CN105695396A - Method for in-vitro isolated culture of chicken fallopian tube epithelial cells - Google Patents
Method for in-vitro isolated culture of chicken fallopian tube epithelial cells Download PDFInfo
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Abstract
The invention provides a method for in-vitro isolated culture of chicken primary fallopian tube epithelial cells.The method includes the following steps that 1, tissue is aseptically separated from the neck of a fallopian funnel, close to a follicle, of a laying hen; 2, the tissue obtained after separation is digested by pancreatic enzymes and EDTA jointly, and the chicken fallopian tube epithelial cells are obtained; 3, at the temperature of 37 DEG C, the chicken fallopian tube epithelial cells are incubated for 2 h; 4, the cells obtained after incubation in the step 3 are cultured.The primary chicken fallopian tube epithelial cells obtained after isolated culture in the method are good in effect, high in growth speed and good in state; digestion time is short, the result is good, fewer parenchyma cells exist, contamination to fibroblasts is avoided, and healthy epithelioid cell populations can be obtained.
Description
Technical field
The present invention relates to animal cell culture field, the method particularly relating to the primary Epithelium Cells Isolation and culture of a kind of chicken。
Background technology
In recent years, the situation that laying hen egg yield declines is increasingly severe, Chicken salpingitis, cyst of fallopian tube etc. are the major reasons of the egg drop reduction causing all kinds of laying hen, this disease is that the laying hen caused by the various causes of disease is with salpingemphraxis, caseous salpingitis, hydrosalpinx and yolk peritonitis in various degree are feature, clinical signs expands for sick chicken abdominal part, egg production declines, lay eggs peak disappearance, eggshell deforms, feeding cost increases, the price of deed is low, carry out very big economic loss to cultivation industrial belt, the people in the industry both at home and abroad but its cause of disease and pathogenesis are puzzled in decades always。Growing and infection mechanism for disclosing cause of disease the determining in natural host body bringing out this disease, being successfully established a kind of Infection in Vitro model is strong technical support。
At present, there is the isolated culture method of bibliographical information primary cell, wherein the most practical is taked magnum tubae uterinae to shear by KasperczykK etc. by confirm final with shearing digestion of cell scraper scraping, tissue digestion 30min is carried out with 1mg/mL Collagenase 37 DEG C, 4 DEG C of centrifugal 20min of 670rcf/min, collect cell through filtering and after cell count according to 1.0 × 106Cells/well is inoculated in the 12 hole polystyrene culture dishs in DMEM/F12 culture medium (the every hole of 1mL/), 5%CO2, 37 DEG C of cultivations all can obtain good chicken salpingo epithelial cell。Additionally, after the chickling fallopian tube growth of SeaverSS hormonal stimulation, intercept fallopian tube cell attachment of inoculation after shearing, digesting fast, energetic。
The method using cell scraper scraping cells in prior art is relatively big to cell injury, and there is fibroblastic interference；Although the cell obtained with hormonal stimulation chickling is adherent soon, energetic, but the cycle is long, and cell pick-up rate is low。Therefore, setting up a kind of rapidly and efficiently pollution-free and to the little chicken salpingo epithelial cell Isolation and culture of cell injury method is this area urgent problem。
Summary of the invention
In view of this, it is an object of the invention to provide one rapidly and efficiently pollution-free and cell injury is little
The method of chicken salpingo epithelial cell Isolation and culture。
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
A kind of method that the invention provides chicken salpingo epithelial cell Isolation and culture, comprises the following steps:
1) from the laying hen aseptic chorista of infundibulum of uterine tube cervical region near follicle；
2) use described in pancreatin and EDTA simultaneous digestion and separate the tissue obtained, obtain chicken salpingo epithelial cell；
3) at the temperature of 37 DEG C, described chicken salpingo epithelial cell 2h is hatched；
4) by described step 3) in hatch after cell carry out cell cultivation。
