Method for separating, culturing and identifying goat hair follicle-derived primary melanocytes

Abstract

The invention provides a method for separating, culturing and identifying goat hair follicle-derived primary melanocytes, and relates to the technical field of cell culture and identification. Comprising the following steps: (1) taking fresh nourishing skin, and putting into protease for digestion culture; (2) taking out the skin cultured in the step (1), blowing away dermis tissues, sieving and centrifuging to obtain hair follicles; (3) carrying out trypsin digestion on the hair follicles obtained in the step (2) to obtain hair follicle-derived melanocytes; (4) inoculating the hair follicle-derived melanocytes obtained in the step (3) into an M-254 culture medium for culture, and changing the culture medium once every 3 days; and (5) identifying the melanocytes cultured in the step (4). The isolated culture method provided by the invention is simple, and the cultured goat hair follicle-derived melanocyte is good in growth condition and stable in form.

Description

Technical Field

[0001] The invention provides a method for separating, culturing and identifying goat hair follicle-derived primary melanocytes, and relates to the technical field of cell culture and identification. Background Art

[0002] Compared with epidermal melanocytes, follicle-derived melanocytes can more directly reveal the root cause of hair hypopigmentation, opening up a new perspective for the treatment of hair pigment disorders. In addition, hair hypopigmentation is difficult to explain with the theory of melanocytes in the epidermis. The separation, cultivation, purification and identification technology of follicle-derived melanin is crucial for subsequent biological research and lays the experimental foundation. The research on melanocytes is relatively extensive. By collecting skin tissue, melanocytes of animals such as mice, rabbits, and black-bone chickens have been successfully obtained, but related research on goat melanocytes has not been reported. The skin of goats is thicker than that of mice, rabbits, and black-bone chickens. Goat skin contains a multi-layer structure and abundant hair follicles, which increases the difficulty of extracting melanocytes, resulting in a smaller number of hair follicles, affecting the subsequent melanocyte extraction amount and purity. Moreover, the distribution of hair follicles in different animals is different, and the melanocytes of different animals are different in morphology, physiological function and metabolic characteristics. These differences lead to poor cell adaptability and low survival rate when the existing melanocyte acquisition methods of mice, rabbits and black-bone chickens are applied to goats. Exploring the mechanism of goat hair color formation is conducive to obtaining the hair color type determined by breeding goals or required by the market, and provides a scientific basis for the genetic improvement, purebred breeding and genetic differentiation research of local goat breeds in my country. Summary of the invention

[0003] The present invention provides a method for isolating, culturing and identifying primary melanocytes derived from goat hair follicles, which solves the technical problems in the prior art that the existing methods for obtaining melanocytes from mice, rabbits and black-bone chickens are poor in cell adaptability and survival rate when applied to goat melanin extraction, and there is a lack of melanocyte extraction methods tailored to the characteristics of goat skin and melanocytes.

[0004] The present invention is achieved by comprising the steps of: (1) Take fresh cultured skin, put it into protease, and digest and culture it at 37℃ for 2 h; (2) separating the dermis and epidermis of the skin cultured in step (1) to obtain hair follicles; (3) digesting the hair follicles obtained in step (2) with trypsin to obtain hair follicle-derived melanocytes; (4) inoculating the hair follicle-derived melanocytes obtained in step (3) in M-254 complete culture medium and changing the medium every 3 days; (5) Identifying the melanocytes cultured in step (4).

[0005] As further preferred, the M-254 complete culture medium comprises the following components and their contents: M-254 culture medium 78%, fetal bovine serum 20%, 50 μg / ml penicillin-streptomycin 2%.

[0006] As a further preference, the protease in step (1) is a type II neutral protease.

[0007] As a further preference, the step of obtaining hair follicles in step (2) comprises: adding type I collagenase to the epidermis for digestion at 37°C for 30 min, repeatedly blowing to form a cell suspension, filtering with a 70 μm cell sieve, centrifuging at 1200 rpm for 10 min, and discarding the supernatant to obtain hair follicles.

[0008] As further preferred, the type I collagenase is 0.25% type I collagenase.

