CN115125194A - Preparation method of yak precursor fat cells - Google Patents

Preparation method of yak precursor fat cells Download PDF

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CN115125194A
CN115125194A CN202210418352.3A CN202210418352A CN115125194A CN 115125194 A CN115125194 A CN 115125194A CN 202210418352 A CN202210418352 A CN 202210418352A CN 115125194 A CN115125194 A CN 115125194A
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阎萍
褚敏
吴晓云
喇永富
马晓明
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Lanzhou Institute of Animal Husbandry and Veterinary Medicine CAAS
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Abstract

The invention relates to a method for in vitro culture and induction of yak precursor adipocytes, and belongs to the technical field of cell and tissue engineering. On the basis of the prior art, the invention carries out primary culture on the kidney periphery and subcutaneous fat of the yak calf in one week of birth in vitro by a collagenase I digestion method, carries out subculture by combining low-concentration pancreatin digestion and microscopic observation with physical shaking, improves an induced differentiation culture medium for induced differentiation culture, and then obtains mature yak fat cells. Establishes a new method for in vitro culture and induced differentiation of yak precursor adipocytes. The method has the advantages of advanced and efficient process, scientific and reasonable culture medium preparation, manpower and material resources saving, good cell morphology, rapid cell proliferation and efficient induced differentiation treatment, is a more effective precursor adipocyte culture method, and provides a necessary carrier for researching the molecular mechanism of yak body fat deposition.

Description

Preparation method of yak precursor fat cells
Technical Field
The invention relates to a method for culturing yak precursor fat cells in vitro, belonging to the field of cell engineering and tissue engineering.
Background
Adipose tissue is an important organ for energy storage and metabolism, and the fat content of livestock affects the meat quality thereof. Fat is derived from the accumulation of adipocytes, which are a type of precursor cells having the ability to proliferate and differentiate into adipocytes, and differentiate into mature adipocytes under the regulation of various factors in vivo, and thus are the best model for studying fat deposition and metabolism in animals. At present, precursor adipocytes have been successfully cultured in humans, mice, pigs, sheep, goats, cattle, etc. The yak is suitable for extremely cold growing environment, and the regulation of fat development and lipid deposition of the yak has obvious specificity in varieties and tissues. Therefore, a proper method for culturing yak precursor adipocytes and inducing differentiation is needed to be established, and a more favorable technical support is provided for exploring the molecular mechanism of yak body fat deposition and improving meat quality.
Adipose tissue contains both adipocytes and preadipocytes. The adipose cells are directly separated from adipose tissues, the adipose cells die within 24 hours after separation, how to improve the survival rate of the adipose cells, especially the adherence rate of non-model animal adipose cells such as yaks, and improving the cell proliferation and differentiation efficiency of the non-model animal adipose cells are the key point of yak precursor adipose cell culture, and the adipose cell culture adopted at the present stage is started from the precursor adipose cells, but the primary adipose cells are difficult to adhere to the walls and slow in growth, are easily polluted by fibroblasts, influence the late-stage induced differentiation process, the survival rate of the cultured precursor cells is low, the time for passability is long, and the number of the obtained cells is small.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: improving the anchorage rate and the proliferation and differentiation efficiency of the precursor adipocytes and establishing a method for in-vitro culture and induced differentiation of the precursor adipocytes of the non-model animal yak. The method for in vitro culture and induced differentiation of yak precursor fat cells comprises the steps of carrying out primary culture on kidney periphery and subcutaneous fat of yaks in vitro by a collagenase I digestion method within one week of birth, carrying out subculture by combining low-concentration pancreatin digestion and microscopic observation with physical shaking, improving an induced differentiation culture medium for induced differentiation culture, and then obtaining mature yak fat cells. Establishes a new method for in vitro culture and induced differentiation of yak precursor adipocytes.
