CN102757939A - Tsingtao amphioxus cell line culture fluid and preparation method thereof - Google Patents
Tsingtao amphioxus cell line culture fluid and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of cytobiology. The successful subculture of Tsingtao amphioxus gill primary cells is implemented by cDNA transfection of human telomere enzyme catalysis subunit hTERT, thereby establishing the Tsingtao amphioxus cell line. The invention provides a cell culture fluid for cell line establishment, which uses a Leiboviz's L15 culture medium as the basal culture medium and contains the following components: at least one essential amino acid and non-essential amino acid, at least one vitamin, at least one salt, at least one buffer solution and saccharide, and at least one phenolic compound. The invention also provides a method for preparing the culture fluid. The invention successfully solves the technical problems in in-vitro subculture of amphioxus cells, and is beneficial to deep research in the fields of genetics, immunology, evolution, auxology, genomics and the like.
Description
The application be that December 29, application number in 2010 are 201010612800.0 the applying date, the application people is Zhongshan University, invention and created name dividing an application for the Chinese invention patent application of " a kind of Qingdao branchiostoma clone and establishment method thereof ".
Technical field
The invention belongs to the cytobiology field, be specifically related to a kind of Qingdao branchiostoma clone nutrient solution, and the method for preparing this nutrient solution.
Background technology
Ocean model animal lancelet, the Latin formal name used at school is Branchiostoma belcheri, belongs to protochordate, has both kept some characteristics of invertebrates, has vertebrate characteristics again, is the transitional type between invertebrates and vertebrates.Lancelet just occurred before 500,000,000 years, was considered to existing vertebrates first ancestor representative, have the title of " living fossil ".Because its unique evolution status, the typical module animal with fetal development of being considered to zoologize to evolve has very high evolution status academicly, is the domestic and international focus of heredity, immunity, evolution, growth and genomics research in recent years.But lancelet is very strict and harsh to the requirement of habitat ecotope, and distribution range is very narrow in the world, and output seldom.It live in sandy in, require transparency of sea water higher, clean water quality, the depth of water be at 5-10 rice, optimal salinity is to contain 14-29 gram salt in every liter of seawater, potential of hydrogen is at 8.1-8.2.It is coastal that lancelet mainly is distributed in Xiamen, Qingdao and Yantai in China.
Be the further research of in-depth lancelet, setting up lancelet clone is a vital link.The 1980s, just the someone attempted cultivating the lancelet cell, but was confined to formerly be commissioned to train fosterly, and cell can't go down to posterity.Support the method for lancelet cell as just disclosing a kind of former being commissioned to train in " Xiamen University's journal (natural science edition) " B05 phase 181-183 page or leaf " the former foster preliminary study of being commissioned to train of lancelet cells in-vitro " in 2006 literary composition.So far also still not having the lancelet cell builds the report that is both at home and abroad, and research lancelet gene function can only carry out through the clone of other species.Its major cause is that the lancelet cell is not bred external, gets into senescence phase rapidly, thereby the cultivation of can't increasing and go down to posterity.
Telomere is the terminal a kind of special construction that placed in-line short segments Tumor-necrosis factor glycoproteins (TTAGGG) n of 6kb and some conjugated protein compositions are arranged of cell chromosome.Cell gets into replicability in the vitro culture process old and feeble relevant with telomere.Each cell fission telomere is lost 30-50 Nucleotide.Telomerase has been avoided the unstable of end of chromosome through keeping and extending the terminal telomere of cell chromosome.Think that at present a key point in the cell immortality process is to keep or prolong the telomere of cell chromosome, Telomerase is expressed again.Telomerase catalytic subunit TERT plays a part to cause the pass in keeping the activity of Telomerase important.An example of the prior art is; The research group of Choy-Pik Chiu imports the HSF with human telomerase catalytic subunit hTERT; This transfectional cell can go down to posterity external continuing, and keeps the biological characteristics of parental cell, has contact to suppress equally.People such as Carmela P.Morales import the HSF with hTERT equally, and they find that the inoblast of high expression level hTERT can carry out 280 population doublings (PD), and parental cell then has only 70-80PD.The rate of propagation and the parental cell of these cells are basic identical.Another example of the prior art is, ataxia-telangiectasia Ataxia-telangiectasia (A-T) patient is very responsive to ionizing radiation, and vitro culture A-T patient's cell is difficulty very, relies on high density serum, and is easy to aging.People such as Wood LD have suppressed cell aging effectively with the inoblast that hTERT imports A-T patient.
