Preparation method of human-derived nasal mucosa mesenchymal stem cell medicine for treating sepsis

Abstract

The invention belongs to the technical field of biological medicine and cell therapy, and particularly relates to a preparation method of a human-derived nasal mucosa mesenchymal stem cell medicine for treating sepsis, which comprises the following steps: cell separation: taking adult nasal mucosa tissues, cutting into pieces, digesting with 0.25% pancreatin, and terminating digestion with a DF12 culture medium containing 20% serum; primary culture: after the tissue adheres to the wall for one week by using the first-step culture medium, replacing a DF12 culture medium containing 10% of serum, and after the cells climb out of the tissue, centrifugally collecting the cells for primary culture; 3, primary culture: identifying a fourth generation of cell surface marker in primary culture by flow cytometry, the invention reveals the deep mechanism of function enhancement of HuNMSCs by inhibiting cell autophagy, maintaining the integrity of a cell membrane structure and improving the survival rate of a sepsis model through molecular means such as lipid metabonomics and the like for the first time, and the deep mechanism of function enhancement of the HuNMSCs is illustrated by using the HuNMSCs to inhibit cell autophagy, maintain the integrity of a cell membrane structure and improve the survival rate of the sepsis model. The possibility of treating sepsis by HuNMSCs is clarified, a brand new cell treatment strategy is provided for sepsis, and a foundation is laid for subsequent clinical application.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and cell therapy technology, specifically relating to a method for preparing human nasal mucosal mesenchymal stem cell drugs for the treatment of sepsis. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome triggered by infection. It is a dangerous condition with a high mortality rate. Its pathological process is complex. In the early stage, it manifests as a "cytokine storm" dominated by M1 macrophages, while in the later stage, it is often accompanied by "immune paralysis" caused by the failure of immune cell function (such as macrophage pyroptosis).

[0003] Mesenchymal stem cells (MSCs) are considered a potential tool for treating sepsis due to their strong immunomodulatory and tissue repair capabilities. However, allogeneic MSCs may cause rejection during differentiation, limiting their efficacy. Therefore, finding a type of MSC with autologous transplantation potential is crucial to overcoming current treatment limitations. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing human nasal mucosal mesenchymal stem cell drugs for the treatment of sepsis. These cells can systematically alleviate the pathological process of sepsis and improve the survival rate by inhibiting autophagy.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing human nasal mucosal mesenchymal stem cell drugs for treating sepsis, comprising the following steps: Step 1, Cell Isolation: Take adult nasal mucosa tissue, cut it into small pieces, digest it with 0.25% trypsin, and terminate the digestion with DF12 medium containing 20% ​​serum; Step 2, Primary Culture: After the tissue adheres to the culture medium from Step 1 for one week, the medium is replaced with DF12 medium containing 10% serum. After the cells crawl out of the tissue, they are collected by centrifugation for primary culture. Step 3, Primary Culture: Flow cytometry was used to identify surface markers in the 4th generation cells of the primary culture, showing that the positive rates of CD29, CD90, and CD105 were ≥95%, and the positive rates of CD34, CD45, and HLA-DR were ≤2%. The HuNMSCs were selected from clinically derived nasal mucosal tissue, and their surface markers were positive for CD29, CD90, and CD105, and negative for CD34, CD45, and HLA-DR.

[0006] Step 4, Drug preparation: The purified human nasal mucosal mesenchymal stem cells (HuNMSCs) from the seed cells are mixed with physiological saline to prepare a cell suspension for intravenous injection.

[0007] Dosage regimen: Administer therapeutically effective doses of HuNMSCs to sepsis-affected individuals via tail vein injection or other methods. Recommended... The cell dose was 1.0 × 10⁻⁶. 6 Cells per kilogram of body weight.

[0008] The active ingredient in this human nasal mucosal mesenchymal stem cell drug used to treat sepsis is human nasal mucosal mesenchymal stem cells.

[0009] As a method for preparing a human nasal mucosal mesenchymal stem cell drug for treating sepsis according to the present invention, preferably, the dosage of the mesenchymal stem cell drug for treating sepsis is 1.0 × 10⁻⁶. 6 Cells per kilogram of body weight.

[0010] The human nasal mucosal mesenchymal stem cells of the present invention effectively combat sepsis by inhibiting macrophage autophagy.

[0011] Compared with the prior art, the beneficial effects of the present invention are: Mechanism innovation: For the first time, molecular methods such as lipid metabolomics have revealed that HuNMSCs improve the survival rate of a sepsis model by inhibiting autophagy, maintaining cell membrane structural integrity, and elucidating the deep mechanism of their enhanced function.

