CN115261303A - Method suitable for mare mammary epithelial cell in-vitro culture - Google Patents
Method suitable for mare mammary epithelial cell in-vitro culture Download PDFInfo
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Abstract
The invention provides a method suitable for culturing mare mammary epithelial cells in vitro. The method suitable for culturing the mare mammary epithelial cells in vitro comprises the following steps: s1: collecting mammary gland tissues; s2: pretreatment of mammary gland tissues; s3: digestion of mammary tissue; s4: inoculating mammary gland tissue; s5: purifying cells; s6: cell passage; s7: freezing and storing the cells; s8: cell recovery; s9: and (4) identifying mammary epithelial cells. The method for in vitro culture of the mare mammary epithelial cells provided by the invention has the advantages that primary mare mammary epithelial cells which can be efficiently, simply and conveniently separated and cultured by an enzyme digestion method, have strong cell activity, can be stably passed and have a normal lactation function are cultured.
Description
Technical Field
The invention relates to the technical field of in-vitro cell culture, in particular to a method suitable for in-vitro culture of mare mammary epithelial cells.
Background
Mammary gland is a special tissue organ of mammals, and a mammary gland epithelial cell line retaining the specific function of the mammary gland can become an ideal model for researching mammary gland development, differentiation, degeneration and a mammary gland bioreactor. Mammary epithelial cell lines of a plurality of animals such as mice, sheep, cattle, pigs and the like are established in sequence at present. A plurality of tissue culture modes including organ culture, primary mammary epithelial cell culture and mammary epithelial cell lines are determined, and the method is used for researching the development mechanism of mammary gland, the secretion mechanism of milk, the construction of a mammary bioreactor and the occurrence of mammary cancer. However, due to the complexity of mammary gland tissue structure, it is very difficult to study the development, secretion function and degeneration of mammary gland epithelial cells, and the study of mammary gland cannot be generalized due to species difference and gland differentiation period difference, and the study of biological characteristics of mammary gland epithelial cells of a certain animal cannot be completely applicable to other animals. Therefore, the types and the number of mammary gland cell lines are far from enough, so the research on establishing mammary gland epithelial cell lines is continued, and more practical cell lines are established.
The equine species are large, herbivorous, monogastric animals and play a significant role in the construction and development of animal husbandry. At present, horse lactation-related research is mainly focused on the aspects of daily ration nutrition regulation and screening of lactation-related genes. However, the research of the breast epithelial in vitro culture system from the level of cytology and molecular biology is only rarely reported at home and abroad. Therefore, the establishment of the in vitro culture system of the equine mammary gland epithelial cells can not only supplement the vacancy of the in vitro culture system of the equine mammary gland epithelial cells, but also increase the variety and the number of the mammary gland cell lines and enrich the content of mammary gland biology. The method has great theoretical significance for further and deeply researching the mammary gland development mechanism and the lactation mechanism of horses and the problem that mammary epithelial cells regulate and control the lactation, the milk yield and the milk components.
In the prior art, the application numbers are: 201710071003.8 patent of a method for separating cultured buffalo mammary epithelial cells from buffalo milk, the main method comprises: the method comprises the following steps of (1) collecting buffalo milk, (2) separating buffalo mammary gland epithelial cells and (3) culturing and purifying the buffalo mammary gland epithelial cells, and has the defects that the mammary gland epithelial cells separated from the buffalo milk are epithelial cells which are exfoliated from mammary glands during lactation, the number of the epithelial cells is small, and the time required for cell climbing-out is long. And the cells have insufficient activity and are easy to die, and the cells grow slowly after passage.
The application numbers are: 201610112297.X patent of a method for isolation and culture of primary cow mammary epithelial cells, the main method comprises: shearing mammary tissues of a lactation cow into tissue blocks, digesting the tissue blocks by using collagenase/hyaluronidase digestive juice, inoculating the digested chyliform tissue blocks into a culture dish for culture, after 1-2 days, dissociating cells from the periphery of the tissue blocks, removing spindle fibroblasts by adopting a mechanical scraping method every day during the culture period until a large number of mammary epithelial cells grow and are fused, and then carrying out passage, and eliminating the possibly mixed fibroblasts by using a poor digestion method during the passage.
The operation process of separating and culturing primary mammary epithelial cells from the mammary gland of a dairy cow, removing mixed fibroblasts by using an instrument scraping method every day during the culture process is complicated, and how to operate is not described in detail. Secondly, with mechanical scraping, the fibroblast scraping is not thorough and requires repeated operations, the probability of scraping mammary epithelial cells is high, and the vitality of mammary epithelial cells can be damaged.
