CN103820381B - A kind of primary culture method of striped rice borer cell - Google Patents
A kind of primary culture method of striped rice borer cell Download PDFInfo
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- CN103820381B CN103820381B CN201410030210.5A CN201410030210A CN103820381B CN 103820381 B CN103820381 B CN 103820381B CN 201410030210 A CN201410030210 A CN 201410030210A CN 103820381 B CN103820381 B CN 103820381B
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Abstract
The invention discloses the primary culture method of a kind of striped rice borer cell.Comprise the steps: that Chilo spp larvae is immersed in ethanol solution by (1), surface sterilization, clean and dry up polypide；(2) polypide is placed in dissection cake wax, dissects striped rice borer, take out and need to set up tissue or the organ of clone, clean；(3) be placed in the Tissue Culture Flask filling cell culture fluid in advance, put in cell culture incubator cultivate, overnight after, wait organize or organ adherent after, add cell culture fluid；(4) every sucking-off in several days with change to cell culture fluid, until extending and the cell bred is full of blake bottle；(5) containing newly breeding the individual cells, put in new blake bottle together with whole cell culture fluid sucking-offs, two blake bottles are put into and incubator is cultivated to Establishment of Cell Line initial success.The present invention contributes to studying striped rice borer in vitro, and the research and development for its controlling way provides help.
Description
Technical field
The present invention relates to belong to Insect cellculture technical field, particularly relate to the primary culture method of a kind of striped rice borer cell.
Background technology
Along with the fast development of life science, in technical field of bioengineering, cell engineering the most more and more comes into one's own.Cultivate insect cell as research material, always cell biology, molecular biology and the importance of the scientific research such as biochemistry and insect toxicology.
Since Grace set up giant silkworm moth clone first in 1962, Insect cellculture technology is developed rapidly, has the most established strain insect cell line more than 800, focus primarily upon Lepidoptera, Diptera (Pan Lizhen etc., 1980,1989；Zeng Qingtao etc., 1998), coleoptera (Lynn et al., 1995; Iwabuchi, 1999;Stiles, 1992), Orthoptera (Heinandez-Crespo et al., 2000), Hymenoptera, Homoptera and Semiptera etc., wherein most is from Lepidoptera and Diptera, derive from other purpose insect cell lines and only account for about 1/10 (Zhang Huan etc., 2007).The foundation of insect cell line, not only promotes the basic research in terms of insect biology, and action oriented research of based on insect cell is also greatly facilitated.Wherein, insect cell-baculovirus expression system (BEVS) is current most efficient eukaryotic expression system, is widely used to the expression of the foreign genes such as beta-interferon.
In the insect cell line set up, study and apply most be Drosophila melanogaster (Drosophila
melanogasteR) S2 clone, cabbage looper (Trichoplusia ni) BTI-Tn-5B1-4(High Five) clone and meadow covet noctuid (Spodoptera frugiperda) Sf21 clone and clone strain Sf9 etc. thereof.For the extensive use of known up to a million kinds of insects and insect cell line, the clone having built up also is nowhere near, it is still necessary to sets up more insect cell line and is actually needed to meet.
Striped rice borerChilo suppressalis(Walker) belonging to Lepidoptera, Pyralidae, is to endanger one of the most serious Occurrence on China paddy rice, be injured in tillering stage and cause withered sheath, withered heart seedling, being injured in the fringe phase and cause insect bite strain and dead ears, the general time underproduction 3%～5%, time serious, the underproduction is more than 3 one-tenth.In addition to hazard rice, striped rice borer also endangers the crops such as corn, sugarcane, grain, broad bean, wild rice stem, Chinese sorghum, rape, wheat, Chinese milk vetch.Striped rice borer has the strongest adaptation ability, can rapidly adapt to adverse environment (such as resistant variety and insecticide), and it causes evil performance and quickly makes a variation.During the preventing and treating of striped rice borer, screening and the qualification of novel biopesticide are increasingly becoming research emphasis.The activity biologicall test of biological pesticide needs the striped rice borer examination worm that a large amount of growth is consistent, strict requirements are wanted to raising place, workload is big, if and have the striped rice borer cell of cultured in vitro, then can study the biological pesticide effect to striped rice borer in vitro, thus contribute to studying the insecticide mechanism of action to striped rice borer at cellular level, contribute to the improvement of current insecticide and the invention of new pesticides.Currently, with respect to Vitro Culture Techniques and the striped rice borer clone of striped rice borer cell, have not been reported.Therefore, by the present invention, set up striped rice borer clone, quantity and the kind of the new insect cell line of China can not only be increased, moreover it is possible to offer reference experience for the foundation of such insect cell line in the future.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that the primary culture method of a kind of striped rice borer cell.
