0022] (2) This invention utilizes hTERT immortalization technology to regulate cell function, effectively enhancing the stability and therapeutic activity of anti-inflammatory miRNAs in Exo cells. Primary MSCs are prone to changes in miRNA expression profiles due to aging during in vitro passage, and may even release pro-inflammatory or pro-fibrotic miRNAs. However, iMSC022 can maintain a stable proliferation state for a long time, ensuring the continuous secretion of key therapeutic miRNAs (miR-146a-5p, miR-29a-3p, miR-29b-3p, let-7b-3p, miR-21-5p). At the same time, the immortalization process can specifically regulate the intracellular miRNA sorting mechanism, promoting the enrichment of the above-mentioned anti-inflammatory and anti-fibrotic miRNAs in Exo cells and reducing the load of harmful miRNAs, significantly improving the therapeutic targeting and efficacy of Exo cells, and providing a higher quality active component guarantee for the treatment of inflammation-related diseases (such as pulmonary fibrosis).
[0023] (3) This invention significantly improves the standardization level of Exo production while reducing production costs and operational complexity. Traditional primary MSC culture requires frequent changes in cell source, and long-term passage can easily lead to changes in cell clonal composition and functional decline, causing fluctuations in key Exo components (proteins, miRNAs, etc.), increasing the difficulty of standardization; while iMSC022 can maintain stable phenotype and secretory function for a long time, ensuring consistency between Exo batches and reducing the cumbersome process of multi-batch validation. In addition, immortalized cells can be continuously and stably expanded without the need for frequent preparation of primary cells, which greatly reduces the costs of culture batches, manpower, and time, simplifies the production operation process, provides economically feasible technical support for the transformation of Exo from laboratory research to clinical application, and promotes the standardized application of Exo-based therapies. Attached Figure Description
[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0025] Figure 1 Imaging images of isolated primary placental mesenchymal stromal cells (A) and iMSC022 (B) provided by this invention.
[0026] Figure 2 This is a diagram illustrating the construction of the lentiviral expression vector provided by the present invention.
[0027] Figure 3 This is a diagram showing the expression of positive markers in primary mesenchymal stromal cells and iMSC022 provided by the present invention.
[0028] Figure 4 This is a diagram showing the expression of negative markers in primary mesenchymal stromal cells and iMSC022 provided by the present invention.
[0029] Figure 5 Chromosomal karyotype characteristics of the immortalized iMSC022 cells provided by this invention.
[0030] Figure 6 The morphology diagram of the iMSC022 exosome provided by this invention.
[0031] Figure 7 Subgroup counting diagrams of exosome markers CD63, CD81 and CD9 provided for this invention.
[0032] Figure 8 Particle size distribution of exosome markers CD63, CD81, and CD9 provided by this invention.
[0033] Figure 9 Immunocolocalization imaging of exosome markers CD63, CD81 and CD9 provided for this invention.
[0034] Figure 10 Expression level maps of miR-146a-5p(A), miR-29a-3p(B), miR-29b-3p(C), let-7b-3p(D), and miR-21-5p(E) provided by this invention.
[0035] Figure 11 The image shows the HE staining results of the Wistar rat model provided by this invention.
[0036] Figure 12 The image shows the Masson staining results of the Wistar rat model provided by this invention.
[0037] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation
[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0039] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.
[0040] Abbreviations that need to be explained:
[0041] iMSC022: hTERT-immortalized human placental chorionic mesenchymal stromal cells (hPCMSCs), hereinafter referred to as iMSC022.
[0042] MSC: mesenchymal stromal cell.
[0043] hTERT: human telomerase reverse transcriptase.
[0044] Exo: Exosomes, Exosomes.
[0045] Example 1: Extraction and culture of human placental mesenchymal stromal cells
[0046] 1. Experimental materials
[0047] This study used placental tissue from a 27-year-old healthy woman who delivered at full term without any complications during her pregnancy. Informed consent was obtained from both parents before sample collection, and the placental collection protocol was approved by the Medical Ethics Committee of the 903 Hospital. All experimental procedures strictly adhered to medical ethical principles and the relevant provisions of the Declaration of Helsinki.