Therefore, there is a need to provide a new method for culturing mare mammary epithelial cells in vitro to solve the above problems.
Disclosure of Invention
The invention aims to provide a method for screening primary mare mammary epithelial cells which can be efficiently, simply and conveniently separated and cultured by an enzyme digestion method, have strong cell activity, can be stably passed and have a normal lactation function, and is suitable for in-vitro culture of the mare mammary epithelial cells.
In order to solve the technical problems, the method for culturing the mare mammary epithelial cells in vitro provided by the invention comprises the following steps: the method comprises the following steps:
s1: collecting mammary gland tissues;
s2: pretreatment of mammary gland tissues;
s3: digestion of mammary tissue;
s4: inoculating mammary gland tissue;
s5: purifying cells;
s6: cell passage;
s7: freezing and storing the cells;
s8: cell recovery;
s9: and (4) identifying mammary epithelial cells.
Preferably, the specific steps in S1 are as follows: the mare adopts healthy Kazakh mares in the middle lactation period, after the mare is anesthetized, the mare breasts are cleaned by iodophor solution, and then the mare breasts are disinfected by 75% alcohol; cutting off superficial skin tissue with a scalpel, separating mammary gland tissue along surface of fascia, and performing circular cutting of mammary gland with circular cutting radius of 2cm and circular cutting depth of subcutaneous 3cm; the cut breast tissue was placed in a 50mL centrifuge tube containing 75% ethanol solution for about 5-10s and immediately removed, washed with sterile 1% PBS buffer, after the blood and milk were washed away, placed in a 50mL sterile sample tube containing 2% PBS buffer, sealed with a sealing film, placed in an ice box, and taken back to the laboratory.
Preferably, the specific steps in S2 are as follows: after the mammary gland tissue is brought back to a laboratory, the outside of a 50mL sterile sample tube containing the mammary gland tissue is disinfected by 75% alcohol and is brought into an intercellular superclean bench; taking out the mammary tissue and placing the mammary tissue in a culture dish containing PBS buffer solution; removing fascia, fat tissue and fibrous fasciculate connective tissue outside the mammary tissue by using ophthalmic scissors and ophthalmic tweezers to obtain parenchyma tissue of mammary epithelial cells; the obtained parenchymal tissue was placed in a 5mL centrifuge tube and cut into paste with an ophthalmic scissors.
Preferably, the specific steps in S3 are as follows: placing the pasty mammary tissue and digestive juice simultaneously containing 0.1% collagenase type I and 0.01% hyaluronidase into a 50mL centrifuge tube according to the volume ratio of 1:3; placing a 50mL centrifuge tube in a constant-temperature shaking water bath kettle, shaking and digesting at the constant temperature of 37 ℃ at the speed of 100r/min, and adding an equal volume of termination culture medium to terminate digestion after most tissues are digested.
Preferably, the specific steps in S4 are as follows: filtering the digested mammary tissue by using a 100-mesh cell sieve, and then filtering by using a 200-mesh cell sieve; putting the filtrate in a 15mL centrifuge tube, centrifuging at 1000r/min for 5min; discarding the supernatant, and leaving the cell layer, wherein the serum in the sediment is added according to the proportion of 1:3, adding erythrocyte lysate in proportion, blowing uniformly, cracking for 2min,1000r/min, and centrifuging for 5min; discarding the supernatant, adding 5 times volume of PBS buffer solution, blowing and beating uniformly at 1000r/min, centrifuging for 3min, and repeating twice; discarding the supernatant, and leaving the cell precipitate; adding 2mL of complete culture medium, uniformly blowing, inoculating into a T25 culture bottle, and supplementing the complete culture medium to 4mL; the T25 flask was incubated at 37 ℃ in an incubator containing 5% of CO2, the cells were observed daily, and the complete medium was replaced every 2 days.