Summary of the invention
The objective of the invention is to solve to realize in the prior art exsomatizing going down to posterity and cultivate the technical barrier of lancelet cell; A kind of stable Qingdao branchiostoma clone and the method for setting up this clone are provided; Set up good experiment porch for the research of lancelet, be beneficial to further deep research heredity, immunity, evolution, growth and genomics.
Another object of the present invention provides a kind of nutrient solution that is used to cultivate said Qingdao branchiostoma clone and preparation method thereof.
Qingdao branchiostoma clone of the present invention is through the cDNA transfection Qingdao branchiostoma cheek primary cell of human telomerase catalytic subunit hTERT.
In the Qingdao branchiostoma clone provided by the invention, the expression vector of described human telomerase catalytic subunit hTERT is retroviral vector pBABE.
In the Qingdao branchiostoma clone provided by the invention, described primary cell is a lymphocytoid cell.
The present invention also provides a kind of method of setting up said Qingdao branchiostoma clone, may further comprise the steps:
1) cultivates Qingdao branchiostoma cheek primary cell: the Qingdao branchiostoma ice bath is extremely gone into a coma 70～75% alcohol disinfecting body surfaces, aseptic dissection; Get lancelet cheek tissue,, be cut into the fine tissue piece with after containing the cell culture fluid rinsing of penicillium mould and Streptomycin sulphate; It is moved to six porocyte culture plates, and coating is even, and 25～29 ℃ of following constant temperature leave standstill to cultivate and make it adherent; Every day, the observation of cell growth conditions and was added 0.5～1ml cell culture fluid; Change the cell culture fluid of 1/2 volume after 3 days;
2) with the cDNA transfection Qingdao branchiostoma cheek primary cell of human telomerase catalytic subunit hTERT: transfection was selected the good 293T cell of growth conditions in preceding 24 hours, after going down to posterity after the trysinization, was inoculated in during 24 holes cultivate every hole 4X10
5Individual cell is treated to be used for transfection after the cell fraction of coverage reaches 60%; The transfection step is following: the cell culture fluid thorough mixing and the room temperature of 8 μ l lipofectamine2000 and 500 μ l antibiotic-frees, serum-free were left standstill 5 minutes; Meanwhile, the cell culture fluid thorough mixing with 2 μ g packaging plasmid pCL-Ampho and 2 μ g pBABE-hTERT and 500 μ l antibiotic-free serum-frees left standstill above-mentioned two kinds of mixed solution mixings and room temperature 20 minutes after 5 minutes.After 20 minutes this mixed solution is joined in the 293T cell, supply nutrient solution, 37 ℃, 5%CO to 2ml
2The middle cultivation was changed to common nutrient solution after 6 hours, collected supernatant after 48 hours, with the membrane filtration of 0.45 μ m.Virus infection liquid 1/4 volume+lancelet cell culture fluid 3/4 volume mixes the back and adds primary cell;
3) obtain survivaling cell, and the cultivation of going down to posterity: after the cell of transfection hTERT began propagation, cell number increased, and when the cell stand density is big, inhaled the method diluted passage suspension cell of beating gently with transfer pipet, and the Dilution ratio that goes down to posterity is 1:4; The visual cell's growth conditions that at every turn goes down to posterity later on went down to posterity once on the 3rd～4;
4) carry out cytobiology and identify, the checking cell characteristic.
