A stem cell co-expressing miR-449a-5p and miR-30c-5p

Abstract

This invention relates to the field of biotechnology and discloses a stem cell co-expressing miR-449a-5p and miR-30c-5p. The stem cell is constructed by cloning a biomimetic polycistronic expression cassette containing miR-449a-5p and miR-30c-5p into a lentiviral vector and infecting the stem cell, enabling stable and synergistic expression of these two miRNAs. The exosomes (miRNAs-Exos) secreted by these cells specifically enrich miR-449a-5p and miR-30c-5p; after local administration, the exosomes are taken up by skin cells, and the two miRNAs synergistically inhibit the expression of the aging-associated secretory phenotype (SASP) core factors CCL2 and PAI-1, downregulate matrix metalloproteinases MMP-1 / MMP-3, reduce collagen degradation, improve the dermal inflammatory microenvironment, and thereby promote collagen synthesis and skin barrier repair.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a stem cell that co-expresses miR-449a-5p and miR-30c-5p. Background Technology

[0002] Photoaging of the skin is an accelerated aging process induced by long-term exposure to ultraviolet radiation in the solar spectrum, particularly UVB (290-320 nm) and UVA (320-400 nm). Clinically, it manifests as deep wrinkles, leathery texture, loss of elasticity, and irregular pigmentation. Its molecular mechanisms exhibit cell type specificity: UVB primarily induces DNA damage in epidermal keratinocytes, activating DNA damage response pathways; UVA, on the other hand, triggers oxidative stress in dermal fibroblasts by generating reactive oxygen species (ROS). Both UVB and UVA synergistically activate key signaling pathways such as MAPK / NF-κB / AP-1, ultimately leading to abnormal extracellular matrix degradation and cellular senescence.

[0003] At the cellular level, UV-induced cellular senescence is accompanied by the establishment of the senescence-associated secretory phenotype (SASP), in which key effector molecules such as CCL2 (monocyte chemoattractant protein-1) and PAI-1 (plasminogen activator inhibitor-1) form a self-reinforcing pathological cycle by promoting chronic inflammation and inhibiting tissue repair. However, current intervention strategies based on small molecule compounds or protein drugs have significant limitations: on the one hand, it is difficult to achieve multi-target synergistic regulation of the SASP network; on the other hand, they face technical bottlenecks such as poor skin permeability, low bioavailability, and insufficient stability.

[0004] Exosomes, as naturally occurring nanoscale extracellular vesicles, have become an important research direction for novel drug delivery systems due to their excellent biocompatibility, low immunogenicity, and ability to cross biological barriers. However, existing technologies have significant drawbacks: while naturally derived exosomes (such as mesenchymal stem cell exosomes) possess certain tissue repair potential, their contents are complex and unstable, their endogenous microRNA (miRNA) loading is limited, and they lack the ability to actively target specific cell types, making it difficult to achieve precise regulation of complex signaling pathways. Especially in the field of skin photoaging treatment, current technologies still cannot overcome the core bottlenecks. How to construct an engineered exosome system that can simultaneously meet the requirements of high-efficiency loading, stable delivery, targeted recognition, and multi-pathway synergistic regulation remains a pressing technical challenge. Therefore, it is urgent to explore new methods to overcome these bottlenecks and promote the development of related research and applications. Summary of the Invention

[0005] This invention constructs engineered hMSCs capable of co-expressing miR-449a-5p and miR-30c-5p and establishes a standardized exosome preparation process. The obtained miRNAs-Exos possess a clear anti-skin photoaging function, providing a new technical solution and experimental basis for the development of related therapeutic products. This technology solves key technical problems in existing exosome intervention strategies for skin photoaging, such as uncertain contents, insufficient target specificity, and limited ability to regulate complex pathological networks.

[0006] On one hand, the present invention provides a stem cell co-expressing miR-449a-5p and miR-30c-5p, wherein the stem cell expresses a chimeric dual miRNA expression unit; the chimeric dual miRNA expression unit expresses pre miR 155 forms the backbone, encoding and processing miR within the same transcript. 449a 5p and miR 30c These two miRNAs are 5p.

[0007] Furthermore, the chimeric dual miRNA expression unit comprises: a first chimeric pre miRNA units, composed of miR 449a 5p mature sequence and its complementary strand are embedded in pre miR Formed in the 155 skeleton; second interlocking pre miRNA units, composed of miR 30c 5p mature sequence and its complementary strand are embedded in pre miR Formed in the 155 skeleton; the first interlocking pre miRNA unit and second chimeric pre miRNA units are transmitted via pre miR The natural flanking sequences of the 155 skeleton are directly connected.

[0008] Furthermore, the first chimera pre The coding sequence of the miRNA unit is shown in SEQ ID NO:9; the second chimeric pre The coding sequence of the miRNA unit is shown in SEQ ID NO:10.

