Cashmere goat hair follicle stem cell isolation culture identification and key marker detection method
Patent Information
- Application Number
- CN202511598132.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-04
- Publication Date
- 2026-01-16
Smart Images
Figure CN121343877A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biotechnology and cell engineering, and more specifically, to a method for isolating, culturing, identifying, and detecting key markers of goat hair follicle stem cells. Background Technology
[0002] Cashmere goats are important economic animals, and their cashmere has extremely high economic value. Cashmere goat follicle stem cells refer to epithelial stem cells located in the ridge of the hair follicle, playing a crucial role in cashmere follicle development, cycle transition, and maintaining cashmere growth. Some researchers have proposed that the biological state of follicle stem cells in the ridge area is one of the main driving forces behind hair follicle morphogenesis. In an intact hair follicle, they are in a quiescent state, but when the skin is damaged or during hair follicle cycle transition, they enter a dividing state, thereby promoting hair follicle morphogenesis.
[0003] Current research on hair follicle stem cells from goat hair follicles has made some progress, but existing technologies still have some shortcomings. In the isolation of hair follicle stem cells, traditional methods may lead to cell damage or low isolation efficiency, making it difficult to obtain sufficient quantities and high purity of hair follicle stem cells, which limits subsequent research and applications. During the culture process, the lack of a culture system that accurately simulates the hair follicle microenvironment affects cell proliferation and stemness maintenance, hindering the full utilization of the biological characteristics of hair follicle stem cells. In identification and detection of key markers, the accuracy and sensitivity of existing technologies need improvement, as misidentification or missed detection may occur, which is detrimental to in-depth research and effective utilization of hair follicle stem cells. Therefore, this paper proposes a method for the isolation, culture, identification, and detection of key markers of goat hair follicle stem cells. Summary of the Invention
[0004] The purpose of this invention is to address the problems raised in the existing background technology. To achieve the above-mentioned objective, this invention provides the following technical solution: a method for isolating, culturing, and identifying cashmere goat follicle stem cells, comprising the following steps: Step 1, tissue acquisition: Select healthy cashmere goat skin tissue, rinse it 3-5 times with sterile physiological saline to remove surface bloodstains and impurities, and cut the skin tissue into small pieces of 1 mm³; Step 2, Enzymatic Digestion: Place the shredded skin tissue pieces into a digestion solution containing 0.25% trypsin and 0.02% EDTA, and digest at 37°C for 30-60 minutes, gently shaking once every 10 minutes. After digestion, add culture medium containing 10% fetal bovine serum to stop the digestion. Step 3, preparation of cell suspension: Filter the digested tissue suspension through a 200-mesh sieve to remove undigested tissue pieces. Centrifuge the filtrate at 1000 r / min for 5-10 minutes, discard the supernatant, and resuspend the cell pellet in culture medium to obtain the cell suspension.
[0005] As a preferred embodiment of the present invention, the digestion time in the enzyme digestion step is 45 minutes.
[0006] As a preferred technical solution of the present invention, the method for culturing goat hair follicle stem cells includes the following steps: Step 1, Primary Culture: The cell suspension obtained in claim 1 is seeded into a culture flask pre-coated with gelatin and placed at a temperature of... Volume fraction is 5% Cultured in an incubator, the culture medium is The culture medium was supplemented with 10% fetal bovine serum (FBS) at a concentration of [missing information]. Epidermal growth factor Concentration is insulin, concentration of Hydrocortisone and 1% (v / v) penicillin and streptomycin, every... Change the culture medium once a day; Step 2, passage culture: When the cell confluence reaches 80%-90%, digest the cells with 0.25% trypsin and 0.02% EDTA, and passage them at a ratio of 1:2-1:3.
[0007] As a preferred technical solution of the present invention, the culture medium is changed every 2 days in the primary culture.
[0008] As a preferred technical solution of the present invention, in the passage culture, passage is performed when the cell confluence reaches 85%, and the passage ratio is 1:2.
