Preparation method of amnion-derived mesenchymal stem cells with high collagen expression

Abstract

The invention aims to provide a preparation method of amnion-derived mesenchymal stem cells with high collagen expression. The preparation method comprises the following steps: step 1, reagents and materials: amnion tissues: obtaining a fresh amnion from a healthy puerpera after delivery; the PBS is used for cleaning amniotic tissues; collagenase: type I collagenase, which is used for digesting amniotic tissues to separate mesenchymal stem cells; a cell culture medium is a DMEM / F12 culture medium, and 10% of fetal calf serum and 1% of penicillin-streptomycin double antibodies are added; the transforming growth factor TGF-beta is used for promoting the expression of mesenchymal stem cell collagen; and the fibroblast growth factor FGF can assist in promoting cell proliferation and collagen expression. The invention aims to provide the preparation method of the amniotic membrane-derived mesenchymal stem cells for highly expressing collagen, and the amniotic membrane-derived mesenchymal stem cells can be widely applied to treatment of immune system diseases, reproductive system diseases, skin system diseases, skeletal system diseases, nervous system diseases, digestive system diseases, respiratory system diseases, circulatory system diseases and urinary system diseases. Particularly, the effects of immunosuppression and tissue repair are achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of regenerative medicine, and in particular to a method for preparing amniotic membrane-derived mesenchymal stem cells that highly express collagen. Background Art

[0002] During the development of the embryo, the fetal membrane is formed. The innermost layer of the fetal membrane is the amnion, which is composed of a thick basement membrane and avascular matrix. Under an electron microscope, the amnion can be divided into five layers from the inside to the outside: epithelial layer, basement membrane layer, compact layer, fibroblast layer and spongy layer. Human amniotic epithelial cells (hAECs) originate from the epithelial layer, which develops from the inner cell mass of the blastocyst. The epithelial layer is in direct contact with the amniotic fluid; the basement membrane layer is composed of narrow acellular reticular fibers; the compact layer determines the tension of the amnion, which is composed of a large number of collagen fibers and reticular fibers, accounting for about 90% of the thickness of the amnion.

[0003] Human amniotic mesenchymal stem cells (hAMSCs) are derived from the fibroblast layer, which is composed of loose fibroblasts and reticular fibers; the spongy layer is composed of wavy reticular fibers and has a certain elasticity. hAECs and hAMSCs have little immune rejection, and amniotic membrane is easy to obtain and is a non-invasive operation. The placenta amniotic membrane and umbilical cord are often "discarded" after the fetus is delivered. Therefore, isolating hAECs and hAMSCs from the epithelial layer and fibroblast layer of the amniotic membrane for cell replacement therapy and regenerative medicine research has become a hot topic in this field.

[0004] The primary hAECs grow in multiple small flakes, and the cells of the P1 to P3 generations grow in a cobblestone-like pattern, arranged closely. After the P4 generation, the cells gradually age, and can be passed to the P5 generation at most. hAECs do not express the telomerase gene, so they cannot proliferate indefinitely and are not tumorigenic. Experimental studies have shown that when non-obese diabetic / severe combined immunodeficient mice were subcutaneously injected with an appropriate amount of hAECs, no tumors formed in the mice. At the same time, hAECs were co-injected with Hela cells and human Burkitt's lymphoma cells into experimental mice, and the results showed that hAECs had almost no effect on tumor formation.

[0005] Similar to hAECs, hAMSCs also have significant plasticity and multidirectional differentiation potential, and can differentiate into cells from different tissues of the three germ layers. hAMSCs were induced to differentiate into ligament cells by transforming growth factor-β1, bFGF and hyaluronic acid combined or alone. The highly specific tendon cell marker Scx was used as a positive regulatory factor to promote the directional differentiation of hAMSCs into tendon cells, and to increase the expression of tendon-related molecules including collagen I and III, fibronectin, matrix metalloproteinase-2, etc. In addition, hAMSCs can also differentiate into osteoblasts and myocytes. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing amniotic membrane-derived mesenchymal stem cells with high collagen expression, which can be widely used in the treatment of immune system diseases, reproductive system diseases, skin system diseases, skeletal system diseases, nervous system diseases, digestive system diseases, respiratory system diseases, circulatory system diseases, and urinary system diseases, especially to play the role of immunosuppression and tissue repair.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing amniotic membrane-derived mesenchymal stem cells that highly express collagen, the method comprising the following steps:

[0009] Step 1. Reagents and materials:

[0010] Amniotic membrane tissue: fresh amniotic membrane obtained from healthy pregnant women after delivery;

[0011] Phosphate buffered saline (PBS): used to wash amniotic tissue;

[0012] Collagenase: Type I collagenase, used to digest amniotic tissue to isolate mesenchymal stem cells;

[0013] Cell culture medium: DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin;

[0014] Step 2: Preparation steps

[0015] Amniotic tissue processing:

[0016] Fresh amniotic tissue was washed several times with PBS under sterile conditions to remove blood and impurities;

[0017] Cut the amniotic membrane into small pieces, put them into a container containing an appropriate amount of collagenase, and digest them in a 37°C constant temperature shaker for 1-2 hours;

[0018] Step 3: Cell isolation and culture:

[0019] The digested tissue suspension was filtered through a strainer to remove undigested tissue debris;

[0020] The filtrate was centrifuged at an appropriate centrifugal speed, 1500 rpm for 5 minutes; the supernatant was discarded and the precipitated cells were resuspended in cell culture medium;

[0021] The cells were inoculated into culture flasks and cultured in an incubator at 37°C and 5% CO2;

[0022] Step 4: Induce high expression of collagen:

[0023] When the cells grow to a certain density, 80% confluence, the culture medium containing specific concentrations of transforming growth factor TGF-β and fibroblast growth factor FGF is replaced;

[0024] The concentration of transforming growth factor TGF-β was set to 5-10 ng / ml, and the concentration of fibroblast growth factor FGF was set to 2-5 ng / ml;

[0025] Continue culturing for 7-10 days, during which time the cell status is regularly observed and the culture medium is replaced;

[0026] Step 5. Cell harvesting and identification:

[0027] After the culture was completed, the cells were collected by trypsin digestion;

[0028] The expression level of collagen in cells was detected by immunofluorescence staining, Western blot and other methods to confirm that the amniotic membrane-derived mesenchymal stem cells with high collagen expression were successfully prepared;

[0029] Step 6. Cell screening and expansion

[0030] After the cells adhere to the wall and grow, replace the culture medium to remove the non-adherent cells and impurities;

[0031] Observe the growth of cells, and when the cell confluence reaches about 80%, perform subculture;

[0032] Mesenchymal stem cells were screened and identified by flow cytometry and other methods.

[0033] The step of screening mesenchymal stem cells by flow cytometry in step 6:

[0034] 1. Cell Preparation

[0035] Isolate mononuclear cell suspension from tissue samples; bone marrow, fat, and separate by density gradient centrifugation and other methods;

[0036] 2. Antibody labeling

[0037] Select appropriate antibody combinations to label cells, including positive marker antibodies for CD73, CD90, and CD105, and negative marker antibodies for CD14, CD19, CD34, CD45, and HLA-DR;

[0038] The cells were incubated with antibodies at 4°C for 30 min;

[0039] Flow cytometer detection: Add the labeled cell suspension to the flow cytometer for detection;

[0040] Flow cytometry classifies and counts cells based on the fluorescent labels carried by antibodies;

[0041] 3. Screening

[0042] According to the results of flow cytometry, cells that simultaneously expressed positive markers and did not express negative markers were screened out, and these cells were considered to be mesenchymal stem cells.

[0043] The transforming growth factor TGF-β stimulates mesenchymal stem cells to synthesize collagen, which is type I and type III collagen.

[0044] The fibroblast growth factor FGF is basic fibroblast growth factor bFGF, which is used to stimulate the activity of mesenchymal stem cells, increase the synthesis of collagen, and may help maintain the stemness of cells.

[0045] The platelet-derived growth factor PDGF is used to induce mesenchymal stem cells to differentiate into fibroblast-like cells, thereby increasing the secretion of collagen.

[0046] The step 4, inducing high expression of collagen, also includes adding inducers: vitamin C, dexamethasone, and hyaluronic acid.

[0047] Compared with the prior art, the method for preparing amnion-derived mesenchymal stem cells with high collagen expression provided by the present invention can prepare amnion-derived mesenchymal stem cells with high collagen expression, providing potential cell resources for the fields of tissue repair and regeneration.