Preferably, step 1) described in the laying hen that laying hen is 25-28 week old。
Preferably, step 1) described in separate and include gathering tissue, shred tissue and cleansing tissue, described shredding is organized as and the tissue collected is cut into the length of side piece of tissue less than 1mm。
Preferably, the cleanout fluid that described cleansing tissue uses includes phosphate buffered solution, and the number of times of described cleaning is 5～8 times。
Preferably, described phosphate buffered solution contains penicillin and streptomycin is dual anti-。
Preferably, step 2) described in the concentration of pancreatin be 0.2～0.3wt%, the concentration of described EDTA is 0.01～0.03wt%。
Preferably, step 2) described in digestion time be 10～20min。
Preferably, step 3) described in the temperature hatched be 37 DEG C, described in hatch CO in environment2Volumetric concentration be 5%。
Preferably, step 4) described in cell cultivate device be the disposable T that 15～25% hyclones were coated25Culture bottle。
Preferably, step 4) described in cell cultivate culture medium include following components: containing the DMEM/F12 of the hyclone that volume fraction is 4～6%, 15～25g/L epidermal growth factor, 80～120mg/L heparin sodium, 2～3mg/L insulin, 180～220mmol/L glutamine, 180～220 μ g/mL penicillins, 180～220 μ g/mL streptomycins。
Beneficial effects of the present invention: the present invention separates delicacy tissue from the infundibulum of uterine tube cervical region near follicle, and the primary chicken salpingo epithelial cell of separation and Culture is effective, and high cell growth speed is in good condition；Digestion time of the present invention is short, and result is good, and heteroproteose cell is few, it is to avoid fibroblastic pollution can obtain the epithelioid cell group perfected, and has the growth in monolayer cluster, has the epithelial cell construction featuress such as close contact between cell。
Figure of description
Fig. 1 is the Growth trends process of the primary Epithelium Cells of chicken of 0.25% pancreatin and 0.02%EDTA simultaneous digestion 15min；
Fig. 2 is the primary Epithelium Cells growth curve of chicken；
Fig. 3 is the cellular morphology that the dyeing of Ji's nurse Sa is identified, wherein a is the primary Epithelium Cells of chicken, and b is fibroblast；
Fig. 4 is chicken salpingo primary epithelial cells transmission electron microscope results (20000 ×)。
Detailed description of the invention
The method that the invention provides chicken salpingo epithelial cell Isolation and culture, comprises the following steps:
1) from the laying hen aseptic chorista of infundibulum of uterine tube cervical region near follicle；
2) use described in pancreatin and EDTA simultaneous digestion and separate the tissue obtained, obtain chicken salpingo epithelial cell；
3) at the temperature of 37 DEG C, described chicken salpingo epithelial cell 2h is hatched；
4) by described step 3) in hatch after cell carry out cell cultivation。
The method good separating effect of the primary Epithelium Cells Isolation and culture of chicken provided by the invention, heteroproteose cell is few, and digestion time is short, high cell growth speed。
The present invention is from the laying hen aseptic chorista of infundibulum of uterine tube cervical region near follicle。In the present invention, the laying hen of described laying hen preferably 25～28 week old, it is more preferably the laying hen of 26～27 weeks；In the present invention, the laying hen of described 25～28 week old reaches peak of laying eggs just, and fallopian tube is reached maturity, and is in the state that function is vigorous, it is not necessary to use hormone, and Epithelium Cells Success rate of virus isolation is high, good separating effect。
After separation obtains Epithelium Cells tissue, the described tissue being separated to is used pancreatin and EDTA simultaneous digestion by the present invention, obtains the primary Epithelium Cells of chicken。The present invention is before carrying out described digestion, it is preferable that the tissue that described separation obtains is shredded and cleaned。In the present invention, shred described in and preferably described tissue is cut into the length of side piece of tissue less than 1mm；The described cleanout fluid used that cleans preferably includes phosphate buffered solution, and the number of times of described cleaning preferably 5～8 times is more preferably 7 times；Described phosphate buffered solution preferably comprises penicillin and streptomycin is dual anti-, penicillin and streptomycin dual anti-can reduce and suppress operation in organize possible germ contamination, the concentration of wherein said penicillin is 180～220 μ g/mL preferably, is more preferably 200 μ g/mL；The concentration of described streptomycin is 180～220 μ g/mL preferably, is more preferably 200 μ g/mL。In the present invention, described cleaning can be removed
