CN109486752B - Method for separating intramuscular fat cells of Qinchuan beef cattle - Google Patents
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Abstract
The invention relates to a separation, purification and identification method of Qinchuan beef cattle intramuscular fat cells, which comprises the steps of digesting the longissimus dorsi of Qinchuan beef cattle by using type II collagenase, neutralizing tissue blocks digested by type II collagenase by using a DMEM/F12 culture medium containing 10% fetal bovine serum, filtering and centrifuging to obtain cells, washing and centrifuging the cells, then suspending the cells in a DMEM/F12 culture medium containing 10% FBS, counting, inoculating the cells in a culture dish, and culturing in CO2Culturing in incubator for 1.5 hr, washing with sterile phosphate buffered saline solution to remove non-adherent cells, and culturing at 37 deg.C under CO2And (3) changing the culture medium every 2 days in an incubator with the concentration of 5%, and identifying the cell immunofluorescence labeling positive cells after the cell confluence reaches 70% -80%. The intramuscular fat cells of the beef cattle obtained by the method have higher positive ratio of preadipocytes, and lay a foundation for the research that the subcutaneous fat deposition and the visceral fat deposition are not increased while the intramuscular fat deposition is improved.
Description
Technical Field
The invention belongs to the technical field of cell biology and cell engineering, and particularly relates to a separation method of intramuscular fat cells of Qinchuan beef cattle.
Background
There are 4 major adipose tissue sites in livestock: visceral, subcutaneous, intramuscular and intramuscular. The accumulation of intramuscular adipose tissue is very important, while the excessive accumulation of adipose tissue elsewhere is a burden on animal production, greatly increasing production costs, with the common phenotype being that some animals are much larger than the same animal, mainly not the accumulation of intramuscular adipose tissue, but mainly the accumulation of subcutaneous or visceral fat. However, to date, the mechanisms that lead to preferential accumulation of intramuscular fat remain undefined.
Adipocytes, particularly intramuscular adipocytes, have a common progenitor cell with myogenic cells during fetal muscle development. Exploring the mechanism by which mesenchymal progenitor cells differentiate early into myogenic or adipoblastic lineages will help understand the mechanism by which intramuscular adipocytes preferentially form, thereby promoting marbling. Consistent with this concept, there is increasing evidence that intramuscular adipocytes behave differently than subcutaneous and visceral adipocytes, and therefore, it is one of the subjects the applicant has studied to explore the unique developmental origin and properties of intramuscular adipocytes, achieving the goal of enhancing beef marbling without increasing the overall obesity of the animal.
Disclosure of Invention
The invention aims to provide a separation method of intramuscular fat cells of Qinchuan beef cattle, and lays a foundation for researching a deposition regulation mechanism of intramuscular fat.
In order to realize the task, the invention adopts the following technical solution:
a method for separating intramuscular fat cells of Qinchuan beef cattle is characterized by comprising the following steps:
1) cleaning longissimus dorsi tissue of 4-day-old Qinchuan beef cattle separated under aseptic condition with PBS and 2% penicillin and streptomycin, and cutting into pieces of 1mm3～2mm3The tissue mass of (a);
2) adding the tissue blocks into a centrifuge tube, digesting with collagenase II, neutralizing the tissue blocks digested with collagenase II with a DMEM/F12 culture medium containing 10% FBS, filtering with a stainless steel screen of 70 meshes and 200 meshes, centrifuging the obtained filtrate at 1500rpm for 10 minutes, and removing supernatant to obtain cells;
3) the obtained cells were resuspended in serum-free DMEM/F12 medium and centrifuged at 1500rpm for 10 minutes, and washed twice after centrifugation;
4) cell resuspension after washingAfter counting in DMEM/F12 medium containing 10% FBS, the cells were plated at 2.5X 105The density of individual cells was seeded in a petri dish, which was placed in CO2An incubator at 5% for 1.5 hours, then washed with sterile PBS to remove non-adherent cells and replaced the original medium in the petri dishes with fresh DMEM/F12 medium containing 10% FBS;
5) culturing the cells at 37 deg.C in CO2In an incubator with the concentration of 5%, a fresh DMEM/F12 culture medium containing 10% FBS is replaced every 2 days, and after the cell confluence reaches 70%, positive cells are identified by using a pre-adipocyte specific marker Pref-1 through a cell immunofluorescence method.
According to the invention, the operation steps of the cell immunofluorescence positive cell identification are as follows:
before use, the cells are inoculated in a 6-well culture plate and added with DMEM/F12 medium containing 10% FBS for culture, when the cell confluence reaches 70%, the DMEM/F12 medium containing 10% FBS is discarded and the cells are washed with PBS, fixed with paraformaldehyde with the concentration of 4% at room temperature for 30 minutes and washed with PBS again;
permeabilizing the cells with 0.1% BSA and 0.5% Triton X-100 for 60 min at room temperature, discarding the liquid, washing again with PBS, and blocking for 60 min at room temperature with 3% BSA;
the blocking solution was discarded and a sufficient amount of diluted murine Pref-1 antibody (primary antibody) was added and incubated overnight at 4 deg.C, then murine Pref-1 antibody was discarded, diluted anti-murine HRP fluorescent secondary antibody was added, incubated for 80min at 37 deg.C, and secondary antibody was discarded and washed 3 times with PBST, 3min each. Then, DAPI was added dropwise and incubated for 5min in the dark, and excess DAPI was removed by washing 4 times with PBST for 5min each time.