[0048] 2. Primary cell extraction procedure
[0049] (1) Sample processing: After delivery, the placenta was immediately placed in high-glucose DMEM medium containing 4500 mg / mL glucose, which contained an antibiotic mixture (0.1% gentamicin, 0.2% streptomycin and 0.12% penicillin).
[0050] (2) Washing and cutting: Use a syringe to draw PBS buffer to rinse the placental tissue repeatedly to remove blood clots, and then cut the placental villi tissue into fragments of 1-2 mm in size.
[0051] (3) Digestion treatment: Add 3-5 mL of trypsin solution to the tissue fragments and incubate at 37℃ and 5% CO2 for 30 min. After digestion, add an equal volume of complete culture medium containing 10% fetal bovine serum (FBS) and antibiotics to terminate the digestion reaction and obtain a mixture.
[0052] (4) Inoculation and culture: Transfer the mixture to a 50 mL centrifuge tube and let it stand for 3 min. After the partially digested tissue fragments precipitate, carefully remove the supernatant. Take 15-20 pieces of partially digested tissue fragments and inoculate them into a cell culture flask. Add an appropriate amount of DMEM + 10% FBS medium (the amount of medium added should be sufficient to fully wet the tissue fragments). Continue to culture at 37℃ for 2-3 days.
[0053] (5) Subsequent culture and observation: Starting from day 3, fresh culture medium was changed daily to remove non-adherent cells, and cell growth around tissue fragments was observed. After 11-14 days of culture, cell proliferation was observed daily using an inverted microscope. When cells migrated from the edge of the tissue fragments and reached approximately 80% confluence, primary chorionic mesenchymal stromal cells (PCMSCs) were harvested for subsequent flow cytometry characterization and immortalization studies.
[0054] 3. Primary and immortalized cell culture
[0055] (1) Culture conditions: All primary PCMSCs and immortalized placental mesenchymal stromal cells were cultured in MSCBM medium (6114021, Shanghai Dakowei, China) without EVs and serum, supplemented with 4% EV-free serum. Human Platelet Lysate (PLTGOLD500R, Sartorius, Germany) and 1% penicillin / streptomycin were used to incubate the cells at 37°C and 5% CO2.
[0056] (2) Passaging and Experimental Analysis: Cells were passaged every 48 hours. When the cell proliferation density reached 80%-90%, cells were separated using 0.5% Trypsin-EDTA (10X) (15400-054, Gibco) for passage and experimental analysis. Primary cells separated at 7 and 14 days were observed to obtain... Figure 1 .
[0057] Example 2: Construction of an immortalized placental mesenchymal matrix cell line
[0058] 1. Construction of hTERT lentiviral expression vector
[0059] This study used the pLV-IRES-hygro vector as a backbone and designed hTERT primers with BamHI (5-ttccatttcaggtgtcgtgaggatccGCCACCatggataaagttttaaacagagag-3) and EcoRI (5-tagagcggccgccctcgaggaattcttatgtttcaggttcaggggg-3) restriction sites. The target fragment (a transfer plasmid overexpressing the hTERT gene synthesized by Quanyang Biotechnology Co., Ltd.) was obtained by PCR amplification. The hTERT fragment was ligated into the digested vector, then transformed into DH5α competent cells. Positive clones were screened, and plasmids were extracted, successfully constructing a lentiviral expression vector containing the hTERT gene. The full-length hTERT gene in this vector is 3399 bp and contains the hygromycin (HYGR) resistance gene (see...). Figure 2 ).
[0060] 2. Lentiviral packaging and titer determination
[0061] (1) Packaging system and materials: Lentiviral packaging was performed in 293T cells using a three-plasmid system (psPAX2, pMD2.G and pLV-hTERT-IRES-hygr). The 293T cell line and the lentiviral packaging plasmids psPAX2 Vector (Addgene, ID#12260) and PMD2.G Vector (Addgene, ID#12259) were provided by Shanghai Bio-Pharmaceutical Technology Co., Ltd.
[0062] (2) Packaging procedure: During virus packaging, 6.5 μg of envelope plasmid (pMD2.G) and 8.5 μg of packaging plasmid (psPAX2) were added for every 10 μg of transfer plasmid. The amounts of virus packaging reagents used are shown in Table 1 below. The viral supernatant was collected after 48-72 hours, filtered through a 0.45 μm filter, and the viral titer was determined.
