Application of hematopoietic stem progenitor cells in treatment of keloid

Abstract

The invention provides application of hematopoietic stem progenitor cells in treatment of keloid. The umbilical cord blood-derived hematopoietic stem cells have an obvious inhibition effect on growth of keloid. Specifically, a PDX (Patient-Derived Xenogram) Balb / c-nu immunodeficient mouse model is adopted to research the treatment effect of human hematopoietic stem cells on keloid, and the result shows that the size and the weight of a graft are effectively reduced through HSCs treatment. In conclusion, the growth of the keloid can be obviously inhibited by the umbilical cord blood-derived hematopoietic stem cells, theoretical support can be provided for clinical application of an umbilical cord blood bank in inflammation-related fibrosis diseases (lung injury, spinal cord injury, myocardial infarction, corneal injury, renal fibrosis, cirrhosis and the like), and the application value is expanded; meanwhile, a new thought and a new prospect can be provided for clinical treatment of the intractable disease, namely keloid in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine technology, and particularly relates to the application of hematopoietic stem and progenitor cells in the treatment of keloid. Background Art

[0002] Wound healing is a dynamic and interactive biological response to tissue injury, involving multiple cells (parenchymal cells, blood cells), extracellular matrix proteins, and soluble proteins to repair the skin barrier, reconstruct skin coherence, prevent infection, and restore tissue integrity and function. After a wound is formed, the body needs to transport various inflammatory cells, chemokines, cytokines, matrix molecules, and nutrients to the wound site, and at the same time, the metabolic demand also increases. The above processes occur simultaneously and are generally divided into three main pathophysiological processes: hemostasis and inflammation stage; cell proliferation stage; wound remodeling stage. Skin tissue wound healing includes two forms: tissue regeneration and scar healing. The former occurs in young individuals or lower organisms, and the latter occurs in adult individuals. The process of wound healing is extremely complex, and the healing outcome depends on the complex interaction between many highly regulated factors, which cooperate with each other to restore damaged skin and repair barrier function. In a healed wound, under the action of certain pathological factors, the wound remodeling mechanism is abnormal, causing severe and persistent local inflammation. Then, fibroblasts in the reticular dermis of the damaged local area respond to inflammatory signals, abnormally proliferate and differentiate, deposit excessive collagen, resulting in extracellular matrix (ECM) disorder, excessive wound healing, and the formation of keloid. Keloid is different from HS (hypertrophic scar), grows and spreads to the surrounding normal skin over time, and is difficult to subside spontaneously.

[0003] Currently, the clinical treatment regimens for keloid mainly target vascular endothelial cells or perivascular cells to inhibit local inflammation, thereby exerting therapeutic effects. These strategies mainly include surgery, local drug injection (steroids, botulinum toxin (BTX), anti-tumor drugs), radiotherapy, biological dressings, lasers, cryotherapy, and compression therapy, etc. Simple surgical treatment is extremely prone to recurrence, so clinically, a combined regimen of multiple strategies is mostly adopted, and the most commonly used is radiotherapy or local steroid injection after surgery. Among them, the radiotherapy regimen after surgery can reduce the recurrence rate by 50%. However, these existing treatment regimens have certain side effects and limitations, and cannot completely prevent the recurrence of keloid. Therefore, further research on the occurrence and development mechanisms of keloid, proposing and improving reasonable treatment strategies for keloid, improving the quality of life of patients, and reducing the recurrence rate of keloid have become important research directions in the field of wound repair. Therefore, new specific treatment strategies for keloid are urgently needed.

[0004] In recent years, the research on stem cells in wound healing and skin regeneration has gradually become a research hotspot. Stem cells are a group of undifferentiated cells with the potential for self-renewal and multi-directional differentiation. They can produce some daughter cells through cell division. These daughter cells can continue to differentiate as stem cells, maintaining the size of their own cell population while further differentiating into various different tissue cells. Stem cells are a continuous source of differentiable cells that make up the tissues and organs of animals and plants. Stem cells play a therapeutic role in various diseases and injuries (Parkinson's disease, heart disease, and diabetes) by replacing defective or damaged cells, so they have been widely concerned by the scientific community. Stem cells are classified into totipotent, pluripotent, and unipotent stem cells according to their differentiation ability, and into embryonic stem cells (ESCs) and adult stem cells (ASCs) according to their source. Hematopoietic stem cells (HSCs) are a type of adult stem cells, isolated from mature organisms, with the ability of self-renewal, and will maintain tissue and organ homeostasis through continuous renewal, also known as tissue stem cells.