[0009] As a preferred technical solution of the present invention, the method for isolating, culturing and identifying cashmere goat hair follicle stem cells includes the following steps: Step 1, morphological observation: observe the morphological characteristics of the cells under an inverted microscope. Cashmere goat hair follicle stem cells are small and round or polygonal, with a large nucleus-to-cytoplasm ratio. The cells adhere to the wall and grow in a colony-like distribution. Step 2, Growth Curve Determination: The growth curve of the cells was determined using the MTT assay. Cells in the logarithmic growth phase were used, and the cell density per well was recorded. Cells were seeded at a density of [number] cells per well in 96-well plates, and [number] cells were seeded on the [number]th day after seeding. Add MTT solution and continue culturing. After hours, discard the supernatant, add DMSO to dissolve the crystals, and use a microplate reader to analyze the crystals at a wavelength of [wavelength value missing]. Measure absorbance value by number of days The x-axis represents absorbance values. Plot the growth curve on the ordinate; Step 3: Divide the cells into cells per well. Cells were seeded at a density of [number] cells per well in 6-well plates and cultured. After the clones are formed and visible to the naked eye, they are fixed with methanol, stained with crystal violet, and the number of clones is counted. Clonal formation rate The calculation formula is: .
[0010] As a preferred embodiment of the present invention, the cloning experiment is carried out in a culture period of 12 days.
[0011] A method for detecting key markers of wool goat hair follicle stem cells includes the following steps: Step 1: Seed cells in a 24-well plate pre-placed with coverslips, culture to an appropriate density, and then fix with 4% paraformaldehyde. Minutes, using a volume fraction of 0.1% Permeation treatment After minutes, it was blocked with 5% goat serum (v / v). Minutes, add anti-cytokeratin ,anti Primary antibodies against β1 integrin, at temperature Incubate overnight. The next day, wash three times with PBS, add fluorescently labeled secondary antibody, and incubate at room temperature. , Incubation Hours later, the nuclei were stained with DAPI, observed and photographed under a fluorescence microscope; Step 2, Real-time quantitative PCR: Extract total RNA from cells, reverse transcribe to synthesize cDNA, use GAPDH as an internal reference gene, design specific primers for CK15, CD34, and β1 integrin, perform real-time quantitative PCR reaction, and calculate the relative expression level of the target gene based on the Ct value.
[0012] As a preferred embodiment of the present invention, in the immunofluorescence staining, fixation is performed with 4% paraformaldehyde for 20 minutes, and then... After permeabilization for 15 minutes, fluorescently labeled secondary antibody was added and incubated at room temperature for 1.5 hours.
[0013] Applications of goat hair follicle stem cells in hair follicle regeneration, animal genetic breeding improvement, or skin tissue engineering.
[0014] Compared with existing technologies, the advantages of this invention are as follows: This invention employs a combined digestion method using trypsin and EDTA, which can precisely target intercellular junction structures. Trypsin specifically hydrolyzes protein junctions, while EDTA loosens cell junctions by chelating calcium ions. The synergistic effect of these two methods enables efficient isolation of hair follicle stem cells from cashmere goat skin tissue. This method not only has high isolation efficiency but also causes minimal damage to cells, preserving cell viability and biological function to the greatest extent possible, providing high-quality cell resources for subsequent culture and research.
[0015] This invention clearly specifies key parameters such as digestion time, temperature, and oscillation interval, making the separation process highly standardized and reproducible. It ensures the stability of the separation effect under different experimental environments and operator conditions, reducing experimental errors and uncertainties, and facilitating the smooth progress of scientific research and the reliability of results.
[0016] The growth factors such as EGF, insulin, and hydrocortisone added to the culture medium of this invention can simulate the in vivo growth microenvironment of hair follicle stem cells. EGF can stimulate cell division and proliferation, accelerating cell growth; insulin participates in the metabolic regulation of cells, promoting the uptake and utilization of nutrients; hydrocortisone helps maintain the normal physiological state of cells and inhibits apoptosis. The synergistic effect of these growth factors provides ideal conditions for the growth and proliferation of hair follicle stem cells, significantly improving cell proliferation capacity and colony formation rate.