Preferably, the specific steps in S5 are as follows: fibroblast cell mixed growth occurred during mammary epithelial cell culture, and according to its sensitivity to trypsin, they were separated, and when the cells were 80% -90% of the bottom of the flask, the medium was aspirated, rinsed 2 times with 1% PBS; adding 1mL of 0.25% trypsin solution, incubating at 37 deg.C and 5% in a CO2 incubator to digest fibroblasts; every 2 minutes; adding a termination culture medium after the fibroblasts are digested to terminate the digestion, and discarding the digestion solution in the bottle; washing with PBS for 2 times, adding 1mL of 0.25% trypsin solution, continuously digesting the cells without the wall, and adding a termination culture medium to terminate digestion when 90% of the cells are subjected to wall removal; repeatedly blowing and beating the cells on the wall of the bottle to remove the wall, putting the cell sap into a 15mL centrifuge tube, centrifuging at 1000r/min for 5min; removing supernatant, adding a proper amount of complete culture medium to resuspend cells, inoculating the cells into a T25 culture flask, and then supplementing the complete culture medium to 4mL; cultured in an incubator containing 5% of CO2 at 37 ℃ and purified several times until mammary epithelial cells were found in the culture flask.
Preferably, the specific steps in S6 are as follows: when mammary epithelial cells in T25 flasks grew to 90% of the area of the bottom of the flask, the cells were subcultured: during subculture, firstly discarding the original culture medium in a T25 culture bottle, adding PBS buffer solution for slight washing, discarding the PBS buffer solution, and repeating twice; adding 1mL of 0.25% trypsin, digesting in an incubator at 37 ℃, and adding an equal volume of termination medium to terminate digestion after the cells have fallen off; transferring the cell suspension to a 15mL centrifuge tube, centrifuging for 5min at 1000r/min, removing the supernatant, adding a complete culture medium, slightly blowing cells by using a pipette gun to break cell masses, dividing the mammary epithelial cells into 3-4 parts, transferring to a new culture bottle, and supplementing the complete culture medium to 4mL; cultured in an incubator containing 5% of CO2 at 37 ℃ and the complete medium was replaced every 2 days.
Preferably, the specific steps in S7 are as follows: taking primary equine mammary gland epithelial cells, sucking off original culture solution, washing for 1-2 times by using PBS buffer solution, adding 1mL of 0.25 trypsin, and digesting dispersed cells; after the cells are fallen off, adding an equal volume of termination culture medium to terminate digestion; sucking into 15mL centrifuge tube, centrifuging for 5min at 1000r/min, discarding supernatant, adding 1mL cell cryopreservation solution, transferring into 2mL cryopreservation tube, labeling, sealing with sealing film, placing into programmed cooling cryopreservation box, standing overnight at-80 deg.C, and transferring into liquid nitrogen for long-term storage on day 2.
Preferably, the specific steps in S8 are as follows: taking out the frozen tube from the liquid nitrogen tank, and quickly putting the frozen tube into a water bath kettle at 37 ℃ for shaking to melt the frozen tube; adding 1mL of complete culture medium when most of the frozen stock solution is melted, and uniformly beating; sucking the mixed solution into a 15mL centrifuge tube, adding a proper amount of complete culture medium, performing centrifugation at 1000r/min for 5min; discarding the supernatant, adding 4mL of complete culture medium, and uniformly blowing; on average 2 new T25 flasks were aspirated and the flasks were filled to 4mL with complete medium and incubated at 37 ℃ in a 5% CO2 incubator.
Preferably, the specific steps in S9 are as follows: when mammary gland epithelial cells in the T25 culture bottle grow to 80% -90%, absorbing the culture solution, and washing for 2-3 times by using PBS buffer solution; adding 1.5-2mL of Trizol solution into each bottle to crack cells, horizontally placing for 15min, repeatedly blowing, collecting the cracking solution to a 1.5mL centrifuge tube, adding 100 mu L of chloroform, shaking for 15s to emulsify, standing for 5min,12000rpm, and centrifuging for 15min at 4 ℃; absorbing 3/4 of the upper-layer homogenized liquid of the centrifuge tube into a new centrifuge tube of 1.5mL, adding isopropanol with the same volume, uniformly mixing, standing for 10min at 10 rpm and 4 ℃, and centrifuging for 10min; white precipitate at the bottom of the centrifuge tube is RNA, supernatant is discarded, 1mL of 75% ethanol is added, the mixture is evenly mixed and then centrifuged at 12000rpm at 4 ℃ for 15min, and the supernatant is discarded; placing the centrifuge tube in a fume hood for 2-5min, adding 5mL of RNA-free enzyme water to dissolve the precipitate, blowing and beating uniformly, carrying out electrophoresis after the RNA is completely dissolved, detecting the quality and concentration of the RNA, and using the RNA to identify the mammary epithelial cell specific gene: keratin 8, keratin 18 and beta-casein.