[0005] Stem cells play an effective role in the process of abnormal wound healing. According to literature reports, mesenchymal stem cells (MSCs) have been proven to have an obvious effect in promoting wound healing. The mechanisms of action include 1) up-regulating anti-inflammatory factors and down-regulating the expression of pro-inflammatory factors, promoting the transformation of macrophages from M1 type to M2 type, and inhibiting the proliferation and activation of DC, NK, T, and B cells mediated by innate immunity and specific immunity; 2) up-regulating angiogenic factors, promoting the proliferation of endothelial cells, and enhancing the promotion of angiogenesis mediated by vascular stability and functional angiogenesis; 3) up-regulating the EMT (Epithelial mesenchymal transition) mediated by PGE2 (Prostaglandin E2), HGF (Hepatocyte growth factor), and IL10 cytokines, down-regulating the proliferation of myofibroblasts, down-regulating the activation of the TGF-β / Smad pathway, promoting the generation of normal ECM (Extracellular matrix), inhibiting the generation of immature ECM, and negatively regulating the anti-fibrotic effect mediated by ECM deposition.

[0006] However, the role of HSCs in the process of wound healing has not been clearly defined. According to the literature reports, as a component of the Stromal Vascular Fraction (SVF), it can directly or indirectly affect the process of wound healing. Kanji et al. found that HSCs can promote the formation of ECM beneficial to wound healing by secreting collagen and downregulating the expression of Matrixmetalloproteinases (MMPs); Kim et al. found that HSCs can stimulate the proliferation of keratinocytes and fibroblasts, significantly accelerating wound healing; Yan et al. found that the differentiation of HSCs into macrophages is an important event in the process of wound healing, but it is damaged to a certain extent in diabetic patients, which may affect the wound healing ability of diabetic patients. The above studies suggest that the role of HSCs in wound healing and skin regeneration needs further research and elaboration, and there is great research potential. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention aims to explore the mechanism of action of HSCs on mature keloids, clarify the curative effect, develop a new treatment plan for this type of refractory disease of keloids, and at the same time clarify and expand the clinical application prospects and scope of the existing umbilical cord blood bank. Specifically, the purpose of the present invention is to provide the application of hematopoietic stem and progenitor cells in the treatment of keloids.

[0008] In the first aspect of the present invention, there is provided the application of hematopoietic stem and progenitor cells in the preparation of a product for treating keloids.

[0009] Further, the product is a drug or a cosmetic.

[0010] Further, the hematopoietic stem and progenitor cells are hematopoietic stem cells, preferably human hematopoietic stem cells.

[0011] Further, the hematopoietic stem and progenitor cells are hematopoietic stem and progenitor cells derived from umbilical cord blood.

[0012] Further, the hematopoietic stem and progenitor cells treat keloids by transforming the collagen fiber morphology of keloid lesion tissues.

[0013] Further, the hematopoietic stem and progenitor cells treat keloids by reducing the ratio of type I collagen to type III collagen in keloid lesion tissues.

[0014] Further, the hematopoietic stem and progenitor cells treat keloids by reducing the proportion of T cells in the immune cell lineage in keloid lesion tissues.

[0015] Further, the hematopoietic stem and progenitor cells treat keloids by inhibiting the IL-17 signaling pathway.

[0016] Furthermore, among the genes related to the IL-17 signaling pathway, the key transcription factor CEBPB was significantly downregulated, and the downstream chemokine CXCL8 was significantly downregulated.

[0017] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be repeated one by one here.

