CN115044541A - High-differentiation-efficiency immortalized pig fat precursor cell line and construction method and application thereof - Google Patents
High-differentiation-efficiency immortalized pig fat precursor cell line and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses an immortalized pig fat precursor cell line with high differentiation efficiency, which is preserved in China Center for Type Culture Collection (CCTCC) in 2022, 5 months and 17 days with the preservation number of CCTCC NO: C2022150. Meanwhile, the invention discloses a construction method, a culture and polyester induction method and application of the cell line. The cell line can be stably passaged and always keeps high differentiation efficiency, and can be used for differentiation research of pig fat precursor cells, physiological research of pig mature fat cells and production and use of cell culture meat.
Description
Technical Field
The invention relates to the technical field of biology and modern agriculture, in particular to a high-differentiation-efficiency immortalized pig fat precursor cell line and a construction method and application thereof.
Background
Fat is an important flavor substance source of meat, so that the mouthfeel and flavor can be effectively improved by adding a proper amount of fat cells into cell culture meat. Therefore, the establishment of the in vitro culture model of the porcine adipose precursor cells can not only provide an effective model for the research of the growth and development of porcine adipose tissues, but also help to research how to improve the production efficiency of pigs, can be used for increasing the flavor of cell culture meat, and has important production and scientific significance.
At present, most of the research and production of pig adipocyte precursor cells are based on the use of primary cells. However, the number of passages of primary cells is limited, and animals need to be continuously slaughtered to obtain the primary cells, so that the cost is high and the primary cells are inconvenient. There have also been some reports of establishment of immortalized porcine adipocyte precursor cell lines, but the differentiation efficiency of these cell lines rapidly decreases with the increase of the number of passages. At present, no porcine adipose precursor cell line which can reliably keep high differentiation efficiency is established.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects and defects of the prior art, the invention provides the immortalized pig fat precursor cell line with high differentiation efficiency and the construction method and the application thereof.
The technical scheme is as follows: the immortalized pig fat precursor cell line with high differentiation efficiency is preserved in China center for type culture Collection of Wuhan university in 5 and 17 months in 2022 with the preservation number of CCTCC NO: C2022150.
The STR characteristics of the cell are as follows: the locus size is 387A12F-226 +/-1 bp, the pigSTR4C-118 +/-1 bp and 122 +/-1 bp, the pigSTR5C-262 +/-1 bp and 266 +/-1 bp, the pigSTR7B-113 +/-1 bp, the pigSTR11B-85 +/-1 bp, the pigSTR13E-125 +/-1 bp and 133 +/-1 bp, the pigSTR14A-186 +/-1 bp, the pigSTR15A-198 +/-1 bp and 202 +/-1 bp, the pigSTR17A-155 +/-1 bp and 163 +/-1 bp, the SW240-93 +/-1 bp and 107 +/-1 bp, and the Amelogenin-232 +/-1 bp and 234 +/-1 bp.
The method for constructing the immortalized pig fat precursor cell line with high differentiation efficiency comprises the following steps:
1) inoculating single cells of immortalized pig fat precursor cells into a 96-well plate by adopting a limiting dilution method, and culturing until the cells are completely converged in the well;
2) carrying out polyester induced differentiation on 1/3 cells in each well from a 96-well plate, a 48-well plate, a 24-well plate and a 12-well plate to a 6-well plate in sequence;
3) the clones with high polyester efficiency were selected, and the remaining 2/3 cells were grown up and stored.
The culture and polyester induction method of the immortalized pig fat precursor cell line with high differentiation efficiency comprises the following steps:
1) cultured in DMEM/F12+ 10% FBS, fibroblast-like in appearance; washing the culture dish with PBS when the cell density is close to 90%, adding pancreatin for 3min, stopping pancreatin reaction with DMEM/F12+ 10% FBS, and passaging 30% of cells to a new culture dish;
2) after the cells reached complete confluence, they were cultured for 3d using induction medium and 4d in maintenance medium, and a large number of lipid droplets were observed.