Compared with the related technology, the method for culturing the mare mammary epithelial cells in vitro provided by the invention has the following beneficial effects:
1. isolating mammary epithelial cells from a mare's mammary gland for a first time;
2. the time for separating the cells is short, the purity of the obtained cells is high, and the number of the cells is large;
3. the method has small cell damage, strong activity of the obtained mammary epithelial cells, good cell growth condition and stable passage, and the mammary epithelial cells have normal lactation function, are convenient for long-term experimental study, and provide stable test materials for the study of equine mammary gland tissue development and lactation mechanism.
Drawings
FIG. 1 is a diagram of the morphology of cells in a culture flask observed under an inverted microscope at a magnification of 100X after inoculation in the process of morphology verification of separation and purification culture of mare mammary epithelial cells in the practice of the present invention;
FIG. 2 is a morphological examination process of separation and purification culture of mare mammary epithelial cells in the practice of the present invention, in which 2d is inoculated and washed with PBS buffer solution, after a new culture medium is replaced, the morphological picture of cells in a culture flask is observed under an inverted microscope with a magnification of 100 × and long spindle fibroblast grows out first;
FIG. 3 is a morphological diagram of the isolation, purification and culture of mare mammary epithelial cells in the practice of the present invention, which is obtained by inoculating 5 d, washing with PBS buffer, replacing with new culture medium, and observing the mixed growth of cells in the culture flask, long spindle fibroblasts and mammary epithelial cells under an inverted microscope with magnification of 100X;
FIG. 4 is a diagram showing the morphology of mammary epithelial cells in a flask in a short spindle or polygonal shape observed under an inverted microscope at a magnification of 100 Xwith collagenase I and hyaluronidase being digested and replaced with a new medium in the procedure of morphological examination of separation and purification of mammary epithelial cells of mare according to the present invention;
FIG. 5 is a morphological diagram of the separation and purification culture of mare mammary epithelial cells in the practice of the present invention, which is observed under an inverted microscope with a magnification of 100 × after digestion and subculture, that the mammary epithelial cells in the culture flask are in the shape of paving stones;
FIG. 6 is a diagram showing the result of electrophoresis of mare mammary epithelial cells in the practice of the present invention, in which CK-8 and CK-18 proteins peculiar to mammary epithelial cells are detected, and beta-casein is confirmed, and the cells have normal lactation function.
Detailed Description
The invention is further described with reference to the following figures and embodiments.
Referring to fig. 1-6, in one embodiment, the method for culturing mare mammary epithelial cells in vitro includes:
materials: an ultra-clean workbench, an electric heating constant-temperature CO2 incubator, an inverted microscope for cells, an ultra-low temperature refrigerator, a constant-temperature shaking water bath kettle, a high-pressure steam sterilization kettle, an electrophoresis apparatus, an ophthalmic scissors, an ophthalmic forceps, a scalpel, gauze, a 200-mesh cell sieve, a 100-mesh cell sieve, a sealing film, a 15mL centrifuge tube, a 50mL sterile sample tube, a 5mL centrifuge tube, a 1.5mL centrifuge tube, a T25 culture bottle and a 9cm culture dish.
Reagent: iodophor, 75% alcohol, DMEM/F12 medium, double antibody, PBS buffer, absolute ethanol, collagenase type I, hyaluronidase, erythrocyte lysate, standard Fetal Bovine Serum (FBS), insulin (IGF), epidermal Growth Factor (EGF), hydrocortisone (Hydrocortisone), dimethyl sulfoxide (DMSO), chloroform, trizol solution, isopropanol, RNase-free water.
And (3) high-pressure sterilization of instruments: ophthalmic scissors, ophthalmic forceps, scalpel, 200-mesh cell sieve, 100-mesh cell sieve, 5mL centrifuge tube, 1.5mL centrifuge tube
Preparing a solution:
(1) Complete medium: each 50mLDMEM/F12 culture medium contains 500 muL double antibody, 250 mug insulin, 500ng epidermal growth factor, 50 mug hydrocortisone and 5mL fetal calf serum.
(2) Termination of the medium: each 50mL of the MEM/F12 medium contained 500. Mu.L of the double antibody and 5mL of fetal bovine serum.
(3) PBS buffer containing 1% double antibody: the double-antibody contained 500 μ L per 50 mLPBS.