[0012] Strategic Innovation: This study clarified the potential of HuNMSCs in treating sepsis, providing a novel cell therapy strategy for sepsis and laying the foundation for subsequent clinical applications. Attached Figure Description

[0013] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 is a schematic diagram of the separation and identification of HuNMSCs provided in the embodiments of this application.

[0014] Figure 2 is a schematic diagram of the survival curve of the HuNMSCs treatment sepsis model provided in the embodiments of this application.

[0015] Figure 3 is a schematic diagram of HE staining of the heart, liver, spleen, lungs and kidneys of mice in different groups 48 hours after CLP using HuNMSCs provided in the embodiments of this application.

[0016] Figure 4 is a schematic diagram of the Venn set of differential lipid metabolites before and after treatment of sepsis model with HuNMSCs provided in the embodiments of this application.

[0017] Figure 5 is a heatmap of the differential metabolites before and after treatment of sepsis model with HuNMSCs provided in the embodiments of this application.

[0018] Figure 6 is a schematic diagram of the pH value of the Rich Factor provided in the embodiments of this application.

[0019] Figure 7 is a schematic diagram of KEGG classification provided in an embodiment of this application.

[0020] Figure 8 is a schematic diagram of immunofluorescence staining of autophagic flux in mitochondria of mouse lung cells from different groups 72 hours after CLP, as provided in the embodiments of this application.

[0021] Figure 9 is a schematic diagram of the Simplewes results of autophagy-related proteins in BMDMs samples after co-culturing HuNMSCs and LPS-treated BMDMs provided in the embodiments of this application.

[0022] Figure 10 is a schematic diagram of the preparation process provided in the embodiments of this application. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0024] Please refer to Figures 1-10. This invention provides the following technical solution: a method for preparing human nasal mucosal mesenchymal stem cell drugs for treating sepsis, comprising the following steps: Step 1, Cell Isolation: Take adult nasal mucosa tissue, cut it into small pieces, digest it with 0.25% trypsin, and terminate the digestion with DF12 medium containing 20% ​​serum; Step 2, Primary Culture: After the tissue adheres to the culture medium from Step 1 for one week, the medium is replaced with DF12 medium containing 10% serum. After the cells crawl out of the tissue, they are collected by centrifugation for primary culture. Step 3, Primary Culture: Flow cytometry was used to identify surface markers in the 4th generation cells of the primary culture, showing that the positive rates of CD29, CD90, and CD105 were ≥95%, and the positive rates of CD34, CD45, and HLA-DR were ≤2%. Step 4, Drug preparation: The purified human nasal mucosal mesenchymal stem cells (HuNMSCs) from the seed cells are mixed with physiological saline to prepare a cell suspension for intravenous injection.

[0025] The dosage of the mesenchymal stem cell drug for the treatment of sepsis is 1.0 × 10⁻⁶. 6 Cells per kilogram of body weight.

[0026] Example 1: Please refer to Figure 1 for the isolation and identification of mesenchymal stem cells derived from human nasal mucosa: Cell isolation: Adult nasal mucosa tissue was taken, minced, and digested with 0.25% trypsin. The digestion was stopped with DF12 medium containing 20% ​​serum. The tissue was allowed to adhere to the culture medium for one week. Then, the medium was replaced with DF12 medium containing 10% serum. After the cells crawled out of the tissue, they were centrifuged and collected for primary culture.

[0027] Cell identification: Flow cytometry was used to identify surface markers of the 4th generation cells, showing that the positive rates of CD29, CD90, and CD105 were ≥ 95%, and the positive rates of CD34, CD45, and HLA-DR were ≤ 2%.

[0028] Example 2: Please see Figure 2 —7. Therapeutic effects of HuNMSCs in a rat model of sepsis: 1. Model Establishment and Grouping: An ICR mouse sepsis model was established using the cecal ligation and puncture method (CLP). One hour after successful model establishment, mice were randomly divided into a sham operation group (cecal ligation without puncture), a CLP model group, and a HuNMSCs treatment group.

[0029] 2. Administration: The HuNMSCs treatment group received 1 × 10 6 200 μl of cell-containing saline suspension was administered via tail vein injection per kg of the sample. The remaining groups received an equal volume of saline solution via tail vein injection.

[0030] 3. Result observation (144 hours): Survival rate: The survival rate was 10% in the model control group and 70% in the HuNMSC group.

[0031] Organ damage: Compared with the sham surgery group, the model group showed varying degrees of damage to all organs, such as hepatocyte fatty degeneration; glomerular edema and unclear renal tubular structure; indistinct red / white pulp boundary and extensive mononuclear cell infiltration in the spleen; pulmonary interstitial edema; and abnormal cardiomyocyte arrangement. After cell therapy, all of the above conditions were significantly alleviated.

