CN113088482A - Method for separating and culturing rumen epithelial cells of calves - Google Patents
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Abstract
The invention provides a method for separating and culturing rumen epithelial cells of calves, which comprises the following steps: (1) selecting rumen tissue at rumen abdominal sac, and cleaning with 4-8 deg.C sterilized water; (2) stripping rumen epithelial tissue, sequentially cleaning with sterilized water, PBS solution I and PBS solution II, cutting into pieces, and digesting with digestive juice for multiple times; (3) filtering, centrifuging and washing the digested rumen epithelial tissue to obtain a rumen epithelial cell mass; (4) the rumen epithelial cell mass is cultured by adopting a complete culture medium. The invention improves and optimizes the rumen epithelial cell separation culture technology, can obtain the rumen epithelial cells with high purity, strong activity and fast growth, can effectively reduce the possibility of pollution of in vitro culture of the rumen epithelial cells, can shorten the culture time to 2-3 days for adherence, and can complete layering in 4-5 days, which is generally completed in about 4 days.
Description
Technical Field
The invention belongs to the technical field of cell separation culture, and particularly relates to a method for separating and culturing calf rumen epithelial cells.
Background
With the development of modern biotechnology, animal cell in vitro culture methods play a crucial role in the field of animal life research. Compared with in vivo animal experiments, animal cell culture has the advantages of low cost, easily controlled conditions, few influencing factors, easy operation and the like, is carried out under artificial conditions, is convenient to regulate, control and observe, is a convenient and effective new method in the fields of researching animal metabolism processes, expression control of genetic materials and the like, and is an important research means for researching animal life laws.
Rumen is a specific digestive organ of ruminant, in which a large number of microorganisms grow, and can effectively degrade and digest roughage with high crude fiber content. Whether the rumen function is healthy or not is directly related to whether the ruminant can grow healthily or not and has better production performance. The feed fed by the ruminant is degraded into volatile fatty acid under the action of rumen microorganisms, the volatile fatty acid provides more than 80% of energy for the ruminant, and the volatile fatty acid produced by the rumen is transported, absorbed and metabolized by rumen epithelial cells for use by organisms, so that the deep understanding of the structure and function of the rumen epithelial cells is helpful for enhancing the utilization rate of the feed in the rumen and improving the production performance of the ruminant. Rumen epithelial cells are cultured in vitro, and the proliferation characteristics, the gastrointestinal function, the absorption, the transportation and the mechanism of nutrient substances and the like of the cells can be researched. The prior method for culturing the rumen usually comprises the steps of digesting rumen epithelial tissues by trypsin and EDTA, then removing microorganisms attached to the rumen by streptomycin, gentamicin, streptomycin or amphotericin and the like, and culturing by using a DMEM complete culture medium. In addition, the existing culture time of the rumen epithelial cells generally begins to adhere to the wall after 3-6 days, and layering can be completed only after 8-10 days, which indicates that the cell development is slow.
At present, the in vitro culture technology of rumen epithelial cells is not mature, and the problems of poor activity of rumen epithelial cells, long adherence time, slow growth, easy pollution and more fibroblasts are generally existed. Therefore, the improvement and optimization of the in vitro culture conditions and methods of the rumen epithelial cells are helpful for the deep understanding of the functions of the rumen epithelial cells and the metabolism of nutrients in the rumen epithelial cells.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for separating and culturing rumen epithelial cells of calves to solve the above problems. The method can obtain rumen epithelial cells with high purity, strong activity and rapid growth. The technical scheme of the invention is as follows:
a method for separating and culturing rumen epithelial cells of calves comprises the following steps:
(1) selecting rumen tissue at rumen abdominal sac, and cleaning with 4-8 deg.C sterilized water;
(2) stripping rumen epithelial tissue, sequentially cleaning with sterilized water, PBS solution I and PBS solution II, cutting into pieces, and digesting with digestive juice for multiple times;
(3) filtering, centrifuging and washing the digested rumen epithelial tissue to obtain a rumen epithelial cell mass;
(4) the rumen epithelial cell mass is cultured by adopting a complete culture medium.