[0018] Compared with the prior art, the present invention has the following advantages and progressiveness:

[0019] The present invention provides the application of hematopoietic stem and progenitor cells in the treatment of keloid. The hematopoietic stem cells derived from umbilical cord blood of the present invention have an obvious inhibitory effect on the growth of keloid. Specifically, the present invention uses a PDX (Patient-derived xenograft) Balb / c-nu immunodeficient mouse model to study the therapeutic effect of human hematopoietic stem cells on keloid. The results show that the graft volume and weight in the HSC only group were reduced compared with the control group (p < 0.05), and there was no significant difference between the MSC only group and the MSC&HSC group and the PBS group; it shows that HSCs can promote the regression of keloid grafts in PDX model mice and have a significant therapeutic effect. In summary, the growth of keloid in the present invention can be significantly inhibited by hematopoietic stem cells derived from umbilical cord blood, which can provide theoretical support for the clinical application of umbilical cord blood banks in inflammation-related fibrotic diseases (such as lung injury, spinal cord injury, myocardial infarction, corneal injury, renal fibrosis, liver cirrhosis, etc.), has the value of expanding application, and can also provide new ideas and prospects for the clinical treatment of the intractable disease keloid in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0021] Figure 1 . Statistical chart of the change in body weight of mice during treatment in Example 2;

[0022] Figure 2 . Statistical chart of the volume and weight of the graft after treatment in Example 2;

[0023] Figure 3 . HE and Masson pathological staining diagrams and statistical analysis diagrams in Example 3;

[0024] Figure 4 . IHC staining diagrams and statistical analysis diagrams in Example 3;

[0025] Figure 5. Single-cell sequencing analysis chart in Example 4;

[0026] Figure 6 . Co-culture results chart of MSC / HSC / MSC&HSC and HDIKFs in Example 5;

[0027] Figure 7 . Results chart of culturing HDIKFs with IL-17A antagonist in Example 5. Detailed implementation mode

[0028] The present invention provides the application of hematopoietic stem and progenitor cells in the treatment of keloid. The present invention will be specifically described below in conjunction with examples to facilitate the further understanding of the present invention by those skilled in the art. However, the examples described below are only a part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be noted that the adjustments and improvements made by those of ordinary skill in the art based on the concept of the present invention should be regarded as the protection scope of the present invention. For the specific technical operation steps and operators not specified in the examples, they are all carried out according to the general technical conditions described in the literature in this field or the relevant product specifications.

[0029] Example 1: Overall plan for in vivo treatment of keloid with human hematopoietic stem cells

[0030] The overall plan for the in vivo treatment of keloid PDX mouse model with human hematopoietic stem cells includes the following steps:

[0031] 1) Place the collected neonatal umbilical cord blood in a clean and sterile 200 ml plasma bottle, add HES according to the volume ratio of hydroxyethyl starch (HES): umbilical cord blood = 1:4, blow and mix evenly, and then let it stand at room temperature for 40 minutes to 1 hour to fully sediment the red blood cells in the blood;

[0032] 2) Use a 25 ml disposable sterile pipette to gently suck out the supernatant of the sedimented umbilical cord blood and place it in a 50 ml centrifuge tube. Add about 30 to 50 ml of supernatant to each centrifuge tube, and centrifuge at 1500 rpm for 7 minutes;

[0033] 3) Discard the supernatant, add 20 ml of sterile red blood cell lysate (ACK) to the cell pellet for resuspension, place the centrifuge tube in a 37 °C water bath to lyse red blood cells for about 10 minutes, invert the centrifuge tube several times during this period to mix evenly, and centrifuge at 1500 rpm for 5 minutes;

[0034] 4) Discard the supernatant, first blow the cell pellet into a single-cell suspension with 1 - 2 ml of sterile PBS buffer, then add 20 ml of PBS buffer to wash the cells. At the same time, take 10 μl of the cell suspension (after diluting 100 times with PBS buffer) for MNC cell counting, and centrifuge at 1500 rpm for 5 minutes;

[0035] 5) Discard the supernatant. Resuspend the cell pellet in 1 ml of PBS buffer. After pipetting to mix evenly, add appropriate amounts of FCR Blocking Reagent (which can block non-specific binding sites) receptor blocker and CD34 Microbeads successively according to the number of MNCs. After thorough mixing, place the centrifuge tube in the dark at 4°C and incubate for 30 minutes (during which, shake and mix every 10 minutes to prevent the formation of cell pellets that may affect the magnetic bead incubation);

