CN103436490A - Construction and ultralow temperature freezing preservation method of fin cell line of schizothorax grahami - Google Patents
Construction and ultralow temperature freezing preservation method of fin cell line of schizothorax grahami Download PDFInfo
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Abstract
The invention relates to construction and ultralow temperature freezing preservation method of a fin cell line of schizothorax grahami, and belongs to the technical field of culture and ultralow temperature ultralow temperature freezing preservation of cells of fresh water aquatic organisms. The method comprises the following steps of: culturing in a DMEM(Dulbecco Modified Eagle Medium)/F12 culture solution which contains fetal calf serum and cell growth factors and has the pH value of 7.0-7.2 by taking the ventral fin tissue of the schizothorax grahami as a material and adopting a tissue explant method; carrying out subculture by adopting a trypsin digestion method. The method particularly comprises the steps of cell culture solution preparation, primary culture and subculture. The construction method disclosed by the invention is short in primary culture time consumption and large in cell quantity and can be used for chromosome analysis; the constructed fin cell line of the schizothorax grahami is in a fiber-like form, can be subjected to serial passage and directly applied to biological characteristic research, meets the requirements for germplasm resource conservation and theoretical research and application of the schizothorax grahami and is suitable for constructing the fin cell lines of other fishes.
Description
Technical field:
The present invention relates to a kind of structure and cryopreservation method of Schizothorax grahami fin clone, belong to fresh water hydrobiont cell cultures and superfreeze Techniques of preserving field.
Background technology:
Fish cell is cultivated and is arised from the sixties in 20th century, and development has formed a set of cell culture system that comprises the comparatively perfects such as substratum, microbiotic, former culture and the cultural method that goes down to posterity so far, up to now, has set up more than 280 strain clones.China's fish cell is cultivated and is gone through more than 30 years, has also only set up more than 50 strain fish cell systems, and the kind related to is had an appointment 30 kinds, mainly take the ocean kind as main.From above example, can find out, the cell culture technology flow process of a set of maturation is not the essential condition of a strain Establishment of Cell Line success, the proven technique flow process can not make cell cultures succeed, cell cultures still need to be started with from the biological characteristics that primary cell species itself are provided, through arduous field study and a large amount of laboratory experiments, culture condition to cell culture system is adjusted, and finds optimum culture condition, just can make cell cultures succeed.Nearly 600 kinds of Yunnan original inhabitants freshwater fish, account for 50% of Freshwater Fishes In China, and therefore, Yunnan original inhabitants' fish germ plasma resource is preserved and seemed particularly important, but the rarely seen report of cell culture studies.
Schizothorax grahami Schizothorax grahami(Ragen; 1904) be under the jurisdiction of Cypriniformes (Cypriniformes) Cyprinidae (Cyprinidae) Schizothorax (Schizothorax); the popular name brachymystax lenok; whitefish; mainly be distributed in tributary, downstream, Jinsha jiang River, in IUCN(World Conservation Union) be listed in critically endangered species in the species Red List.Through making great efforts for many years, artificial propagation technology for schizothorax grahami was researched and developed successfully in 2013, and plan will be bred the fry basins such as Dian Chi and cattle pen river of releasing go back to, but the limitation due to cultured fishes itself, the pool is supported artificial propagation under environment and is easily caused chromosome abnormalty, as polyploidization and different times of change, need the cell Short-term Culture for karyotyping to detect the quality of artificial propagation kind, guarantee to release individual surviving rate and breeding potential; On the other hand, field is introduced a fine variety individual need for some time and could be adapted to the pool and support environment, and this laundering period is the outbreak period of various diseases, may bring extinction to introducing a fine variety individuality, how to carry out disease control and seem particularly important, and the resistant effect mechanism of drugs be the important step of disease control, resistant effect mechanism at the horizontal drugs of molecular cell is indispensable, therefore, need the construction construction method of a kind of Schizothorax grahami clone of research, set up Schizothorax grahami clone.