[0063] Table 1. Dosage of Virus Packaging Reagent
[0064] Reagent Name Volume usage OPTI-MEM (1×) 200μL hTERT vector plasmid 10μg PMD2.G 6.5μg psPAX2 8.5μg PEI transfection reagent 75μL
[0065] (3) Titer Calculation: Viral titer was calculated by detecting the integration copy number using qPCR. The formula is: IU / mL=(C×N×D×1000) / V, where C is the average integration copy number and N is the number of infected cells (2.5×10⁻⁶). 5 (D represents the dilution factor, and V represents the viral volume.) The viral titer, calculated by qPCR, is approximately 3 × 10⁻⁶. 8 The concentration of TU / mL was measured, and experiments showed that the infection efficiency of the target cells could reach 80%.
[0066] 3. Target cell infection and screening
[0067] (1) Infection procedure: Before viral infection of the target cells, primary placental mesenchymal stem cells were seeded in six-well plates and passaged twice to achieve optimal cell growth. Each plate contained 3 × 10⁻⁶ cells. 6 Each cell was infected with 500 μL of recombinant lentivirus, thoroughly shaken, and incubated at 37°C for 12-24 h. Then, the cells were incubated with fresh MSC medium for a longer period.
[0068] (2) Resistance screening: 72 h after infection, resistance gene screening was initiated using 1 μg / mL hygromycin, with primary cells serving as the control group. The screening process lasted for 15 days, with the complete culture medium containing hygromycin being replaced every 3-4 days. After all control group cells died, the resulting target cells were named iMSC022 and used for subsequent experiments.
[0069] Example 3: Functional and Phenotypic Detection of iMSC022
[0070] 1. Flow cytometry detection of surface markers using iMSC022
[0071] (1) Detection procedure: When the cells are in the logarithmic growth phase, they are digested and separated with 0.25% trypsin, gently washed three times with PBS, and then prepared into 1×10⁻⁶ cells. 6 Cell suspensions of 10 cells / mL were prepared. Antibody incubation medium containing 1% fetal bovine serum was added, and the mesenchymal stem cell markers CD90 (FITC), CD73 (APC), CD105 (PE), and epithelial cell markers CD34 (APC) and CD45 (FITC) were incubated with iMSC022 at room temperature in the dark for 20 min. The antibody concentration was 5 μL / 10 mL as recommended. 6 Cells were added, and all antibodies were purchased from BioLegend (San Diego, CA, USA). The cell suspension was then washed twice with PBS and resuspended. Data on the expression ratios of cell surface markers were collected using flow cytometry (BD FACS Celesta, BD, USA). Experimental results were recorded and analyzed using FlowJo software.
[0072] (2) Detection results and conclusions: The results showed that the expression rates of positive markers (CD90, CD73, CD105) in primary cells and iMSC022 were as high as 99%. Figure 3 The expression levels of negative markers (CD34, CD45) were both below 1%. Figure 4 iPMSCs (immortalized placental mesenchymal stromal cells) exhibit the same surface marker pattern as parental cells, indicating that during continuous proliferation, the surface phenotype expression of immortalized cells is not affected by exogenously expressed hTERT genes, and they retain the stem cell characteristics of parental cells.
[0073] 2. Genetic stability - karyotype testing
[0074] (1) Detection Procedure: Primary cells were cultured to P3, and iMSC022 cells were cultured to P60. Cells in the logarithmic growth phase were selected, and colchicine was added to a final concentration of 0.2 μg / ml. Cells were incubated at 37°C for 1-2 hours to arrest cell division. Cells were then digested with trypsin, centrifuged at 1200 r / min to collect the cells, and resuspended in preheated 0.075 M KCl hypotonic solution. The cells were then treated with hypotonic solution at 37°C for 40 min. Next, 2 ml of methanol:acetic acid (3:1) fixative was added and mixed. After standing at room temperature for 10 min, the cells were centrifuged. This fixation was repeated three times to thoroughly remove the cytoplasm. Finally, the cell suspension was dropped onto a glass slide, baked at 75°C for 2-3 hours, digested with 0.25% trypsin for 1 min, stained with Giemsa for 5-10 min, and examined under a microscope to obtain the desired cell structure. Figure 5 .