The formula of the induction culture medium is as follows: DMEM/F12, 10% FBS, 1ug/mL Insulin, 5nM T3, 100uM IBMX, 2ug/mL Dexamethane, 125uM indomethcin, 1uM Rosiglitazone, 33uM Biotin, 17uM Pantothenic Acid, 1ug/uL transferase;
the formula of the maintenance medium is as follows: DMEM/F12, 10% FBS, 1ug/mL Insulin, 5nM T3, 1uM Rosiglitazone.
The culture and polyester induction method of the immortalized pig fat precursor cell line with high differentiation efficiency comprises the following steps:
culturing immortalized pig fat precursor cells;
obtaining monoclonal cells by a limiting dilution method;
inducing the obtained monoclonal cells to differentiate;
screening out a monoclonal cell line with the highest polyester differentiation efficiency;
the monoclonal with the highest differentiation efficiency of the polyester is Pig # 4. It is preserved.
And carrying out continuous passage and differentiation performance identification on the obtained monoclonal cell line.
The invention relates to application of the immortalized pig fat precursor cell line with high differentiation efficiency in food development.
The invention relates to an application of the immortalized pig fat precursor cell line with high differentiation efficiency in virus culture or detection.
The invention relates to application of the high-differentiation-efficiency immortalized pig fat precursor cell line in vaccine development and production.
The immortalized pig fat precursor cell line with high differentiation efficiency is applied to drug screening.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the invention constructs a pig fat precursor cell line which can be stably passaged for more than 35 generations and always keeps high differentiation efficiency for the first time through a large amount of creative labor of an inventor: pig # 4. Through optimization, a culture solution formula which can exert Pig # 4 polyester differentiation capacity to the maximum extent is obtained.
The immortalized Pig adipose precursor cell line Pig # 4 with high differentiation efficiency can be stably passaged, the use of Pig primary adipose cells is reduced, and the production and experiment cost is reduced; pig # 4 can always keep extremely high differentiation capability, can be used for research on a differentiation polyester mechanism of the adipocyte precursor cells, and can also be used for production of cell culture meat.
The immortalized pig fat precursor cell line with high differentiation efficiency has been passaged for more than 35 generations up to now; more than 90% of the cells can be changed into mature adipocytes with a large number of lipid droplets by using 9-factor induction medium. The cell line can be used for the research on the differentiation of pig fat precursor cells, the physiological research of pig mature fat cells and the production and use of cell culture meat.
Drawings
FIG. 1 is a morphological diagram of 107 monoclonal porcine adipose precursor cells;
FIG. 2 is a 86 strain monoclonal oil red O staining map;
FIG. 3 is a graph showing the OD values of the monoclonal oil red staining;
FIG. 4 is a graph of Pig # 4 growth for different generations;
FIG. 5 is a graph of the effect of varying insulin concentrations in induction medium on differentiation; panel A is a cell culture image; panel B is a plot of concentration versus differentiation;
FIG. 6 is a graph showing the effect of varying IBMX concentration in the induction medium on differentiation; panel A is a cell culture image; panel B is a graph of concentration versus differentiation;
FIG. 7 is a graph of the effect of varying dexamethasone concentration in the induction medium on differentiation; panel A is a cell culture image; panel B is a plot of concentration versus differentiation;
FIG. 8 is a graph showing the effect of varying indomethacin concentrations in induction medium on differentiation; panel A is a cell culture image; panel B is a plot of concentration versus differentiation;
FIG. 9 is a polyester differentiation map of different generations of Pig # 4 cells; panel A is a cell culture image; panel B is a differentiation diagram;
FIG. 10 is a graph of the detection results of Pig #4STR loci in FIG. 1;
FIG. 11 is a graph of the detection results of Pig #4STR loci in FIG. 2;
FIG. 12 is a graph of the detection results of Pig #4STR loci in FIG. 3;
FIG. 13 is a graph of the detection results of Pig #4STR loci in FIG. 4;
FIG. 14 is a graph of the detection results of Pig #4STR loci in FIG. 5;
FIG. 15 is a graph of the detection results of Pig #4STR loci shown in FIG. 6;
FIG. 16 is a graph of the detection results of Pig #4STR loci in FIG. 7;
FIG. 17 is a graph of the detection results of Pig #4STR loci, FIG. 8;
FIG. 18 is a graph of the detection results of Pig #4STR loci in FIG. 9;
FIG. 19 is a graph of the detection results of Pig #4STR loci shown in FIG. 10;
fig. 20 is a graph 11 showing the detection results of Pig #4STR loci.