By literature search, have no and identical report of the present invention.
Summary of the invention:
The structure and the cryopreservation method that the purpose of this invention is to provide a kind of Schizothorax grahami fin clone easy to operation, preserve and the needs of theoretical investigation the quick karyotyping of Schizothorax grahami, germ plasm resource to meet.
The structure of Schizothorax grahami fin clone of the present invention and cryopreservation method, its concrete steps are as follows:
(1) prepare PBS thimerosal and cell culture fluid
The PBS thimerosal: add microbiotic in PBS, making penicillin concn is 200IU/ml, and Streptomycin sulphate concentration is 200 μ g/ml, and gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Basic culture solution: add foetal calf serum in the DMEM/F12 nutrient solution, make the foetal calf serum volume account for 10% of cumulative volume;
Primary nutrient solution: in the DMEM/F12 nutrient solution, add foetal calf serum, cell growth factor bFGF and microbiotic, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 10ng/ml, penicillin concn is 200IU/ml, Streptomycin sulphate concentration is 200 μ g/ml, gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Nutrient solution goes down to posterity: in the DMEM/F12 nutrient solution, add foetal calf serum, cell growth factor bFGF and microbiotic, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 6ng/ml, penicillin concn is 100IU/ml, Streptomycin sulphate concentration is 100 μ g/ml, and amphotericin B concentration is 10 μ g/ml;
The pH value of regulating above-mentioned nutrient solution is 7.0-7.2, is positioned in 4 ℃ of refrigerators and saves backup;
(2) former culture
Alcohol disinfecting Schizothorax grahami fish body with 75% three times, be placed in Bechtop, with aseptic disscting instrument, gets its abdomeinal fin, is placed in sterile petri dish, and the PBS thimerosal is cut into and organizes fritter with aseptic apparatus, and it is inoculated in to 25cm after cleaning abdomeinal fin tissue 5 times
2in Tissue Culture Flask, add primary nutrient solution 5ml to start former culture in 20 ℃ of incubators, every three and half amounts are changed nutrient solution, and within the 2nd day, cell starts to move from tissue block, within 20 days, grows up to cell monolayer;
(3) cultivation of going down to posterity
At the bottom of being paved with bottle, primary abdomeinal fin cell starts to go down to posterity after 80%, going down to posterity, it is as follows to cultivate concrete grammar, and the primary nutrient solution in first sucking-off culturing bottle adds 0.1% trypsinase-EDTA solution 1ml, digestion 2min, after cell rounding, add go down to posterity nutrient solution 1ml with in and pancreatin reaction, a piping and druming bottle end, attached cell is come off, the 1:1 by volume that goes down to posterity first passes, and after this 1:2 or 1:3 are gone down to posterity by volume, in 20 ℃ of incubators, continues to cultivate; Within every 5 days, go down to posterity once, while reaching for the 10th generation, cell culture fluid changes basic culture solution into, now, and the Establishment of Cell Line success;
(4) cell cryopreservation and recovery
(a) preparation cell cryopreservation protection liquid: frozen storing liquid comprises the DMEM/F12 nutrient solution, and foetal calf serum and DMSO press the volume ratio of 7:2:1 and mix, matching while using;
(b) cell cryopreservation: the cell in the vegetative period of taking the logarithm, after above-mentioned trysinization, the centrifugal 8min of cell suspension 1200rpm, discard supernatant liquor, and to the cells frozen storing liquid that adds preparation in cell precipitation, resuspended, the concentration that makes cell is 1 * 10
6individual/ml, be transferred to the 1ml cell suspension in the 1.8ml cryopreservation tube, and cryopreservation tube is placed in to program temperature reduction box, and-80 ℃ are spent the night, and then puts into the medium-term and long-term preservation of liquid nitrogen;
(c) cell recovery: above-mentioned cryopreservation tube is taken out from liquid nitrogen, putting into 37 ℃ of water-baths rocks fast to thawing, then in aseptic operating platform, the cell that will thaw is transferred in the 15ml centrifuge tube, and adds the equivalent basic culture solution, the centrifugal 8min of 1000rpm, except supernatant liquor, use the basic culture solution re-suspended cell, and be transferred in Tissue Culture Flask, in 20 ℃ of incubators, cultivate, within 7 days, cell grows up to individual layer.