[0009] Furthermore, the method for preparing the stem cells includes: Step 1: The chimeric dual miRNA expression unit was cloned into a lentiviral expression vector to obtain a recombinant plasmid; Step 2: Co-transfect the recombinant plasmid, packaging plasmid, and envelope plasmid into host cells, culture and collect the transfection products, and purify them to obtain lentiviral particles; Step 3: Infect human mesenchymal stem cells with the lentiviral particles and screen and culture them to obtain stem cells that co-express miR-449a-5p and miR-30c-5p.

[0010] On the other hand, the present invention provides a method for preparing the above-mentioned stem cells, the method comprising: Step 1: The chimeric dual miRNA expression unit was cloned into a lentiviral expression vector to obtain a recombinant plasmid; Step 2: Co-transfect the recombinant plasmid, packaging plasmid, and envelope plasmid into host cells, culture and collect the transfection products, and purify them to obtain lentiviral particles; Step 3: Infect human mesenchymal stem cells with the lentiviral particles and screen and culture them to obtain stem cells that co-express miR-449a-5p and miR-30c-5p; The chimeric dual miRNA expression unit uses pre miR 155 forms the backbone, encoding and processing miR within the same transcript. 449a 5p and miR 30c These two miRNAs are 5p; Furthermore, the packaging plasmid includes: psPAX2; Furthermore, the envelope plasmid includes pMD2.G.

[0011] On the other hand, the present invention provides an exosome for preventing and / or treating photoaging of the skin, wherein the exosome is obtained by secretion from the above-described stem cells or stem cells prepared by the above-described preparation method.

[0012] Furthermore, the exosomes encapsulate two miRNAs, miR-449a-5p and miR-30c-5p.

[0013] On the other hand, the present invention provides a pharmaceutical composition for preventing and / or treating photoaging of the skin, the pharmaceutical composition comprising the above-described stem cells or stem cells prepared by the above-described preparation method or the above-described exosomes.

[0014] Furthermore, the administration method of the pharmaceutical composition includes local delivery, wherein the local delivery is applied to the target administration site via a local delivery carrier; Furthermore, the local delivery carrier comprises a thermosensitive hydrogel or a soluble microneedle array.

[0015] On the other hand, the present invention provides the use of the above-described stem cells or stem cells prepared by the above-described preparation method or the above-described exosomes or the above-described pharmaceutical compositions in the preparation of products for the prevention and / or treatment of skin photoaging. Furthermore, the product works by inhibiting the age-related secretory phenotype SASP factor in skin tissue; Furthermore, the SASP factor includes CCL2 and / or PAI-1.

[0016] The technical solution of this invention has the following advantages: 1. Enhancing the determinacy of therapeutic components in exosomes and achieving synergistic co-expression of miR-449a-5p and miR-30c-5p: The composition of existing natural exosome contents is greatly affected by cell state and is difficult to standardize. This invention constructs an engineered human mesenchymal stem cell line that stably co-expresses miR-449a-5p and miR-30c-5p, thereby achieving deterministic programming of functional miRNA components in secretory exosomes and avoiding membrane damage and efficiency instability caused by physical loading. Furthermore, this invention uses a polycistronic vector design to simulate the processing mechanism of natural miRNA clusters, ensuring the efficient synergistic expression of the two miRNAs.

[0017] 2. Achieving multi-target synergistic regulation of core pathways of skin photoaging and verifying anti-aging and repair effects: Addressing the limited effectiveness of single-target intervention, this invention utilizes a combination of miRNAs carried by engineered exosomes to synergistically inhibit two key nodes in the SASP signaling network, CCL2 and PAI-1, thereby intervening in key driving pathways of photoaging from upstream, providing a new potential strategy for achieving superior anti-aging effects. Furthermore, this invention has demonstrated in a skin photoaging model that these miRNAs-Exos can significantly inhibit the expression of key SASP factors (CCL2, PAI-1), improve the dermal inflammatory microenvironment, and promote collagen synthesis, verifying its anti-aging and repair effects.

[0018] 3. Establish a stable and scalable standardized production process and form a standardized exosome preparation system: This invention constructs a production system with an engineered cell bank as the core, integrating linearly scalable downstream purification technologies (such as tangential flow filtration and size exclusion chromatography) to overcome the consistency challenges faced by traditional methods (such as ultracentrifugation) in large-scale production, thereby improving the batch-to-batch consistency of engineered exosomes in key quality attributes (such as specific miRNA loading and particle size distribution). In practice, a standardized exosome preparation process has been established through bioreactors and chromatography technology, and multiple quality control standards such as particle size distribution, biomarker expression, and functional miRNA content have been formulated to ensure a high degree of consistency between product batches.