The step of the primary culture method of striped rice borer cell is as follows:
(1) aseptically, Chilo spp larvae is immersed in 70% or 75% ethanol solution, carries out surface sterilization 10～20 minutes, clean Chilo spp larvae 3～5 times with sterile distilled water, blot polypide with aseptic filter paper or dry up polypide with hair-dryer；
(2) polypide is placed in the dissection cake wax crossed with 70% or 75% ethanol disinfection, to fix end to end with dissecting needle, dissect striped rice borer, take out and need to set up tissue or the organ of clone, tissue or organ are put in the culture dish filling HBSS cleaning fluid, clean 2～3 times, the most again with cell culture fluid cleansing tissue or organ；
(3) cleaned tissue or organ are placed in fill the T-25cm of 0.5mL～1mL cell culture fluid in advance2In Tissue Culture Flask, put in the cell culture incubator of 27 DEG C of unglazed photographs cultivate, overnight after, wait organize or organ adherent after, add 2mL cell culture fluid, make tissue block wholly or largely be immersed in this nutrient solution；
(4) every sucking-off in 7～10 days and the cell culture fluid that changes to 50%～70% amount, until extending and the cell bred is full of blake bottle；
(5) containing newly breeding the individual cells, put in new blake bottle together with whole cell culture fluid sucking-offs, add new cell nutrient solution, and the former blake bottle containing tissue block adds same sucking-off amount new cell nutrient solution as much, two blake bottles are put into and incubator is cultivated to Establishment of Cell Line initial success.
Described cell culture fluid is Insect cellculture liquid and penicillin, streptomysin, amphotericin B and the mixture of animal blood serum, and pH value is 6.0-6.8.Described Insect cellculture liquid is selected from TNM-FH, and described animal blood serum is hyclone.Described Penicillin Content is 200U/ml, and content of streptomycin is 0.2 μ g/ml, and amphotericin B content is 0.5 μ g/ml.Described animal blood serum volume ratio content is 10-20%.
The invention has the beneficial effects as follows: use such scheme, striped rice borer Various Tissues has been carried out original cuiture, has achieved preferable culture effect.The present invention is quickly, effective, repeatability is strong, is the important supplement cultivating lepidopteran insect cell.Striped rice borer is paddy rice important pests, and the present invention contributes to studying striped rice borer in vitro, provides help for the research of its controlling way and the exploitation of novel biopesticide.
Accompanying drawing explanation
Fig. 1 is to cultivate the cultivation aspect graph of fat body cells after 24 hours；
Fig. 2 is to cultivate the cultivation aspect graph of fat body cells after 15 days；
Fig. 3 is to cultivate the cultivation aspect graph of fat body cells after 30 days；
Fig. 4 is to cultivate the cultivation aspect graph of blood lymphocyte after 2 days；
Fig. 5 is to cultivate the cultivation aspect graph of blood lymphocyte after 16 days;
Fig. 6 is to cultivate the cultivation aspect graph of blood lymphocyte after 30 days.
Detailed description of the invention
The step of the primary culture method of striped rice borer cell is as follows:
(1) aseptically, Chilo spp larvae is immersed in 70% or 75% ethanol solution, carries out surface sterilization 10～20 minutes, clean Chilo spp larvae 3～5 times with sterile distilled water, blot polypide with aseptic filter paper or dry up polypide with hair-dryer；
(2) polypide is placed in the dissection cake wax crossed with 70% or 75% ethanol disinfection, to fix end to end with dissecting needle, dissect striped rice borer, take out and need to set up tissue or the organ of clone, tissue or organ are put in the culture dish filling HBSS cleaning fluid, clean 2～3 times, the most again with cell culture fluid cleansing tissue or organ；
(3) cleaned tissue or organ are placed in fill the T-25cm of 0.5mL～1mL cell culture fluid in advance2In Tissue Culture Flask, put in the cell culture incubator of 27 DEG C of unglazed photographs cultivate, overnight after, wait organize or organ adherent after, add 2mL cell culture fluid, make tissue block wholly or largely be immersed in this nutrient solution；
(4) every sucking-off in 7～10 days and the cell culture fluid that changes to 50%～70% amount, until extending and the cell bred is full of blake bottle；
(5) containing newly breeding the individual cells, put in new blake bottle together with whole cell culture fluid sucking-offs, add new cell nutrient solution, and the former blake bottle containing tissue block adds same sucking-off amount new cell nutrient solution as much, two blake bottles are put into and incubator is cultivated to Establishment of Cell Line initial success.