A preparation method of yak precursor adipocytes comprises the following steps:
step 1, taking fat tissues around the kidney and under the skin of a low-day-old yak, removing blood vessels and connective tissues visible in the fat tissues, adding a collagenase I culture medium for digestion treatment, and adding a growth culture medium for terminating digestion reaction;
step 2, after the reaction product is filtered to remove undigested tissue blocks and clustered fat cells, centrifuging the cell suspension to remove supernatant, adding erythrocyte lysate for incubation treatment, centrifuging to remove supernatant, adding a growth medium added with cell factors EGF and IGF1, and then culturing;
step 3, when the yak precursor fat cells are cultured until the density reaches 80% -90%, cleaning the cells obtained in the step 2, adding a pancreatin solution for digestion treatment, adding a growth culture medium to stop digestion reaction, adding the growth culture medium added with cell factors EGF and IGF1, and then culturing;
and 4, when the cell density in the culture bottle is 60-70%, cleaning the cells obtained in the step 3, and culturing by using an induced differentiation culture medium added with rosiglitazone to obtain differentiated cells.
In the step 1, the digestion treatment temperature is 35-40 ℃, and the time is 60-90 min.
In step 1, the preparation method of the collagenase I culture medium comprises the following steps: 50-500mg collagenase I is dissolved in 50-200 ml D-Hanks solution with 2-8% NaHCO 3 Adjusting the pH value of the solution to 7.2-7.4, and filtering.
In the step 2, the rotation speed in the centrifugal treatment process is 1000-.
In step 2, the preparation method of the erythrocyte lysate comprises the following steps: taking KHCO 3 5-30g、NH 4 Cl 50-150g、Na 2 EDTA 1-10g, dissolving in 3000mL double distilled water 500-5 to prepare a storage solution, adjusting the pH value of the solution to 7.2-7.4 by using HCl or NaOH, filtering, and diluting for use.
The dilution factor is 5-30 times.
In the step 3, the concentration of the pancreatin solution is 0.05-0.5%.
In step 1 and step 3, the preparation method of the growth medium is as follows: mixing completely DMEM/F12 complete medium containing 1-4% double antibody and 5-15% FBS, filtering and sterilizing.
In the step 4, the concentration of the rosiglitazone in the culture medium is 10 to 300 nmol/L.
In the step 4, the preparation method of the induced differentiation medium added with rosiglitazone comprises the following steps: mixing growth culture medium containing 5-25 μ g/mL insulin, 0.5-4 μmol/L dexamethasone, 0.1-1mmol/L IBMX, and 50-300nmol/L rosiglitazone.
Advantageous effects
1. According to the method for in vitro culture and induced differentiation of the yak precursor adipocytes, provided by the invention, the yak precursor adipocytes with high anchorage rate, strong proliferation capacity and obvious differentiation efficiency can be cultured by adding the cell factors EGF and IGF 1.
2. According to the method for in-vitro culture and induced differentiation of the yak precursor adipocytes, the number of washing times is increased when adipose tissues are treated, after 0.1% collagenase I is added, the digestion time is prolonged and controlled to be 60-90 min, 1 oscillation time is carried out every 5min, erythrocytes in a cell suspension are removed by using erythrocyte lysate, and the obtained primary precursor adipocytes are more pure.
3. The method for in vitro culture and induced differentiation of yak precursor adipocytes, provided by the invention, adopts a 70-mesh and 40-mesh cell sieve to collect cell filtrate; when the cells are subcultured, the concentration of 0.25% pancreatin solution is reduced to 0.1%, 1mL of pancreatin solution is added into a culture bottle, the digestion time is controlled within 2min, a microscope observation method is combined, the retraction of cell cytoplasm is observed after the pancreatin is added, physical mechanical oscillation is carried out when the edge of the cell surface is irregularly serrated, the cell surface is gently oscillated for 60 to 80 times, the digestion time is shortened, and the transitional digestion of the pancreatin to the cells is reduced.
4. According to the method for inducing differentiation of yak precursor adipocytes, provided by the invention, rosiglitazone is added to an induced differentiation medium on the basis of the original medium, so that the induced differentiation efficiency is improved.