[0019] 4. Providing therapeutic products with clear mechanisms and controllable quality, as well as flexible application solutions: This invention provides a topical formulation with engineered exosomes as the active ingredient, offering a novel biological treatment solution with a clear mechanism of action and industrializability for the clinical prevention and improvement of skin photoaging; at the same time, it constructs a flexible application system, in which the engineered exosomes can be adapted to various transdermal delivery systems such as thermosensitive hydrogels and soluble microneedle arrays, and the appropriate dosage form can be selected according to clinical needs, providing a product form with transformational value for skin anti-aging. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 The image shows the screening results of different miRNAs in UVB-induced human dermal fibroblasts for inhibiting CCL2 and PAI-1 mRNA expression, where a represents the effect of inhibiting CCL2 mRNA expression and b represents the effect of inhibiting PAI-1 mRNA expression. Figure 2 The figure shows the effects of miR-449a-5p and miR-30c-5p alone and in combination on the expression of CCL2 and PAI-1 mRNA, secreted proteins and collagen. In the figure, a is the relative expression level of CCL2 and PAI-1 mRNA, b is the secretion level of CCL2 and PAI-1 proteins and c is the expression level of type I collagen. Figure 3 This is a schematic diagram illustrating the construction of a polycistronic pri-miRNA expression vector. Figure 4The image shows the results of detecting the expression levels of miR-449a-5p and miR-30c-5p in engineered cells (miRNAs-hMSCs); Figure 5 The image shows the characterization results of engineered exosomes (miRNAs-Exos). In the image, a is the exosome particle size analysis, b is the Western blotting image used to detect characteristic exosome markers (CD63, TSG101) and the negative control protein Calnexin, and c is the transmission electron microscope (TEM) image showing the morphology, size and structural features of the exosomes. Scale bar: 50 nm. Figure 6 The graph shows the expression levels of functional miRNAs (miR-449a-5p and miR-30c-5p) in miRNAs-Exos. Figure 7 The figure shows the effects of miRNAs-Exos on the expression of SASP-related factors (CCL2, PAI-1) and the activity of SA-β-gal in UVB-induced senescent dermal fibroblasts. Figure a shows the expression results of SASP-related factors (CCL2, PAI-1), and figure b shows the effect on SA-β-gal activity. Figure 8 The image shows the therapeutic effects of miRNAs-Exos on photoaged mouse skin. In the image, a is a photograph of mouse skin, b is the dermal thickness of mice in different treatment groups, c is the protein expression levels of type I collagen, type III collagen, and internal reference GAPDH in the skin of mice in different treatment groups, and d is the result of relative quantitative analysis of the expression of type I collagen and type III collagen. Figure 9 The figure shows the effect of miRNAs-Exos treatment on the mRNA levels of CCL2, PAI-1, and key collagen degradation enzymes MMP-1 / 3 in skin tissue. In the figure, a represents the relative mRNA expression levels of CCL2 and PAI-1, and b represents the mRNA expression levels of key collagen degradation enzymes MMP-13 / 3. Detailed Implementation

[0022] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0023] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0024] Materials or reagents used in this invention: 1. miRNA-related reagents: miRNA mimics were mainly synthesized by Shanghai Jima Company.

[0025] Transfection reagent: Lipofectamine™ 2000 transfection reagent, catalog number 11668019 (Thermo Fisher).

[0026] RNA extraction: TRIzol reagent, catalog number 15596026 (Thermo Fisher).

[0027] cDNA synthesis: miScript II RT Kit, catalog number 218161 (Qiagen).

[0028] qPCR Master Mix miScript SYBR® Green PCR Kit, Cat. No. 218073 (Qiagen).

[0029] 2. Stem cell source and construction / culture: Cells: Human umbilical cord mesenchymal stem cells (hMSC), catalog number PCS-500-010 (ATCC).

[0030] Culture medium: Mesenchymal stem cell culture medium, catalog number PT-3001 (Lonza).

[0031] High-glucose DMEM medium, catalog number 11965 092 (Gibco™).

[0032] Exosome-free culture medium (for exosome collection): Exosome-free fetal bovine serum, catalog number EXO-FBS-50A-1 (System Biosciences), was prepared with DMEM / F12 basal medium at a final concentration of 10% (v / v). That is, the exosome-free serum and DMEM / F12 basal medium were mixed at a volume ratio of 1:9 (v / v) and then filtered through a 0.22 μm filter membrane for sterilization before use.

[0033] SA β Gal staining kit, catalog number 9860S (Cell Signaling Technology).

[0034] Western Blot Cell / Tissue Lysis Buffer (RIPA), Catalog No. 89901 (Thermo Fisher).