Detailed Description
The technical solution of the present invention is further described with reference to the accompanying drawings and the detailed description.
The immortalized pig fat precursor cells used in the present invention were established by the inventors.
Example 1: the limiting dilution method obtains the monoclonal cell line.
1. Seeding immortalized porcine adipose precursor cells in a 96-well plate at a density of 0.5 cells per well;
2. observing the cell state every day, and carrying out passage to a 48-pore plate when the cells reach 90% confluence;
3. observing the cells in the step 2, and carrying out passage to a 24-pore plate when the cells reach 90% confluence;
4. observing the cells in the step 3, and carrying out passage to a 12-pore plate when the cells reach 90% confluence;
5. observing the cells in the step 4, and carrying out passage to a 6-pore plate when the cells reach 90% confluence;
as a result: a total of 107 single clones were obtained, see FIG. 1.
Example 2: screening the monoclonal with highest induced differentiation efficiency.
1. Cells in the 6-well plate in step 5 of example 1 were trypsinized, 2/3 cells were frozen in liquid nitrogen, and 1/3 cells were passaged into one well of a 12-well plate;
2. when the monoclonal cells in the 12-pore plate grow to be completely confluent;
2. replacing the culture solution in the step 2 with an induction culture solution, and placing the culture solution in a cell culture box for culturing for 3 days;
3. the culture solution in step 2 was changed to a maintenance culture solution, and the culture solution was changed every 2 days for 2 times.
4. Fixing the cells in the step 3 by using 4% paraformaldehyde, performing oil red O staining and photographing, and measuring the light absorption value at 500nm by using a spectrophotometer.
The components of the induction culture solution are as follows: to the complete medium of DMEM/F12 containing 10% FBS was added 1. mu.g/mL Insulin, 5nM T3, 100. mu.M IBMX, 2. mu.g/mL Dexamethasone, 125. mu.M Indomothacin, 1. mu.M Rosiglitazone, 33. mu.M Biotin, 17. mu.M pantothic Acid, 1. mu.g/. mu.L Transferrin.
The components of the maintenance culture solution are as follows: add 1. mu.g/mL Insulin, 5nM T3, 1. mu.M Rosglitazone to DMEM/F12 complete medium containing 10% FBS.
And (3) dyeing and identifying oil red O: after differentiation of the lipogenic precursor cells is completed, removing maintenance cell culture solution in each hole, adding 4% paraformaldehyde, and fixing the cells for 15 min; washing once with 60% isopropanol; drying at room temperature; adding 1mL of oil red O working solution into each hole, and standing for 20min at room temperature; quickly discarding oil red O working solution in each hole, and cleaning for 4 times by using deionized water; standing and drying at room temperature, and taking an oil red O dyeing picture; adding 1.5mL of isopropanol into each hole, and incubating for 20min at room temperature; sucking the supernatant; the absorbance at a wavelength of 500nm was measured using a spectrophotometer.
As a result: the cell death during differentiation was removed, and induced differentiation was completed by 85 clones, 60 of which had differentiation ability, as shown in FIG. 2.
The single clone with obvious oil red staining was selected to measure its OD500 value and sorted according to differentiation ability, see FIG. 3. Clone4 was found to have the highest differentiation efficiency and was named Pig # 4.
Example 3: and detecting the proliferation activity of the Pig # 4.