Drawings
FIG. 1 is a picture of primary yak precursor adipocytes; improving the form of the fat cells of the yak precursors: a, culturing preadipocytes for 0h and B, culturing preadipocytes for 24h and C, culturing preadipocytes for 4D and D, and culturing preadipocytes for 10D. The form of the yak precursor fat cells without adding the cytokines EGF and IGF1 in the culture medium is shown in a figure (E-H): e, culturing the precursor adipocytes for 0H and F, culturing the precursor adipocytes for 24H and G, culturing the precursor adipocytes for 4d and H, and culturing the precursor adipocytes for 10 d; compared with the modified group, the cell culture time is 24H, the cell adherence amount is relatively less (F-G), and the cell growth and fusion rate is slower (H). The cell suspension is in a yak precursor fat cell form without adding erythrocyte lysate: i, culturing precursor adipocytes for 0h and J, culturing the precursor adipocytes for 24h and K, culturing the precursor adipocytes for 4d and L, and culturing the precursor adipocytes for 10 d; the cell suspension was relatively more contaminated (I), less adherent (J), less adherent (K) pure (K) and slower (L) in cell growth rate compared to the modified group.
Fig. 2 is a graph of growth curves of yak precursor adipocytes, and the proliferation conditions of 0d, 2d, 4d, 6d, 8d, 10d, 12d and 14d yak precursor adipocytes are detected by a cck8 method (n is 3);
fig. 3 is a diagram of differentiation effect of yak precursor adipocytes through induced differentiation culture, and lipid drop generation effects of yak precursor adipocytes from 0 th to 10 th of induced differentiation are detected by adopting an oil red O staining method;
FIG. 4 shows expression of PPAR γ, CEBP α and FABP4 genes of yaks under induced differentiation culture conditions, and RT qPCR detection is adopted.
FIG. 5 is a graph showing a comparison of the expression levels of PPAR γ, C/EBP α and FABP 4.
Detailed Description
The reagents used in the following examples are as follows:
1.0.1% collagenase I medium: 100mg of collagenase I was dissolved in 100 ml of D-Hanks solution with 5.6% NaHCO 3 Adjusting the pH value of the solution to 7.2-7.4, filtering by a 0.22 mu m filter, subpackaging and storing at-20 ℃ for later use.
2. Growth medium: DMEM/F12 complete medium containing 2% double antibody and 10% FBS is mixed uniformly and filtered by a 0.22-micron filter for sterilization, subpackaged by a 50-mL centrifuge tube and sealed, and stored at 4 ℃;
3. induction differentiation culture medium: uniformly mixing a growth culture medium containing 10 mu g/mL insulin, 1 mu mol/L dexamethasone, 0.5mmol/L IBMX and 100nmol/L rosiglitazone, sealing, and storing at 4 ℃;
10 μ g/mL insulin: dissolving 10mg of insulin dry powder in 5mL of HCL solution with the pH value of 2, wherein the concentration is 2 mg/mL, subpackaging 250ul of insulin dry powder into a 1.5mL of EP tube, storing the tube at-20 ℃ in a dark place, adding one part of insulin dry powder into 50mL of DMEM growth medium when the tube is used, and finally, the concentration of a working solution is 10 ug/mL;
b. dexamethasone: dissolving 25mg of dexamethasone dry powder in 6.37mL of 95% ethanol, filtering, sterilizing, packaging, and storing at-20 deg.C;
c.50mM IBMX stock: dissolving 11.5mg IBMX in 60 μ L of 1M KOH, adding 940 μ L of ultrapure water, filtering for sterilization at 0.22M, packaging, and storing at-20 deg.C;
d. rosiglitazone: weighing 4mg of Rogelinone powder, dissolving in DMSO (dimethylsulfoxide) 400ul, filtering to remove bacteria, adding 40ul of the obtained solution into 11.2ml of culture medium to obtain 100uM stock solution, subpackaging the stock solution, and storing at-20 ℃; adding 50ul of stock solution into 49.95ml of culture solution when in use, wherein the concentration of the obtained solution is 100 nM;
4. maintenance of differentiation medium: adding insulin into the growth medium to a final concentration of 10 μ g/mL, subpackaging and sealing with a 50mL centrifuge tube, and storing at 4 ℃;
5. erythrocyte lysate: taking KHCO 3 10g、NH 4 Cl 80g、Na 2 3.7g of EDTA is dissolved in 1000mL of double distilled water to prepare a storage solution. Adjusting pH to 7.2-7.4 with HCl or NaOH, filtering, sterilizing, packaging, and storing at 2-8 deg.C. When the working solution was prepared, the stock solution was diluted 10 times and used.