[0035] 3. Lentiviral construction and packaging: Lentiviral expression vector: pCDH CMV MCS EF1α Puro lentiviral expression vector, catalog number CD510B 1 (SystemBiosciences).

[0036] Lentiviral packaging plasmids: psPAX2 (packaging plasmid), catalog number 12260 (Addgene); pMD2.G (enveloping plasmid), catalog number 12259 (Addgene).

[0037] Transfection reagent for packaging: linear polyethyleneimine (PEI), catalog number 408727 (Polysciences).

[0038] Packaging cells: HEK293T cell line, catalog number CRL 3216 (ATCC).

[0039] Screening drug: Puromycin, catalog number P8833 (Sigma) Aldrich.

[0040] 4. Exosome isolation and purification: Material: 100 kDa filtration membrane, item number C02R015A01 (Millipore Ultracel).

[0041] SEC columns used for purification: HiPrep™ Sephacryl™ S-400 HR columns, catalog number 28935604 (Cytiva).

[0042] PBS buffer, catalog number 10010023 (Gibco).

[0043] BCA Protein Quantitative Kit, Catalog No. 23225 (Thermo Fisher).

[0044] Example 1. Screening for miRNA combinations that can simultaneously and efficiently inhibit CCL2 and PAI-1 1. Bioinformatics screening and identification of high-confidence candidate molecules First, the TargetScan v8.0, miRDB v6.0, miRTarBase 2023, and TarBase v9.0 databases were used for systematic screening. The screening criteria were as follows: (1) TargetScan ≤ -0.35 (indicating evolutionary conservation); (2) miRDB prediction score ≥ 85 (high confidence prediction); (3) direct experimental evidence in the miRTarBase or TarBase databases. Based on these criteria, high-confidence candidate molecules targeting CCL2 were screened, including miR-449a-5p, miR-124-3p, and miR-181b-5p; high-confidence candidate molecules targeting PAI-1 were screened, including miR-30c-5p, miR-30b-5p, miR-30a-5p, miR-29b-3p, and miR-145-5p. The sequences are shown in Table 1.

[0045] Table 1: Mature sequences of 8 candidate miRNAs

[0046] 2. Screening for optimal single-target miRNAs in cell models To further screen for the most effective single-target molecules targeting CCL2 and PAI-1, this embodiment established a UVB-induced premature senescence model of human dermal fibroblasts (see reference: Yoon et al., UVB-induced premature senescence in human dermal fibroblasts: Role of ROS and MAPK signaling. JDermatol Sci, 2010). Human dermal fibroblasts (HDF) were cultured in 6-well plates at 37°C and 5% CO2 at a density of 80% (approximately 1 × 10⁻⁶ cells / well) using Gibco™ high-glucose DMEM as the basal medium supplemented with 10% (v / v) fetal bovine serum (FBS) to 80% density (approximately 1 × 10⁻⁶ cells / well). 5Cells / well, culture medium volume 2 mL / well. In the miRNA transfection experiment, the culture medium was replaced with an equal volume of high-glucose DMEM containing 2% (v / v) FBS to reduce serum shielding. Using Lipofectamine™ 2000 transfection reagent, three miRNAs targeting CCL2 (miR-449a-5p, miR-124-3p, and miR-181b-5p) and five miRNAs targeting PAI-1 (miR-30c-5p, miR-30b-5p, miR-30a-5p, miR-29b-3p, and miR-145-5p) mimics were transfected into cells in 6-well plates at a transfection rate of 100 pmol per well. Cells were collected 24 h after transfection, and total RNA was extracted. The mRNA levels of the corresponding target genes were detected by qRT-PCR (qRT-PCR primers used are shown in Table 2) to analyze the regulatory effects of each miRNA mimic on the target genes. Results are as follows. Figure 1 As shown in a, among the high-confidence candidate molecules targeting CCL2, miR-449a-5p exhibited the most significant inhibitory effect (inhibition rate approximately 65%, P < 0.01); Figure 1 As shown in b, among the candidate molecules targeting PAI-1, miR-30c exhibited the most significant inhibitory effect (inhibition rate approximately 60%, P < 0.001). Notably, miR-449a-5p alone showed no significant inhibitory effect on PAI-1 (P > 0.05), and miR-30c-5p alone also showed no significant inhibitory effect on CCL2 (P > 0.05). Figure 2 (a and b).