1. Pig # 4 generation 10 (P10), generation 20 (P20), generation 30 (P30) and generation 10 of immortalized porcine adipose precursor cells (PSPA P10) were combined at 2.5 × 10 4 One/well is seeded in 24-well plates.
2. Standing at 37 deg.C with 5% CO 2 Culturing in a cell culture box.
3. Cells were digested with pancreatin every 24 hours and counted using a hemocytometer plate.
4. The inoculation time was used as the first day (1d), the culture time (d) was used as the abscissa, and the number of cells (x 10) 4 ) The ordinate is plotted.
As a result: pig # 4 in passage 10, 20, 30 and immortalized porcine adipose precursor cells in passage 10 had the same growth trend, see FIG. 6. The cells grew slowly in the first 48 hours of cell inoculation, the number of cells began to double at day 3, the cells entered the logarithmic growth phase, the cell rate began to slow down by day 6, the cells were contact-inhibited at day 9, and the cells almost stopped growing. It was shown that Pig # 4 has a proliferative capacity consistent with that of immortalized porcine adipocyte precursor cells, and that Pig # 4 still maintained the proliferative activity when passaging times exceeded 30 generations.
Example 4: and determining the optimal culture medium formula for Pig # 4 polyester differentiation.
1. On the basis of the formulation of the induction medium in step 4 of example 2, the concentrations of the components were individually changed as follows:
Insulin:0.35ug/mL,0.7ug/mL,1ug/mL,3.5ug/mL；
IBMX:0.05mM，0.1mM，0.25mM，0.5mM；
Dexamethasone:0.5ug/mL，0.66ug/mL，1ug/mL，2ug/mL；
Indomethacin:125uM，62.5uM，0uM；
preparing an induction culture medium;
2. pig # 4 was passaged to 12-well plates, and after the cells were confluent in growth, the cells were cultured for 3 days using the above induction medium.
3. Replacing the induction culture medium in the step 2 with a maintenance culture solution for culturing for 4 days, and replacing the culture solution once every 48 hours, wherein the maintenance culture solution comprises the following components: add 1. mu.g/mL Insulin, 5nM T3, 1. mu.M Rosglitazone to DMEM/F12 complete medium containing 10% FBS.
4. Fixing the cells in the step 3 by using 4% paraformaldehyde, performing oil red O staining and photographing, and measuring the light absorption value at 500nm by using a spectrophotometer.
As a result: the optimal concentration of insulin is 1ug/mL, shown in figure 5, the optimal concentration of IBMX is 100uM, shown in figure 6, the optimal concentration of dexamethasone is 2ug/mL, shown in figure 7, and the optimal concentration of indomethacin is 125uM, shown in figure 8.
Example 5: and (4) detecting the differentiation capability of different generations of Pig # 4 polyester.
1. Pig # 4 generation 10 (P10), generation 20 (P20), generation 30 (P30) was replaced by 2.5 × 10 4 Planting in 24-well plate at 37 deg.C with 5% CO 2 Culturing in a cell culture box.
2. After the cells are converged, replacing the culture solution in the step 1 with an induction culture solution, and placing the culture solution in a cell incubator for 3 days;
3. the culture solution in step 2 was changed to a maintenance culture solution, and the culture solution was changed every 2 days for 2 times.
The formulation of the induction culture medium and the maintenance culture medium is the same as that in example 2.
4. Fixing the cells in the step 3 by using 4% paraformaldehyde, performing oil red O staining and photographing, and measuring the light absorption value at 500nm by using a spectrophotometer.
As a result: pig # 4 from generations 10, 20 and 30 had similar polyester differentiation capacity, as shown in FIG. 9.
Example 6: STR characterization of Pig # 4.
STR (short repeat repeats) is a specific sequence widely found in the genome characterized by the presence of multiple contiguous repeats of bases. The number of such repeats is highly genetically diverse, and thus the STR combinations for each individual are all different. STR detection has been widely used for paternity testing and forensic personal gene fingerprinting.