[0009] As a further preference, the identification in step (5) comprises one or more of DOPA staining, immunofluorescence staining, and tissue section immunofluorescence method.

[0010] Beneficial effects of the present invention: (1) The present invention establishes an in vitro separation and culture system for goat hair follicle-derived primary melanocytes. The cells grow well and fast under M-254 complete culture medium. Growth factors or human melanin growth additives are often used to separate and culture mouse, mink, and black-bone chicken melanocytes to promote the growth of separated melanocytes. The culture medium of the present application has few components and has the advantage of low culture cost. The present application obtains goat hair follicles by blowing and freeing. The blowing and freeing method is gentle, reduces damage to hair follicles and surrounding tissues, helps to maintain the integrity and vitality of hair follicles, effectively releases hair follicles from tissues, and is beneficial to improving the efficiency of hair follicle acquisition and maintaining hair follicle quality. The operation method is simple, energy-saving, and easy to implement, and can achieve large-scale production of goat hair follicle-derived melanocytes.

[0011] (2) The present invention identifies melanocytes based on cell morphology by DOPA staining and immunofluorescence staining, and determines that the melanocytes are located above the hair papilla and in the outer root sheath of the hair follicle.

[0012] (3) The present invention uses type II neutral protease and type I collagenase. Type II neutral protease is a fast, effective and mild cell digestion enzyme that causes little damage to cells and can maintain the integrity of cell membranes. It is derived from bacteria and is free of mycoplasma or other animal virus contamination. It has strong stability and is not affected by temperature, pH and serum components. Type I collagenase can hydrolyze collagen in connective tissue and has a good digestive effect on intercellular matrix. It is gentle and efficient, and has a mild digestive effect. It does not require mechanical vibration, will not damage cells, and maintains cell integrity. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 These are microscopic images of the primary melanocytes in Example 1 at the 6th and 8th days after separation, with the left image being the 6th day and the right image being the 8th day; Figure 2 This is a microscopic picture of the growth state of the melanocytes in Example 1 during the 4th, 5th and 6th passages, and on the 5th day of growth. From left to right, it is the melanocytes at the 4th, 5th and 6th passages; Figure 3 It is a microscopic picture of the growth state of melanocytes isolated and cultured in Example 2 and Comparative Example 1 on the 5th day; Figure 4 This is a microscopic picture of the growth state of the 11th generation of melanocytes isolated and cultured in Comparative Example 1 and Example 1; Figure 5 The DOPA staining method was used to identify the 11th generation melanocytes cultured in Example 1 and Example 2, respectively; Figure 6 This is a graph showing the melanin content of the 11th generation melanocytes cultured in Example 1 and Example 2 (* indicates P <0.05); Figure 7 The figure is an identification diagram of melanocytes cultured in Example 1. The left figure is a test group, which is identified by using the DOPA staining method. The right figure is a control group, which is different from the left figure in that the DOPA reagent is replaced with PBS, serving as the control group. Figure 8 The marker genes MITF and TYR for melanocyte production are selected, and the melanocytes cultured in Example 1 are identified by immunofluorescence; Fig. 9 These are observation pictures of skin tissue cross-sections and longitudinal sections after immunohistochemistry of paraffin sections. Picture A shows the melanin in melanocytes of goat skin tissue longitudinal sections (200×); Picture B shows the melanin in melanocytes of goat skin tissue transverse sections (200×); Picture C shows the location of melanin after immunohistochemical staining of longitudinal sections (200×); Picture D shows the location of melanin after immunohistochemical staining of transverse sections (200×); Fig.10 The marker gene TYR for melanocyte production was selected, and the localization of hair follicle-derived melanocytes was analyzed by immunofluorescence under 200 and 400 times microscope. DETAILED DESCRIPTION