[0047] Table 2: Primer sequences for detecting 8 candidate miRNAs

[0048] 3. Verification of the combined effect Based on the above results, to simultaneously inhibit CCL2 and PAI-1, the above method was repeated, and miR-449a-5p and miR-30c-5p mimics were simultaneously transfected into cells in 6-well plates at a transfection rate of 50 pmol per well for combined application, resulting in transfected cells. 24 h after transfection, total RNA was extracted from the transfected cells, and the mRNA levels of the corresponding target genes in the total RNA of the transfected cells were detected by qRT-PCR to analyze the regulatory effect of the combined miRNA mimics (miR-449a-5p + miR-30c-5p) on the target genes. The results are as follows: Figure 2As shown in Figure a, qRT-PCR results indicated that the combined use of both treatments effectively suppressed the mRNA expression of CCL2 and PAI-1 to low levels (inhibition rates of 75% and 65%, respectively, *p<0.001), both significantly superior to their respective optimal single-treatment groups. To confirm that the above mRNA inhibition effectively translates into a reduction in functional protein secretion, the concentrations of CCL2 and PAI-1 in the cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). The results are as follows... Figure 2 As shown in b, miR-449a-5p alone significantly reduced CCL2 protein secretion (P<0.001), but had no significant effect on PAI-1 secretion. miR-30c-5p alone significantly reduced PAI-1 protein secretion (P<0.001), but had no significant effect on CCL2 secretion. Combined treatment with miR-449a-5p and miR-30c-5p simultaneously suppressed the secretion of both CCL2 and PAI-1 to lower levels, and the effects were significantly better than either miRNA mimic alone (P<0.01).

[0049] To evaluate the effect of this combination on reversing the core phenotype of photoaging—extracellular matrix loss—this embodiment used Western blotting to detect the expression level of intracellular type I collagen. The results are as follows: Figure 2 As shown in Figure c, the expression level of type I collagen in the UVB-induced aging model group was significantly lower than that in the normal control group. The miR-449a-5p monotherapy group had no significant effect on collagen enhancement, while the miR-30c-5p monotherapy group showed an enhancement effect. However, the combined treatment of miR-449a-5p and miR-30c-5p could restore the expression of type I collagen to the greatest extent, and this effect was significantly better than that of any miRNA mimic monotherapy group.

[0050] In summary, this embodiment is the first to combine miR-449a-5p and miR-30c-5p, which specifically inhibit CCL2 and PAI-1 respectively, and confirms that this combination can not only inhibit the secretion of harmful SASP factors (CCL2 and PAI-1), but also positively promote the synthesis of collagen, the core supporting structure of the dermis, thereby achieving an anti-photoaging effect.

[0051] Example 2: Construction and Identification of Engineered Human Mesenchymal Stem Cells 1. Construction of polycistronic pre-miRNA expression vector This embodiment aims to construct a lentiviral expression vector (e.g., [vector name missing]) that can efficiently co-express miR-449a-5p and miR-30c-5p in human mesenchymal stem cells (hMSCs). Figure 3The vector uses a human pre-miR-155 expression backbone, which significantly enhances the processing and generation efficiency of exogenous miRNAs. According to pre-miR-155... miR The natural stem-ring structure of 155, respectively, will incorporate miR 449a 5p and miR 30c The mature 5p sequences (SEQ ID NO.1 and SEQ ID NO.4) and their complementary strands were inserted into the backbone after replacing the original mature miR-155 sequences and their complementary strands, resulting in sequences SEQ ID NO.26 and SEQ ID NO.27. Sequences SEQ ID NO.26 and SEQ ID NO.27 were tandemly linked to form a polycistronic pre-miRNA expression unit. EcoRI and NotI restriction endonuclease recognition sites were introduced at the 5′ and 3′ ends of the tandem sequences, respectively, to facilitate directional cloning (as shown in SEQ ID NO.28). The entire sequence was then synthesized in vitro. The sequences of the two chimeric units are shown in Table 3.

[0052] Table 3. Sequences of two chimeric units

[0053] Subsequently, the synthesized pre miRNA expression unit and lentiviral expression vector pCDH CMV MCS EF1α Puro was ligated after double digestion with EcoRI and NotI to ligate pre The miRNA expression unit was cloned into the multiple cloning site of the vector, and transcribed under the CMV promoter to obtain a miRNA expression unit containing tandem pre-... Recombinant plasmids containing miRNA expression units. This vector system utilizes the host cell's endogenous Drosha / Dicer processing pathway to ensure the expression of two chimeric pre-... miRNAs are sequentially recognized and cleaved, efficiently generating mature miRNAs. 449a 5p and miR 30c 5p, and a stable co-expressing cell line was obtained through puromycin resistance screening.