Mucus in piece of tissue。
In the present invention, described digestion uses pancreatin and EDTA to carry out, and the concentration of described pancreatin preferably 0.2～0.3wt% is more preferably 0.25wt%；The concentration of described EDTA preferably 0.01～0.03wt%, is more preferably 0.02wt%。In the present invention, the temperature of described digestion is 37 DEG C；The time of described digestion preferably 10～20min, is more preferably 15min；Described digestion preferably carries out when water bath with thermostatic control。
Primary for the chicken obtained Epithelium Cells after to tissue digestion, is hatched 2h by the present invention at the temperature of 37 DEG C。The tissue obtained first is centrifuged by the present invention preferably in after described digestion, then is hatched by the product after described being centrifuged。
Described postdigestive tissue is preferably sequentially carried out and is centrifuged for the first time and centrifugal for the second time by the present invention, is specially after being centrifuged for the first time and removes supernatant, precipitation is carried out centrifugal for the second time。In the present invention, in the present invention, rotating speed preferably 650～700rcf/min that described first time is centrifugal, it is more preferably 670rcf/min；Time preferably 15～25min that described first time is centrifugal, is more preferably 20min。Rotating speed preferably 650～700rcf/min that described second time is centrifugal, is more preferably 670rcf/min, time preferably 5～15min that second time is centrifugal, is more preferably 10min。
Complete to after digestion tissue centrifugal after, the present invention preferably obtains suspended tissue with DMEM/F12 culture medium suspension precipitate。In the present invention, it is 4～5% hyclones that described DMEM/F12 culture medium preferably adds volume fraction, and preferred interpolation volume fraction is 5% hyclone；The volume preferably 5～15ml of described DMEM/F12 culture medium, is more preferably 10ml。
After obtaining suspended tissue, the present invention is centrifugal after preferably described suspended tissue being filtered。In the present invention, the sieved filter of cell that described suspended tissue preferably uses diameter to be 100 μm, collect the cell in suspended tissue。The described cell collected is centrifuged by the present invention, described centrifugal rotating speed preferably 650～700rcf/min, is more preferably 670rcf/min；Described preferably 5～15min of centrifugal time, is more preferably 10min, described centrifugal number of times preferably 1～3 time, is more preferably 2 times；
After centrifugal, present invention preferably uses DMEM/F12 culture medium and the cell after described being centrifuged is carried out resuspended, obtain chicken salpingo epithelial cell。
After obtaining chicken salpingo epithelial cell, described chicken salpingo epithelial cell is hatched by the present invention。In the present invention, the temperature hatched described in is 37 DEG C；The described time hatched is 1～3h, it is preferred that for 2h；The described CO hatched in environment2Volumetric concentration preferably 5%。
The present invention is after described chicken salpingo epithelial cell is hatched, it is preferred that by centrifugal after the cell harvesting of adherent growth non-in the process of hatching。In the present invention, described centrifugal rotating speed preferably 650～700rcf/min, it is more preferably 670rcf/min；The described centrifugal time is preferably 5～15min, more preferably 10min；