[0048] The method for preparing amniotic membrane-derived mesenchymal stem cells with high collagen expression of the present invention has the following beneficial effects:

[0049] 1. Medical applications

[0050] Promotes tissue repair:

[0051] Amniotic mesenchymal stem cells that highly express collagen can provide damaged tissues with abundant collagen, which is essential for the structural support and repair of tissues. For example, in skin injury repair, collagen can promote the formation of new extracellular matrix, accelerate wound healing, and reduce scar formation.

[0052] In bone tissue repair, collagen can combine with calcium salts to provide a scaffold for osteoblasts and promote new bone formation.

[0053] Enhance tissue regeneration ability:

[0054] Mesenchymal stem cells have multidirectional differentiation potential, and the high expression of collagen makes them more advantageous in tissue regeneration. They can secrete a variety of growth factors and cytokines to stimulate the proliferation and differentiation of surrounding cells. At the same time, the presence of collagen provides a stable structural basis for regenerated tissue.

[0055] For example, in the repair of myocardial damage, this type of stem cell can differentiate into cardiomyocyte-like cells and improve the elasticity and contractile function of myocardial tissue by secreting substances such as collagen.

[0056] 2. Advantages of Material Sources

[0057] Amniotic membrane is abundant and has no ethical controversy:

[0058] The amniotic membrane is a waste product after childbirth, which is abundant and easy to obtain. Compared with other stem cell sources such as embryonic stem cells, mesenchymal stem cells derived from the amniotic membrane do not have ethical controversy and are more likely to be widely used in clinical research and treatment.

[0059] Amniotic tissue can be legally collected from healthy pregnant women after childbirth, and stem cells can be isolated and prepared to provide a sustainable source of cells for medical needs.

[0060] Low immunogenicity:

[0061] Amnion-derived mesenchymal stem cells have low immunogenicity, which means that they are less likely to cause host immune rejection in transplantation therapy. This allows them to be used for autologous or allogeneic transplantation, expanding their clinical application range.

[0062] Even when transplants are performed between different individuals, the use of immunosuppressants can be reduced, reducing treatment risks and side effects.

[0063] 3. Advantages of preparation method

[0064] Strong controllability:

[0065] Through specific preparation methods, such as adding transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF), the expression level of collagen in mesenchymal stem cells can be precisely regulated. This enables us to customize stem cell products with specific collagen expression levels according to different treatment needs.

[0066] The concentration of growth factors and culture time can be adjusted according to the repair requirements of different tissues to obtain the best collagen expression effect and improve the targetedness and effectiveness of treatment.

[0067] High stability:

[0068] This preparation method is relatively stable and reliable, and can guarantee the quality and consistency of stem cell products to a certain extent. By strictly controlling each link in the preparation process, such as the processing of amniotic tissue, cell culture conditions and the use of induction factors, the variation in the preparation process can be reduced and the stability of the product can be improved.

[0069] This will help to achieve standardization and regularization of stem cell therapy and provide reliable cell products for clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is an electron micrograph of human amniotic mesenchymal stem cells. DETAILED DESCRIPTION

[0071] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0072] The method for preparing amnion-derived mesenchymal stem cells with high collagen expression of the present invention comprises the following steps:

[0073] 1. Material preparation

[0074] Placental tissue from healthy pregnant women ensures that the placenta is free from infectious diseases and other abnormalities.

[0075] Sterile surgical instruments, including scalpels, tweezers, scissors, etc.

[0076] Reagents and consumables required for cell culture, such as culture medium (such as DMEM / F12 medium), serum, trypsin, culture flasks, culture dishes, etc.

[0077] Reagents and Materials:

[0078] Amniotic membrane tissue: fresh amniotic membrane obtained from healthy pregnant women after delivery.

[0079] Phosphate buffered saline (PBS): used to wash the amniotic membrane tissue.

[0080] Collagenase: such as type I collagenase, used to digest amniotic tissue to isolate mesenchymal stem cells.

[0081] Cell culture medium: such as DMEM / F12 culture medium, supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin double antibody, etc.

[0082] Transforming growth factor-β (TGF-β): used to promote the expression of collagen in mesenchymal stem cells.