[0036]

[0037] 6) After incubation, wash the cells in each tube with 20 ml of PBS buffer (to wash away the magnetic beads that did not bind to the cells). If there is precipitation, filtration is required. Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and resuspend with 1 ml of PBS buffer;

[0038] 7) Install the LS column on the magnetic stand. Cover the column opening with a 40-μm cell filter (to prevent cell clumps from blocking the sorting column), and rinse the column 3 times successively with 1 ml of PBS buffer;

[0039] 8) Slowly add the cell suspension drop by drop and pass it through the magnetic field. After all the cells in the sorting column have drained, add 1 ml of PBS buffer to the column to wash the column and remove the non-target cells remaining in the column. Wash the column 3 times;

[0040] 9) Remove the column from the magnetic field, place it on the collection tube, add 4 ml of PBS buffer, quickly push out the cells labeled with magnetic beads using the piston, and take 10 μl for counting;

[0041] 10) Resuspend the obtained cells with PBS buffer to a concentration of 2×10 6 cells / mL, and locally inject them subcutaneously into the graft on the shoulders of the successfully constructed keloid PDX model mice, injecting 1×10 5 cells / 50 μL for each mouse;

[0042] 11) Administration of human hematopoietic stem cell therapy starts on the 12th day after transplantation of the model mice, and the treatment is given once every week.

[0043] Example 2: Preparation of keloid PDX mouse model

[0044] Use the PDX (Patient-derived xenograft) Balb / c-nu immunodeficient mouse model to study the therapeutic effect of human hematopoietic stem cells on keloids. The results show that HSCs treatment effectively reduces the volume and weight of the graft.

[0045] Experimental method for preparing the keloid PDX mouse model:

[0046] 1) Order female Balb / c-nu mice at 6-7 weeks of age, adaptively raise them for one week, irradiate them 4-5 hours before transplantation, and the irradiation dose is 2 Cy; randomly group the irradiated mice according to body weight into a PBS group (solvent control group), an MSC only group, an HSC only group, and an MSC&HSC group;

[0047] 2) Obtain fresh samples of keloids from patients and store them in physiological saline supplemented with gentamicin to ensure that the transplantation experiment is carried out within 8 hours after the specimens are removed from the body. Cut the samples into small pieces of about 1 cm 3 in advance for constructing a keloid PDX mouse model.

[0048] 3) In a laminar flow hood, intraperitoneally inject 200 μl of avertin anesthetic into each mouse; perform a transplantation operation after it is completely anesthetized, implant the small pieces of keloid samples subcutaneously and suture them, and apply aureomycin ointment to the wound to prevent infection;

[0049] Place the transplanted mice on an electric heating pad until they wake up, and put them back into the cage for continued breeding if they are active normally;

[0050] 4) Observe the growth of the grafts after transplantation. It can be seen that obvious blood circulation is established in the grafts, and there is no necrosis, shrinkage, or obvious immune rejection. That is, the model mice are successfully constructed.

[0051] 5) After the model mice are successfully constructed, perform in-situ injection for cell therapy. The PBS group is injected with 50 μL of PBS, the MSC only group and the HSC only group are injected with 1×10 5 cells / 50 μL, and the MSC&HSC group is injected with 1×10 5 MSCs&1×10 5 HSCs / 50 μL; monitor the vital signs, body weight changes, and graft growth of the mice in real time during the treatment process.

[0052] 6) Sacrifice the mice 8 weeks after transplantation, take out the grafts, count and compare the weights and volumes of the grafts in each group, and leave the specimens fixed in 10% formaldehyde solution by volume for pathological phenotype analysis; or take the specimens in tissue protective solution, prepare single-cell suspensions for 10x Genomic single-cell sequencing.