Further, the PBS solution had a pH of 7.4 and contained potassium dihydrogen phosphate at a final concentration of 0.1mol/L and disodium hydrogen phosphate at a final concentration of 0.1 mol/L.
Further, the PBS solution II contains a mixed solution of penicillin-streptomycin-amphotericin B (100 Xthree antibody) (i.e. PBS solution containing 2% ABAM (v/v)) with the volume concentration of 2%, wherein the mixed solution of penicillin-streptomycin-amphotericin B (100 Xthree antibody) contains 10kU/ml of penicillin, 10 mg/ml of streptomycin and 25 mu g/ml of amphotericin B.
Further, the digestive juice is pressedThe final concentration of the composition is 2.5-5% (w/v) of trypsin and CaCl21.08 mM, HEPES25mM, ABAM 2% (v/v), solvent Krebs-Ringer buffer.
Further, the control parameters for culturing the rumen epithelial cell mass by using the complete culture medium are as follows: at 37 deg.C, 5% CO2And culturing for 4-5 days under the condition of 95% humidity, replacing the complete culture medium every 1-3 d, and continuously culturing until more than 85% of cell layering is completed.
Further, the complete medium is a MEM basal medium.
Compared with the prior art, the invention can obtain the following technical effects:
the invention improves and optimizes the rumen epithelial cell separation culture technology, can obtain the rumen epithelial cells with high purity, strong activity and fast growth, can effectively reduce the possibility of pollution of in vitro culture of the rumen epithelial cells, can shorten the culture time to 2-3 days for adherence, and can complete layering in 4-5 days, which is generally completed in about 4 days.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a diagram of rumen epithelial cells of calves cultured for 24h according to the isolation method of example 1 of the present invention.
FIG. 2 is a diagram of the rumen epithelial cells of calves cultured for 48h according to the isolation method of example 1 of the present invention.
FIG. 3 is a diagram showing the state of rumen epithelial cells of calves cultured for 72 hours according to the isolation method of example 1.
FIG. 4 is a diagram of the rumen epithelial cells of calves cultured for 96h according to the isolation method of example 1.
FIG. 5 is a diagram of the rumen epithelial cells of calves cultured for 96h according to the isolation method of example 2 of the present invention.
Fig. 6 is a diagram showing the state of cells after 3 passages of rumen epithelial cells of calves according to example 1 of example 3 of the present invention.
Fig. 7 is a diagram showing the state of the rumen epithelial cells of calves frozen and thawed 12h according to example 1 of example 3 of the present invention.
Fig. 8 is a diagram showing the state of the rumen epithelial cells of calves frozen and thawed 24h according to example 1 of example 3 of the present invention.
Fig. 9 is a diagram showing the state of the rumen epithelial cells of calves after cryopreservation-resuscitation for 48 hours according to example 1 of the present invention in example 3.
FIG. 10 is a growth curve of rumen epithelial cells of calves separately cultured according to example 1 of the present invention after cryopreservation and recovery.
Detailed Description
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1
The embodiment provides a method for separating and culturing rumen epithelial cells of calves, which comprises the following steps:
(1) selecting rumen tissue at a rumen abdominal sac after calf slaughtering through laparotomy, washing rumen contents and rumen tissue with refrigerated sterilized water, soaking the obtained rumen tissue in refrigerated PBS solution, and returning to a laboratory;
(2) peeling the rumen epithelial layer from the muscle layer using a blunt instrument, and rinsing the obtained rumen epithelial layer with sterile water;
(3) sequentially rinsing the obtained rumen epithelial layer in sterilized water and a sterilized PBS solution I, and repeating for 3 times;
(4) rinsing the rinsed rumen epithelial tissue with a PBS (phosphate buffer solution) solution II containing 2% ABAM, cutting into pieces, and digesting the tissue for multiple times by using digestive juice until the tissue becomes white and sticky;
(5) filtering and centrifuging the digested tissue to obtain a degraded rumen epithelial cell mass;
(6) suspending and collecting rumen epithelial cell mass, and transferring the rumen epithelial cell mass into a culture bottle of an MEM basal medium for adherent culture;
(7) and replacing the culture solution after the cells are cultured for 24 hours in an adherent manner, then replacing the culture solution every 2 days to continuously culture cell layering, and collecting the cultured rumen epithelial cells or using the rumen epithelial cells for passage after more than 85% of the layering is completed.