(4) PBS buffer containing 2% double antibody: 1000 muL of the double antibody was contained in each 50 mLPBS.
(5) 75% of ethanol: each 50mL of 75% absolute ethanol contains 37.5mL of absolute ethanol and 12.5mL of PBS buffer.
(6) Freezing and storing liquid: DMEM/F12 medium: FBS: DMSO =7:2:1.
the method comprises the following specific steps:
s1: and (4) collecting mammary gland tissues. The mare adopts healthy Kazakh mares in the middle lactation period, after the mare is anesthetized, the mare breasts are cleaned by iodophor solution, and then the mare breasts are disinfected by 75% alcohol; cutting surface skin tissue with a scalpel, separating mammary gland tissue along the surface of fascia, and performing circular cutting on mammary gland with a circular cutting radius of 2cm and a circular cutting depth of subcutaneous 3cm; the cut breast tissue was placed in a 50mL centrifuge tube containing 75% ethanol solution for about 5-10s and immediately removed, washed with sterile 1% PBS buffer, after the blood and milk were washed away, placed in a 50mL sterile sample tube containing 2% PBS buffer, sealed with a sealing film, placed in an ice box, and quickly brought back to the laboratory in a short time.
S2: pretreatment of mammary gland tissue. After the mammary gland tissue is brought back to a laboratory, the outside of a 50mL sterile sample tube containing the mammary gland tissue is disinfected by 75% alcohol and is brought into an intercellular superclean workbench; taking out the mammary tissue and placing the mammary tissue in a culture dish containing PBS buffer solution; removing fascia, fat tissue (soft and uniform) and fibrous bundle connective tissue outside the mammary tissue by using ophthalmic scissors and forceps to obtain parenchyma tissue of mammary epithelial cells; the obtained parenchymal tissue was placed in a 5mL centrifuge tube and cut into a paste by ophthalmic cutting.
S3: digestion of mammary tissue. Placing pasty mammary gland tissue and digestive juice simultaneously containing 0.1% type I collagenase and 0.01% hyaluronidase into a 50mL centrifuge tube according to the volume ratio of 1:3 (mammary gland tissue: digestive juice); placing a 50mL centrifuge tube in a constant-temperature shaking water bath kettle, shaking and digesting at the constant temperature of 37 ℃ at the speed of 100r/min, and adding an equal volume of termination culture medium to terminate digestion after most tissues are digested (the culture medium contains protein and competitively inhibits enzyme digestion cells).
S4: inoculation of mammary tissue. Filtering the digested mammary tissue by using a 100-mesh cell sieve, and then filtering by using a 200-mesh cell sieve; putting the filtrate in a 15mL centrifuge tube, centrifuging at 1000r/min for 5min; discarding supernatant, and leaving cell layer (if there is serum in the precipitate, adding erythrocyte lysate according to 1:3 ratio, blowing and beating uniformly, cracking for 2min,1000r/min, and centrifuging for 5 min); discarding the supernatant, adding 5 times volume of PBS buffer solution, blowing and beating uniformly at 1000r/min, centrifuging for 3min, and repeating twice; discarding the supernatant, and leaving the cell precipitate; adding 2mL of complete culture medium, uniformly blowing, inoculating into a T25 culture bottle, and supplementing the complete culture medium to 4mL; the T25 flask was incubated in an incubator at 37 ℃ and 5% CO2, cells were observed daily, and the complete medium was changed every 2 days.
S5: and (4) purifying the cells. Fibroblasts grow in a mixed way in the mammary epithelial cell culture process, and can be separated according to different sensibility to trypsin. When the cells were about 85% of the bottom of the flask, the medium in the flask was aspirated and rinsed 2 times with 1% PBS; adding 1mL of 0.25% trypsin solution, adding to 37 deg.C, and digesting fibroblast cells in 5% CO2 incubator; every 2 minutes; adding a termination culture medium after the fibroblasts are digested to terminate the digestion, and removing the digestion solution in the bottle; washing with PBS for 2 times, adding 1mL of 0.25% trypsin solution, continuously digesting the cells without removing the wall, and adding a termination culture medium to terminate digestion when 90% of the cells are removed from the wall; repeatedly blowing and beating the cells on the wall of the bottle to remove the wall, putting the cell sap into a 15mL centrifuge tube, centrifuging at 1000r/min for 5min; removing supernatant, adding a proper amount of complete culture medium to resuspend cells, inoculating the cells into a T25 culture flask, and then supplementing the complete culture medium to 4mL; cultured in an incubator containing 5% of CO2 at 37 ℃ and purified several times until mammary epithelial cells were found in the culture flask.