[0083] Fibroblast Growth Factor (FGF): can help promote cell proliferation and collagen expression.

[0084] 2. Preparation steps

[0085] Amniotic tissue processing:

[0086] The fresh amniotic membrane tissue was washed several times with PBS under sterile conditions to remove blood and impurities.

[0087] Cut the amniotic membrane into small pieces, put them into a container containing an appropriate amount of collagenase, and digest them in a 37°C constant temperature shaker for a certain period of time, usually 1-2 hours.

[0088] Cell isolation and culture:

[0089] The digested tissue suspension was filtered through a strainer to remove undigested tissue fragments.

[0090] The filtrate is centrifuged at an appropriate centrifugal speed, such as 1500 rpm for 5 minutes.

[0091] The supernatant was discarded and the pelleted cells were resuspended in cell culture medium.

[0092] The cells were inoculated into culture flasks and cultured in an incubator at 37°C and 5% CO2.

[0093] Inducing high expression of collagen:

[0094] 3. Extraction of Mesenchymal Stem Cells

[0095] When the cells grow to a certain density, such as 80% confluence, the culture medium containing specific concentrations of TGF-β and FGF is replaced.

[0096] The concentration of TGF-β can be set to 5-10 ng / ml, and the concentration of FGF can be set to 2-5 ng / ml.

[0097] Continue culturing for a period of time, usually 7-10 days, during which time the cell status is regularly observed and the culture medium is replaced.

[0098] Cell harvesting and characterization:

[0099] After the culture was completed, the cells were collected by trypsinization.

[0100] The expression level of collagen in cells can be detected by immunofluorescence staining, Western blot and other methods to determine whether the amniotic membrane-derived mesenchymal stem cells with high collagen expression are successfully prepared.

[0101] IV. Cell Screening and Expansion

[0102] After the cells adhere to the wall and grow, replace the culture medium and remove the non-adherent cells and impurities.

[0103] The growth of cells was observed, and when the cell confluence reached about 80%, subculture was performed.

[0104] Mesenchymal stem cells can be screened and identified by methods such as flow cytometry.

[0105] Steps for screening mesenchymal stem cells by flow cytometry:

[0106] 1. Cell Preparation

[0107] Isolate mononuclear cell suspension from tissue samples (such as bone marrow, fat, etc.). Separation can be performed by density gradient centrifugation or other methods.

[0108] 2. Antibody labeling

[0109] Select the appropriate antibody combination to label the cells. Common labeling antibodies include positive labeling antibodies such as CD73, CD90, CD105, and negative labeling antibodies such as CD14, CD19, CD34, CD45, and HLA-DR.

[0110] Incubate the cells with the antibody under appropriate conditions for a period of time, usually at 4°C for about 30 minutes.

[0111] Flow cytometry

[0112] The labeled cell suspension was added to the flow cytometer for detection.

[0113] Flow cytometry classifies and counts cells based on the fluorescent labels attached to the antibodies.

[0114] 3. Screening

[0115] According to the results of flow cytometry, cells that simultaneously expressed positive markers and did not express negative markers were screened out, and these cells were considered to be mesenchymal stem cells.

[0116] Table 1. Screening of mesenchymal stem cells by flow cytometry

[0117]

[0118] In order to enhance the expression of collagen, appropriate growth factors or other inducers may be added to the culture medium.

[0119] 5. Detection of collagen expression

[0120] Immunofluorescence staining and Western blot were used to detect the expression of collagen in mesenchymal stem cells.

[0121] Table 2. Detection of collagen expression in mesenchymal stem cells by immunofluorescence staining

[0122]

[0123]

[0124] Table 3. Detection of collagen expression in mesenchymal stem cells by Western blot

[0125]

[0126]

[0127] VI. Cell Preservation and Application

[0128] Once mesenchymal stem cells with high collagen expression are obtained, they can be frozen and preserved for subsequent use.

[0129] When used, the cells are thawed and appropriately processed, such as injected into damaged tissue sites to promote tissue repair and regeneration.

[0130] 7. Growth Factors

[0131] Transforming Growth Factor-β (TGF-β):

[0132] TGF-β can stimulate mesenchymal stem cells to synthesize collagen, especially type I and type III collagen. It promotes the transcription and translation of collagen genes by regulating intracellular signaling pathways.