[0054] 2. Lentiviral Packaging and Preparation HEK293T cells were cultured in serum-free, high-glucose DMEM at 37°C and 5% CO2 in 10cm dishes to 80% confluence (8-10 mL of culture medium). Packaging was performed using a mature third-generation lentiviral packaging system. The successfully constructed pre-contained lentiviral cells containing tandem pre-containers were then packaged. Recombinant lentiviral plasmids containing miRNA expression units were mixed with packaging plasmid psPAX2 and envelope plasmid pMD2.G at a mass ratio of 4:3:1 (8 μg:6 μg:2 μg) to obtain DNA. Then, a transfection complex was prepared by mixing DNA and PEI at a mass-to-volume ratio of 1:3 (total DNA 16 μg, PEI 48 μL) in serum-free high-glucose DMEM. The complex was incubated at room temperature for 20 minutes, and then directly added to cells in 10 cm culture dishes for transfection. Eight hours after transfection, the culture medium was replaced with an equal volume of complete medium (high-glucose DMEM + 10% FBS), and the cells were cultured at 37°C and 5% CO2 for another 60 hours. The cell culture supernatant was then collected, filtered through a 0.45 μm filter to remove cell debris, and concentrated and purified by ultracentrifugation to obtain high-titer lentiviral particles. The functional titer of high-titer lentiviral particles was verified by infecting target cells with these particles (the physical titer of the high-titer viral particles was determined using qPCR targeting specific regions of the lentiviral genome). All prepared viruses had a functional titer of at least 1 × 10⁻⁶. 8 TU / mL. The viral stock solution was aliquoted and stored at -80°C for long-term use. Simultaneously, a control virus containing only the empty vector (NC-lentivirus) was prepared following the exact same procedure.

[0055] 3. hMSC infection and screening of stable transgenic strains Human umbilical cord mesenchymal stem cells (hMSCs) of passage 3-5, in good growth condition, were cultured in a mesenchymal stem cell-specific culture medium to approximately 70% confluence to obtain an hMSC culture system. High-titer lentiviral particles (titer ≥ 1 × 10⁻⁶) prepared in the previous step were then used. 8 A concentrated viral solution (TU / mL) was directly added to the hMSC culture system. Virus was added at a multiplicity of infection (MOI) of 20, along with an infection enhancer (Polybrene, final concentration 8 μg / mL), and cultured at 37°C and 5% CO2 for 24 hours. After 24 hours of infection, the culture medium was replaced with fresh mesenchymal stem cell-specific medium, and cultured for another 72 hours to ensure viral integration and the initiation of miR expression. 449a-5p and miR 30c-5p. Next, the cells were cultured in a puromycin-containing medium for 10-14 days for selection, with the medium changed every 2-3 days, to obtain a stable cell population expressing both miRNAs. Ultimately, a stable hMSC transfected cell line overexpressing miR-449a-5p and miR-30c-5p was obtained, named miRNAs-hMSC. Simultaneously, a control cell line transduced with an empty vector (NC-hMSC) was established. Single clones were isolated and amplified using the limiting dilution method.

[0056] 4. Verification at the cellular and molecular level to stabilize the cell line The expression levels of mature miR-449a and miR-30c-5p in stable cell lines were detected by real-time quantitative PCR using the stem-loop method (qRT-PCR). The primer sequences used for the stem-loop method are shown in Table 2. Figure 4 As shown, compared with control cells (NC-hMSCs), the expression levels of miR-449a-5p and miR-30c-5p in engineered cells (miRNAs-hMSCs) were significantly upregulated. The relative expression levels of miR-449a-5p were (28.5±3.2) times that of NC-hMSCs, and miR-30c-5p were (31.6±3.8) times (p<0.001). Figure 4 This result demonstrates that the vector system can efficiently and synergistically overexpress miR-449a and miR-30c in hMSCs.

[0057] Example 3: Large-scale preparation and characterization of engineered exosomes (miRNAs-Exos) 1. Large-scale cell culture and collection of culture supernatant miRNAs-hMSCs were seeded into a stirred bioreactor and cultured in batches under strictly controlled culture parameters (temperature: 37.0℃, pH: 7.2±0.1). When the cell density reached 80-90%, the original culture medium was discarded, and the cells were gently washed twice with pre-warmed (37.0℃) phosphate-buffered saline (PBS), then replaced with exosome-free medium and cultured for another 48 hours. After culture, all culture supernatant was collected and immediately placed at 4℃ for subsequent processing to maximize exosome activity. Control exosomes (NC-Exos) were prepared from NC-hMSCs using the same process.

[0058] 2. Isolation and purification of exosomes The collected culture supernatant was centrifuged at 400×g (10 min, 4 °C) and 2,000×g (20 min, 4 °C) successively to remove cells and debris, and the centrifuged supernatant was obtained; then the centrifuged supernatant was concentrated 20-fold using a tangential flow filtration system (100 kDa molecular weight cut-off) to obtain a concentrated solution; and then the concentrated solution was replaced with PBS (pH 7.4) buffer to obtain a replacement solution. The replacement solution was purified by size exclusion chromatography column (Sephacryl S-400 HR), and the exosome characteristic elution peak was collected. The final product was sterile filtered through a 0.22 μm filter membrane to obtain miRNAs-Exos. The control NC-Exos was prepared from NC-hMSC by the same process.