After centrifugal, the present invention collects cell, and the cell after hatching is carried out cell cultivation。In the present invention, described cell is cultivated preferably in T25Culture bottle carries out；Described T25Culture bottle preferably uses the disposable T that 15～25% hyclones were coated25Culture bottle, is more preferably the disposable T using 20% hyclone to be coated25Culture bottle。In the present invention, the cell culture medium that described cell is cultivated preferably includes the component of following content: containing the DMEM/F12 of the hyclone that volume fraction is 4～6%, 15～25g/L epidermal growth factor, 80～120mg/L heparin sodium, 2～3mg/L insulin, 180～220mmol/L glutamine, 180～220 μ g/mL penicillins, 180～220 μ g/mL streptomycins；The preferred component including following content: volume fraction is DMEM/F12, the 20g/L epidermal growth factor containing hyclone of 5%, 100mg/L heparin sodium, 2.5mg/L insulin, 200mmol/L glutamine, 200 μ g/mL penicillins, 200 μ g/mL streptomycins。
T of the present invention25Culture bottle culture bottle is placed in cell culture incubator to be cultivated, the temperature of cultivation preferably 37 DEG C, is more preferably 37 DEG C, the CO in described culture environment2Volumetric concentration preferably 5%。
Below in conjunction with embodiment, the method for the primary Epithelium Cells Isolation and culture of chicken provided by the invention is described in detail, but they can not be interpreted as limiting the scope of the present invention。
Embodiment 1
From the laying hen that week old the is 26 weeks aseptic chorista of infundibulum of uterine tube cervical region near follicle, the tissue shear being separated to is broken into the piece of tissue of length of side 0.8mm, use containing penicillin 200 μ g/mL, the phosphate buffered solution cleansing tissue block of streptomycin 200 μ g/mL 7 times is with after removing mucus, the pancreatin of 0.25wt% and the EDTA of 0.02wt% is added, at the thermostat water baths of 37 DEG C to tissue digestion 15min in tissue。Then the centrifugal 20min of 670rcf/min, after collecting sedimentation cell, again by centrifugal for cell precipitation 670rcf/min 10min, after two times centrifugal completes, obtain suspended tissue with the DMEM/F12 culture medium suspension precipitate of 10mL, use the sieved filter of cell of diameter 100 μm, collect the cell in suspended tissue, the cell 670rcf/min collected is centrifuged 10min, repeated centrifugation twice, cell is carried out resuspended by rear use DMEM/F12 culture medium, obtains chicken salpingo epithelial cell。By the chicken salpingo epithelial cell that obtains at 37 DEG C, 5%CO2Cell culture incubator in hatch 2h。After hatching, 670rcf/min, 10min after the cell harvesting of adherent growth non-in the process of hatching is centrifugal, and collected after centrifugation cell with containing volume fraction is
DMEM/F12, the 20g/L epidermal growth factor containing hyclone of 5%, 100mg/L heparin sodium, 2.5mg/L insulin, 200mmol/L glutamine, 200 μ g/mL penicillins, the disposable T that the culture medium of 200 μ g/mL streptomycins was coated at 20% hyclone25Culture bottle carries out cell cultivation。
Take above-mentioned cultured cell suspension to count, the disposable T being then coated by cell suspension inoculation to 20% hyclone25In cell bottle, in 37 DEG C, 5%CO2Cultivate in incubator。First observed cell growth state after 24h, then measured cell viability from the 2nd～10 day every day with mtt assay, sets 3 holes every time and repeats, measures OD value by microplate reader in 490nm, and draw cell growth curve。Cell viability measurement result is shown in Fig. 1, wherein a is for cultivating 24h chicken salpingo epithelium primary cell (100 ×), b is for cultivating 48h chicken salpingo epithelium primary cell (200 ×), c is for cultivating 60h chicken salpingo epithelium primary cell (200 ×), d is for cultivating 72h chicken salpingo epithelium primary cell (200 ×), e is for cultivating 7d chicken salpingo epithelium primary cell (200 ×), f for cultivating 10d chicken salpingo epithelium primary cell (200 ×)；As seen from Figure 1, the chicken salpingo Epithelial cell alive that the present invention cultivates is good。
The growth curve result drawn is shown in Fig. 2, and as shown in Figure 2, the chicken salpingo epithelial cell that this method is cultivated OD value when cultivating the 3rd day is the highest, and after cultivating 7 days, OD value is thrown away and maintained 0.35, and this method cultured cells fast growth is in good condition。
Take above-mentioned cultured cell suspension and carry out the dyeing of Ji's nurse Sa, the form of observation of cell, fibroblast is also carried out the dyeing of Ji's nurse Sa simultaneously, the epithelial morphocytology of chicken salpingo carrying out cultivating is identified, result is shown in that Figure of description 3, Fig. 3-a shows: relies closely between the primary Epithelium Cells of chicken that this method is cultivated, overlaps, cell nuclear hyperchromatism is high-visible, circular or oval。Fig. 3-b display is different from epithelial cell, and fibroblast radially grows, and cell space is spindle shape or star, and nucleus is oval, is positioned in the middle of cell。