[0014] Experimental instruments, equipment, consumables and reagents: CO2 constant temperature incubator (ESCO), inverted microscope (Zen), low-speed centrifuge (TDL5M, Hunan Xiangli Scientific Instrument Co., Ltd.), refrigerator (Midea), pipette (Epend), microscissors (Kanghua Medical Beauty Equipment, 14 cm straight head), 6-well cell plate (Scottje), anhydrous ethanol (Sinopharm), fetal bovine serum (Gibco), 0.25% trypsin (Hyclone), double antibody (Hyclone), pancreatic enzyme (Hyclone), M-254 culture medium (Thermo), DMEM / F12 culture medium (Hyclone), TESCA buffer (Solebao), collagenase I (Solebao), 0.25% type II neutral protease (Hyclone), 40 μm cell sieve (Solebao), 70 μm cell sieve (Solebao), phosphate buffer (Hyclone). Example

[0015] A method for efficiently isolating and culturing goat hair follicle-derived primary melanocytes comprises the following steps: (1) After sampling, the skin tissue was placed in DMEM-F12 medium containing 50 ug / ml penicillin-streptomycin, stored at 4°C, and brought to the intercellular space within 1 hour for the isolation and culture of melanocytes. After completing the intercellular culture, the fresh skin tissue was removed from the DMEM-F12 medium and washed 6 times with PBS to remove impurities and bacteria as much as possible to avoid interference. The skin was cut into strips (2 mm x 3 mm) with ophthalmic scissors; placed in 0.25% type II neutral protease for digestion at 37°C for 2 hours, and the amount of protease used for each skin strip was 1 ml. Type II neutral protease is a fast, effective and mild cell digestion enzyme that causes little damage to cells and can maintain the integrity of cell membranes; it is derived from bacteria and is not contaminated by mycoplasma or other animal viruses; it is highly stable and is not affected by temperature, pH and serum components.

[0016] (2) Separate the epidermis and subcutaneous adipose tissue under a stereomicroscope, add type I collagenase and digest at 37°C for 30 min. The amount of collagenase used for each piece of skin is 1 ml. Beat repeatedly to form a cell suspension, filter with a 70 μm cell sieve, and centrifuge at 1200 rpm for 10 min. Discard the supernatant and obtain the hair follicles.

[0017] (3) The hair follicles obtained in step (2) were washed 2-3 times with PBS for 2 min each time, centrifuged at 1200 rpm for 10 min, the supernatant was discarded, and the hair follicles were incubated in a CO2 incubator at 37°C for 3 min after being treated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). The hair follicles were sieved with a 40 μm filter and centrifuged at 1000 rpm for 10 min. The upper culture medium was discarded to isolate the hair follicle-derived melanocytes.

[0018] (4) The hair follicle-derived melanocytes obtained in step (3) are inoculated into M-254 complete medium containing serum, placed in a CO2 incubator, and cultured at 37°C. After 3 days, M-254 complete medium is used for selective culture, and the medium is changed every 3 days. Figure 1 Observe the adhesion status of primary melanocytes on the 6th and 8th days after separation, and it can be seen that the melanocytes grow well. Figure 2 The melanocytes in the culture medium grew to the state of the 5th day during the 4th, 5th, and 6th passages, showing that the melanocytes grew fast and in good condition. The melanocytes were still stable and did not change significantly after being cultured in M-254 complete medium until the 18th passage, which was conducive to further obtaining melanocytes with higher purity.

[0019] The M-254 complete medium used in Example 1 contains the following components and their contents: M-254 medium 78%, fetal bovine serum 20% and 50 μg / ml penicillin-streptomycin 2%. Example

[0020] A method for efficiently isolating and culturing goat hair follicle-derived primary melanocytes comprises the following steps: (1) After the skin tissue is sampled, it is placed in DMEM-F12 culture medium containing 50 ug / ml penicillin-streptomycin, stored at 4°C, and brought to the intercellular space within 1 hour for the isolation and culture of melanocytes. After the intercellular culture is completed, the fresh skin tissue is removed from the DMEM-F12 culture medium, washed with PBS, and the skin is cut into strips (2 mm x 3 mm) again with ophthalmic scissors; placed in 0.25% type II neutral protease for digestion at 37°C for 1.5 hours, and the amount of protease used for each skin strip is 1 ml.