Finally, the liquid was blotted dry and the results were observed under a fluorescent microscope.
Compared with the prior art, the separation method of the intramuscular fat cells of the Qinchuan beef cattle has the following advantages:
(1) the obtained intramuscular fat cells of the Qinchuan beef cattle have high positive rate of preadipocytes;
(2) no pollution is caused;
(3) the intramuscular fat cells obtained by digesting the longest muscle tissue of the back of the Qinchuan beef cattle aged 4 days by adopting type II collagenase obviously improve the positive cell rate of the intramuscular fat cells obtained from the muscle tissue and greatly reduce the risk of fat cell pollution. Meanwhile, the pref-1 antibody is used for immunofluorescent labeling of preadipocytes, a new method is provided for separation and identification of preadipocytes, and a foundation is laid for research on improvement of intramuscular fat deposition without increase of subcutaneous and visceral fat deposition.
Drawings
FIG. 1 is a photograph (200X) of the intramuscular fat cells of the Qinchuan beef cattle observed under a microscope at the 0D, 2D and 4D (marked as D0, D2 and D4 in the figure) after the intramuscular fat cells inoculation;
FIG. 2 is an immunofluorescence photograph (40X) of preadipocytes identified using preadipocyte specific expression factor Pref-1;
FIG. 3 shows the expression level of the preadipocyte specific expression factor Pref-1 protein detected by Western-blot.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Detailed Description
The applicant finds that more positive preadipocytes can be obtained by using collagenase II to digest the longissimus dorsi of the Qinchuan beef cattle calf in the research process through experimental comparison, and the differences exist between the method and other methods such as collagenase I digestion or the method and the method for obtaining intramuscular adipocytes by taking the longissimus dorsi of the adult cattle as a sample.
Comparing the longissimus dorsi tissue of the newborn cattle (0-1 week of birth) with different collagenase, different digestion time and different differential adherence time, the applicant finds that more positive preadipocytes can be obtained by digesting the longissimus dorsi tissue of the newborn cattle (0-1 week of birth) with collagenase type II for 1.5 hours and replacing the fresh culture medium after 1.5 hours after inoculation. Meanwhile, the reliability of identifying the intramuscular precursor fat cells is further improved by using methods such as immunofluorescence and the like.
In the following examples, the animal tissues and sources of reagents involved are as follows:
4-day-old calves from national center for beef improvement experimental farms (Shaanxi Yang Ling, China);
collagenase type II, DAPI, was purchased from GIBCO.
FBS, DMEM/F12, PBS medium was purchased from Hyclone.
Cell culture dishes and centrifuge tubes were purchased from Corning.
Paraformaldehyde and PBST at a concentration of 4% were purchased from solibao corporation.
4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole, DAPI), BSA and a nonionic surfactant Triton X-100 (hereinafter referred to as Triton X-100) were purchased from Sigma.
Murine Pref-1 antibody (primary antibody) and anti-murine HRP fluorescent secondary antibody were purchased from abcam.
The method for separating intramuscular fat cells of the Qinchuan beef cattle, which is provided by the embodiment, is implemented by the following steps:
1) cleaning longissimus dorsi tissue of 4-day-old Qinchuan beef cattle separated under aseptic condition with PBS and 2% penicillin and streptomycin, and cutting into pieces of 1mm3～2mm3The tissue mass of (a);
2) adding the tissue blocks into a centrifuge tube, digesting with collagenase II, neutralizing the tissue blocks digested with collagenase II with a DMEM/F12 culture medium containing 10% FBS, filtering with a stainless steel screen of 70 meshes and 200 meshes, centrifuging the obtained filtrate at 1500rpm for 10 minutes, and removing supernatant to obtain cells;
3) the obtained cells are centrifuged at 1500rpm for 10 minutes by using serum-free DMEM/F12 medium, and the cells are washed twice after centrifugation;
4) the washed cells were resuspended in DMEM/F12 medium containing 10% FBS, and after counting, the cells were cultured at 2.5X 105The density of individual cells was seeded in a petri dish, which was placed in 5% CO2After 1.5 hours in the incubator, a small amount of cells were observed to adhere under a microscope, and then washed with sterile PBS to remove non-adherent cells, and the original medium in the petri dish was replaced with fresh DMEM/F12 medium containing 10% FBS; 2d post inoculation ObservationCells were found to grow exponentially, and after 4 days of inoculation, cells were observed to reach substantially 100% confluence without contamination (FIG. 1).