Described cell culture fluid is Insect cellculture liquid and penicillin, streptomysin, amphotericin B and the mixture of animal blood serum, and pH value is 6.0-6.8.Described Insect cellculture liquid is selected from TNM-FH, and described animal blood serum is hyclone.Described Penicillin Content is 200U/ml, and content of streptomycin is 0.2 μ g/ml, and amphotericin B content is 0.5 μ g/ml.Described animal blood serum volume ratio content is 10-20%.
Below in conjunction with example, the present invention is described in further detail:
Embodiment 1
The original cuiture of Chilo spp larvae fat body cells
Through the following step:
(1) in aseptic operating platform, (operation is the most below, institute's appliance will through sterilizing or disinfect) striped rice borer mature larva 1 is immersed in the ethanol solution of 70% 10 minutes, carry out surface sterilization, clean striped rice borer mature larva 3 times with sterile distilled water, blot striped rice borer mature larva body with aseptic filter paper or dry up striped rice borer mature larva body with hair-dryer；
(2) it is placed in in the 70% dissection cake wax sterilized by drying up striped rice borer mature larva body, to fix end to end with dissecting needle, striped rice borer is cut off from back with sterilization dissecting scissors, with dissecting needle, polypide is fixed again, fat-body tissue is taken out with elbow tweezers, keep it complete during operation as far as possible, note touching brokenly the part being joined directly together outside the alimentary canal equivalents of insect；
(3) fat-body is put into fill HBSS cleaning fluid (penicillin containing 200U/ml, 0.2 μ g
The streptomysin of/ml, the amphotericin B of 0.5 μ g/ml) culture dish in, clean fat-body 2 times with HBSS cleaning fluid, clean fat-body 1 time with cell culture fluid the most again；
(4) cleaned fat-body is placed in fills the T-25cm of 0.5mL cell culture fluid in advance2In Tissue Culture Flask, put in the cell culture incubator of 27 DEG C of unglazed photographs cultivate, overnight after, after fat-body is adherent, add 2mL cell culture fluid, make fat-body be mostly submerged in this nutrient solution, note not making fat-body be suspended in cell culture fluid liquid feeding when；
(5) every the cell culture fluid of 7 days sucking-off 50% amounts, and change to the new nutrient solution of sucking-off amount simultaneously, tissue block can be observed after 24 hours in vitro and under above-mentioned condition of culture, quickly from the cell (see figure 1) dissociating the most single about after aforesaid operations；See cell after 15th day constantly rise in value and be gradually filled with whole blake bottle (see figure 2)；After 30th day, proliferative cell is full of whole blake bottle (see figure 3), the cell containing added value, put in new blake bottle together with whole cell culture fluid sucking-offs, and add the new cell culture fluid of 2mL, and the former blake bottle containing tissue block adds same sucking-off amount new cell culture fluid as much.Two blake bottles are put in incubator and are cultivated, and indicate original cuiture success, and cell starts to pass on.Chilo spp larvae fat body cells system is tentatively successfully established.
Embodiment 2
The original cuiture of Chilo spp larvae blood lymphocyte
(1) in aseptic operating platform, (operation is the most below, institute's appliance will through sterilizing or disinfect) striped rice borer mature larva 1 is immersed in the ethanol solution of 75% 20 minutes, carry out surface, clean striped rice borer mature larva 5 times with sterile distilled water, blot striped rice borer mature larva body with aseptic filter paper or dry up striped rice borer mature larva body with hair-dryer；
(2) it is placed in in the 75% dissection cake wax sterilized by drying up striped rice borer mature larva body, to fix end to end with dissecting needle, cut off striped rice borer with sterilization dissecting scissors from back, draw hemolymph with 20 μ L pipettors and put into equipped with HBSS cleaning fluid (penicillin containing 200U/ml, 0.2 μ g
The streptomysin of/ml, the amphotericin B of 0.5 μ g/ml) blake bottle in, tighten lid, static placement 15 minutes.Note touching brokenly the part being joined directly together outside the alimentary canal equivalents of insect；
(3) after blood lymphocyte is adherent, HBSS cleaning fluid is absorbed with pipettor, add fresh HBSS cleaning fluid, it is repeated 3 times, to prevent the generation of melanism, after absorbing HBSS cleaning fluid at the 3rd time, add 2mL cell culture fluid, tighten bottle cap, blake bottle is put in the cell culture incubator of 27 DEG C of unglazed photographs and cultivate；
(4) every the cell culture fluid of 10 days sucking-off 70% amounts, and change to the new nutrient solution of sucking-off amount simultaneously.