1 Primary culture of Yak precursor adipocytes
Taking perirenal and subcutaneous adipose tissues of 5-day-old yaks under aseptic condition, transferring into a dish washed with Phosphate Buffer Solution (PBS) containing (anti-penicillin-streptomycin), rinsing for 6 times, and removing blood vessels and connective tissues visible in the adipose tissues by using an ophthalmic scissors; then cut the tissue mass to 1mm 3 Adding 0.1% collagenase I into the small blocks, digesting the small blocks in water bath at 37 ℃ for 60-90 min, and oscillating the small blocks for 1 time every 5 min; DMEM/F12 growth medium was then added to stop the collagenase digestion reaction. Filtering with 70 um and 40um cell sieve to remove undigested tissue mass and clustered fat cells, transferring the cell suspension into a 15mL centrifuge tube, and centrifuging at 1400g/min for 5 min; discarding the supernatant after centrifugation, adding equal volume of erythrocyte lysate to incubate cell precipitation at room temperature, continuing centrifugation under the same condition again, discarding the supernatant after centrifugation, adding DMEM/F12 growth medium containing cytokines EGF and IGF1 to suspend the cell precipitation, fully mixing the cell suspension, counting under a microscope, and counting at 5 × 10 4 cells/mL were inoculated to 25cm 2 Culturing in a cell culture bottle to obtain yak precursor fat cells, and culturing at 37 deg.C with saturated humidity and 5% CO 2 Culturing under the condition, and replacing the culture medium every 24 hours.
Subculturing yak fat cells
When the yak precursor fat cells are cultured until the density reaches 80% -90%, removing original culture solution, and washing twice by using PBS containing 10% double antibodies; adding 1mL of 0.1% pancreatin solution into a culture bottle, digesting at 37 ℃ for 2 min; after pancreatin is added, observing the cell state under a microscope, finding that the cytoplasm of the cell retracts, changing the cell shape from a spindle shape to a circular shape or an oval shape, and when the edge of the cell surface is irregularly jagged, carrying out physical oscillation, beating the cell bottle in the palm for 60-80 times, and observing under the microscope, wherein if the cell is in a circular shape with smooth edge, the oscillation frequency can be shortened until the cell is completely suspended, and adding a small amount of DMEM/F12 growth medium to terminate the digestion reaction; adding DMEM/F12 growth medium added with cell factors EGF and IGF1, and repeatedly blowing the bottle wall to ensure that the cells completely fall off from the bottle wall and form cell suspension; after counting, the cells were inoculated at a certain density into new flasks and continued to be cultured in DMEM/F12 growth medium supplemented with the cytokines EGF and IGF1, with the medium being changed every 2 d.
Inducing differentiation of 3 yak precursor fat cell
When the cell density in the culture bottle is 60% -70%, removing the original culture medium, washing twice with PBS containing 2% double antibody, and washing the originalReplacing the growth medium added with the adherence factors EGF and IGF1 with the improved induced differentiation medium added with 100nmol/L rosiglitazone, and keeping the temperature at 37 ℃, the saturation humidity and the 5% CO 2 The culture is continued under the condition (1); culturing for 2 days, removing induced differentiation culture medium, changing to maintenance culture medium, maintaining at 37 deg.C, saturated humidity, and 5% CO 2 Continuing culturing under the condition; the differentiation culture was maintained until the cells were completely differentiated.