[0133] There are different isoforms of TGF-β, such as TGF-β1, TGF-β2, and TGF-β3, which may have different strengths and specificities in regulating collagen expression.

[0134] Fibroblast Growth Factor (FGF):

[0135] Some members of the FGF family, such as basic fibroblast growth factor (bFGF), can promote cell proliferation and collagen production.

[0136] bFGF can stimulate the activity of mesenchymal stem cells, increase collagen synthesis, and may help maintain the stemness of cells.

[0137] Platelet-derived growth factor (PDGF):

[0138] PDGF can induce mesenchymal stem cells to differentiate into fibroblast-like cells, thereby increasing the secretion of collagen.

[0139] It plays an important role in tissue repair and regeneration, promoting collagen deposition and wound healing.

[0140] 8. Inducers

[0141] Vitamin C:

[0142] Vitamin C is an important cofactor in the process of collagen synthesis. It can promote the hydroxylation of proline and lysine, which is a key step in the formation of collagen.

[0143] Adding an appropriate amount of vitamin C to the culture medium can significantly increase the expression level of collagen in mesenchymal stem cells.

[0144] Dexamethasone:

[0145] Dexamethasone is a glucocorticoid drug that can induce mesenchymal stem cells to differentiate into osteoblasts or fibroblasts at a certain concentration, thereby increasing the synthesis of collagen.

[0146] However, attention should be paid to concentration control when using dexamethasone, as too high a concentration may have toxic effects on cells.

[0147] Hyaluronic Acid:

[0148] Hyaluronic acid can bind to receptors on the surface of mesenchymal stem cells, activate intracellular signaling pathways, and promote the production of collagen.

[0149] It can also improve the microenvironment of the extracellular matrix and provide good conditions for the synthesis of collagen.

[0150] Table 4. Comparison of collagen expression levels under different inducer treatment conditions

[0151]

[0152]

[0153] Table 5. Comparison of collagen expression levels under different growth factor treatment conditions

[0154]

[0155]

[0156] IX. Analysis of collagen expression levels under different treatment conditions

[0157] EGF treatment:

[0158] The expression level of collagen after EGF treatment was 30% higher than that of the control group, which indicated that epidermal growth factor (EGF) had a significant promoting effect on the synthesis of collagen.

[0159] The possible mechanism is that EGF stimulates cell proliferation and differentiation, thereby increasing collagen production.

[0160] FGF treatment:

[0161] The collagen expression level after FGF treatment was 125% of that in the control group, indicating that fibroblast growth factor (FGF) can also effectively promote the synthesis of collagen.

[0162] FGF may promote the production of collagen by activating related signaling pathways.

[0163] 10. In addition, the expression level of collagen in mesenchymal stem cells is affected by many factors, as follows:

[0164] 1. Extracellular factors

[0165] Growth factors and cytokines

[0166] Transforming growth factor-β (TGF-β): It can significantly promote the synthesis of collagen by mesenchymal stem cells, especially type I and type III collagen. It regulates the transcription and translation of collagen genes by activating specific signaling pathways.

[0167] Fibroblast growth factor (FGF): It can stimulate mesenchymal stem cells to produce collagen to a certain extent. Different FGF subtypes may have different effects on collagen expression.

[0168] Platelet-derived growth factor (PDGF): It also regulates the synthesis of collagen and can work synergistically with other growth factors.

[0169] 2. Mechanical stimulation

[0170] Tensile stress: Appropriate tensile stress can upregulate the expression of collagen in mesenchymal stem cells. For example, in tissue engineering, applying mechanical stretching to cultured mesenchymal stem cells can simulate the mechanical environment of in vivo tissues, promote the synthesis of collagen and enhance the mechanical properties of tissues.

[0171] Fluid shear force: In a fluid environment, the fluid shear force to which mesenchymal stem cells are subjected will also affect the expression of collagen. Appropriate fluid shear force may regulate the production of collagen by activating intracellular signal transduction pathways.

[0172] 3. Oxygen concentration

[0173] Hypoxia: Moderate hypoxia can increase the expression of collagen in mesenchymal stem cells. Hypoxia may induce the activation of specific transcription factors, thereby promoting the expression of collagen genes.