[0021] (2) Separate the epidermis and subcutaneous adipose tissue under a stereomicroscope, add 1 ml of type I collagenase and digest at 37°C to form a cell suspension, filter with a 70 μm cell sieve and centrifuge at 1200 rpm for 10 min, discard the supernatant and obtain the hair follicles.

[0022] (3) The hair follicles obtained in step (2) were washed 2-3 times with PBS for 2 min each time, centrifuged at 1200 rpm for 10 min, the supernatant was discarded, and the hair follicles were incubated in a CO2 incubator at 37°C for 3 min after being treated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA). The hair follicles were sieved with a 40 μm filter and centrifuged at 1000 rpm for 10 min. The upper culture medium was discarded to isolate the hair follicle-derived melanocytes.

[0023] (4) The hair follicle-derived melanocytes obtained in step (3) are inoculated into M-254 complete medium containing serum, placed in a CO2 incubator, and cultured at 37°C. After 5 days, DMEM-F12 complete medium is used for selective culture, and the medium is changed every 3 days. The M-254 complete medium in this step contains the following components and their contents: M-254 medium 78%, fetal bovine serum 20% and 50 μg / ml penicillin-streptomycin 2%. The DMEM-F12 complete medium in this step contains the following components and their contents: DMEM-F12 medium 78%, fetal bovine serum 20% and 50 μg / ml penicillin-streptomycin 2%.

[0024] Comparative Example 1 The difference from Example 2 is that, in the process of culturing goat hair follicle-derived primary melanocytes, only the DMEM-F12 culture medium commonly used in laboratories is selected, and the components of the culture medium used are different.

[0025] The growth of goat hair follicle-derived primary melanocytes obtained by the culture methods of Example 2 and Comparative Example 1 were observed respectively.

[0026] See also Figure 3 Compared with Example 1, the growth rate of melanocytes in Example 1 in DMEM-F12 complete medium was significantly faster than that in the control group (Example 2), but the cell stability was poor in the later stage. Figure 4 In Example 2, the cell morphology obtained by culturing with DMEM-F12 complete medium gradually changed, and cell differentiation occurred; while in Example 1, the cell morphology of the 11th generation melanocytes cultured with M-254 complete medium was stable, and cell subculture and purification were achieved.

[0027] In summary, the melanocytes cultured in M-254 complete medium during the culture process in Example 1 grew well and had a stable morphology. In Example 2, after the fifth day of cell culture, DMEM-F12 complete medium was used for subsequent cell subculture. Although the growth rate in the early stage was not as good as that in Comparative Example 1, the cell stability in the later stage was better than that in Comparative Example 1. Example

[0028] A method for morphologically identifying the goat hair follicle-derived primary melanocytes obtained in Example 1, comprising the following steps: (1) Cell climbing: Place a glass slide in a six-well plate in advance, digest the cells with trypsin and move them to the six-well plate, and wash with PBS three times, 3 min each time; (2) Add an appropriate amount of 4% paraformaldehyde as a cell tissue fixative to fix the cells at room temperature for 30 min, discard the fixative, and wash with PBS three times, 5 min each time; (3) Add 0.3% Triton-x100 to permeabilize for 30 min, and then wash with PBS three times, 5 min each time; (4) Add 0.1% L-Dopa staining solution and incubate in a 37°C incubator for 3 h. Rinse with PBS three times and observe under a microscope.

[0029] The reagents used in Example 3 include the following components and their contents: DOPA staining solution, trypsin, 0.1% L-DOPA incubation solution, Triton X-100, and 4% paraformaldehyde, wherein 0.1% L-DOPA is prepared using 0.5 mol / L hydrochloric acid as a mother solution. Example

[0030] A method for identifying goat hair follicle-derived primary melanocytes obtained in Example 1 from proteins, comprising the following steps: (1) Cell slides: Place a glass slide in a six-well plate in advance and inoculate the cells. When the cell slide confluence reaches 70%, discard the M-254 culture medium and wash with PBS three times, 1 min each time. (2) Fixation: Add an appropriate amount of 4% paraformaldehyde cell tissue fixative to each well and fix at 37°C for 10 min. Wash with PBS three times, each time for 1 min.