The present invention also provides a kind of said nutrient solution of setting up the method for Qingdao branchiostoma clone that is exclusively used in; This nutrient solution is a basic medium with Leiboviz ' s L15 substratum, and contains following component: at least a necessary amino acid and nonessential amino acid, at least a VITAMINs, at least a salt, at least a damping fluid and carbohydrate, at least a phenolic cpd;
Said amino acid is glycocoll, L-Ala, l-arginine, l-asparagine, halfcystine, Stimulina, Histidine, Isoleucine, leucine, Methionin, phenylalanine(Phe), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan;
Described VITAMINs is choline chloride 60, VA, folic acid, vitamin PP, Y factor, vitamin G, thiamine monophosphate chloride, i-Inositol;
Described salt is calcium chloride, magnesium chloride, sal epsom, Repone K, potassium primary phosphate, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, Sodium.alpha.-ketopropionate;
Described carbohydrate is a D-galactose;
Said phenolic cpd is phenol red;
The osmotic pressure of said nutrient solution is 750-800mOsm/kg;
The pH of said nutrient solution is 7.2-7.4;
Add in said nutrient solution before using that to account for volume percent be 10% foetal calf serum.
The nutrient solution that is exclusively used in the method for setting up Qingdao branchiostoma clone of the present invention, each component concentration is: glycocoll is 380-420mg/L, and the L-L-Ala is 400-480mg/L, and the L-L-arginine is 800-1100mg/L; Altheine acid is 450-530mg/L, and the L-halfcystine is 200-260mg/L, and L-glutaminate is 570-620mg/L, and the L-Histidine is 480-520mg/L; The L-Isoleucine is 480-520mg/L, and the L-leucine is 220-270mg/L, and L-Methionin is 120-170mg/L, and the L-methionine(Met) is 120-170mg/L; The L-phenylalanine(Phe) is 230-280mg/L, and the L-Serine is 360-420mg/L, and the L-Threonine is 550-610mg/L, and the L-tryptophane is 36-43mg/L; L-tyrosine is 530-620mg/L, and the L-Xie Ansuan is 160-220mg/L, and choline chloride 60 is 1.7-2.2mg/L, and the D-VA is 1.8-2.3mg/L; Folic acid is 1.7-2.2mg/L, and vitamin PP is 1.6-2.2mg/L, and Y factor is 1.8-2.3mg/L, and vitamin G is 0.18-0.22mg/L; Thiamine monophosphate chloride is 1.9-2.1mg/L, and i-Inositol is 3.6-4.3mg/L, and calcium chloride is 250-300mg/L; Magnesium chloride is 170-198mg/L, and sal epsom is 180-200mg/L, and Repone K is 770-810mg/L; Potassium primary phosphate is 110-130mg/L, and sodium-chlor is 19500-20100mg/L, and SODIUM PHOSPHATE, MONOBASIC is 370-390mg/L; Sodium.alpha.-ketopropionate is 1050-1120mg/L, and D-galactose is 1650-1900mg/L, and phenol red is 18-21mg/L.
The present invention also provides a kind of method for preparing said nutrient solution, and it may further comprise the steps:
It is in the ultrapure water of 18.2 Ω that each component is added 0.8 liter of resistivity; Osmotic pressure is allocated to 750-800mOsm/kg, potential of hydrogen is transferred to 7.0-7.2, using resistivity is that the ultrapure water of 18.2 Ω transfers to 1 liter with the TV of nutrient solution; Abundant mixing, 4 ℃ of preservations behind the filtration sterilization; Add before using that to account for volume percent be 10% foetal calf serum.
The present invention builds system, method and special culture solution provides a kind of Qingdao branchiostoma clone through above-mentioned; Successfully having solved stripped going down to posterity and having cultivated the technical barrier of lancelet cell; Help scientific research to Qingdao branchiostoma, and the corresponding further investigation that promotes fields such as heredity, immunity, evolution, growth and genomics.