S6: and (5) carrying out cell passage. When mammary epithelial cells in T25 flasks grew to 90% of the area of the bottom of the flask, the cells were subcultured: during subculture, firstly discarding the original culture medium in a T25 culture bottle, adding PBS buffer solution for slight washing, discarding the PBS buffer solution, and repeating twice; adding 1mL of 0.25% trypsin, digesting in an incubator at 37 ℃, and adding an equal volume of termination medium to terminate digestion after cells are detached; transferring the cell suspension to a 15mL centrifuge tube, centrifuging for 5min at 1000r/min, removing the supernatant, adding a complete culture medium, slightly blowing cells by using a pipette gun to break cell masses, dividing the mammary epithelial cells into 3-4 parts, transferring to a new culture bottle, and supplementing the complete culture medium to 4mL; cultured in an incubator containing 5% of CO2 at 37 ℃ and the complete medium was replaced every 2 days.
S7: and (5) freezing and storing the cells. Taking primary equine mammary gland epithelial cells, sucking off an original culture medium, washing for 1-2 times by using a PBS buffer solution, adding 1mL of 0.25 trypsin, and digesting dispersed cells; after the cells are fallen off, adding an equal volume of termination culture medium to terminate digestion; sucking into 15mL centrifuge tube, centrifuging for 5min at 1000r/min, discarding supernatant, adding 1mL cell cryopreservation solution, transferring into 2mL cryopreservation tube, labeling, sealing with sealing film, placing into programmed cooling cryopreservation box, standing overnight at-80 deg.C, and transferring into liquid nitrogen for long-term storage on day 2.
S8: and (5) recovering the cells. Taking out the freezing tube from the liquid nitrogen tank, and quickly putting the tube into a water bath kettle at 37 ℃ to shake and melt the tube; adding 1mL of complete culture medium when most of the frozen stock solution is melted, and uniformly beating; sucking the mixed solution into a 15mL centrifuge tube, adding a proper amount of complete culture medium, performing centrifugation at 1000r/min for 5min; discarding the supernatant, adding 4mL of complete culture medium, and uniformly blowing; on average 2 new T25 flasks were pipetted and the flasks were replenished to 4mL with complete medium and incubated at 37 ℃ in a 5% CO2 incubator.
S9: and (4) identifying mammary epithelial cells. When mammary gland epithelial cells in the T25 culture bottle grow to about 80% -90%, sucking culture solution, and washing with PBS buffer solution for 2-3 times; adding 1.5-2mL of Trizol solution into each bottle to crack cells, horizontally placing for 15min, repeatedly blowing, collecting the cracking solution to a 1.5mL centrifuge tube, adding 100 mu L of chloroform, shaking for 15s to emulsify, standing for 5min,12000rpm, and centrifuging for 15min at 4 ℃; absorbing 3/4 of the upper-layer homogenized liquid of the centrifuge tube into a new centrifuge tube of 1.5mL, adding isopropanol with the same volume, uniformly mixing, standing for 10min at 10 rpm and 4 ℃, and centrifuging for 10min; white precipitate at the bottom of the centrifuge tube is RNA, supernatant is discarded, 1mL of 75% ethanol is added, the mixture is evenly mixed and then centrifuged at 12000rpm at 4 ℃ for 15min, and the supernatant is discarded; placing the centrifuge tube in a fume hood for 2-5min, adding 5mL of RNA-free enzyme water to dissolve the precipitate, blowing and beating uniformly, carrying out electrophoresis after the RNA is completely dissolved, detecting the quality and concentration of the RNA, and using the RNA to identify the mammary epithelial cell specific gene: keratin 8, keratin 18 and beta-casein.
Compared with the related technology, the method for culturing the mare mammary epithelial cells in vitro provided by the invention has the following beneficial effects:
1. isolating mammary epithelial cells from mare mammary glands for a first time;
2. the time for separating the cells is short, the purity of the obtained cells is high, and the number of the cells is large;
3. the method has small cell damage, strong activity of the obtained mammary epithelial cells, good cell growth condition and stable passage, and the mammary epithelial cells have normal lactation function, are convenient for long-term experimental study, and provide stable test materials for the study of equine mammary gland tissue development and lactation mechanism.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method suitable for culturing mare mammary epithelial cells in vitro is characterized by comprising the following steps:
s1: collecting mammary gland tissues;
s2: pretreatment of mammary gland tissues;
s3: digestion of mammary tissue;
s4: inoculating mammary gland tissue;
s5: purifying cells;
s6: cell passage;
s7: freezing and storing the cells;
s8: cell recovery;
s9: and (4) identifying mammary epithelial cells.