[0075] (2) Detection results and conclusions: The results showed that iMSC022 was diploid (2n=46, XY), with normal chromosome number and structure, indicating that immortalization treatment did not cause genetic variation, and the newly established cell line had stable karyotype characteristics.
[0076] Example 4: Isolation and Identification of iMSC-Exo
[0077] 1. Main reagents and instruments
[0078] Table 2 Main Reagents
[0079] name factory Exosome detection kit Exo view PEG8000 Merck Biotechnology
[0080] Table 3 Main Instruments
[0081] name factory Fully Automated Exosome Fluorescence Detection and Analysis System Exo view FEI Tecnai G2 TEM Thermofisher high-speed centrifuge Xiangyi Instruments Co., Ltd.
[0082] 2. Extraction and purification of exosomes
[0083] Immortalized placental mesenchymal stem cells (passages 3 and 60) in the logarithmic growth phase (cell density 70%-80%) were selected and cultured in fresh medium for 48 hours. The supernatant was collected. Cell debris and apoptotic bodies were removed by centrifugation at 300×g (10 min, room temperature), 2000×g (10 min, 4℃), and 10000×g (30 min, 4℃) sequentially. The supernatant was stored at -80℃ for later use. 25 mL of 10% PEG solution was added to every 100 mL of supernatant, gently mixed 4-5 times, and incubated at 4℃ for >12 h. The next day, the cells were centrifuged at 4℃ at maximum speed for 40 min, the supernatant was discarded, and the precipitate was resuspended in PBS to obtain the exosome solution.
[0084] 3. Detection of exosome morphology, phenotype and particle size
[0085] Morphological observation: The morphology of exosomes purified by the PEG method was observed using a FEI Tecnai G2 transmission electron microscope (TEM) to verify their typical structural characteristics. The results showed that the isolated iMSC022-derived exosomes (iMSC022-Exo) had a typical cup-shaped morphology (see...). Figure 6 ).
[0086] Multi-parameter detection: For concentration, phenotypic analysis, and particle size determination, a single-particle interferometric reflection imaging sensing analysis (SP-IRIS) system was used, and samples were processed according to the requirements of the ExoView exosome detection kit (#EV-TE-TRA-C, Sigma, Germany). The results showed high expression of transmembrane protein markers, including common markers CD63, CD81, and CD9; MIgG was not expressed as a negative control (see [link to relevant documentation]). Figure 7 The particle size distribution of exosomes ranges from 50 to 200 nm (see...). Figure 8 Immune colocalization imaging better illustrates these results (see...). Figure 9 ).
[0087] Immune colocalization imaging: The above results were further verified by immune colocalization imaging.
[0088] Example 4: miRNA component analysis and content determination of iMSC-Exo
[0089] 1. Experimental materials:
[0090] Cell culture supernatant containing exosomes; MicroRNA extraction kit (Beyotime); RNA reverse transcription kit (Beyotime); qPCR SYBRGREEN dye (Genstar); 10KD ultrafiltration tubes (Millipore).
[0091] 2. Exosome enrichment
[0092] Cell culture supernatant containing exosomes was concentrated by ultrafiltration using 10KD ultrafiltration tubes at 4000 rpm for 20 min per centrifugation. The supernatant was concentrated to 200 μL and then transferred to new RNase-free centrifuge tubes for storage at -80°C.
[0093] 3. RNA extraction
[0094] (1) Take the processed cell supernatant enrichment, discard the culture medium, and gently wash once with an appropriate amount of PBS.
[0095] (2) Add 1 ml of lysis buffer, pipette the lysis buffer, and let stand on ice for 1 min;
[0096] (3) Add an equal volume of binding solution I to the lysis solution, let it stand on ice for 1 min, then transfer it to a centrifuge column and centrifuge at 12000g for 1 min.
[0097] (4) Collect the centrifuged liquid into a new centrifuge tube, add an equal volume of binding liquid II, invert and mix well, then transfer to a centrifuge column and centrifuge at 12000g at room temperature for 1 min.