[0017] The optimized culture system of this invention provides a long-term stable growth environment for hair follicle stem cells, prolonging their lifespan and maintaining their favorable biological characteristics and functions during in vitro culture. This is of great significance for in-depth research into the biological behavior and signaling pathways of hair follicle stem cells, as well as the development of related therapeutic methods.
[0018] This invention comprehensively utilizes multiple methods, including morphological observation, growth curve measurement, and clonogenic assays, to identify cells from different perspectives. Morphological observation allows for a direct assessment of whether the cell morphological characteristics conform to the typical features of hair follicle stem cells; growth curve measurement reflects cell growth viability and proliferative capacity; and clonogenic assays detect the cell's self-renewal and clonogenic abilities. This multi-dimensional identification significantly improves the accuracy and reliability of the results, avoiding potential misjudgments that may arise from relying on a single method.
[0019] The identification method of this invention can accurately identify hair follicle stem cells in the early stages of cell culture, and promptly screen out cell populations with research and application value. This helps save experimental time and resources, improve research efficiency, and accelerate the translation of relevant scientific research results.
[0020] This invention employs a combination of immunofluorescence staining and real-time quantitative PCR to comprehensively detect key markers of hair follicle stem cells, such as CK15, CD34, and β1 integrin. Immunofluorescence staining can visually display the distribution and expression of these markers within cells, while real-time quantitative PCR can accurately measure the gene expression levels of these markers. The combined detection of multiple markers allows for a more comprehensive and in-depth understanding of the biological characteristics and molecular mechanisms of hair follicle stem cells.
[0021] This invention accurately detects the expression of key markers, providing crucial evidence for in-depth research on the differentiation regulation mechanisms, signal transduction pathways, and relationship between hair follicle stem cells and hair follicle development and regeneration. Simultaneously, it also provides important reference indicators for developing treatment methods and screening drugs based on hair follicle stem cells.
[0022] The method of this invention helps to gain a deeper understanding of the biological characteristics and regulatory mechanisms of hair follicle stem cells in cashmere goats, providing a theoretical basis and technical support for improving cashmere yield and quality. By optimizing the culture and application of hair follicle stem cells, it is hoped that high-yielding and high-quality cashmere goat breeds can be cultivated, promoting the sustainable development of the cashmere goat industry.
[0023] The hair follicle stem cells of this invention possess multi-directional differentiation potential and have broad application prospects in the field of regenerative medicine. The method of this invention provides an effective technical means for the research and application of hair follicle stem cells, which helps to promote the research and clinical application of hair follicle stem cells in skin repair, hair regeneration, tissue engineering, and other fields, and provides new ideas and methods for solving related medical problems. Attached Figure Description
[0024] Figure 1 This is a flowchart of the steps for isolating and culturing goat hair follicle stem cells provided by the present invention; Figure 2 A data block diagram of operating parameters provided for this invention; Figure 3 This is a data block diagram of the core parameters provided for this invention. Detailed Implementation
[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, not all, of the embodiments of the present invention.
[0026] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. It should be noted that, in the absence of conflict, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.
[0027] Example 1: Method for isolating, culturing and identifying cashmere goat follicle stem cells, including the following steps: Step 1, tissue acquisition: Select healthy cashmere goat skin tissue, rinse with sterile physiological saline 3-5 times to remove bloodstains and impurities on the surface, and cut the skin tissue into small pieces of 1mm³; Step 2, Enzymatic Digestion: Place the shredded skin tissue pieces into a digestion solution containing 0.25% trypsin and 0.02% EDTA, and digest at 37°C for 30-60 minutes, gently shaking once every 10 minutes. After digestion, add culture medium containing 10% fetal bovine serum to stop the digestion. Step 3, Cell Suspension Preparation: Filter the digested tissue suspension through a 200-mesh sieve to remove undigested tissue fragments. Centrifuge the filtrate at 1000 rpm for 5-10 minutes, discard the supernatant, and resuspend the cell pellet in culture medium to obtain the cell suspension. In the enzymatic digestion step, the digestion time is 45 minutes.