5) Culturing the cells in an incubator at 37 ℃ and with the concentration of CO2 of 5%, replacing fresh DMEM/F12 culture medium containing 10% FBS once every 2 days, and after the cell confluence reaches 70%, using a preadipocyte specific marker Pref-1 to identify positive cells by a cell immunofluorescence method.
The operation steps of the cell immunofluorescence positive cell identification are as follows:
before use, the cells are inoculated in a 6-well culture plate and added with DMEM/F12 medium containing 10% FBS for culture, when the cell confluence reaches 70%, the DMEM/F12 medium containing 10% FBS is discarded and the cells are washed with PBS, fixed with paraformaldehyde with the concentration of 4% at room temperature for 30 minutes and washed with PBS again;
permeabilizing the cells with 0.1% bovine serum albumin and 0.5% Triton X-100 for 60 minutes at room temperature, discarding the liquid, washing again with PBS, and blocking for 60 minutes at room temperature with 3% BSA;
the blocking solution was discarded and a sufficient amount of diluted murine Pref-1 antibody (primary antibody) was added and incubated overnight at 4 deg.C, then murine Pref-1 antibody was discarded, diluted anti-murine HRP fluorescent secondary antibody was added, incubated for 80min at 37 deg.C, and secondary antibody was discarded and washed 3 times with PBST, 3min each. Then, DAPI was added dropwise and incubated for 5min in the dark, and excess DAPI was removed by washing 4 times with PBST for 5min each time.
And finally, sucking the liquid to be dry, observing the result under a fluorescence microscope, and identifying the proportion of the precursor fat cells in the separated cells.
After counting, the immune positive cells of the precursor fat cells reach 91.33% + -0.03 (figure 2). The cells which reach the contact inhibition are induced to differentiate, the total cell proteins of 0D, 2D, 4D and 6D (marked as D0, D2, D4 and D6 in the figure) are respectively collected, the expression level of Pref-1 protein which is a specific marker of precursor fat cells is detected, and the Pref-1 protein is found to be in a descending trend (figure 3), which indicates that the separated cells are precursor fat cells.
Claims (1)
1. A method for separating intramuscular fat cells of Qinchuan beef cattle is characterized by comprising the following steps:
1) cleaning longissimus dorsi tissue of 4-day-old Qinchuan beef cattle separated under aseptic condition with PBS and 2% penicillin and streptomycin, and cutting into pieces of 1mm3～2mm3The tissue mass of (a);
2) adding the tissue blocks into a centrifuge tube, digesting with collagenase II, neutralizing the tissue blocks digested with collagenase II with a DMEM/F12 culture medium containing 10% FBS, filtering with a stainless steel screen of 70 meshes and 200 meshes, centrifuging the obtained filtrate at 1500rpm for 10 minutes, and removing supernatant to obtain cells;
3) the obtained cells were resuspended in serum-free DMEM/F12 medium and centrifuged at 1500rpm for 10 minutes, and washed twice after centrifugation;
4) the washed cells were resuspended in DMEM/F12 medium containing 10% FBS, and after counting, the cells were resuspended at 2.5X 105The density of each cell is inoculated in a culture dish; placing the culture dish in CO2Cultured in a 5% incubator for 1.5 hours, then washed with sterile PBS to remove non-adherent cells, and the original medium in the petri dish was replaced with fresh DMEM/F12 medium containing 10% FBS;
5) culturing the cells at 37 deg.C in CO2Changing a fresh DMEM/F12 culture medium containing 10% FBS once every 2 days in an incubator with the concentration of 5%, and identifying the cell immunofluorescence labeling positive cells by using a preadipocyte specific label Pref-1 after the cell confluence reaches 70% -80%;
the operation steps of the cell immunofluorescence labeling positive cell identification are as follows:
before use, the cells are inoculated in a 6-well culture plate and added with DMEM/F12 medium containing 10% FBS for culture, when the cell confluence reaches 70%, the DMEM/F12 medium containing 10% FBS is discarded and the cells are washed with PBS, fixed with paraformaldehyde with the concentration of 4% at room temperature for 30 minutes and washed with PBS again;
permeabilizing the cells with 0.1% bovine serum albumin and 0.5% nonionic surfactant Triton X-100 for 60 minutes at room temperature, discarding the liquid, washing again with PBS, and blocking for 60 minutes at room temperature with 3% BSA;
discarding the blocking solution, adding a sufficient amount of diluted murine Pref-1 antibody, incubating at 4 ℃ overnight, discarding murine Pref-1 antibody, adding diluted anti-murine HRP fluorescent secondary antibody, incubating at 37 ℃ for 80min, discarding secondary antibody, washing with PBST for 3 times, each time for 3 min; then 4',6-diamidino-2-phenylindole is dripped to be incubated for 5min in a dark place, and PBST is used for washing for 4 times to remove redundant 4',6-diamidino-2-phenylindole for 5min each time;
finally the liquid was blotted dry and the image was observed under a fluorescent microscope.
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