[0098] (5) After centrifugation, discard the waste liquid, add 500 μl of washing solution I and washing solution II in sequence, and centrifuge at 12000g for 1 min at low temperature;
[0099] (6) Transfer the centrifuge column to a new RNase-free 1.5 ml collection tube, add 50 μl of elution buffer, and let stand for 2 min;
[0100] (7) Centrifuge at 12000g for 2 min, discard the centrifuge column, and the resulting eluent is the target microRNA sample. Take 2 μl and quantify it on a UV spectrophotometer. Store the sample at -80℃ for later use.
[0101] 4. RNA reverse transcription synthesis of the first strand
[0102] (1) Refer to the instructions of the cDNA first strand synthesis kit, take 0.1-1 μg of the prepared RNA sample, and use it for reverse transcription to synthesize the first strand of cDNA;
[0103] (2) Reverse transcription reaction system (20 μl): 0.5-1 μg total RNA, 1 μl RNase inhibitor, 2 μl ldNTP mix, 4 μl reaction buffer, 1 μl reverse transcriptase, 1 μl stem-loop primer, and DEPC-water to make up to 20 μl;
[0104] (3) Reverse transcription reaction procedure: 25℃ for 10 min; 45℃ for 50 min; 80℃ for 5 min;
[0105] (4) After reverse transcription is completed, take 2 μl of cDNA sample for qPCR detection.
[0106] 5. Real-time quantitative PCR reaction
[0107] (1) Gene expression was detected using the SYBR Green Mix kit;
[0108] (2) Reaction system: 1 μl of reverse transcribed cDNA first strand product, 10 μl of SYBR Green Mix, 50 pmol of gene primers, and 20 μl of sterile deionized water;
[0109] (3) The reaction system was placed in a real-time PCR instrument for detection. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 sec, 60℃ annealing for 15 sec, for a total of 40 cycles; and finally incubation at 4℃ for 1 h.
[0110] (4) According to 2 -△△Ct The method calculates the relative expression level of the target gene and obtains... Figure 10 .
[0111] 5. Experimental Results
[0112] In hTERT immortalized placental mesenchymal stromal cells (PMSCs), the expression levels of miR-146a-5p, miR-29a-3p, miR-29b-3p, let-7b-3p, and miR-21-5p were significantly higher than in primary cells. Among them, miR-29a-3p, miR-29b-3p, and miR-21-5p are closely related to the fibrosis process: miR-29a-3p and miR-29b-3p can directly target fibrosis-related genes, such as collagen genes (COL1A1, COL3A1, COL4A1), and ECM regulatory factors (TGF-β1, CTGF, MMP2), and exert anti-fibrotic effects by inhibiting the core fibrosis signaling pathway TGF-β / Smad3; miR-21-5p has been proven effective as a clinical intervention target in animal experiments. Furthermore, miR-146a-5p can alleviate inflammatory fibrosis (such as pulmonary fibrosis) by inhibiting NF-κB / IL-6. Given that these key components are all associated with fibrosis, further animal experiments will be conducted to verify its anti-fibrotic ability.
[0113] Example 5: Rat experiment to verify the anti-fibrotic effect of iMSC022-Exo
[0114] 1. Laboratory animals and their housing conditions
[0115] The Wistar rats used in this experiment were 6-week-old males, weighing 200-220g, SPF grade, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., Tongxiang Branch (Quality Certificate: 20230712Aazz0600000640), and housed at Shanghai Pharmaceutical Industry Research Institute Co., Ltd. All experimental procedures complied with relevant Chinese regulations on laboratory animals. The rats were housed in standard animal facilities under the following conditions: temperature 18-24℃, relative humidity approximately 45%, noise <60dB, working illumination >200lx, and a 12-hour day-night cycle. They were fed and watered freely under normal conditions.
[0116] 2. Induction of pulmonary fibrosis model and experimental grouping
[0117] Pulmonary fibrosis in rats was induced by intratracheal administration: Bleomycin was injected into Wistar rats via a needle inserted centripetally through the interstitial space of the tracheal cartilage rings at a dose of 2.5 mg / kg. The treated rats were then randomly divided into stratified groups according to body weight, with 10 rats in each group:
[0118] Blank control group (control): injected with normal saline alone.