2. The method for culturing mare mammary epithelial cells in vitro according to claim 1, wherein the specific steps in S1 are as follows: the mare adopts healthy Kazakh mares in the middle lactation period, after the mare is anesthetized, the mare breasts are cleaned by iodophor solution, and then the mare breasts are disinfected by 75% alcohol; cutting surface skin tissue with a scalpel, separating mammary gland tissue along the surface of fascia, and performing circular cutting on mammary gland with a circular cutting radius of 2cm and a circular cutting depth of subcutaneous 3cm; the cut breast tissue was placed in a 50mL centrifuge tube containing 75% ethanol solution for about 5-10s and immediately removed, washed with sterile 1% PBS buffer, after the blood and milk were washed away, placed in a 50mL sterile sample tube containing 2% PBS buffer, sealed with a sealing film, placed in an ice box, and taken back to the laboratory.
3. The method for culturing mare mammary epithelial cells in vitro according to claim 2, wherein the specific steps in S2 are as follows: after the mammary gland tissue is brought back to a laboratory, the outside of a 50mL sterile sample tube containing the mammary gland tissue is disinfected by 75% alcohol and is brought into an intercellular superclean workbench; taking out the mammary tissue and placing the mammary tissue in a culture dish containing PBS buffer solution; removing fascia, fat tissue and fibrous fasciculate connective tissue outside the mammary tissue by using ophthalmic scissors and ophthalmic forceps to obtain parenchyma tissue of mammary epithelial cells; the obtained parenchymal tissue was placed in a 5mL centrifuge tube and cut into a paste by ophthalmic cutting.
4. The method for culturing mare mammary epithelial cells in vitro according to claim 3, wherein the specific steps in S3 are as follows: placing the pasty mammary tissue and digestive juice simultaneously containing 0.1% collagenase type I and 0.01% hyaluronidase into a 50mL centrifuge tube according to the volume ratio of 1:3; placing a 50mL centrifuge tube in a constant-temperature shaking water bath kettle, shaking and digesting at the constant temperature of 37 ℃ at the speed of 100r/min, and adding an equal volume of termination culture medium to terminate digestion after most tissues are digested.
5. The method for culturing mare mammary epithelial cells in vitro according to claim 4, wherein the specific steps in S4 are as follows: filtering the digested mammary tissue by using a 100-mesh cell sieve, and then filtering by using a 200-mesh cell sieve; putting the filtrate in a 15mL centrifuge tube, centrifuging at 1000r/min for 5min; and (4) discarding the supernatant, and reserving a cell layer, wherein if serum exists in the sediment, the volume of the cell layer is measured according to the following ratio of 1:3, adding erythrocyte lysate, blowing uniformly, cracking for 2min,1000r/min, and centrifuging for 5min; discarding the supernatant, adding 5 times volume of PBS buffer solution, blowing and beating uniformly at 1000r/min, centrifuging for 3min, and repeating twice; discarding the supernatant, and leaving the cell precipitate; adding 2mL of complete culture medium, uniformly blowing and beating, inoculating into a T25 culture bottle, and supplementing the complete culture medium to 4mL; the T25 flask was incubated in an incubator at 37 ℃ and 5% CO2, cells were observed daily, and the complete medium was changed every 2 days.
6. The method for culturing mare mammary epithelial cells in vitro according to claim 5, wherein the specific steps in S5 are as follows: mixing growth of fibroblast in mammary epithelial cell culture process, separating two cells according to their sensitivity to trypsin, sucking out culture medium when the cell is 80% -90% of bottle bottom, and rinsing with 1% PBS for 2 times; adding 1mL of 0.25% trypsin solution, incubating at 37 deg.C and 5% in a CO2 incubator to digest fibroblasts; every 2 minutes; adding a termination culture medium after the fibroblasts are digested to terminate the digestion, and discarding the digestion solution in the bottle; washing with PBS for 2 times, adding 1mL of 0.25% trypsin solution, continuously digesting the cells without removing the wall, and adding a termination culture medium to terminate digestion when 90% of the cells are removed from the wall; repeatedly blowing and beating the cells on the wall of the bottle to remove the wall, putting the cell sap into a 15mL centrifuge tube, centrifuging at 1000r/min for 5min; removing supernatant, adding a proper amount of complete culture medium to resuspend cells, inoculating the cells into a T25 culture flask, and then supplementing the complete culture medium to 4mL; cultured in an incubator containing 5% of CO2 at 37 ℃ and purified several times until mammary epithelial cells were found in the culture flask.