In each step of the present invention, per-cent used is volume percent
Unusual effect of the present invention is:
1, easy to operation.
2, the cell just moved out in the abdomeinal fin tissue block has activity, and cell concentration is large, can be used for chromosome analysis.
3, the Schizothorax grahami abdomeinal fin clone form built is for becoming fiber-like, can continuous passage and directly apply to biological characteristic research, met the needs to the preservation of Schizothorax grahami germ plasm resource and theoretical investigation and application;
4, this construction process also is applicable to other fish and builds abdomeinal fin clone.
Embodiment:
Structure and the cryopreservation method of the Schizothorax grahami fin clone of the present embodiment, its concrete steps are as follows:
(1) prepare PBS thimerosal and cell culture fluid
The PBS thimerosal: add microbiotic in PBS, making penicillin concn is 200IU/ml, and Streptomycin sulphate concentration is 200 μ g/ml, and gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Basic culture solution: add foetal calf serum in the DMEM/F12 nutrient solution, make the foetal calf serum volume account for 10% of cumulative volume;
Primary nutrient solution: in the DMEM/F12 nutrient solution, add foetal calf serum, cell growth factor bFGF and microbiotic, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 10ng/ml, penicillin concn is 200IU/ml, Streptomycin sulphate concentration is 200 μ g/ml, gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Nutrient solution goes down to posterity: in the DMEM/F12 nutrient solution, add foetal calf serum, cell growth factor bFGF and microbiotic, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 6ng/ml, penicillin concn is 100IU/ml, Streptomycin sulphate concentration is 100 μ g/ml, and amphotericin B concentration is 10 μ g/ml;
The pH value of regulating above-mentioned nutrient solution is 7.0 or 7.2, is positioned in 4 ℃ of refrigerators and saves backup;
(2) former culture
Alcohol disinfecting Schizothorax grahami fish body with 75% three times, be placed in Bechtop, with aseptic disscting instrument, gets its abdomeinal fin, is placed in sterile petri dish, and the PBS thimerosal is cut into and organizes fritter with aseptic apparatus, and it is inoculated in to 25cm after cleaning abdomeinal fin tissue 5 times
2in Tissue Culture Flask, add primary nutrient solution 5ml to start former culture in 20 ℃ of incubators, every three and half amounts are changed nutrient solution, and within the 2nd day, cell starts to move from tissue block, within 20 days, grows up to cell monolayer;
(3) cultivation of going down to posterity
At the bottom of being paved with bottle, primary abdomeinal fin cell starts to go down to posterity after 80%, going down to posterity, it is as follows to cultivate concrete grammar, and the primary nutrient solution in first sucking-off culturing bottle adds 0.1% trypsinase-EDTA solution 1ml, digestion 2min, after cell rounding, add go down to posterity nutrient solution 1ml with in and pancreatin reaction, a piping and druming bottle end, attached cell is come off, the 1:1 by volume that goes down to posterity first passes, and after this 1:2 or 1:3 are gone down to posterity by volume, in 20 ℃ of incubators, continues to cultivate; Within every 5 days, go down to posterity once, while reaching for the 10th generation, cell culture fluid changes basic culture solution into, now, and the Establishment of Cell Line success;
(4) cell cryopreservation and recovery
(a) preparation cell cryopreservation protection liquid: frozen storing liquid comprises the DMEM/F12 nutrient solution, and foetal calf serum and DMSO press the volume ratio of 7:2:1 and mix, matching while using;
(b) cell cryopreservation: the cell in the vegetative period of taking the logarithm, after above-mentioned trysinization, the centrifugal 8min of cell suspension 1200rpm, discard supernatant liquor, and to the cells frozen storing liquid that adds preparation in cell precipitation, resuspended, the concentration that makes cell is 1 * 10
6individual/ml, be transferred to the 1ml cell suspension in the 1.8ml cryopreservation tube, and cryopreservation tube is placed in to program temperature reduction box, and-80 ℃ are spent the night, and then puts into the medium-term and long-term preservation of liquid nitrogen;
(c) cell recovery: above-mentioned cryopreservation tube is taken out from liquid nitrogen, putting into 37 ℃ of water-baths rocks fast to thawing, then in aseptic operating platform, the cell that will thaw is transferred in the 15ml centrifuge tube, and adds the equivalent basic culture solution, the centrifugal 8min of 1000rpm, except supernatant liquor, use the basic culture solution re-suspended cell, and be transferred in Tissue Culture Flask, in 20 ℃ of incubators, cultivate, within 7 days, cell grows up to individual layer.