The PBS solution in the step (1) has a pH value of 7.4 and contains potassium dihydrogen phosphate with a final concentration of 0.1mol/L and disodium hydrogen phosphate with a final concentration of 0.1 mol/L.
The blunt instrument used in step (2) needs to be sterilized and the adhered serosal and muscular layers should be separated cleanly.
The sterilized water and PBS used in step (3) are not reused every time they are replaced.
And (3) the PBS solution II used in the step (4) is a PBS solution containing a mixed solution (100 multiplied by three antibodies) of penicillin-streptomycin-amphotericin B with the volume concentration of 2%, the penicillin content in the mixed solution (100 multiplied by three antibodies) of penicillin-streptomycin-amphotericin B is 10kU/ml, the streptomycin content is 10 mg/ml, and the amphotericin B content is 25 mu g/ml. The solution was prepared in PBS and the working concentration of penicillin, streptomycin and amphotericin B used in the cell culture medium was 100U/ml, 0.1mg/ml and 0.25. mu.g/ml, respectively.
The proposed size of the sheared tissue in step (4) is<1 cm2Tissue patch of (2). The composition of the digestive juice used for the minced rumen epithelial tissue small pieces according to the final concentration is as follows: 2.5% (w/v) Trypsin +1.08 mM CaCl2+ 25mM HEPES solution +2% (v/v) ABAM in Krebs-Ringer buffer for about 15min per digestion.
The filtration and centrifugation conditions in the step (5) are 70 Xg centrifugation at 4 ℃ for 6 min.
And (4) flushing the rumen epithelial cell mass obtained after centrifugation in the step (6) by using a complete culture medium, and then carrying out transfer culture and adherent culture in a penning medium. Taking rumen epithelial cell mass obtained after complete culture medium is re-suspended, observing cell morphology uniformity under microscope, and regulating cell density to be more than 106Per ml, trypan blue staining detects that the cell survival rate reaches more than 95 percent, then the cell is inoculated into a culture bottle containing complete culture medium and placed in a 37 ℃,containing 5% CO2The incubator is used for 4 d. Observing 24h, 48h, 72h and 96h rumen epithelial cell state diagrams of calves in the culture process, which are respectively shown in figures 1-4, and showing that the rumen epithelial cells have strong activity and fast growth in an in vitro culture system.
The complete medium used in steps (6) and (7) is MEM minimal medium.
Example 2
The embodiment provides a method for separating and culturing rumen epithelial cells of calves, which comprises the following steps:
(1) selecting rumen tissue at a rumen abdominal sac after calf slaughtering through laparotomy, washing rumen contents and rumen tissue with refrigerated sterilized water, soaking the obtained rumen tissue in refrigerated PBS solution, and returning to a laboratory;
(2) peeling the rumen epithelial layer from the muscle layer using a blunt instrument, and rinsing the obtained rumen epithelial layer with sterile water;
(3) sequentially cleaning the obtained rumen epithelial layer in sterilized water and a sterilized PBS solution I, and repeating for 3 times;
(4) rinsing the cleaned rumen epithelial tissue with a PBS (phosphate buffer solution) solution II containing 2% ABAM, cutting into pieces, and digesting the tissue for multiple times by using digestive juice until the tissue becomes white and sticky;
(5) filtering and centrifuging the digested tissue to obtain a degraded rumen epithelial cell mass;
(6) suspending and collecting rumen epithelial cell mass, and transferring the rumen epithelial cell mass into a culture bottle containing a complete culture medium for adherent culture;
(7) and replacing the culture solution after the cells are cultured for 24 hours in an adherent manner, then replacing the culture solution every 2 days to continuously culture cell layering, and collecting the cultured rumen epithelial cells or using the rumen epithelial cells for passage after more than 85% of the layering is completed.