[0031] (3) Permeabilization: Add an appropriate amount of 0.25% Triton-x100 to each well, fix at 37°C for 15 min, and wash with PBS three times, 1 min each time.

[0032] (4) Blocking: Block with 5% BSA at room temperature for 1 h.

[0033] (5) Primary antibody incubation: Add MITF antibody, incubate overnight at 4°C, and wash three times with PBS, each time for 1 min.

[0034] (6) Secondary antibody incubation: Add secondary antibody goat anti-rabbit / mouse IgG (HRP) to the cells, incubate at 37°C for 1 h, and wash with PBS three times, 1 min each time.

[0035] (7) Incubate the DAPI plate at room temperature for 2 min and then wash with PBS three times, 1 min each time.

[0036] (8) Take a glass slide, add a drop of anti-fluorescence quencher, and perform laser focusing observation. Example

[0037] A method for identifying goat hair follicle-derived primary melanocytes obtained in Example 1 from proteins, comprising the following steps: (1) Cell slides: Place a glass slide in a six-well plate in advance and inoculate the cells. When the cell confluence reaches 70%, discard the M-254 culture medium and wash with PBS three times, 3 min each time. (2) Fixation: Add an appropriate amount of 4% paraformaldehyde cell tissue fixative to each well and fix at 37°C for 10 min. Wash with PBS three times, 2 min each time.

[0038] (3) Permeabilization: Add an appropriate amount of 0.25% Triton-x100 to each well, fix at 37°C for 15 min, and wash with PBS three times, 2 min each time.

[0039] (4) Blocking: Block with 5% BSA at room temperature for 1 h.

[0040] (5) Primary antibody incubation: Add TYR antibody, incubate overnight at 4°C, and wash three times with PBS, 2 min each time.

[0041] (6) Secondary antibody incubation: Add secondary antibody goat anti-rabbit / mouse IgG (HRP) to the cells, incubate at 37°C for 1 h, and wash with PBS three times, 2 min each time.

[0042] (7) Incubate the DAPI plate at room temperature for 2 min and then wash with PBS three times, 2 min each time.

[0043] (8) Take a glass slide, add a drop of anti-fluorescence quencher, turn the glass slide upside down on the anti-fluorescence quencher, and perform laser focusing observation.

[0044] Comparative Example 3 In the identification of goat hair follicle-derived primary melanocytes, the method of morphologically identifying goat hair follicle-derived primary melanocytes is selected. The melanocytes observed under a microscope should be distributed in two or three levels and are preliminarily identified as melanocytes.

[0045] The hair follicles obtained in Example 1 and Example 2 were dyed. The experimental results are as follows: Figure 5-Figure 7 .

[0046] See also Figure 5 , indicating that after subculture to the 11th generation, the cells in the F12 medium have differentiated, while the cells in Example 1 using the M254 medium are morphologically stable melanocytes.

[0047] See also Figure 6After culturing the 11th generation of melanocytes in Example 1 and Example 2, 0.25% trypsin was added to the melanocytes for digestion at 37°C for 3 min, and the digestion was terminated with complete culture medium. The melanocytes were lysed by adding 0.2 mol / L NaOH solution, and the melanocytes were water bathed at 80°C for 15 min to fully lyse the melanocytes; the lysed melanocyte suspension was transferred to a 96-well plate, with 5 replicates per group; the absorbance value at a wavelength of 490 nm was measured using an ELISA instrument to calculate the melanin content of each group of cells. The results showed that the melanin content of the goat melanocytes in Example 1 was significantly higher than that in Example 2 (P<0.05). The test results confirmed that the cells grew well and fast under the M-254 complete culture medium.

[0048] Figure 7 This is a control test picture of the 18th generation M254 complete culture medium. The test group is Example 1, and the control group is under the same conditions, except that the DOPA reagent in Example 3 was replaced with PBS as a control test, which can further illustrate that the melanocytes cultured in the M254 culture medium are morphologically stable.