[0119] Bleomycin treatment group (MC): After successful model establishment, physiological saline was administered.
[0120] Positive control groups: including the 0.6 mg / kg dexamethasone group (Dex), the 200 mg / kg pirfenidone group (PFD), and the iMSC022-Exo group (Exo: divided into high, medium, and low dose groups, with doses of 5 × 10⁻⁶ mg / kg and 5 × 10⁻⁶ mg / kg respectively). 8 / each, 5×10 7 / each, 5×10 6 / Only).
[0121] Based on previous laboratory results, pirfenidone alone was not effective, so dexamethasone was added as a positive control.
[0122] Administration method and cycle:
[0123] iMSC022-Exo group: Drug administration began 7 days after modeling, and was repeated every other day for a total of 10 doses until 21 days after modeling.
[0124] Dexamethasone and pirfenidone groups: Dosing began 7 days after modeling, once daily, for 21 days after modeling.
[0125] 3. Experimental Results
[0126] After HE staining (see) Figure 11 ) and Masson staining (see Figure 12 Following observation of pathological changes in rat lung tissue, HE staining and Masson staining results in the Wistar rat model showed that intratracheal administration of iMSC022-Exo reduced collagen deposition and fibrosis in rat lung tissue. The intervention of iMSC022-Exo significantly weakened the phenotypes of two fibrosis-related substances in lung tissue. The experimental report showed that immortalized cell-derived exosomes (iMSC022-Exo) significantly resisted bleomycin-induced pulmonary fibrosis in rats, playing a protective and therapeutic role in lung inflammation and histological damage. These results indicate that placental mesenchymal stromal cell-derived extracellular vesicles (iMSC022-Exo) immortalized with the hTERT gene have a good anti-fibrotic effect.
[0127] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.
Claims
1. A method for stably producing mesenchymal matrix cell exosomes containing effective anti-inflammatory miRNA components, characterized in that, Includes the following steps: S1. Obtain placental tissue and isolate and culture primary placental mesenchymal matrix cells; S2. Immortalized placental mesenchymal matrix cell line was constructed by transducing the hTERT gene with lentivirus; S3. The immortalized placental mesenchymal matrix cell line is cultured in serum-free medium, and the cell supernatant is collected. S4. The obtained supernatant was successively subjected to centrifugation, PEG precipitation and ultrafiltration concentration to extract exosomes.
2. The preparation method according to claim 1, characterized in that, In step S1, the isolation and culture of primary placental mesenchymal matrix cells includes: cutting placental villus tissue into fragments of 1-2 mm in size, digesting with trypsin, and then inoculating and culturing until the cell confluence reaches 75-85%.
3. The preparation method according to claim 1, characterized in that, In step S3, the serum-free culture medium is MSCBM medium containing 4% human platelet lysate.
4. A mesenchymal matrix cell exosome prepared by the method according to any one of claims 1 to 3.
5. The mesenchymal matrix cell exosomes according to claim 4, characterized in that, The mesenchymal matrix cell exosomes include miR-146a-5p, miR-29a-3p, miR-29b-3p, let-7b-3p, and miR-21-5p.
6. The mesenchymal matrix cell exosomes according to claim 5, characterized in that, The exosomes containing miR-146a-5p, miR-29a-3p, miR-29b-3p, let-7b-3p, and miR-21-5p have a particle size distribution between 50 and 200 nm.
7. A pharmaceutical composition, characterized in that, The active ingredient of the pharmaceutical composition is the mesenchymal matrix cell exosome as described in claim 4.
8. The use of the mesenchymal matrix cell exosomes as described in claim 4 in the preparation of antifibrotic drugs.
9. The application according to claim 8, characterized in that, The fibrosis includes pulmonary fibrosis, liver fibrosis, or skin fibrosis.
10. The application according to claim 9, characterized in that, The fibrosis is pulmonary fibrosis, and the application includes the following steps: intratracheal injection of the exosomes as described in claim 4 into rats, wherein the dose of the exosomes is 5 × 10⁻⁶. 6 ~5×10 8 Item / time.