The PBS solution in the step (1) has a pH value of 7.4 and contains a mixed solution of monopotassium phosphate with a final concentration of 0.1mol/L and disodium phosphate with a final concentration of 0.1 mol/L.
The blunt instrument used in step (2) needs to be sterilized and the adhered serosal and muscular layers should be separated cleanly.
The sterilized water and PBS used in step (3) are not reused every time they are replaced.
And (3) the PBS solution II used in the step (4) is a PBS solution containing a mixed solution (100 multiplied by three antibodies) of penicillin-streptomycin-amphotericin B with the volume concentration of 2%, the penicillin content in the mixed solution (100 multiplied by three antibodies) of penicillin-streptomycin-amphotericin B is 10kU/ml, the streptomycin content is 10 mg/ml, and the amphotericin B content is 25 mu g/ml. The solution was prepared in PBS and the working concentration of penicillin, streptomycin and amphotericin B used in the cell culture medium was 100U/ml, 0.1mg/ml and 0.25. mu.g/ml, respectively.
The proposed size of the sheared tissue in step (4) is<1 cm2Tissue patch of (2). The composition of the digestive juice used for the minced rumen epithelial tissue small pieces according to the final concentration is as follows: 5% (w/v) Trypsin +1.08 mM CaCl2+ 25mM HEPES solution +2% (v/v) ABAM in Krebs-Ringer buffer for about 15 min.
The filtration and centrifugation conditions in the step (5) are 70 Xg centrifugation at 4 ℃ for 6 min.
And (4) flushing the rumen epithelial cell mass obtained after centrifugation in the step (6) by using a complete culture medium, and then carrying out transfer culture and adherent culture in a penning medium. Taking rumen epithelial cell mass obtained after complete culture medium is re-suspended, observing cell morphology uniformity under microscope, and regulating cell density to be more than 106Perml, after trypan blue staining detection cell survival rate reaches more than 95%, inoculating the cell into a culture bottle containing complete culture medium, placing the culture bottle at 37 ℃, and containing 5% CO2The incubator is used for 4 d.
The complete medium used in steps (6) and (7) is MEM minimal medium. Fig. 5 provides a state diagram of rumen epithelial cells of calves cultured for 96 hours in the present example, and it can be clearly observed that rumen epithelial cells are uniformly distributed in an in vitro culture system, and are large in number and strong in activity.
Example 3
The rumen epithelial cells of the calves obtained in example 1 are sequentially subjected to passage, cryopreservation and recovery, and the specific operation is as follows:
1. cell passage
Pouring the culture medium of the rumen epithelial cells of the calf obtained in the example 1, adding preheated PBS for cleaning twice, adding 0.25% trypsin into an incubator at 37 ℃ for 5-8min according to the volume of 1/10 added with the culture medium, observing the digestion condition by using a microscope, ensuring that no obvious adherent cells (if the cells can be incubated at 37 ℃ for 1-2min continuously, beating a culture bottle dish by hands), after ensuring that the digestion is complete, timely adding an equal volume of serum-containing culture medium to stop the digestion (ensuring that the cells are digested and the cell membrane structure of the damaged cells is as little as possible), sucking the suspension by using a pipette, adding 2-3ml of PBS for rinsing, and repeating 1-2 times to collect the cells in the bottle as much as possible. The cell suspension is centrifuged at 1200rpm for 5min, the obtained cell sediment is added into the culture medium for basic suspension, then the cell sediment is transferred into a new culture flask dish (the cell passage is generally 1: 3, namely, one flask of cell is transferred into three flasks), the cell is passaged for 3 times according to the steps, and the cell state after the passage is shown in figure 6, so that the rumen epithelial cells can be clearly observed to be uniformly distributed, have large quantity and have strong activity.