[0049] Comparative Example 4 The difference from Example 5 is that in the identification of goat hair follicle-derived primary melanocytes, the melanocyte marker gene MITF was selected for immunofluorescence staining. Under a microscope, it was observed that both antibodies showed positive reactions, and they were further identified as melanocytes. The experimental results refer to Figure 8 and Fig.10 . Select marker genes related to coat color formation MITF and TYR , the results show MITF and TYR All of them expressed positive, so through identification, it was determined that the isolated and cultured cells were goat melanocytes. The identifications in Example 4 and Example 5 together showed that melanocytes were obtained.

[0050] In summary, it was identified from the morphology and protein that the cells cultured using M-254 complete medium in Example 1 were melanocytes. Example

[0051] A method for localizing the expression of hair follicle-derived melanocytes in goat skin tissue by paraffin section immunohistochemistry, comprising the following steps: (1) Melting wax: Turn on the constant temperature drying oven and adjust it to 70℃. Put the wax block into the wax cup to melt the wax.

[0052] (2) Sampling: Cut the tissue into 2×2 cm2 pieces, wrap it with gauze and rinse it in running water for 1 h.

[0053] (3) Dehydration: 50% alcohol for 30 min, 70% alcohol for 30 min, 80% alcohol for 30 min, 90% alcohol for 30 min, 95% alcohol I for 20 min, 95% alcohol II for 20 min, 100% alcohol I for 30 min, 100% alcohol II for 30 min, alcohol:xylene (1:1) for 30 min.

[0054] (4) Transparency: Xylene I is transparent for 5 min, and xylene II is transparent for 5 min. The transparency time is determined according to the size of the tissue.

[0055] (5) Wax dipping: Melt the wax in a drying oven, and then place the transparent tissue into the pre-melted liquid paraffin. The wax dipping time for each two times is 1-1.5 h.

[0056] (6) Embedding: Pour the melted wax into the embedding box and place it at room temperature to allow the liquid paraffin to solidify.

[0057] (7) Sectioning: Use a paraffin slicer to cut the tissue block into 10 μm thick slices. Spread the slices in a 37°C water bath and remove the tissue slices with a glass slide.

[0058] (8) Observation: Observe under an inverted microscope.

[0059] (9) Baking: Place the slices in a 37°C constant temperature drying oven and bake for 1 h. Example

[0060] A method for localizing the expression of hair follicle-derived melanocytes in goat skin tissue by immunohistochemistry, comprising the following steps: (1) Tissue fixation: Before dewaxing, bake the sections in a 60°C incubator for 1 h.

[0061] (2) Dewaxing: Soak the sections in xylene for 10 min, then change to xylene and soak for another 10 min.

[0062] (3) Hydration: Soak in anhydrous ethanol for 5 min; soak in 95% ethanol for 5 min; soak in 85% ethanol for 5 min; soak in 75% ethanol for 5 min.

[0063] (4) Washing: Soak in ddH2O for 5 min and rinse three times.

[0064] (5) Antigen retrieval (boiling method): Prepare a renaturing agent, add 1.25 g to 400 ml of pure water, stir and heat, and remove the slices after the solution cools to room temperature.

[0065] (6) Washing: Soak in ddH2O for 5 min, wash twice; soak in PBS for 5 min, wash twice.

[0066] (7) Inactivation of enzyme: Add 50 μl of inactivation enzyme reagent to each slice and incubate at room temperature in the dark for 15 min.

[0067] (8) Washing: Soak in PBS for 5 min, wash three times (discard the first wash quickly).

[0068] (9) Blocking: Add 50 μl of 3% BSA-PBS to each section and block in a wet box at room temperature for 30 min.

[0069] (10) Add primary antibody: discard the blocking solution, add 50 μl of MITF antibody to each section, and incubate overnight at 4°C.

[0070] (11) Rewarming: Remove the slices the next day and rewarm them at room temperature for 40 min.

[0071] (12) Washing: Soak in PBS for 5 min, wash 3 times. (Pour out the first wash quickly).