The culture effect is as follows:
1. morphological observation of yak precursor fat cells
The cells obtained in FIG. 1 were suspended in the medium in the form of small spheres at the time of inoculation (FIG. 1, A, E), and it was observed that a part of the cells began to adhere 24h after inoculation (FIG. 1, B, F). Adherent cells began to expand and deform after 5 days of culture, mostly in the form of elongated spindle-shaped fibroblast-like cells (fig. 1, C, G), and precursor adipocytes began to proliferate in a spindle-shape at 9 days, with a cell density of about 70% of the bottom area of the flask (fig. 1, D, H).
Detection of proliferation Activity of Yak precursor adipocytes by CCK8 method
At 1 × 10 4 /cm 2 The precursor adipocytes are inoculated to a 96-well culture plate, and cck8 is added at 0d, 2d, 4d, 6d, 8d and 10d of culture, and the absorbance value of each well is measured by a 450nm wavelength enzyme-linked detector. Fig. 2 shows that the growth curve of yak precursor adipocytes shows that the proliferation efficiency of cells in the improved group is higher compared to cells in the control group (precursor adipocytes obtained without adding EGF and IGF1 to the culture medium and without adding erythrocyte lysate to the cell suspension).
3. Oil red O dyeing
Continuously culturing the induced and differentiated precursor adipocytes for 10 days, and performing oil red O staining every two days
Mixing oil red O storage solution with distilled water at a ratio of 3:2, standing at room temperature for 10min, and filtering with filter paper; removing the original culture medium in the 6-well plate, washing with preheated PBS for 2 times, adding 4% paraformaldehyde cell fixing solution, and fixing at room temperature for 60 min; removing the fixing solution in the holes after 60min, rinsing with PBS for 10min, adding a proper amount of oil red O working solution, and dyeing at room temperature for 30 min; removing oil red O staining solution, washing cells with distilled water for 2 times, 1min for one time, and removing excess staining solution; the staining was observed under a microscope.
Results of oil red O staining fig. 3 and 4 show that lipid droplets are produced more rapidly in modified yak precursor adipocytes and more at each stage as induced differentiation proceeds. Small lipid droplets begin to appear in the cytoplasm of the cells on the induction day 2, more lipid droplets appear in the adipocytes on the day 4, and the small lipid droplets in the cytoplasm are increased obviously by the day 6, so that the cells are full of a large number of lipid droplets on the differentiation day 8. The lipid droplets began to fuse on day 10.
4. Detection of adipogenic differentiation marker genes PPAR gamma, C/EBP alpha and FABP4
The differentiation of the adipocyte is regulated by multiple differentiation factors, and PPAR gamma, C/EBP alpha and FABP4 are used as key regulation factors of adipogenic differentiation and are important to adipogenic differentiation. Respectively collecting cells of 6 different time points of 0d, 2d, 4d, 6d, 8d and 10d of induced differentiation, adding Trizol to blow and collect the cells, extracting total RNA, and detecting the expression changes of beta-actin, PPAR gamma, C/EBP alpha and FABP4 in the differentiation process of the yak precursor cells. Fluorescent quantitative PCR primers were designed based on the mRNA sequences of genes of yak β -actin, PPAR γ, C/EBP α, FABP4 published by GeneBank (https:// www.ncbi.nlm.nih.gov /), using Primer Premier 5.0 software. The primer information is shown in tables 2-5, and the primer synthesis service is completed by Biotech limited of Beijing Optimalaceae.
Figure RE-GDA0003831128290000061
beta-Actin (NM-001009784) was used as an internal reference gene, and the expression levels of the target gene and the internal reference gene were measured using a Bio-Rad CFX 96qRT-PCR apparatus, and biological and technical repeats (3X 3) were set for each group. Reaction system 10 μ L: SYBR Premix Ex TaqTM II 5. mu.L, F/R (10. mu. mol/L) each 0.4. mu.L, cDNA template 0.8. mu.L, ddH 2 O3.4. mu.L. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 30s, annealing for 30s, extension at 95 ℃ for 30s, and 45 cycles; extension at 55 ℃ for 2 s.