[0028] The method for culturing wool goat hair follicle stem cells includes the following steps: Step 1, primary culture: The cell suspension obtained in claim 1 is seeded into a culture flask pre-coated with gelatin and placed at a temperature of... Volume fraction is 5% Cultured in an incubator, the culture medium is The culture medium was supplemented with 10% fetal bovine serum (FBS) at a concentration of [missing information]. Epidermal growth factor Concentration is insulin, concentration of Hydrocortisone and 1% (v / v) penicillin and streptomycin, every... Change the culture medium once a day; Step 2, passage culture: When the cell confluence reaches 80%-90%, digest the cells with 0.25% trypsin and 0.02% EDTA, and passage them at a ratio of 1:2-1:3.
[0029] In primary culture, the culture medium should be changed every 2 days. In subculture, subculture should be performed when the cell confluence reaches 85%, with a subculture ratio of 1:2.
[0030] The method for isolating, culturing and identifying cashmere goat hair follicle stem cells includes the following steps: Step 1, morphological observation: observe the morphological characteristics of the cells under an inverted microscope. Cashmere goat hair follicle stem cells are small and round or polygonal, with a large nucleus-to-cytoplasm ratio. The cells adhere to the wall and grow in a colony-like distribution. Step 2, Growth Curve Determination: The growth curve of the cells was determined using the MTT assay. Cells in the logarithmic growth phase were used, and the cell density per well was recorded. Cells were seeded at a density of [number] cells per well in 96-well plates, and [number] cells were seeded on the [number]th day after seeding. Add MTT solution and continue culturing. After hours, discard the supernatant, add DMSO to dissolve the crystals, and use a microplate reader to analyze the crystals at a wavelength of [wavelength value missing]. Measure absorbance value by number of days The x-axis represents absorbance values. Plot the growth curve on the ordinate; Step 3: Divide the cells into cells per well. Cells were seeded at a density of [number] cells per well in 6-well plates and cultured. After the clones are formed and visible to the naked eye, they are fixed with methanol, stained with crystal violet, and the number of clones is counted. Clonal formation rate The calculation formula is: In the clone formation experiment, the culture time was 12 days.
[0031] A method for detecting key markers of wool goat hair follicle stem cells includes the following steps: Step 1: Seed cells in a 24-well plate pre-placed with coverslips, culture to an appropriate density, and then fix with 4% paraformaldehyde. Minutes, using a volume fraction of 0.1% Permeation treatment After minutes, it was blocked with 5% goat serum (v / v). Minutes, add anti-cytokeratin ,anti Primary antibodies against β1 integrin, at temperature Incubate overnight. The next day, wash three times with PBS, add fluorescently labeled secondary antibody, and incubate at room temperature. , Incubation Hours later, the nuclei were stained with DAPI, observed and photographed under a fluorescence microscope; Step 2, Real-time quantitative PCR: Extract total RNA from cells, reverse transcribe to synthesize cDNA, use GAPDH as an internal reference gene, design specific primers for CK15, CD34, and β1 integrin, perform real-time quantitative PCR reaction, and calculate the relative expression level of the target gene based on the Ct value.
[0032] In immunofluorescence staining, fixation was performed with 4% paraformaldehyde for 20 minutes, and then... After permeabilization for 15 minutes, fluorescently labeled secondary antibody was added and incubated at room temperature for 1.5 hours.
[0033] Applications of goat hair follicle stem cells in hair follicle regeneration, animal genetic breeding improvement, or skin tissue engineering.