2. Cell cryopreservation
The subculture cells obtained in step 1 were digested in the same manner as in step 1 and the cell pellet was obtained, and after the cell pellet was obtained, the supernatant was removed and a suitable amount of cryopreservation solution (10% DMSO: trimethylmaple, 90% medium containing 10% FBS: fetal bovine serum; one tube of cell cryopreservation in a common culture flask) was added. The cryopreservation tube is placed in a cryopreservation box special for cryopreserved cells, the cryopreservation box is kept at minus 80 ℃ overnight, then liquid nitrogen is used for long-term preservation, and the subculture cells of the embodiment can be preserved for at least more than 1 year.
3. Cell resuscitation
Taking out the frozen cells in the liquid nitrogen from the liquid nitrogen, recovering the cells in a water bath at 37 ℃, transferring the cell suspension in the frozen tube into a centrifuge tube on a super clean bench, collecting the residual cells in the tube as much as possible by using PBS (phosphate buffer solution) in the same way as the cell suspension in the process of passage and freezing, centrifuging to remove the supernatant, adding a proper amount of culture medium, and culturing in a flask culture box (the survival rate of the recovered cells is 70 percent in general, and the recovered tube can be cultured in 2-3 flasks). Fig. 7 to 9 provide cell state diagrams of calf rumen epithelial cells of example 1 after cryopreservation-resuscitation for 12h, 24h and 48h, respectively. The growth curve of the calf rumen epithelial cells of example 1 after cryopreservation and recovery is shown in fig. 10. With reference to fig. 7-10, it can be shown that the cell activity after cryopreservation recovery is in an S-shape, which proves that the cell activity is better, and the requirement of in vitro culture of rumen epithelial cells is met. The invention also proves that the rumen epithelial cells can be successfully cultured in vitro and subjected to passage-cryopreservation-recovery.
Comparative example 1
The difference from example 1 is that the medium used in steps (6) and (7) is DMEM medium. The result shows that the adherence rate of the fibroblast is high in the culture process. In addition, compared with DMEM complete culture medium, the MEM basic culture medium has simpler components, low cost and convenient operation.
Comparative example 2
The difference from example 2 is that the medium used in steps (6) and (7) is DMEM medium. The result shows that the adherence rate of the fibroblast is high in the culture process. In addition, compared with DMEM complete culture medium, the MEM basic culture medium has simpler components, low cost and convenient operation.
In conclusion, the simple and feasible rumen epithelial cell separation culture technology is designed, rumen epithelial cells with high purity, strong activity and fast growth can be obtained by the method, the possibility of pollution of in-vitro culture of the rumen epithelial cells can be effectively reduced, the culture time can be shortened to 2-3d for adherence, layering can be completed in 4-5d, and the culture process is convenient and fast.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (6)
1. A method for separating and culturing rumen epithelial cells of calves is characterized by comprising the following steps: the method comprises the following steps:
(1) selecting rumen tissue at rumen abdominal sac, and cleaning with 4-8 deg.C sterilized water;
(2) stripping rumen epithelial tissue, sequentially cleaning with sterilized water, PBS solution I and PBS solution II, cutting into pieces, and digesting with digestive juice for multiple times;
(3) filtering, centrifuging and washing the digested rumen epithelial tissue to obtain a rumen epithelial cell mass;
(4) the rumen epithelial cell mass is cultured by adopting a complete culture medium.
2. The method for separating and culturing rumen epithelial cells of calves according to claim 1, wherein: the PBS solution had a pH of 7.4 and contained potassium dihydrogen phosphate at a final concentration of 0.1mol/L and disodium hydrogen phosphate at a final concentration of 0.1 mol/L.
3. The method for separating and culturing rumen epithelial cells of calves according to claim 1, wherein: and the PBS solution II contains a mixed solution (100 multiplied by three antibodies) of penicillin-streptomycin-amphotericin B with the volume concentration of 2 percent, wherein the mixed solution (100 multiplied by three antibodies) of penicillin-streptomycin-amphotericin B contains 10kU/ml of penicillin, 10 mg/ml of streptomycin and 25 mu g/ml of amphotericin B.