Description of drawings
Fig. 1: the cheek tissue of lancelet is carried out formerly being commissioned to train fosterly, and cell free goes out tissue block after 12 hours.
Fig. 2: the part cell can stretch out long feeler under adherent state.
Fig. 3: the third generation passage cell that utilizes the lancelet clone that the present invention sets up.
Fig. 4: utilize lancelet clone that the present invention sets up the 6th generation passage cell.
Fig. 5: utilize lancelet clone that the present invention sets up the 8th generation passage cell.
Fig. 6: Wright-Gimsa coloration result.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit range of application of the present invention.
Qingdao branchiostoma clone provided by the invention is through the cDNA transfection Qingdao branchiostoma cheek primary cell of human telomerase catalytic subunit hTERT.The expression vector of described human telomerase catalytic subunit hTERT is retroviral vector pBABE.Described primary cell is a lymphocytoid cell.
Embodiment 1
The concrete steps of setting up said Qingdao branchiostoma clone provided by the invention are following:
1) cultivates Qingdao branchiostoma cheek primary cell: the Qingdao branchiostoma ice bath is extremely gone into a coma 70～75% alcohol disinfecting body surfaces, aseptic dissection; Get lancelet cheek tissue,, be cut into the fine tissue piece with after containing the cell culture fluid rinsing of penicillium mould and Streptomycin sulphate; It is moved to six porocyte culture plates, and coating is even, and 25～29 ℃ of following constant temperature leave standstill to cultivate and make it adherent; Every day, the observation of cell growth conditions and was added 0.5～1ml cell culture fluid; Change the cell culture fluid of 1/2 volume after 3 days.As shown in Figure 1, utilize lancelet cheek tissue carry out former be commissioned to train support after, visible attached cell dissociates out from organize with suspension cell, suspension cell is rounded, attached cell is polygon, but primary cell does not divide.
2) with the cDNA transfection Qingdao branchiostoma cheek primary cell of human telomerase catalytic subunit hTERT.The expression vector of described human telomerase catalytic subunit hTERT is that retroviral vector pBABE is (available from Addgene company, Plasmid1771#).The good 293T cell of growth conditions (available from typical case's culture collection council of Chinese Academy of Sciences cell bank) was selected in transfection in preceding 24 hours, after going down to posterity after the trysinization, was inoculated in the cultivation of 24 holes every hole 4X10
5Individual cell is treated to be used for transfection after the cell fraction of coverage reaches 60%; The transfection step is following: the cell culture fluid thorough mixing and the room temperature of 8 μ l lipofectamine2000 and 500 μ l antibiotic-frees, serum-free are left standstill 5min; Meanwhile, with the cell culture fluid thorough mixing of 2 μ g packaging plasmid pCL-Ampho (available from Imgenex company), after 5 minutes above-mentioned two kinds of mixed solution mixings and room temperature were left standstill 20 minutes with 2 μ g pBABE-hTERT and 500 μ l antibiotic-free serum-frees.After 20 minutes this mixed solution is joined in the 293T cell, supply nutrient solution, 37 ℃, 5%CO to 2ml
2The middle cultivation was changed to common nutrient solution after 6 hours, collected supernatant after 48 hours, with the membrane filtration of 0.45 μ m.Virus infection liquid 1/4 volume+lancelet cell culture fluid 3/4 volume mixes the back and adds primary cell.