[0034] Working principle: Healthy cashmere goat skin tissue is an important source of hair follicle stem cells. The surface of the skin tissue often has bloodstains and impurities. Rinsing with sterile saline removes these external contaminants, ensuring a sterile environment and cell purity for subsequent procedures. Cutting the skin tissue into small pieces increases the contact area between the tissue and digestive enzymes, allowing the enzymes to function more effectively and thus more efficiently separating hair follicle stem cells from the tissue.
[0035] The digestion solution, composed of 0.25% trypsin and 0.02% EDTA, was chosen based on their effects on intercellular junction structures. Trypsin specifically hydrolyzes protein junctions between cells, disrupting cell-cell and cell-matrix adhesion. EDTA chelates calcium ions in the extracellular fluid; calcium ions play a crucial role in maintaining intercellular junctions and normal cell morphology. Removing calcium ions loosens intercellular junctions, allowing trypsin to function more effectively. Digestion was performed at 37°C because this temperature is close to the physiological temperature of organisms, maintaining optimal enzyme activity. Gentle agitation every 10 minutes ensures sufficient contact between the enzyme and tissue, guaranteeing uniform and effective digestion. After digestion, culture medium containing 10% fetal bovine serum was added to terminate the process, as the proteins in the serum neutralize trypsin activity, preventing over-digestion and cell damage.
[0036] Filtering through a 200-mesh sieve removes undigested tissue fragments, leaving only single cells or small cell clusters, resulting in a relatively pure cell suspension. Centrifugation utilizes the differences in cell mass and density to cause cells to settle to the bottom of the tube, while the supernatant contains some impurities and digestion fluids. After discarding the supernatant, the cell pellet is resuspended in culture medium to provide a suitable environment for cell survival, facilitating subsequent culture procedures.
[0037] The isolated cell suspension was inoculated into culture flasks pre-coated with gelatin. Gelatin mimics the extracellular matrix environment of cells in vivo, providing a scaffold for cell attachment and growth, which is beneficial for cell adhesion. DMEM / F12 medium is a commonly used cell culture medium containing various nutrients required for cell growth, such as amino acids, vitamins, and minerals. Adding 10% fetal bovine serum (FBS) provides various growth factors, hormones, nutrients, and regulatory factors that promote cell growth, proliferation, and survival. Epidermal growth factor (EGF) stimulates cell division and proliferation, accelerating cell growth; insulin participates in cellular metabolic regulation, promoting cellular uptake and utilization of nutrients; hydrocortisone has anti-inflammatory and cell growth-regulating effects, helping to maintain normal cellular physiological states; and dual antibiotics (penicillin and streptomycin) prevent bacterial and fungal contamination, ensuring a sterile culture environment. The culture medium was changed every 2-3 days to replenish nutrients consumed by the cells, remove metabolic waste, and maintain a stable cell growth environment.
[0038] When cell confluence reaches 80%-90%, it means the cells have grown to a certain density in the culture flask. At this point, the space between cells becomes limited, and the supply of nutrients gradually becomes insufficient, inhibiting cell growth. Subculturing is then performed, using trypsin and EDTA to digest the cells and detach them from the culture flask wall. The cells are then passaged at a specific ratio (1:2-1:3) and seeded into new culture flasks, providing them with new growth space and sufficient nutrients, thus enabling them to continue to maintain good growth and proliferation.
[0039] Hair follicle stem cells possess unique morphological characteristics. Their small, round or polygonal cell shape, along with a relatively high nucleus-to-cytoplasm ratio, are key features distinguishing them from other cells. These cells adhere to the culture surface and grow in colony-like patterns because they possess the ability to self-renew and proliferate; under suitable culture conditions, they continuously divide, forming cell colonies. Observing these morphological characteristics using an inverted microscope can provide a preliminary assessment of whether the cells are hair follicle stem cells.