4. The method for separating and culturing rumen epithelial cells of calves according to claim 1, wherein: the digestive juice comprises 2.5-5% (w/v) of trypsin and CaCl according to final concentration21.08 mM, HEPES25mM, ABAM 2% (v/v), solvent Krebs-Ringer buffer.
5. The method for separating and culturing rumen epithelial cells of calves according to claim 1, wherein: the control parameters for culturing the rumen epithelial cell mass by adopting a complete culture medium are as follows: at 37 deg.C, 5% CO2And culturing for 4-5 days under the condition of 95% humidity, replacing the complete culture medium every 1-3 d, and continuously culturing until more than 85% of cell layering is completed.
6. The method for separating and culturing rumen epithelial cells of calves according to claim 1 or 5, wherein: the complete culture medium is an MEM basic culture medium.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113930383A (en) * | 2021-09-28 | 2022-01-14 | 黑龙江八一农垦大学 | Method for constructing in-vitro culture system of rumen epithelial tissue of dairy cow |
| CN117503800A (en) * | 2024-01-04 | 2024-02-06 | 北京益华生物科技有限公司 | Gastric mucosa epithelial cell extract and preparation method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105238741A (en) * | 2015-11-14 | 2016-01-13 | 河南农业大学 | Complete medium for culturing ruminal epithelium cells of dairy cows and application thereof |
| CN105308176A (en) * | 2013-03-15 | 2016-02-03 | 迪斯吉尼科斯有限公司 | Isolated discogenic cells, methods of using, and methods of preparing same from mammalian tissue |
| CN105316279A (en) * | 2014-07-17 | 2016-02-10 | 北京大北农科技集团股份有限公司动物医学研究中心 | Method for efficiently separating and purifying mammary epithelial cells |
| CN106967674A (en) * | 2017-05-22 | 2017-07-21 | 江西农业大学 | A kind of isolated culture method of sheep rumen epithelial cell |
| CN107881197A (en) * | 2017-11-17 | 2018-04-06 | 扬州大学 | One kind immortalizes cow rumen epithelial cell line and its construction method |
| CN109097320A (en) * | 2018-07-23 | 2018-12-28 | 南京农业大学 | A kind of sheep lamb cud epithelial cell cultural method |
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
-
2021
- 2021-04-16 CN CN202110411660.9A patent/CN113088482B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105308176A (en) * | 2013-03-15 | 2016-02-03 | 迪斯吉尼科斯有限公司 | Isolated discogenic cells, methods of using, and methods of preparing same from mammalian tissue |
| CN105316279A (en) * | 2014-07-17 | 2016-02-10 | 北京大北农科技集团股份有限公司动物医学研究中心 | Method for efficiently separating and purifying mammary epithelial cells |
| CN105238741A (en) * | 2015-11-14 | 2016-01-13 | 河南农业大学 | Complete medium for culturing ruminal epithelium cells of dairy cows and application thereof |
| CN106967674A (en) * | 2017-05-22 | 2017-07-21 | 江西农业大学 | A kind of isolated culture method of sheep rumen epithelial cell |