Take above-mentioned cultivation 72h chicken salpingo epithelial cell and carry out electron microscopic observation, result is as shown in Figure 4, wherein a is microvillus, and b is mitochondrion, and c is secretion cavity, d is endoplasmic reticulum, e is nucleus, visible nucleus clearly in cell, and cell surface has a large amount of microvillus, cell is contained within the mitochondrion, endoplasmic reticulum and the secretion cavity that enrich, identifies the primary Epithelium Cells of chicken further。
Embodiment 2
From the laying hen that week old the is 27 weeks aseptic chorista of infundibulum of uterine tube cervical region near follicle, the tissue shear being separated to is broken into the piece of tissue of length of side 0.9mm, use containing penicillin 190U/mL, the phosphate buffered solution cleansing tissue block of streptomycin 190U/mL 5 times is with after removing mucus, the EDTA of the pancreatin of 0.25wt% and 0.02wt% in tissue, at the thermostat water baths of 37 DEG C to tissue digestion 15min。Then the centrifugal 20min of 670rcf/min, after collecting sedimentation cell, then by centrifugal for cell precipitation 670rcf/min 10min, after two times centrifugal completes, is hanged with the DMEM/F12 culture medium suspension precipitate of 10mL
Huckaback weave, uses the sieved filter of cell of diameter 100 μm, collects the cell in suspended tissue, the cell 670rcf/min collected is centrifuged 10min, repeated centrifugation twice, cell is carried out resuspended by rear use DMEM/F12 culture medium, obtains chicken salpingo epithelial cell。By the chicken salpingo epithelial cell that obtains at 37 DEG C, 5%CO2Cell culture incubator in hatch 1.5h。After hatching, by 670rcf/min after the cell harvesting of adherent growth non-in the process of hatching, 10min is centrifuged, collected after centrifugation cell is with containing DMEM/F12, the 20g/L epidermal growth factor containing hyclone that volume fraction is 5%, 100mg/L heparin sodium, 2.5mg/L insulin, 200mmol/L glutamine, 200 μ g/mL penicillins, the disposable T that the culture medium of 200 μ g/mL streptomycins was coated at 20% hyclone25Culture bottle carries out cell cultivation。
Embodiment 3
From the laying hen that week old the is 25 weeks aseptic chorista of infundibulum of uterine tube cervical region near follicle, the tissue shear being separated to is broken into the piece of tissue of length of side 0.8mm, use containing penicillin 210U/mL, the phosphate buffered solution cleansing tissue block of streptomycin 210U/mL 5 times is with after removing mucus, the EDTA of the pancreatin of 0.25wt% and 0.02wt% in tissue, at the thermostat water baths of 37 DEG C to tissue digestion 15min。Then the centrifugal 20min of 670rcf/min, after collecting sedimentation cell, again by centrifugal for cell precipitation 670rcf/min 10min, after two times centrifugal completes, obtain suspended tissue with the DMEM/F12 culture medium suspension precipitate of 10mL, use the sieved filter of cell of diameter 100 μm, collect the cell in suspended tissue, the cell 670rcf/min collected is centrifuged 10min, repeated centrifugation twice, cell is carried out resuspended by rear use DMEM/F12 culture medium, obtains chicken salpingo epithelial cell。By the chicken salpingo epithelial cell that obtains at 37 DEG C, 5%CO2Cell culture incubator in hatch 2.5h。After hatching, by 670rcf/min after the cell harvesting of adherent growth non-in the process of hatching, 10min is centrifuged, collected after centrifugation cell is with containing DMEM/F12, the 20g/L epidermal growth factor containing hyclone that volume fraction is 5%, 100mg/L heparin sodium, 2.5mg/L insulin, 200mmol/L glutamine, 200 μ g/mL penicillins, the disposable T that the culture medium of 200 μ g/mL streptomycins was coated at 20% hyclone25Culture bottle carries out cell cultivation。
As seen from the above embodiment, the primary chicken salpingo epithelial cell that method culture of isolated of the present invention is cultivated is effective, and high cell growth speed is in good condition；Heteroproteose cell is few, it is to avoid fibroblastic pollution can obtain the epithelioid cell group perfected。