(5) in each step, per-cent used is volume percent.
Claims (1)
1. the structure of a Schizothorax grahami fin clone and cryopreservation method is characterized in that the concrete steps of the method are as follows:
(1) prepare PBS thimerosal and cell culture fluid
The PBS thimerosal: add microbiotic in PBS, making penicillin concn is 200IU/ml, and Streptomycin sulphate concentration is 200 μ g/ml, and gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Basic culture solution: add foetal calf serum in the DMEM/F12 nutrient solution, make the foetal calf serum volume account for 10% of cumulative volume;
Primary nutrient solution: in the DMEM/F12 nutrient solution, add foetal calf serum, cell growth factor bFGF and microbiotic, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 10ng/ml, penicillin concn is 200IU/ml, Streptomycin sulphate concentration is 200 μ g/ml, gentamicin concentration is 20 μ g/ml, and amphotericin B concentration is 20 μ g/ml;
Nutrient solution goes down to posterity: in the DMEM/F12 nutrient solution, add foetal calf serum, cell growth factor bFGF and microbiotic, make the foetal calf serum volume account for 15% of cumulative volume, bFGF concentration is 6ng/ml, penicillin concn is 100IU/ml, Streptomycin sulphate concentration is 100 μ g/ml, and amphotericin B concentration is 10 μ g/ml;
The pH value of regulating above-mentioned nutrient solution is 7.0-7.2, is positioned in 4 ℃ of refrigerators and saves backup;
(2) former culture
Alcohol disinfecting Schizothorax grahami fish body with 75% three times, be placed in Bechtop, with aseptic disscting instrument, gets its abdomeinal fin, is placed in sterile petri dish, and the PBS thimerosal is cut into and organizes fritter with aseptic apparatus, and it is inoculated in to 25cm after cleaning abdomeinal fin tissue 5 times
2in Tissue Culture Flask, add primary nutrient solution 5ml to start former culture in 20 ℃ of incubators, every three and half amounts are changed nutrient solution, and within the 2nd day, cell starts to move from tissue block, within 20 days, grows up to cell monolayer;
(3) cultivation of going down to posterity
At the bottom of being paved with bottle, primary abdomeinal fin cell starts to go down to posterity after 80%, going down to posterity, it is as follows to cultivate concrete grammar, and the primary nutrient solution in first sucking-off culturing bottle adds 0.1% trypsinase-EDTA solution 1ml, digestion 2min, after cell rounding, add go down to posterity nutrient solution 1ml with in and pancreatin reaction, a piping and druming bottle end, attached cell is come off, the 1:1 by volume that goes down to posterity first passes, and after this 1:2 or 1:3 are gone down to posterity by volume, in 20 ℃ of incubators, continues to cultivate; Within every 5 days, go down to posterity once, while reaching for the 10th generation, cell culture fluid changes basic culture solution into, now, and the Establishment of Cell Line success;
(4) cell cryopreservation and recovery
(a) preparation cell cryopreservation protection liquid: frozen storing liquid comprises the DMEM/F12 nutrient solution, and foetal calf serum and DMSO press the volume ratio of 7:2:1 and mix, matching while using;