The results are shown in FIG. 5, where the expression levels of PPAR γ, C/EBP α and FABP4 were significantly higher than 0 day at 4d, 6d, 8d and 10 d. The method shows that the yak precursor fat cells are successfully induced and differentiated into the fat cells, and the induced differentiation method is efficient and reasonable.

Claims (10)

1. A preparation method of yak precursor adipocytes is characterized by comprising the following steps:
step 1, taking fat tissues around the kidney and under the skin of a low-day-old yak, removing blood vessels and connective tissues visible in the fat tissues, adding a collagenase I culture medium for digestion treatment, and adding a growth culture medium for terminating digestion reaction;
step 2, after the reaction product is filtered to remove undigested tissue blocks and clustered fat cells, centrifuging the cell suspension to remove supernatant, adding erythrocyte lysate for incubation treatment, centrifuging to remove supernatant, adding a growth medium added with cell factors EGF and IGF1, and then culturing;
step 3, when the yak precursor fat cells are cultured until the density reaches 80% -90%, cleaning the cells obtained in the step 2, adding a pancreatin solution for digestion treatment, adding a growth culture medium to stop digestion reaction, adding the growth culture medium added with cell factors EGF and IGF1, and then culturing;
culturing after the culture medium is long;
and 4, when the cell density in the culture bottle is 60-70%, cleaning the cells obtained in the step 3, and culturing by using an induced differentiation culture medium added with rosiglitazone to obtain differentiated cells.
2. The method for preparing yak precursor adipocytes of claim 1, wherein in step 1, the digestion treatment temperature is 35-40 ℃ and the time is 60-90 min.
3. The method for preparing yak precursor adipocytes according to claim 1, wherein in step 1, the collagenase I medium is prepared by: 50-500mg collagenase I was dissolved in 50-200 ml D-Hanks solution with 2-8% NaHCO 3 Adjusting the pH value of the solution to 7.2-7.4, and filtering.
4. The method for preparing yak precursor adipocytes, as recited in claim 1, wherein in step 2, the rotation speed of the centrifugation process is 1000-.
5. The method for preparing yak precursor adipocytes of claim 1, wherein in step 2, the erythrocyte lysate is prepared by: taking KHCO 3 5-30g、NH 4 Cl 50-150g、Na 2 EDTA 1-10g, dissolved in 500-3000mL double distilled water to prepare a storage solution, using HCl or NaOH to adjust the pH value of the solution to 7.2-7.4, filtering, and diluting for use.
6. The method for preparing yak precursor adipocytes according to claim 5, wherein the dilution factor is 5-30 times.
7. The method for preparing yak precursor adipocytes according to claim 1, wherein in step 3, the pancreatin solution has a concentration of 0.05-0.5%.
8. The method for preparing yak precursor adipocytes of claim 1, wherein in step 1 and step 3, the growth medium is prepared by: mixing completely DMEM/F12 complete medium containing 1-4% double antibody and 5-15% FBS, filtering and sterilizing.
9. The method for preparing yak precursor adipocytes according to claim 1, wherein in step 4, the concentration of rosiglitazone in the culture medium is 10-300 nmol/L.
10. The method for preparing yak precursor adipocytes according to claim 1, wherein in step 4, the method for preparing the rosiglitazone-added differentiation-inducing medium is: mixing growth culture medium containing 5-25 μ g/mL insulin, 0.5-4 μmol/L dexamethasone, 0.1-1mmol/L IBMX, and 50-300nmol/L rosiglitazone.
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CN117625527A (en) * 2023-12-20 2024-03-01 中国肉类食品综合研究中心 Method for separating, identifying and adipogenic differentiating mammalian fat precursor cells, composition and application

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