(5) after aforesaid operations, the individual cells (see figure 4) in a large number bred after 2 days, be can be observed and extend the most to the periphery；See cell continuous wire drawing growth after 16th day, rise in value and be full of whole blake bottle (see figure 5)；See proliferative cell after 30th day and be full of whole blake bottle (see figure 6)；The cell containing added value, put in new blake bottle together with whole cell culture fluid sucking-offs, according to 1:5(cell volume: last nutrient solution volume) carry out Secondary Culture.Chilo spp larvae blood lymphocyte system is tentatively successfully established.
Claims (1)
1. the primary culture method of a striped rice borer cell, it is characterised in that comprise the steps:
(1) aseptically, Chilo spp larvae is immersed in 70% or 75% ethanol solution, carries out surface sterilization 10～20 minutes, clean Chilo spp larvae 3～5 times with sterile distilled water, blot polypide with aseptic filter paper or dry up polypide with hair-dryer；
(2) polypide is placed in the dissection cake wax crossed with 70% or 75% ethanol disinfection, to fix end to end with dissecting needle, dissect striped rice borer, take out and need to set up tissue or the organ of clone, tissue or organ are put in the culture dish filling HBSS cleaning fluid, clean 2～3 times, the most again with cell culture fluid cleansing tissue or organ；
(3) cleaned tissue or organ are placed in fill the T-25cm of 0.5mL～1mL cell culture fluid in advance2In Tissue Culture Flask, put in the cell culture incubator of 27 DEG C of unglazed photographs cultivate, overnight after, wait organize or organ adherent after, add 2mL cell culture fluid, make tissue block wholly or largely be immersed in this nutrient solution；
(4) every sucking-off in 7～10 days and the cell culture fluid that changes to 50%～70% amount, until extending and the cell bred is full of blake bottle；
(5) containing newly breeding the individual cells, put in new blake bottle together with whole cell culture fluid sucking-offs, add new cell culture fluid, and the former blake bottle containing tissue block adds same sucking-off amount new cell culture fluid as much, two blake bottles are put into and incubator is cultivated to Establishment of Cell Line initial success；
Described cell culture fluid is Insect cellculture liquid and penicillin, streptomysin, amphotericin B and the mixture of animal blood serum, and pH value is 6.0-6.8；
Described Insect cellculture liquid is selected from TNM-FH, and described animal blood serum is hyclone；
Described Penicillin Content is 200U/ml, and content of streptomycin is 0.2 μ g/ml, and amphotericin B content is 0.5 μ g/ml；
Described animal blood serum volume ratio content is 10-20%；
Containing the penicillin of 200U/ml, the streptomysin of 0.2 μ g/ml and the amphotericin B of 0.5 μ g/ml in described HBSS cleaning fluid.
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| CN105820994A (en) * | 2015-01-06 | 2016-08-03 | 中国计量学院 | Primary culture method of ampullaria gigas hemocyte |
| CN105176911A (en) * | 2015-01-07 | 2015-12-23 | 中国计量学院 | Chilo suppressalis larva midgut cell line with high yield of baculovirus |
| CN105820995A (en) * | 2015-01-07 | 2016-08-03 | 中国计量学院 | Method for collecting Chilo suppressalis fat body used for cell culture |
| CN105861417A (en) * | 2016-06-06 | 2016-08-17 | 中国计量大学 | Establishment method for pomacea canaliculata ovary cell line |
| CN107129962B (en) * | 2017-05-18 | 2019-03-05 | 中国计量大学 | A kind of primary culture method of Hirudo japonica salivary gland cell |
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| CN101182487A (en) * | 2007-11-14 | 2008-05-21 | 山东大学 | Insect epidermis cell line as well as construction method and use thereof |
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| CN101182487A (en) * | 2007-11-14 | 2008-05-21 | 山东大学 | Insect epidermis cell line as well as construction method and use thereof |
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| 昆虫细胞系的培养和建立技术;张寰等;《昆虫学报》;20070831;第50卷(第8期);第834 -839页 * |
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