7. The method for culturing the mare mammary epithelial cells in vitro according to claim 6, wherein the specific steps in S6 are as follows: when mammary epithelial cells in T25 flasks grew to 90% of the area of the bottom of the flask, the cells were subcultured: during subculture, firstly discarding the original culture medium in a T25 culture bottle, adding PBS buffer solution for slight washing, discarding the PBS buffer solution, and repeating twice; adding 1mL of 0.25% trypsin, digesting in an incubator at 37 ℃, and adding an equal volume of termination medium to terminate digestion after the cells have fallen off; transferring the cell suspension to a 15mL centrifuge tube, centrifuging for 5min at 1000r/min, removing the supernatant, adding a complete culture medium, slightly blowing cells by using a pipette gun to break cell masses, dividing the mammary epithelial cells into 3-4 parts, transferring to a new culture bottle, and supplementing the complete culture medium to 4mL; cultured in an incubator containing 5% of CO2 at 37 ℃ and the complete medium was replaced every 2 days.
8. The method for culturing mare mammary epithelial cells in vitro according to claim 7, wherein the specific steps in S7 are as follows: taking primary equine mammary gland epithelial cells, sucking off an original culture medium, washing for 1-2 times by using a PBS buffer solution, adding 1mL of 0.25 trypsin, and digesting dispersed cells; after the cells are exfoliated, adding an equal volume of termination medium to terminate digestion; sucking into 15mL centrifuge tube, centrifuging for 5min at 1000r/min, discarding supernatant, adding 1mL cell cryopreservation solution, transferring into 2mL cryopreservation tube, labeling, sealing with sealing film, placing into programmed cooling cryopreservation box, standing overnight at-80 deg.C, and transferring into liquid nitrogen for long-term storage on day 2.
9. The method for culturing mare mammary epithelial cells in vitro according to claim 8, wherein the specific steps in S8 are as follows: taking out the freezing tube from the liquid nitrogen tank, and quickly putting the tube into a water bath kettle at 37 ℃ to shake and melt the tube; adding 1mL of complete culture medium when most of the frozen stock solution is melted, and uniformly beating; sucking the mixed solution into a 15mL centrifuge tube, adding a proper amount of complete culture medium, performing centrifugation at 1000r/min for 5min; discarding the supernatant, adding 4mL of complete culture medium, and uniformly blowing; on average 2 new T25 flasks were aspirated and the flasks were filled to 4mL with complete medium and incubated at 37 ℃ in a 5% CO2 incubator.
10. The method for culturing mare mammary epithelial cells in vitro according to claim 9, wherein the specific steps in S9 are as follows: when mammary gland epithelial cells in the T25 culture bottle grow to 80% -90%, absorbing the culture solution, and washing for 2-3 times by using PBS buffer solution; adding 1.5-2mL of Trizol solution into each bottle to crack cells, horizontally placing for 15min, repeatedly blowing, collecting the cracking solution to a 1.5mL centrifuge tube, adding 100 mu L of chloroform, shaking for 15s to emulsify, standing for 5min,12000rpm, and centrifuging for 15min at 4 ℃; absorbing 3/4 of the upper-layer homogenized liquid of the centrifuge tube into a new centrifuge tube of 1.5mL, adding isopropanol with the same volume, uniformly mixing, standing for 10min at 10 rpm and 4 ℃, and centrifuging for 10min; white precipitate at the bottom of the centrifuge tube is RNA, supernatant is discarded, 1mL of 75% ethanol is added, the mixture is evenly mixed and then centrifuged at 12000rpm at 4 ℃ for 15min, and the supernatant is discarded; placing the centrifuge tube in a fume hood for 2-5min, adding 5mL of RNA-free enzyme water to dissolve the precipitate, blowing and beating uniformly, carrying out electrophoresis after the RNA is completely dissolved, detecting the quality and concentration of the RNA, and using the RNA to identify the mammary epithelial cell specific gene: keratin 8, keratin 18 and beta-casein.
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