[0059] 3. Quality characterization of exosomes The following quality controls were performed on the isolated miRNAs-Exos: Particle size and concentration: Nanoparticle tracking analysis (NTA) showed that the main peak of its particle size distribution was in the range of 80 - 150 nm, and the results were as Figure 5 shown in a of

[0060] Marker proteins: Western Blot detection confirmed the expression of CD63 and TSG101 and the non-expression of Calnexin, and the results were as Figure 5 shown in b of

[0061] Morphological structure: Transmission electron microscopy (TEM) observation showed that it had a typical cup-shaped vesicle structure, and the results were as Figure 5 shown in c of

[0062] miRNA level detection: Determined by qPCR, compared with NC-Exos, the relative enrichment multiples of miR-449a and miR-30c in miRNAs-Exos were both >10-fold, and the results were as Figure 6 shown in

[0063] Example 4: Application of miRNAs-Exos in skin photoaging repair 1. In vitro functional verification Human dermal fibroblasts (HDF) were irradiated with UVB (30 mJ / cm 2 ²) to induce senescence, and senescent cells were obtained. Four experimental groups were set up, namely the normal control group (human dermal fibroblasts), the UVB model group (senescent cells), the NC-Exos treatment group, and the miRNAs-Exos treatment group. In the NC-Exos treatment group and the miRNAs-Exos treatment group, 50 μg / mL of NC-Exos or miRNAs-Exos was added to the culture medium and allowed to act for 48 hours. Subsequently, the expression levels of SASP factors in the four experimental groups were detected by qRT-PCR respectively, and the results were as Figure 7 As shown in Figure a, miRNAs-Exos treatment significantly downregulated the mRNA expression levels of key SASP factors CCL2 and PAI-1 in aging HDF, with inhibition rates of 55% and 60%, respectively (p<0.001), significantly better than the NC-Exos treatment group (inhibition rate <15%). Then, aging-related β-galactosidase staining (SA-β-gal staining) was performed on the four experimental groups, and the results are shown in Figure a. Figure 7 As shown in b, the proportion of SA-β-gal positive cells in the miRNAs-Exos treatment group was significantly lower than that in the model group and the NC-Exos treatment group.

[0064] 2. Evaluation of therapeutic effects in animals Using SKH-1 hairless mice, chronic UVB irradiation (60 mJ / cm²) was applied. 2 A mouse model of skin photoaging was established by administering the treatment three times a week for eight weeks. Four experimental groups were set up: a normal control group (SKH-1 hairless mice), a UVB model group (skin photoaging model mice), an NC-Exos treatment group, and a miRNAs-Exos treatment group. Thermosensitive PEG-HPMC hydrogel precursor solutions were prepared according to classic methods (see: L. Klouda, AG Mikos, Eur. J. Pharm. Biopharm., 2008, 68, 34–45). PEG and HPMC were dissolved separately in deionized water to prepare 2% (w / v, g / 100 mL) solutions, filtered sterilely, and stored at 4°C. Equal volumes were mixed immediately before use to obtain a final 1% (w / v, g / 100 mL) PEG-HPMC precursor solution, which was rapidly gelled at 37°C for local loading and delivery of exosomes. Purified miRNAs-Exos or NC-Exos (50 μg / dose) were uniformly dispersed in a hydrogel matrix, gently mixed, and kept at a low temperature to prevent a decrease in exosome activity. The NC-Exos treatment group and the miRNAs-Exos treatment group were respectively treated locally with a thermosensitive hydrogel (50 μg exosomes / dose) loaded with NC-Exos or miRNAs-Exos exosomes in photoaged mice, three times a week for four weeks. After treatment, the therapeutic effect on the skin of the photoaged mice was statistically analyzed. The miRNAs-Exos treatment group showed significant improvement in skin roughness and wrinkles (results are shown in Figure 1). Figure 8As shown in Figure a); compared with the normal control group (218.5±12.7 μm), chronic UVB irradiation caused significant dermal atrophy in mice, with the thickness reduced to 142.3±10.5 μm (p<0.01). After treatment with miRNAs-Exos, the dermal thickness was significantly restored, reaching 198.6±13.1 μm, recovering to 90% of the normal level. Its efficacy was significantly better than that of the NC-Exos treatment group (results are shown in Figure a). Figure 8 As shown in b); Western blotting showed that miRNAs-Exos treatment significantly increased the levels of type I and type III collagen in the skin, with significantly better efficacy than the NC-Exos group (results are shown in b). Figure 8 (as shown in c and d).