(b) cell cryopreservation: the cell in the vegetative period of taking the logarithm, after above-mentioned trysinization, the centrifugal 8min of cell suspension 1200rpm, discard supernatant liquor, and to the cells frozen storing liquid that adds preparation in cell precipitation, resuspended, the concentration that makes cell is 1 * 10
6individual/ml, be transferred to the 1ml cell suspension in the 1.8ml cryopreservation tube, and cryopreservation tube is placed in to program temperature reduction box, and-80 ℃ are spent the night, and then puts into the medium-term and long-term preservation of liquid nitrogen;
(c) cell recovery: above-mentioned cryopreservation tube is taken out from liquid nitrogen, putting into 37 ℃ of water-baths rocks fast to thawing, then in aseptic operating platform, the cell that will thaw is transferred in the 15ml centrifuge tube, and adds the equivalent basic culture solution, the centrifugal 8min of 1000rpm, except supernatant liquor, use the basic culture solution re-suspended cell, and be transferred in Tissue Culture Flask, in 20 ℃ of incubators, cultivate, within 7 days, cell grows up to individual layer.
(5) in each step, per-cent used is volume percent.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894050A (en) * | 2015-05-08 | 2015-09-09 | 西南大学 | Cryopreservation resuscitation method for silkworm embryonic cells infected with nosema bombycis |
| CN106508891A (en) * | 2016-10-27 | 2017-03-22 | 中国科学院昆明动物研究所 | Construction method of kidney cell line of Schizothorax taliensis |
| CN107641612A (en) * | 2017-09-30 | 2018-01-30 | 湖南师范大学 | Triploid cruciancarp caudal fin cell system 3nFC and its construction method and application |
| CN113943693A (en) * | 2021-09-27 | 2022-01-18 | 广东海洋大学 | Construction method of tilapia ventral fin cell line |
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| CN101372682A (en) * | 2008-10-15 | 2009-02-25 | 中国海洋大学 | Construction method of Epinephelus fuscoguttatus fin cell line |
| CN102492650A (en) * | 2011-11-24 | 2012-06-13 | 大连海洋大学 | Construction method for Hexagrammos otakii cell line |
| CN102757934A (en) * | 2012-08-07 | 2012-10-31 | 中国科学院昆明动物研究所 | Construction method of fin cell line of anabarilius grahami |
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2013
- 2013-09-05 CN CN201310400065.0A patent/CN103436490B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101372682A (en) * | 2008-10-15 | 2009-02-25 | 中国海洋大学 | Construction method of Epinephelus fuscoguttatus fin cell line |
| CN102492650A (en) * | 2011-11-24 | 2012-06-13 | 大连海洋大学 | Construction method for Hexagrammos otakii cell line |
| CN102757934A (en) * | 2012-08-07 | 2012-10-31 | 中国科学院昆明动物研究所 | Construction method of fin cell line of anabarilius grahami |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104894050A (en) * | 2015-05-08 | 2015-09-09 | 西南大学 | Cryopreservation resuscitation method for silkworm embryonic cells infected with nosema bombycis |
| CN106508891A (en) * | 2016-10-27 | 2017-03-22 | 中国科学院昆明动物研究所 | Construction method of kidney cell line of Schizothorax taliensis |
| CN106508891B (en) * | 2016-10-27 | 2019-09-20 | 中国科学院昆明动物研究所 | A kind of construction method of Dali schizothoracin kidney cell system |
| CN107641612A (en) * | 2017-09-30 | 2018-01-30 | 湖南师范大学 | Triploid cruciancarp caudal fin cell system 3nFC and its construction method and application |
| CN113943693A (en) * | 2021-09-27 | 2022-01-18 | 广东海洋大学 | Construction method of tilapia ventral fin cell line |
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