| CN107881197A (en) * | 2017-11-17 | 2018-04-06 | 扬州大学 | One kind immortalizes cow rumen epithelial cell line and its construction method |
| CN109097320A (en) * | 2018-07-23 | 2018-12-28 | 南京农业大学 | A kind of sheep lamb cud epithelial cell cultural method |
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
Non-Patent Citations (18)
| Title |
|---|
| C.KENT-DENNIS 等: "Effects of lipopolysaccharide exposure in primary bovine ruminal epithelial cells", 《JOURNAL OF DAIRY SCIENCE》 * |
| C.KENT-DENNIS 等: "Effects of lipopolysaccharide exposure in primary bovine ruminal epithelial cells", 《JOURNAL OF DAIRY SCIENCE》, vol. 103, no. 10, 31 October 2020 (2020-10-31), pages 3 - 4 * |
| KENT-DENNIS 等: "Effects of a proinflammatory response on metaboloc function of cultured，promary ruminal epithelial cells", 《JOURNAL OF DAIRY SCIENCE》 * |
| KENT-DENNIS 等: "Effects of a proinflammatory response on metaboloc function of cultured，promary ruminal epithelial cells", 《JOURNAL OF DAIRY SCIENCE》, vol. 104, no. 1, 12 January 2021 (2021-01-12), pages 1002 - 1017 * |
| R.L.BALDWIN 等: "Developmental Changes in Glucose and Butyrate Metabolism by Isolated Sheep Ruminal Cells", 《THE JOURNAL OF NUTRITION》 * |
| R.L.BALDWIN 等: "Developmental Changes in Glucose and Butyrate Metabolism by Isolated Sheep Ruminal Cells", 《THE JOURNAL OF NUTRITION》, vol. 122, no. 5, 1 May 1992 (1992-05-01), pages 1150 * |
| 姜茂成 等: "奶牛瘤胃上皮细胞分离培养与鉴定", 《中国农业大学学报》 * |
| 姜茂成 等: "奶牛瘤胃上皮细胞分离培养与鉴定", 《中国农业大学学报》, vol. 23, no. 1, 15 January 2018 (2018-01-15), pages 80 - 86 * |
| 姜雅慧 等: "亚急性瘤胃酸中毒对反刍动物瘤胃生理功能及物质吸收转运的影响", 《动物营养学报》 * |
| 姜雅慧 等: "亚急性瘤胃酸中毒对反刍动物瘤胃生理功能及物质吸收转运的影响", 《动物营养学报》, vol. 33, no. 2, 19 March 2021 (2021-03-19), pages 637 - 643 * |
| 孙志洪 等: "山羊瘤胃上皮细胞和空肠粘膜上皮细胞原代培养技术研究", 《动物营养学报》 * |
| 孙志洪 等: "山羊瘤胃上皮细胞和空肠粘膜上皮细胞原代培养技术研究", 《动物营养学报》, vol. 22, no. 3, 15 June 2010 (2010-06-15), pages 602 - 610 * |
| 成永旭等: "不同脂肪酸对中华绒螯蟹肝胰腺组织三种FABP表达的影响", 《基因组学与应用生物学》 * |
| 成永旭等: "不同脂肪酸对中华绒螯蟹肝胰腺组织三种FABP表达的影响", 《基因组学与应用生物学》, no. 10, 25 October 2017 (2017-10-25), pages 4100 - 4107 * |
| 金鑫等: "绵羊瘤胃上皮细胞的体外分离培养、冻存及复苏方法", 《中国农业大学学报》 * |
| 金鑫等: "绵羊瘤胃上皮细胞的体外分离培养、冻存及复苏方法", 《中国农业大学学报》, no. 05, 15 May 2019 (2019-05-15), pages 63 - 72 * |
| 闫磊: "日粮精料水平对瘤胃上皮SCFA吸收相关载体表达的影响及其机理研究", 《万方学位论文》 * |
| 闫磊: "日粮精料水平对瘤胃上皮SCFA吸收相关载体表达的影响及其机理研究", 《万方学位论文》, 9 August 2016 (2016-08-09), pages 1 - 122 * |
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| CN113930383A (en) * | 2021-09-28 | 2022-01-14 | 黑龙江八一农垦大学 | Method for constructing in-vitro culture system of rumen epithelial tissue of dairy cow |
| CN117503800A (en) * | 2024-01-04 | 2024-02-06 | 北京益华生物科技有限公司 | Gastric mucosa epithelial cell extract and preparation method and application thereof |
| CN117503800B (en) * | 2024-01-04 | 2024-04-05 | 北京益华生物科技有限公司 | Gastric mucosa epithelial cell extract and preparation method and application thereof |
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