[0174] Normoxic environment: Under normal oxygen concentration, the expression level of collagen is relatively stable, but different culture conditions and cell states will also affect it.

[0175] 4. Intracellular factors

[0176] Transcription Factors

[0177] Specific transcription factors such as the Smad protein family play a key role in the TGF-β signaling pathway and can regulate the transcription of collagen genes. The activation of transcription factors such as Smad2 and Smad3 can promote the synthesis of collagen.

[0178] Other transcription factors such as AP-1 and NF-κB may also be involved in regulating the expression of collagen, and their activities are regulated by multiple signaling pathways.

[0179] signaling pathway

[0180] MAPK signaling pathway: including branches such as ERK, JNK and p38. These signaling pathways can be activated by growth factors, mechanical stimulation and other factors, thereby affecting the expression of collagen in mesenchymal stem cells.

[0181] PI3K / Akt signaling pathway: It plays an important role in cell survival, proliferation and metabolism, and may also be involved in regulating collagen synthesis. Activating this signaling pathway can promote the survival of mesenchymal stem cells and the production of collagen.

[0182] Epigenetic regulation

[0183] DNA methylation: The DNA methylation status of the collagen gene promoter region can affect its transcriptional activity. Hypermethylation may inhibit the expression of collagen, while hypomethylation may promote its expression.

[0184] Histone modification: Histone modifications such as acetylation and methylation can also affect the transcription of collagen genes. For example, histone acetylation is often associated with gene activation and may promote collagen expression.

[0185] In mesenchymal stem cells, there are multiple transcription factors involved in the regulation of collagen expression, mainly including the following:

[0186] 1) Smad protein family

[0187] Smad2 and Smad3:

[0188] It plays a key role in the transforming growth factor-β (TGF-β) signaling pathway. When TGF-β binds to receptors on the cell membrane, it activates Smad2 and Smad3 and phosphorylates them.

[0189] Phosphorylated Smad2 and Smad3 form a complex with Smad4, enter the cell nucleus, bind to the promoter region of the collagen gene, and regulate the transcription of the collagen gene.

[0190] It promotes the synthesis of type I and type III collagen by mesenchymal stem cells, which plays an important role in maintaining the stability of the extracellular matrix and tissue repair.

[0191] Smad7:

[0192] As an inhibitory Smad, it negatively regulates the TGF-β signaling pathway.

[0193] It can bind to activated TGF-β receptors, prevent the phosphorylation of Smad2 and Smad3, and thus inhibit the expression of collagen.

[0194] 2) AP-1 (Activator Protein-1) transcription factor

[0195] Composition and activation:

[0196] It is composed of dimers of proteins from the Jun, Fos and ATF families.

[0197] It can be activated by a variety of signaling pathways, such as MAPK (Mitogen-activated protein kinase) signaling pathway, growth factors and cytokines.

[0198] In mesenchymal stem cells, activation of AP-1 transcription factor may be involved in regulating collagen expression.

[0199] Regulation of collagen:

[0200] On the one hand, AP-1 can bind to specific sequences in the promoter region of the collagen gene and directly regulate the transcription of collagen.

[0201] On the other hand, AP-1 interacts with other transcription factors such as the Smad protein family to synergistically regulate collagen expression. In some cases, AP-1 may promote collagen expression; in other cases, it may inhibit collagen expression, depending on the cell environment and signal stimulation.

[0202] 3) NF-κB (Nuclear factor kappa-light-chain-enhancer of activated Bcells) transcription factor

[0203] Activation mechanism:

[0204] NF-κB usually exists in the cytoplasm in an inactive form and binds to the inhibitory protein IκB. When stimulated by inflammatory factors, oxidative stress, etc., IκB is phosphorylated and degraded, releasing NF-κB.

[0205] Activated NF-κB enters the cell nucleus and regulates the transcription of target genes.

[0206] Effects on collagen:

[0207] NF-κB can regulate the expression of multiple genes, including those involved in inflammatory response, cell proliferation and differentiation. In mesenchymal stem cells, NF-κB may affect the expression of collagen by regulating inflammatory response and extracellular matrix metabolism.

[0208] Under inflammatory conditions, NF-κB may promote collagen degradation, while under certain specific conditions, it may also be involved in the regulation of collagen synthesis.