[0053] Result analysis:

[0054] As Figure 1 can be seen, the state of the keloid PDX mice is stable after modeling, and the body weights of each group gradually recover during the treatment period. Compared with the PBS group, the body weight of the mice in the MSC only group is lower, and there is a significant difference (p < 0.01); there is no significant difference in the body weight of the mice in the HSC only group and the MSC&HSC group compared with the PBS group, indicating that the treatment of keloid PDX mice with HSCs has good safety.

[0055] Depend on Figure 2 It can be seen that the mice were killed 8 weeks after transplantation to obtain the grafts. The graft volume and weight of the HSC only group were reduced compared with the control group (p < 0.05), and there was no significant difference between the MSC only group and the MSC&HSC group and the PBS group; this shows that HSCs can promote the regression of keloid grafts in PDX model mice and has a significant therapeutic effect.

[0056] Example 3: Transplant Pathological Phenotyping

[0057] Hematoxylin-eosin staining and Masson staining were used to detect and analyze the collagen content and inflammatory infiltration of the grafts, and IHC staining was used to analyze the changes in different collagen types in the grafts. The results showed that HSCs treatment changed the fiber morphology and reduced the ratio of type I collagen to type III collagen.

[0058] Experimental Methods for Phenotyping of Transplant Pathology:

[0059] 1) The keloid specimen obtained is fixed with formaldehyde for more than 24 hours, dehydrated step by step from low to high concentrations of alcohol, and then dissolved in paraffin clearing agent xylene to make it transparent; embedded in paraffin with a melting point of 52-60 degrees, and cooled and solidified into a block;

[0060] 2) First, use a blade to trim the excess paraffin around the tissue block, leaving about 2mm of paraffin, and trim the tissue block into a trapezoidal shape to facilitate the separation of the wax band when spreading the slices; the thickness of the tissue slices is 5μm, and the temperature of the spreading slices is generally 42-45 degrees. Put the sliced ​​slices into the baking machine for baking, the baking temperature is 60 degrees, and the baking time is 2 hours;

[0061] 3) Before staining, the sections must be placed in xylene to remove the paraffin, then passed through high-concentration to low-concentration alcohol, and finally rehydrated in distilled water;

[0062] 4) Hematoxylin-eosin staining (HE staining) or Masson staining (Masson staining), xylene transparent, sealing, and microscopic examination;

[0063] 5) After antigen retrieval, the rehydrated sections were subjected to immunohistochemical staining (IHC staining) to stain type I and type III collagen to characterize the distribution of collagen in the extracellular matrix.

[0064] Result analysis:

[0065] Depend on Figure 3It can be seen that the HE staining results showed that there was a certain amount of collagen deposition in each group. In the HSC only group and the MSC&HSC group, the collagen morphology changed from mature thick and homogeneous red-stained collagen fiber bundles with parallel or staggered distribution to finer collagen fibers, and the inflammatory infiltration increased; the MT staining results showed that the blue area positive for collagen in the HSC only group and the MSC&HSC group was smaller than that in the PBS group, and the collagen volume fraction (CVF) tended to decrease.

[0066] From Figure 4 It can be seen that the IHC staining results showed that compared with the PBS group, the MSC only group and the MSC&HSC group, the HSC only group significantly decreased the ratio of COLⅠ to COLⅢ (p < 0.05), reflecting the change in the microenvironment and extracellular matrix composition in the graft after HSC treatment, and the repair and remodeling process inside the scar lesion has been triggered.

[0067] Example 4: Single-cell sequencing analysis of grafts

[0068] The method of 10×Genomic single-cell sequencing was used to analyze the cell subset composition of the graft and its enriched pathways. The results showed that after HSCs treatment, the proportion of T cells in the immune cell lineage decreased significantly, and the IL-17 signaling pathway was significantly enriched.