[0072] (13) Add enzyme-labeled secondary antibody: Add 25 μl of HRP-labeled secondary antibody (anti-rabbit / anti-mouse) to each section and incubate at 37°C for 30 min.

[0073] (14) Washing: Soak in PBS for 5 min, wash three times (discard the first wash quickly).

[0074] (15) Color development (DAB method): Add 50 μl of color development solution to each section and develop the color in a wet box for 5 min.

[0075] (16) Stop color development: Use distilled water to stop the color development reaction.

[0076] (17) Restaining: Add 50 μl of hematoxylin somatic cell rapid staining solution to each section, stain for 30 s, and rinse with distilled water.

[0077] (18) Decolorization and anti-blueing: Place the slices in 1% hydrochloric acid-ethanol for 3-5 seconds, then quickly remove them and place them in distilled water to stop the decolorization. Then place them in PBS for anti-blueing for 5 minutes.

[0078] (19) Sealing: Soak in 75% ethanol for 5 min; 85% ethanol for 5 min; 95% ethanol for 5 min; and anhydrous ethanol for 5 min. Replace with xylene and soak for another 10 min. Add neutral gum and cover glass.

[0079] (20) Observation: Observe under a microscope. Example

[0080] A method for localizing the expression of hair follicle-derived melanocytes in goat skin tissue by immunofluorescence technology comprises the following steps: (1) Tissue fixation: Before dewaxing, bake the sections in a 60°C incubator for 1 h.

[0081] (2) Dewaxing: Soak the sections in xylene for 10 min. (3) Hydration: Soak in anhydrous ethanol for 5 min; soak in 95% ethanol for 5 min; soak in 85% ethanol for 5 min; soak in 75% ethanol for 5 min.

[0082] (4) Washing: Soak in ddH2O and wash 3 times.

[0083] (5) Antigen retrieval (boiling method): Prepare the renaturing agent, add 1.25 g to 400 ml of pure water, stir and heat, and remove the slices after the solution cools to room temperature (about 30 min).

[0084] (6) Washing: Soak in ddH2O for 5 min, then soak in PBS for 2 times.

[0085] (7) Inactivation of enzyme: Add 50 μl of inactivation enzyme reagent to each slice and incubate at room temperature in the dark for 15 min.

[0086] (8) Washing: Soak and wash three times in PBS.

[0087] (9) Blocking: Add 50 μl of 3% BSA-PBS to each section and block in a wet box at room temperature for 30 min.

[0088] (10) Add primary antibody: discard the blocking solution, add 50 μl of MITF antibody to each section, and incubate overnight at 4°C.

[0089] (11) Rewarming: Remove the slices the next day and rewarm them at room temperature for 40 min.

[0090] (12) Washing: Soak and wash three times in PBS.

[0091] (13) Add fluorescent secondary antibody: Add 25 μl of goat anti-rabbit IgG (H+L) secondary antibody to each section and incubate at room temperature for 2 h.

[0092] (14) Add DAPI and place at room temperature for 2 min. Soak in PBS for 5 min and wash twice.

[0093] (15) Remove the coverslip and apply sealing film.

[0094] Comparative Example 6 The difference from Example 7 is that the skin is not subjected to additional treatment, and the melanocytes are directly located through paraffin sections. Fig. 9, under the microscope, the cross-section and longitudinal section of paraffin skin were observed, and melanin granules were visible above the hair papilla and the outer root sheath of the hair follicles before staining, which interfered with the expression of immunohistochemistry after staining and made it impossible to clearly locate the expression of melanocytes in the hair follicles.

[0095] Comparative Example 7 The difference from Example 8 is that the fluorescent secondary antibody used for immunofluorescence marks the marker gene of melanin production through the specific binding between the antibody and the antigen, and emits green fluorescence under a fluorescence microscope to determine the location of melanocytes. The histochemical secondary antibody is HRP. The marker genes MITF and TYR of melanocytes are marked by the method of tissue immunofluorescence for immunofluorescence staining, and the expression and localization of melanocytes are observed under a microscope. The experimental results are shown in FIG. Fig. 9 , Fig. 9 In Figures A, B, and C, green fluorescence is the marker gene TYR for melanocytes (200×); in Figures D, E, and F, green fluorescence is the marker gene TYR for melanocytes (400×); blue fluorescence indicates all cells in the hair follicle. It is positively expressed above the hair papilla and in the outer root sheath of the hair follicle.