[0065] 3. Preliminary analysis of molecular mechanisms Skin tissues from the four experimental groups after treatment were subjected to qPCR detection (primers are shown in Table 4). The results showed that the mRNA expression levels of CCL2 and PAI-1 were significantly downregulated in the miRNAs-Exos treatment group (results are shown in Table 4). Figure 9 As shown in a), it suggests that the key mechanism by which it exerts its repair effect is to synergistically inhibit the expression level of local SASP factors by delivering miR-449a-5p and miR-30c-5p.

[0066] Table 4: Primers for gene detection in mouse skin tissue

[0067] To further investigate the mechanism by which miRNAs-Exos promote collagen restoration, this experiment detected the expression of key collagen degradation enzymes MMP-1 and MMP-3. The results are as follows: Figure 9 As shown in b, UVB irradiation significantly upregulated the expression levels of MMP-13 and MMP-3 mRNA, while miRNAs-Exos treatment significantly inhibited their levels, with a significantly better inhibitory effect than the NC-Exos group (p<0.01). This suggests that miRNAs-Exos may reduce collagen degradation by inhibiting MMP expression, thereby increasing skin collagen content. in conclusion: This invention constructed engineered hMSCs capable of co-expressing miR-449a-5p and miR-30c-5p and established a standardized preparation process for the corresponding exosomes. The obtained miRNAs-Exos exhibit clear anti-photoaging function, providing new technical solutions and experimental evidence for the development of related therapeutic products.

[0068] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A stem cell co-expressing miR-449a-5p and miR-30c-5p, characterized in that, The stem cells express a chimeric dual miRNA expression unit; the chimeric dual miRNA expression unit is expressed as a pre- miR 155 forms the backbone, encoding and processing miR within the same transcript. 449a 5p and miR 30c These two miRNAs are 5p.

2. The stem cells according to claim 1, characterized in that, The chimeric dual miRNA expression unit comprises: a first chimeric pre miRNA units, composed of miR 449a 5p mature sequence and its complementary strand are embedded in pre miR Formed in the 155 skeleton; second interlocking pre miRNA units, composed of miR 30c 5p mature sequence and its complementary strand are embedded in pre miR Formed in the 155 skeleton; the first interlocking pre miRNA unit and second chimeric pre miRNA units are transmitted via pre miR The natural flanking sequences of the 155 skeleton are directly connected.

3. The stem cells according to claim 2, characterized in that, The first interlocking pre The coding sequence of the miRNA unit is shown in SEQ ID NO:9; the second chimeric pre The coding sequence of the miRNA unit is shown in SEQ ID NO:

10.

4. The method for preparing stem cells according to any one of claims 1-3, characterized in that, The method includes: Step 1: The chimeric dual miRNA expression unit was cloned into a lentiviral expression vector to obtain a recombinant plasmid; Step 2: Co-transfect the recombinant plasmid, packaging plasmid, and envelope plasmid into host cells, culture and collect the transfection products, and purify them to obtain lentiviral particles; Step 3: Infect mesenchymal stem cells with the lentiviral particles and screen and culture them to obtain stem cells that co-express miR-449a-5p and miR-30c-5p; The chimeric dual miRNA expression unit uses pre miR 155 forms the backbone, encoding and processing miR within the same transcript. 449a 5p and miR 30c These two miRNAs are 5p; Preferably, the packaging plasmid comprises: psPAX2; Preferably, the envelope plasmid includes pMD2.G.

5. An exosome for the prevention and / or treatment of photoaging of the skin, characterized in that, The exosomes are obtained by secretion from stem cells prepared by any one of claims 1-3 or by the preparation method of claim 4.

6. The exosome according to claim 5, characterized in that, The exosomes are loaded with two miRNAs, miR-449a-5p and miR-30c-5p.

7. A pharmaceutical composition for preventing and / or treating photoaging of the skin, characterized in that, The pharmaceutical composition comprises the stem cells of any one of claims 1-3, stem cells prepared by the preparation method of claim 4, or exosomes of claim 5.

8. The pharmaceutical composition according to claim 7, characterized in that, The drug composition is administered via local delivery, wherein the local delivery is applied to the target administration site via a local delivery carrier; Preferably, the local delivery carrier comprises a thermosensitive hydrogel or a soluble microneedle array.

9. The use of the stem cells according to any one of claims 1-3, or the stem cells prepared by the preparation method according to claim 4, or the exosomes according to claim 5, or the pharmaceutical composition according to any one of claims 7-8, in the preparation of products for the prevention and / or treatment of photoaging of the skin.

10. The application according to claim 9, characterized in that, The product exerts its effect of preventing and / or treating photoaging of the skin by inhibiting the age-related secretory phenotype SASP factor in skin tissue; Preferably, the SASP factor includes CCL2 and / or PAI-1.

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