[0040] The MTT assay is a commonly used method for assessing cell proliferation. MTT is a yellow tetrazolium salt. Mitochondrial succinate dehydrogenase in living cells reduces MTT to insoluble blue-purple formazan crystals. Upon addition of DMSO, the formazan crystals dissolve, forming a colored solution. The absorbance of this solution is directly proportional to the number of living cells. By measuring absorbance at different time points and plotting growth curves, cell growth can be visually assessed. The latency phase represents the time it takes for cells to adapt to the new culture environment; the logarithmic growth phase is the stage of rapid cell proliferation, during which the cell number increases exponentially; the plateau phase indicates that cell growth has reached saturation, and the rate of cell proliferation slows down. Analyzing the growth curves allows for determination of whether the cell viability and proliferation capacity are consistent with the characteristics of hair follicle stem cells.
[0041] Clonogenic assays are important methods for detecting cell self-renewal and proliferation capabilities. A small number of cells (100 cells per well) are seeded into a culture plate. If the cells possess self-renewal and proliferation capabilities, they will continuously divide to form visible clones. The clonogenic rate reflects the proportion of cells with clonogenic capacity. Hair follicle stem cells have a high clonogenic capacity; therefore, by calculating the clonogenic rate, it is possible to further identify whether the cells are hair follicle stem cells.
[0042] Immunofluorescence staining utilizes the principle of specific binding between antigens and antibodies. Cytokeratin 15 (CK15), CD34, and β1 integrin are key markers for hair follicle stem cells. Primary antibodies targeting these markers are added to the cells, and these antibodies specifically bind to the corresponding antigens within the cells. Then, fluorescently labeled secondary antibodies are added, which bind to the primary antibodies, causing the antigen-antibody complexes within the cells to become fluorescently labeled. Staining the nucleus with DAPI reveals its location, and observation under a fluorescence microscope shows that cells with fluorescent labels indicate the presence of the corresponding markers, thus determining whether the cells are hair follicle stem cells.
[0043] Real-time quantitative PCR (qPCR) is a technique used to detect gene expression levels. First, total RNA is extracted from cells and reverse transcribed to synthesize cDNA. Then, GAPDH is used as an internal reference gene, as its expression in cells is relatively stable and can be used as a reference to correct for differences between different samples. Specific primers targeting CK15, CD34, and β1 integrin are designed. During the PCR reaction, the fluorescence signal continuously increases with each amplification cycle. By detecting the intensity of the fluorescence signal and the number of cycles reaching the threshold (Ct value), the relative expression level of the target gene can be calculated. High expression levels of these key markers in cells indicate that the cells possess characteristics of hair follicle stem cells.
[0044] Test case Experimental Objective The effectiveness and reliability of the method for isolating, culturing, identifying, and detecting key markers of goat hair follicle stem cells provided by this invention are verified, and the performance of the method in terms of cell isolation efficiency, culture effect, identification accuracy, and marker detection sensitivity are evaluated.
[0045] Experimental Materials and Methods Test materials Healthy cashmere goat skin tissue was selected as the test sample. The reagents used included DMEM / F12 medium, fetal bovine serum, epidermal growth factor (EGF), insulin, hydrocortisone, penicillin and streptomycin, trypsin, EDTA, gelatin, MTT solution, DMSO, methanol, crystal violet, primary antibodies against cytokeratin 15 (CK15), anti-CD34, and anti-β1 integrin, as well as fluorescently labeled secondary antibodies and DAPI. The equipment used included an inverted microscope, an enzyme-linked immunosorbent assay (ELISA) reader, a fluorescence microscope, and a real-time quantitative PCR instrument.
[0046] Test methods The procedure shall be performed in accordance with the method for isolating, culturing, identifying, and detecting key markers of goat hair follicle stem cells provided in this invention: Separation method: Select healthy cashmere goat skin tissue, rinse and cut it into small pieces, then digest it with enzymes, and then prepare a cell suspension through steps such as filtration and centrifugation.
[0047] Culture method: The cell suspension was inoculated into gelatin-coated culture flasks for primary culture. After the cells reached a certain degree of confluence, they were passaged.