The above is only the preferred embodiment of the present invention; it should be pointed out that, for those skilled in the art, under the premise without departing from the principles of the invention; can also making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention。
Claims (10)
1. a method for chicken salpingo epithelial cell Isolation and culture, comprises the following steps:
1) from the laying hen aseptic chorista of infundibulum of uterine tube cervical region near follicle；
2) use described in pancreatin and EDTA simultaneous digestion and separate the tissue obtained, obtain chicken salpingo epithelial cell；
3) described chicken salpingo epithelial cell 2h is hatched；
4) by described step 3) in hatch after cell carry out cell cultivation。
2. method according to claim 1, it is characterised in that step 1) described in the laying hen that laying hen is 25-28 week old。
3. method according to claim 1, it is characterised in that step 1) described in separate include gather tissue, shred tissue and cleansing tissue, described shredding is organized as and the tissue collected is cut into the length of side piece of tissue less than 1mm。
4. method according to claim 3, it is characterised in that the cleanout fluid that described cleansing tissue uses includes phosphate buffered solution, and the number of times of described cleaning is 5～8 times。
5. method according to claim 3, it is characterised in that described phosphate buffered solution contains penicillin and streptomycin is dual anti-。
6. method according to claim 1, it is characterised in that step 2) described in the concentration of pancreatin be 0.2～0.3wt%, the concentration of described EDTA is 0.01～0.03wt%。
7. method according to claim 1, it is characterised in that step 2) described in digestion time be 10～20min。
8. method according to claim 1, it is characterised in that step 3) described in the temperature hatched be 37 DEG C, described in hatch CO in environment2Volumetric concentration be 5%。
9. method according to claim 1, it is characterised in that step 4) described in cell cultivate device be the disposable T that 15～25% hyclones were coated25Culture bottle。
10. method according to claim 1, it is characterized in that, step 4) described in cell cultivate culture medium include following components: containing the hyclone DMEM/F12 that volume fraction is 4～6%, 15～25g/L epidermal growth factor, 80～120mg/L heparin sodium, 2～3mg/L insulin, 180～220mmol/L glutamine, 180～220 μ g/mL penicillins, 180～220 μ g/mL streptomycins。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106167788A (en) * | 2016-07-15 | 2016-11-30 | 四川农业大学 | Laying hen magnum tubae uterinae epithelial cell is cultivated and oxidative stress method for establishing model |
| CN116465700A (en) * | 2023-04-21 | 2023-07-21 | 上海腾瑞制药股份有限公司 | Method for detecting biological activity of active protein and polypeptide in medicine gel |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL218931B1 (en) * | 2011-11-18 | 2015-02-27 | Univ T Przyrodniczy Im Jana I Jędrzeja Śniadeckich | Method for obtaining epithelial cells from the chicken oviduct in primary culture in vitro |
| CN104818240A (en) * | 2015-04-27 | 2015-08-05 | 山东省农业科学院畜牧兽医研究所 | In-vitro culture method of mouse oviduct epithelial cells |
-
2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| PL218931B1 (en) * | 2011-11-18 | 2015-02-27 | Univ T Przyrodniczy Im Jana I Jędrzeja Śniadeckich | Method for obtaining epithelial cells from the chicken oviduct in primary culture in vitro |
| CN104818240A (en) * | 2015-04-27 | 2015-08-05 | 山东省农业科学院畜牧兽医研究所 | In-vitro culture method of mouse oviduct epithelial cells |
Non-Patent Citations (1)
| Title |
|---|
| 张秀平 等: "鸡原代输卵管上皮细胞体外分离培养与鉴定", 《中国兽药学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106167788A (en) * | 2016-07-15 | 2016-11-30 | 四川农业大学 | Laying hen magnum tubae uterinae epithelial cell is cultivated and oxidative stress method for establishing model |
| CN106167788B (en) * | 2016-07-15 | 2019-08-23 | 四川农业大学 | Laying hen magnum tubae uterinae epithelial cell culture and oxidative stress method for establishing model |
| CN116465700A (en) * | 2023-04-21 | 2023-07-21 | 上海腾瑞制药股份有限公司 | Method for detecting biological activity of active protein and polypeptide in medicine gel |
| CN116465700B (en) * | 2023-04-21 | 2023-11-28 | 上海腾瑞制药股份有限公司 | Method for detecting biological activity of active protein and polypeptide in medicine gel |
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