[0032] Lipid metabolomics analysis: Inguinal lymph nodes from each group were collected for lipid metabolomics analysis. Differential analyses were performed between the sham-operated group and the model group, and between the model group and the treatment group. Venn set analysis was conducted on the two differential sets, yielding 69 differentially expressed lipid metabolites. Further heatmaps showed that the selected differentially expressed metabolites exhibited the same trend in the control and treatment groups compared to the model group. KEGG enrichment of these differentially expressed metabolites revealed that 70.31% were involved in cholesterol metabolism, and 7.81% were involved in autophagy. The trends of these metabolites indicate that HuNMSCs can inhibit autophagy, maintain cell membrane structure, and reduce cell damage.

[0033] In vivo mitochondrial autophagy: Lung tissues from each model group were subjected to immunofluorescence staining. Compared with the model group, the autophagic flux in the treatment group was significantly reduced (atp5a1+ / LC3b+ cells), indicating that cell therapy can weaken mitochondrial autophagy.

[0034] Example 3: Please see Figure 8 The in vitro regulatory effect of HuNMSCs on macrophages: 1. Macrophage acquisition: Bone marrow from the femoral bone of ICR mice was prepared into a single-cell suspension by passing it through a 40 μm filter and then induced in medium containing M-CSF for 7 days to obtain bone marrow-derived macrophages (BMDMs).

[0035] 2. Co-culture and stimulation: A Transwell co-culture system was established. BMDMs were seeded in the lower layer, and HuNMSCs prepared in Example 1 were seeded in the upper chamber. The co-culture system was stimulated with 1 μg / mL LPS for 24 hours to simulate a sepsis environment. In vitro macrophage mitochondrial autophagy: 3. Effect detection: Simple wes: Total protein extracted from BMDMs was analyzed, and the results showed that PINK1, LC3I, and LC3II proteins were significantly reduced in the treatment group, indicating that HuNMSCs can significantly inhibit autophagy, consistent with in vivo results.

[0036] Please refer to Figures 2 and 3 for the in vivo therapeutic effects of HuNMSCs: Significantly improved survival rate: In a rat model of sepsis induced by cecal ligation and puncture, tail vein injection of HuNMSCs increased the 144-hour survival rate of the model animals from 10% (model group) to a maximum of 70%. Organ sections also showed that HuNMSCs treatment has a multi-organ protective function.

[0037] Please see Figure 4—6. Lipid metabolomics mechanisms reveal: As shown in Example 2, lipid metabolomics analysis was performed on the inguinal lymph nodes of the model before and after HuNMSCs treatment. The results showed that there were a total of 69 differential metabolites, which were enriched in cholesterol metabolism and autophagy-related pathways.

[0038] Please refer to Figure 7, Autophagy Flow Detection (Core Mechanism): In vivo immunofluorescence showed that after HuNMSCs treatment, mitochondrial autophagy in the model lung tissue was significantly reduced.

[0039] Please refer to Figure 8, HuNMSCs remodel macrophage homeostasis: SimpleWest showed that HuNMSCs significantly reduced the expression of autophagy-related proteins such as PINK and LC3II in LPS-treated BMDMs, indicating that HuNMSCs can maintain macrophage homeostasis.

[0040] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing human nasal mucosal mesenchymal stem cell drugs for the treatment of sepsis, characterized in that, Includes the following steps: Step 1, Cell Isolation: Take adult nasal mucosa tissue, cut it into small pieces, digest it with 0.25% trypsin, and terminate the digestion with DF12 medium containing 20% ​​serum; Step 2, Primary Culture: After the tissue adheres to the culture medium from Step 1 for one week, the medium is replaced with DF12 medium containing 10% serum. After the cells have crawled out of the tissue, they are collected by centrifugation for primary culture. Step 3, Seeding Cells: Flow cytometry was used to identify surface markers in the fourth generation of primary cultured cells, showing that the positive rates of CD29, CD90, and CD105 were ≥95%, and the positive rates of CD34, CD45, and HLA-DR were ≤2%. Step 4, Drug preparation: The purified human nasal mucosal mesenchymal stem cells (HuNMSCs) from the seed cells are mixed with physiological saline to prepare a cell suspension for intravenous injection.

2. The method for preparing a human nasal mucosal mesenchymal stem cell drug for treating sepsis according to claim 1, characterized in that: The dosage of the mesenchymal stem cell drug for the treatment of sepsis is 1.0 × 10⁻⁶. 6 Cells per kilogram of body weight.

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