[0209] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only preferred examples of the invention and are not intended to limit the present invention. Without departing from the novel spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing amniotic membrane-derived mesenchymal stem cells that highly express collagen, characterized in that: The method comprises the following steps: Step 1. Reagents and materials: Amniotic membrane tissue: fresh amniotic membrane obtained from healthy pregnant women after delivery; Phosphate buffered saline (PBS): used to wash amniotic tissue; Collagenase: Type I collagenase, used to digest amniotic tissue to isolate mesenchymal stem cells; Cell culture medium: DMEM / F12 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin; Step 2: Preparation steps Amniotic tissue processing: Fresh amniotic tissue was washed several times with PBS under sterile conditions to remove blood and impurities; Cut the amniotic membrane into small pieces, put them into a container containing an appropriate amount of collagenase, and digest them in a 37°C constant temperature shaker for 1-2 hours; Step 3: Cell isolation and culture: The digested tissue suspension was filtered through a strainer to remove undigested tissue debris; The filtrate was centrifuged at an appropriate centrifugal speed, 1500 rpm for 5 minutes; the supernatant was discarded and the precipitated cells were resuspended in cell culture medium; The cells were inoculated into culture flasks and cultured in an incubator at 37°C and 5% CO2; Step 4: Induce high expression of collagen: When the cells grow to a certain density, 80% confluence, the culture medium containing specific concentrations of transforming growth factor TGF-β and fibroblast growth factor FGF is replaced; The concentration of transforming growth factor TGF-β was set to 5-10 ng / ml, and the concentration of fibroblast growth factor FGF was set to 2-5 ng / ml; Continue culturing for 7-10 days, during which time the cell status is regularly observed and the culture medium is replaced; Step 5. Cell harvesting and identification: After the culture was completed, the cells were collected by trypsin digestion; The expression level of collagen in cells was detected by immunofluorescence staining, Western blot and other methods to confirm that the amniotic membrane-derived mesenchymal stem cells with high collagen expression were successfully prepared; Step 6. Cell screening and expansion After the cells adhere to the wall and grow, replace the culture medium to remove the non-adherent cells and impurities; Observe the growth of cells, and when the cell confluence reaches about 80%, perform subculture; Mesenchymal stem cells were screened and identified by flow cytometry and other methods.

2. The method for preparing amnion-derived mesenchymal stem cells that highly express collagen according to claim 1, characterized in that: The step of screening mesenchymal stem cells by flow cytometry in step 6:

1. Cell Preparation Isolate mononuclear cell suspension from tissue samples; bone marrow, fat, and separate by density gradient centrifugation and other methods; 2. Antibody labeling Select appropriate antibody combinations to label cells, including positive marker antibodies for CD73, CD90, and CD105, and negative marker antibodies for CD14, CD19, CD34, CD45, and HLA-DR; The cells were incubated with antibodies at 4°C for 30 min; Flow cytometer detection: Add the labeled cell suspension to the flow cytometer for detection; Flow cytometry classifies and counts cells based on the fluorescent labels carried by antibodies; 3. Screening According to the results of flow cytometry, cells that simultaneously expressed positive markers and did not express negative markers were screened out, and these cells were considered to be mesenchymal stem cells.

3. The method for preparing amnion-derived mesenchymal stem cells that highly express collagen according to claim 1, characterized in that: The transforming growth factor TGF-β stimulates mesenchymal stem cells to synthesize collagen, which is type I and type III collagen.

4. The method for preparing amnion-derived mesenchymal stem cells that highly express collagen according to claim 1, characterized in that: The fibroblast growth factor FGF is basic fibroblast growth factor bFGF, which is used to stimulate the activity of mesenchymal stem cells, increase the synthesis of collagen, and may help maintain the stemness of cells.

5. The method for preparing amnion-derived mesenchymal stem cells that highly express collagen according to claim 1, characterized in that: The platelet-derived growth factor PDGF is used to induce mesenchymal stem cells to differentiate into fibroblast-like cells, thereby increasing the secretion of collagen.

6. The method for preparing amnion-derived mesenchymal stem cells that highly express collagen according to claim 1, characterized in that: The step 4, inducing high expression of collagen, also includes adding inducers: vitamin C, dexamethasone, and hyaluronic acid.

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