[0069] Experimental method for single-cell sequencing analysis of grafts:

[0070] 1) Prepare the graft samples in the tissue preservation solution into single-cell suspensions, load them into the 10×Genomic single-cell capture system, construct cDNA libraries with 10× tags, and perform short-read NGS sequencing on the Illumina sequencer;

[0071] 2) After obtaining the raw sequencing data, extract cell-barcode, UMI and RNA sequences, align them with the reference genome, filter and correct UMI and perform counting, evaluate the number of cells, and finally obtain the gene expression matrix of each cell;

[0072] 3) Use principal component analysis to reduce the dimensionality of the data, perform unsupervised uniform manifold approximation and projection (UMAP) clustering to display the distribution of cells, and divide cell populations according to the established lineage-specific marker genes. Among them, the characteristics of the fibroblast lineage are COL1A1 and COL1A2, and the characteristics of the immune cell lineage are LYZ and HLA-DRA;

[0073] 4) Isolate and characterize immune cell lineages using specific cell markers, enrich differential signaling pathways of immune cell subsets in immune cell lineage subsets, and perform differential expressed genes (DEGs) analysis.

[0074] Result analysis:

[0075] According to gene sets, UMAP clustering was performed, and the graft cells were divided into 16 clusters ( Figure 5 a, Figure 5 b). After MSC or HSC treatment, the proportions of type 2 and type 3 fibroblasts decreased sharply, while the proportion of type 1 fibroblasts increased; after combined treatment with HSC and MSC, the proportion of type 4 fibroblasts increased ( Figure 5 d). We calculated the ratio of COL1A1 to COL3A1 in the four types of fibroblasts. The COL1A1 / COL3A1 of type 1 fibroblasts was significantly lower than the other three types (p < 0.001), and the COL1A1 / COL3A1 of type 4 fibroblasts was significantly higher than the other three types (p < 0.01) ( Figure 5 c). The results showed that after HSC treatment, the biological behavior of KFs was redefined, and the ratio of COLⅠ to COLⅢ in keloid grafts was significantly reduced by increasing the proportion of type 1 fibroblasts.

[0076] We isolated and identified myeloid cells (macrophages and monocytes), proliferating immune cells, NK cells, and T cells in the immune cell line using specific cell markers ( Figure 5 e). MSC treatment and combined treatment with HSC and MSC showed a significant increase in the proportion of T cells, while after HSC treatment, the proportion of T cells was close to that of the control group ( Figure 5 f). The results showed that the role of T cell subsets in promoting keloid regression by HSC deserves further investigation.

[0077] We analyzed immune cell lineage subsets using the Kyoto Encyclopedia of Genes and Genomes (KEGG). The IL-17 signaling pathway was significantly enriched in the HSC treatment group ( Figure 5 h). Analyzing the differentially expressed genes in the IL-17 signaling pathway, NFKB1, CEBPB, CXCL2, and CXCL8 were significantly decreased in the HSC treatment group ( Figure 5 g), and CEBPB and CXCL8 were the core molecules with significant downregulation ( Figure 5 i). Using the graft samples for qRT-PCR verification, the genes related to the IL-17 signaling pathway, IL-17 molecules, and receptors in the HSC treatment group were significantly downregulated compared with the control group ( Figure 5j). The results showed that HSCs had the ability to regulate the biological behavior of KFs by inhibiting the IL-17 signaling pathway.

[0078] Example 5: In vitro cell function analysis

[0079] Human-derived immortalized keloid fibroblasts (HDIKFs) (purchased from Hefei Wanwu Biotechnology Co., Ltd.) were used to study the effects of HSCs on HDIKFs. The results showed that HSCs could change the cell morphology of HDIKFs, inhibit growth, significantly reduce the gene expression of the IL-17 pathway; and further verified that inhibiting the gene expression of the IL-17 pathway could inhibit HDIKFs by antagonizing IL-17 to culture HDIKFs.

[0080] Experimental methods for in vitro cell function analysis:

[0081] 1) Inoculate HDIKFs in plastic culture dishes, use the primary fibroblast basal medium containing 10% fetal bovine serum, culture at 37°C and 5% CO2, passage at a ratio of 1:2 every 3 days, and use the P3 generation for subsequent experiments;

[0082] 2) Select a 0.4 μm PC membrane cell embedding dish for co-culture experiments, divided into Control group, MSC only group, HSC only group, MSC&HSC group; spread 5×10 4 HDIKFs cells in each well of a 24-well plate, add 800 μl of medium, and after culturing in an incubator for 6 h until the HDIKFs adhered, add 200 μl of liquid to the Control group; put the remaining groups into the embedding dish, add 1×10 5 cells to the MSC only group and the HSC only group, and add 1×10 5 MSCs & 1×10 5 HSCs to the MSC&HSC group, and add 200 μl of medium into the dish; after adding the liquid, co-culture for 72 h;