The pig STR detection method adopted in the embodiment is disclosed in the text of construction of a pig 12 STR locus multiplex amplification system (DOI: 10.16467/j.1008-3650.2019.04.006).
1. And culturing the cells until the cells are confluent, and extracting the genome DNA by using an Ezup column type animal tissue genome DNA extraction kit.
2. The PCR system was configured as follows:
the primer sequences used and their modifications were as follows:
3. PCR amplification was performed as follows:
4. performing STR detection
4.1 Take 96-well reaction plate, use marker to mark plate name and experiment date.
4.2, making an electronic STR detection table and automatically generating a loading table.
4.3 Using a continuous applicator, a mixture of 990. mu.l HIDI and 10ul LIZ500 was pipetted into a 96-well reaction plate at 10. mu.l per well.
4.4 Place 96-well plates in a plate centrifuge and centrifuge at 1200rmp for 15 s.
4.5 Using 12 rows of 10. mu.L rifles, 1. mu.L of the above PCR product was added to the corresponding wells of a 96-well plate against the STR detection Table.
4.6 Place 96-well plates in a plate centrifuge, centrifuge for 15s at 1200 rmp.
4.7 seal 96-well plates with a coversheet membrane, shake, place 96-well plates in a plate centrifuge, centrifuge at 1200rmp for 30 s. Placing in a PCR instrument.
4.8 denaturation procedure 98 ℃ for 5min, without heating the hot lid, the 96-well plate was rapidly cooled on ice-water mixture immediately after the procedure was finished.
4.9 Place 96-well plates in a plate centrifuge, centrifuge for 15s at 1200 rmp.
4.10 STR samples were tested using an ABI 3730xl instrument.
4.11 reading the number of bases of the peak according to the off-line data.
Through detection, the immortalized Pig fat precursor cell line Pig # 4 with high differentiation efficiency can be stably passaged, the use of Pig primary fat cells is reduced, and the production and experiment cost is reduced; the Pig # 4 can always keep extremely high differentiation capability, can be used for researching the mechanism of differentiation polyester of the adipocyte precursor cells, and can also be used for producing cell culture meat.
Claims (10)
1. An immortalized pig fat precursor cell line with high differentiation efficiency, which is preserved in China Center for Type Culture Collection (CCTCC) No. C2022150 at 5/17/2022.
2. The high differentiation efficiency immortalized porcine adipose precursor cell line according to claim 1, characterized in that: the STR characteristics of the cell are as follows: the locus size is 387A 12-12F +/-1 bp, the pigSTR4C-118 +/-1 bp and 122 +/-1 bp, the pigSTR5C-262 +/-1 bp and 266 +/-1 bp, the pigSTR7B-113 +/-1 bp, the pigSTR11B-85 +/-1 bp, the pigSTR13E-125 +/-1 bp and 133 +/-1 bp, the pigSTR14A-186 +/-1 bp, the pigSTR15A-198 +/-1 bp and 202 +/-1 bp, the pigSTR17A-155 +/-1 bp and 163 +/-1 bp, the SW240-93 +/-1 bp and 107 +/-1 bp, the Amelogenin-232 +/-1 bp and 234 +/-1 bp.
3. The construction method of the immortalized pig fat precursor cell line with high differentiation efficiency is characterized in that: the method comprises the following steps:
1) inoculating single cells of immortalized pig fat precursor cells into a 96-well plate by adopting a limiting dilution method, and culturing until the cells are completely converged in the well;
2) carrying out polyester induced differentiation on 1/3 cells in each well from a 96-well plate, a 48-well plate, a 24-well plate and a 12-well plate to a 6-well plate in an expansion culture manner;
3) the clones with high polyester efficiency were selected, and the remaining 2/3 cells were grown up and stored.