[0096] In summary, immunofluorescence technology was used to detect MITF and TYR proteins in goat hair follicle-derived melanocytes, and their expression was mainly located above the hair follicle papilla and in the outer root sheath.

[0097] Beneficial effects of the present invention: (1) The present invention establishes an in vitro separation and culture system for goat hair follicle-derived primary melanocytes. The cells grow well and fast under M-254 complete culture medium. Growth factors or human melanin growth additives are often used to separate and culture mouse, mink, and black-bone chicken melanocytes to promote the growth of separated melanocytes. The culture medium of the present application has few components and has the advantage of low culture cost. The present application obtains goat hair follicles by blowing and freeing. The blowing and freeing method is gentle, reduces damage to hair follicles and surrounding tissues, helps to maintain the integrity and vitality of hair follicles, effectively releases hair follicles from tissues, and is beneficial to improving the efficiency of hair follicle acquisition and maintaining hair follicle quality. The operation method is simple, energy-saving, and easy to implement, and can achieve large-scale production of goat hair follicle-derived melanocytes.

[0098] (2) The present invention identifies melanocytes based on cell morphology by DOPA staining and immunofluorescence staining, and determines that the melanocytes are located above the hair papilla and in the outer root sheath of the hair follicle.

[0099] (3) The present invention uses type II neutral protease and type I collagenase. Type II neutral protease is a fast, effective and mild cell digestion enzyme that causes little damage to cells and can maintain the integrity of cell membranes. It is derived from bacteria and is free of mycoplasma or other animal virus contamination. It has strong stability and is not affected by temperature, pH and serum components. Type I collagenase can hydrolyze collagen in connective tissue and has a good digestive effect on intercellular matrix. It is gentle and efficient, and has a mild digestive effect. It does not require mechanical vibration, will not damage cells, and maintains cell integrity.

Claims

1. A method for isolating, culturing and identifying goat hair follicle-derived primary melanocytes, characterized in that: The steps include: (1) Take fresh cultured skin, put it into protease, and digest and culture it at 37℃ for 2 h; (2) separating the dermis and epidermis of the skin cultured in step (1) to obtain hair follicles; (3) digesting the hair follicles obtained in step (2) with trypsin to obtain hair follicle-derived melanocytes; (4) inoculating the hair follicle-derived melanocytes obtained in step (3) in M-254 complete culture medium and changing the medium every 3 days; (5) Identifying the melanocytes cultured in step (4).

2. The method for isolating, culturing and identifying goat hair follicle-derived primary melanocytes according to claim 1, characterized in that: The M-254 complete culture medium includes the following components and their contents: M-254 culture medium 78%, fetal bovine serum 20%, 50 μg / ml penicillin-streptomycin 2%.

3. The method for isolating, culturing and identifying goat hair follicle-derived primary melanocytes according to claim 1, characterized in that: The protease in step (1) is a type II neutral protease.

4. The method for isolating, culturing and identifying goat hair follicle-derived primary melanocytes according to claim 1, characterized in that: Step (2) of obtaining hair follicles includes: adding type I collagenase to the epidermis for digestion at 37°C for 30 minutes, repeatedly blowing and beating to form a cell suspension, filtering through a 70 μm cell sieve, centrifuging at 1200 rpm for 10 minutes, discarding the supernatant, and obtaining hair follicles.

5. The method for isolating, culturing and identifying goat hair follicle-derived primary melanocytes according to claim 4, characterized in that: The type I collagenase is 0.25% type I collagenase.

6. The method for isolating, culturing and identifying goat hair follicle-derived primary melanocytes according to claim 1, characterized in that: The identification in step (5) includes one or more of DOPA staining, immunofluorescence staining, and tissue section immunofluorescence method.

Images