[0083] 3) Use IL-17A antagonist 1 (HY-101913, MedChemExpress) to set up the anti-IL17A treated group to culture HDIKFs, with a concentration of 0.57 μM / ml; spread 1×10 4 HDIKFs cells in each well of a 24-well plate, add 400 μl of medium, and after culturing in an incubator for 6 h until the HDIKFs adhered, add 100 μl of medium to the Control group and add 100 μl of medium supplemented with the antagonist to the anti-IL17A treated group; culture for 72 h after adding the drug;

[0084] 4) Incubate for 72 h, photograph and record the morphology of HDIKFs cells every 24 h, digest and harvest the cells for counting, extract RNA and reverse transcribe to obtain cDNA, and perform qPCR to examine the expression of genes in the IL-17 pathway in each group.

[0085] Result analysis:

[0086] When MSC / HSC / MSC&HSC were co-cultured with HDIKFs, microscopic photography showed that HDIKFs adhered and grew in a spindle shape. In the 72-h co-culture group, the number of round cells was significantly increased compared with the Control group, and the total number of cells in the field of view decreased ( Figure 6 A). The counting results showed that there were no differences among the groups at 48 h; at 72 h, the MSC co-culture group and the MSC&HSC co-culture group were significantly lower than the Control group (p < 0.01), and the HSC co-culture group was significantly lower than the Control group (p < 0.001) ( Figure 6 B). The qPCR results showed that in the HSC co-culture group compared with the Control group, among the genes related to the IL17 pathway, the key transcription factor CEBPB was significantly downregulated (p < 0.0001), and the downstream chemokine CXCL8 was significantly downregulated (p < 0.01) ( Figure 6 C).

[0087] Results of culturing HDIKFs with the IL-17A antagonist. Microscopic photography showed that HDIKFs adhered and grew in a spindle shape. There was no obvious change in the cell morphology of the anti-IL17A treated group compared with the Control group, and the total number of cells in the field of view decreased ( Figure 7 A). The counting results showed that the anti-IL17A treated group was significantly lower than the Control group at both 48 h and 72 h (p < 0.01) ( Figure 7 B). The qPCR results showed that in the anti-IL17A treated group compared with the Control group, among the genes related to the IL17 pathway, the key transcription factor CEBPB was significantly downregulated (p < 0.0001), and the downstream chemokine CXCL8 was significantly downregulated (p < 0.0001) ( Figure 7 C).

[0088] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. For those skilled in the art of the present technology, any modifications and changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. Use of hematopoietic stem and progenitor cells in the preparation of a product for treating keloid.

2. The application according to claim 1, wherein The product is a drug or a cosmetic.

3. The application according to claim 1, wherein The hematopoietic stem and progenitor cells are hematopoietic stem cells, preferably human hematopoietic stem cells.

4. The application according to claim 1, characterized in that, The hematopoietic stem and progenitor cells are hematopoietic stem and progenitor cells derived from umbilical cord blood.

5. The application according to claim 1, wherein The hematopoietic stem and progenitor cells treat keloid by transforming the collagen fiber morphology of keloid lesion tissues.

6. The application according to claim 1, characterized in that The hematopoietic stem and progenitor cells treat keloid by reducing the ratio of type I collagen to type III collagen in keloid lesion tissues.

7. The application according to claim 1, characterized in that The hematopoietic stem and progenitor cells treat keloid by reducing the proportion of T cells in the immune cell lineage in keloid lesion tissues.

8. The application according to claim 1, characterized in that, The hematopoietic stem and progenitor cells treat keloid by inhibiting the IL-17 signaling pathway.

9. The application according to claim 8, characterized in that Among the genes related to the IL-17 signaling pathway, the key transcription factor CEBPB is significantly downregulated, and the downstream chemokine CXCL8 is significantly downregulated.

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