[0048] Identification methods: Cells were identified by morphological observation, growth curve measurement, and clonogenic assay.
[0049] Key marker detection methods: Immunofluorescence staining and real-time quantitative PCR were used to detect the expression of key markers in cells.
[0050] A control group was set up, which used traditional methods for hair follicle stem cell isolation, culture, identification, and marker detection.
[0051] Test data
[0052] Cell culture effect Cell proliferation capacity Cell growth curves were determined using the MTT assay, and cell doubling time was calculated.
[0053] Clonal formation rate
[0054] Accuracy of identification The accuracy of identification was calculated by comprehensively judging whether the cells were hair follicle stem cells through morphological observation, growth curve measurement, and clonogenic assay.
[0055] Sensitivity of key marker detection Immunofluorescence staining The proportion of positive cells was counted.
[0056] Real-time quantitative PCR Calculate the relative expression level of the target gene:
[0057] Experimental conclusions The separation method of this invention is significantly superior to traditional methods in terms of cell separation efficiency and cell viability. A greater number of cells are separated per gram of skin tissue, and the cells exhibit higher viability, indicating that this method can more effectively obtain high-quality hair follicle stem cells from cashmere goat skin tissue, reducing cell damage and providing ample cell resources for subsequent culture and research.
[0058] During cell culture, the cell proliferation capacity and colony formation rate of the experimental group were significantly higher than those of the control group. The shorter cell doubling time and higher colony formation rate indicate that the culture system of this invention can better simulate the growth microenvironment of hair follicle stem cells, promote cell growth and proliferation, maintain cell stemness, and is beneficial for the long-term culture and application of hair follicle stem cells.
[0059] The identification method of this invention has high accuracy and can more accurately determine whether cells are hair follicle stem cells. By using multi-dimensional identification indicators to comprehensively evaluate the biological characteristics of cells, the possibility of misidentification is reduced, providing a reliable cell source for subsequent research and applications.
[0060] Regarding the detection of key biomarkers, the proportion of immunofluorescence-positive cells and the relative expression level of the target gene by real-time quantitative PCR in the experimental group were significantly higher than those in the control group. This indicates that the biomarker detection method of the present invention has higher sensitivity and can more accurately detect the expression of key biomarkers in hair follicle stem cells, which is helpful for in-depth research on the biological characteristics and regulatory mechanisms of hair follicle stem cells.
[0061] The method for isolating, culturing, identifying, and detecting key markers of cashmere goat follicle stem cells provided by this invention has significant advantages, providing strong technical support for the research and application of cashmere goat follicle stem cells, and has broad application prospects in improving cashmere yield and quality and promoting the development of related biotechnology.
[0062] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described herein. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present invention, as well as all technical solutions and improvements that do not depart from the spirit and scope of the invention, are covered within the scope of the claims of the present invention.
Claims
1. A method for isolating, culturing and identifying caprine hair follicle stem cells, characterized in that, It comprises the following steps: Step 1, tissue acquisition: healthy cashmere goat skin tissue is selected, washed with sterile normal saline for 3-5 times to remove bloodstains and impurities on the surface, and cut into small pieces of 1mm3; Step 2, enzyme digestion: the cut skin tissue pieces are placed in a digestion solution containing 0.25% trypsin and 0.02% EDTA, and digested at 37℃ for 30-60 minutes, with gentle shaking every 10 minutes during the digestion process. After the digestion is completed, a culture solution containing 10% fetal bovine serum is added to terminate the digestion. Step 3, cell suspension preparation: the digested tissue suspension is filtered through a 200-mesh sieve to remove undigested tissue pieces, and the filtrate is centrifuged at 1000r / min for 5-10 minutes, and the supernatant is discarded. The cell pellet is resuspended with culture solution to obtain a cell suspension.
2. The method for isolating, culturing, and identifying cashmere goat hair follicle stem cells according to claim 1, characterized in that, In the enzyme digestion step, the digestion time is 45 minutes.