4. The culture and polyester induction method of high differentiation efficiency immortalized pig fat precursor cell line is characterized in that: the method comprises the following steps:
1) cultured in DMEM/F12+ 10% FBS, fibroblast-like in appearance; when the cell density is close to 90%, washing the culture dish by using PBS, adding pancreatin for digestion for 3min, stopping the pancreatin reaction by using DMEM/F12+ 10% FBS, and passaging 30% of cells to a new culture dish;
2) after the cells reached complete confluence, cultured for 3d using induction medium and 4d in maintenance medium, large lipid droplets were observed.
5. The method for culturing and polyester-inducing immortalized porcine adipose precursor cell lines with high differentiation efficiency according to claim 4, wherein: the formula of the induction culture medium is as follows: DMEM/F12, 10% FBS, 1ug/mL Insulin, 5nM T3, 100uM IBMX, 2ug/mL Dexamethasone, 125uM Indomothacin, 1uM Rosiglitazone, 33uM Biotin, 17uM pantothic Acid, 1ug/uL Transferin.
6. The method for culturing and polyester-inducing immortalized porcine adipose precursor cell lines with high differentiation efficiency according to claim 4, wherein: the formula of the maintenance medium is as follows: DMEM/F12, 10% FBS, 1ug/mL Insulin, 5nM T3, 1uM Rosiglitazone.
7. The application of the immortalized pig fat precursor cell line with high differentiation efficiency in food development.
8. The application of the immortalized pig fat precursor cell line with high differentiation efficiency in virus culture or detection.
9. The application of the immortalized pig fat precursor cell line with high differentiation efficiency in the development and production of vaccines.
10. The application of the immortalized pig fat precursor cell line with high differentiation efficiency in drug screening.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010026299A1 (en) * | 2008-09-08 | 2010-03-11 | Timo Ylikomi | Methods and means for soft tissue engineering |
| CN102382800A (en) * | 2011-12-08 | 2012-03-21 | 吉林大学 | Method for inducing formation of porcine fat cells by cell signal channel inhibitor |
| CN103060267A (en) * | 2012-12-26 | 2013-04-24 | 上海市内分泌代谢病研究所 | An induced differentiation method for 3T3-L1 preadipocytes |
| CN105087476A (en) * | 2015-08-31 | 2015-11-25 | 中国医学科学院北京协和医院 | Induced differentiation method of 3T3-L1 preadipocytes line |
| CN107858326A (en) * | 2017-12-12 | 2018-03-30 | 上海银海圣生物科技有限公司 | Murine preadipocyte cell 3T3 L1 differentiation-inducing agents and method of inducing differentiation |
| CN109628404A (en) * | 2018-12-18 | 2019-04-16 | 浙江大学 | The construction method and purposes of Preadipocyte immortalized cell line under pigskin |
-
2022
- 2022-05-24 CN CN202210568452.4A patent/CN115044541B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010026299A1 (en) * | 2008-09-08 | 2010-03-11 | Timo Ylikomi | Methods and means for soft tissue engineering |
| CN102143755A (en) * | 2008-09-08 | 2011-08-03 | 蒂莫·伊利科米 | Methods and means for soft tissue engineering |
| CN102382800A (en) * | 2011-12-08 | 2012-03-21 | 吉林大学 | Method for inducing formation of porcine fat cells by cell signal channel inhibitor |
| CN103060267A (en) * | 2012-12-26 | 2013-04-24 | 上海市内分泌代谢病研究所 | An induced differentiation method for 3T3-L1 preadipocytes |
| CN105087476A (en) * | 2015-08-31 | 2015-11-25 | 中国医学科学院北京协和医院 | Induced differentiation method of 3T3-L1 preadipocytes line |
| CN107858326A (en) * | 2017-12-12 | 2018-03-30 | 上海银海圣生物科技有限公司 | Murine preadipocyte cell 3T3 L1 differentiation-inducing agents and method of inducing differentiation |
| CN109628404A (en) * | 2018-12-18 | 2019-04-16 | 浙江大学 | The construction method and purposes of Preadipocyte immortalized cell line under pigskin |
Non-Patent Citations (1)
| Title |
|---|
| 王友升等: "前体脂肪细胞3T3-L1诱导分化条件的优化" * |
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