3. The method for isolating, culturing, and identifying cashmere goat hair follicle stem cells according to claim 2, characterized in that, The cashmere goat hair follicle stem cell culture method comprises the following steps: Step 1, Primary Culture: The cell suspension obtained in claim 1 is seeded into a culture flask pre-coated with gelatin and placed at a temperature of... Volume fraction is 5% Cultured in an incubator, the culture medium is The culture medium was supplemented with 10% fetal bovine serum (FBS) at a concentration of [missing information]. Epidermal growth factor Concentration is insulin, concentration of Hydrocortisone and 1% (v / v) penicillin and streptomycin, every... Change the culture medium once a day; Step 2, subculture: when the cell confluence reaches 80%-90%, the cells are digested with 0.25% trypsin and 0.02% EDTA, and subcultured at a ratio of 1:2-1:
3.
4. The method of claim 3, wherein the hair follicle stem cells are obtained from a cashmere goat. 5 In the primary culture, the culture medium is replaced every 2 days.
5. The method for isolating, culturing, and identifying cashmere goat hair follicle stem cells according to claim 4, characterized in that, In the subculture, the cells are subcultured when the cell confluence reaches 85%, and the subculture ratio is 1:
2.
6. The cashmere goat hair follicle stem cell separation, culture and identification method according to claim 5, comprising the following steps: Step 1, morphological observation: observe the morphological characteristics of the cells under an inverted microscope. The cashmere goat hair follicle stem cells are small and round or polygonal, have a large nucleus-to-cytoplasm ratio, grow adherently, and are distributed in a colony-like manner. Step 2, Growth Curve Determination: The growth curve of the cells was determined using the MTT assay. Cells in the logarithmic growth phase were used, and the cell density per well was recorded. Cells were seeded at a density of [number] cells per well in 96-well plates, and [number] cells were seeded on the [number]th day after seeding. Add MTT solution and continue culturing. After hours, discard the supernatant, add DMSO to dissolve the crystals, and use a microplate reader to analyze the crystals at a wavelength of [wavelength value missing]. Measure absorbance value by number of days The x-axis represents absorbance values. Plot the growth curve on the ordinate; Step 3, cells were seeded in 6-well plates at a density of 100 cells per well and cultured for 7 days, then fixed with methanol and stained with crystal violet to count the number of colonies The colony formation rate was calculated as follows: . 7. The method of claim 6, wherein the hair follicle stem cells are obtained from a cashmere goat. In the colony formation experiment, the culture time is 12 days. 8. A method for detecting key markers of caprine hair follicle stem cells, characterized by, It comprises the following steps: Step 1: Seed cells into 24-well plates pre-placed with coverslips, culture to an appropriate density, and then fix with 4% paraformaldehyde. Minutes, using a volume fraction of 0.1% Permeation treatment After minutes, it was blocked with 5% goat serum (v / v). Minutes, add anti-cytokeratin ,anti Primary antibodies against β1 integrin, at temperature Incubate overnight. The next day, wash three times with PBS, add fluorescently labeled secondary antibody, and incubate at room temperature. , Incubation Hours later, the nuclei were stained with DAPI, observed and photographed under a fluorescence microscope; Step 2, real-time fluorescent quantitative PCR: total RNA is extracted, and cDNA is synthesized by reverse transcription. GAPDH is used as an internal reference gene, and specific primers for CK15, CD34 and β1 integrin are designed for real-time fluorescent quantitative PCR reaction. The relative expression amount of the target gene is calculated according to the Ct value.
9. The method for detecting key markers of cashmere goat follicle stem cells according to claim 8, characterized in that, For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and incubated with fluorescently labeled secondary antibodies for 1.5 hours at room temperature. For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 for 10. The cashmere goat hair follicle stem cells obtained by the method of any one of claims 1 to 9 for use in hair follicle regeneration, animal genetic breeding improvement or skin tissue engineering.