Attached cell and suspension cell are carried out the hTERT transfection after 5 days simultaneously, and suspension cell begins division to occur, and has most of cell to be tending towards half adherent state gradually, few part cell attachment, and adhesion is closely bottom Tissue Culture Dish.Partly adherent and the adherent cellular form and the suspension cell in former generation are basic identical.Under inverted microscope, observe, all rounded though cell is not of uniform size, nucleus is positioned at the central authorities of cell.As shown in Figure 2, attached cell stretches out long feeler sometimes, but the longest lasting 2 days, feeler can fade away afterwards, the cellular-restoring original state.Along with the increase of passage number, the difference in size of cell has diminishing trend, but the phenomenon that cell differs in size still exists.Adherent polygon necrocytosis after the transfection has only circular suspension cell to survive.
3) obtain survivaling cell, and the cultivation of going down to posterity.After the cell of transfection hTERT began propagation, cell number increased, and when the cell stand density is big, inhaled the method for the beating suspension cell that goes down to posterity gently with transfer pipet, and the Dilution ratio that goes down to posterity is 1: 4; The visual cell's growth conditions that at every turn goes down to posterity later on went down to posterity once on the 3rd～4.Fig. 3 is a third generation passage cell, Fig. 4 be the 6th generation passage cell, Fig. 5 be the 8th generation passage cell.This shows that the cellular form in eight generations of the first-generation to the does not have fairly obvious difference, but proved that this cell successfully realized the cultivation of going down to posterity, is the lancelet clone that can go down to posterity and cultivate.To passing to for the 16th generation at present, cell proliferation is vigorous.
4) carry out cytobiology and identify, the checking cell characteristic.Cell suspension dripped to slide glass carry out smear, carry out Wright-Gimsa dyeing after the drying.The result is as shown in Figure 6.Judge that from coloration result this clone is lymphocytoid cell.
Embodiment 2～5
The nutrient solution component that is exclusively used in the above-mentioned method of setting up Qingdao branchiostoma clone provided by the invention is preferred:
Preparation embodiment
The preparation that is exclusively used in the cell culture fluid of setting up said Qingdao branchiostoma clone method provided by the invention:
In the ultrapure water (resistivity 18.2 Ω) with 0.8 liter of the adding of component shown in the table; Osmotic pressure is allocated to 750-800mOsm/kg, potential of hydrogen is transferred to 7.0-7.2, the TV of nutrient solution is transferred to 1 liter with ultrapure water (resistivity 18.2 Ω); Abundant mixing, 4 ℃ of preservations behind the filtration sterilization; Add before using that to account for volume percent be 10% foetal calf serum.
The method of setting up said Qingdao branchiostoma clone provided by the invention has the repetition reproducibility, for the person of ordinary skill in the field, as long as according to above-described concrete steps operation, can obtain Qingdao branchiostoma clone of the present invention.
Claims (3)
1. Qingdao branchiostoma clone nutrient solution; With Leiboviz ' s L15 substratum is basic medium, it is characterized in that also containing following component: at least a necessary amino acid and nonessential amino acid, at least a VITAMINs, at least a salt, at least a damping fluid and carbohydrate, at least a phenolic cpd;
Said amino acid is glycocoll, L-Ala, l-arginine, l-asparagine, halfcystine, Stimulina, Histidine, Isoleucine, leucine, Methionin, phenylalanine(Phe), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan;
Described VITAMINs is choline chloride 60, VA, folic acid, vitamin PP, Y factor, vitamin G, thiamine monophosphate chloride, i-Inositol;
Described salt is calcium chloride, magnesium chloride, sal epsom, Repone K, potassium primary phosphate, sodium-chlor, SODIUM PHOSPHATE, MONOBASIC, Sodium.alpha.-ketopropionate;
Described carbohydrate is a D-galactose;
Said phenolic cpd is phenol red;
The osmotic pressure of said nutrient solution is 750-800mOsm/kg;
The pH of said nutrient solution is 7.2-7.4;
Add in said nutrient solution before using that to account for volume percent be 10% foetal calf serum.
2. nutrient solution according to claim 1, it is characterized in that its each component concentration is: glycocoll is 380-420mg/L, and the L-L-Ala is 400-480mg/L, and the L-L-arginine is 800-1100mg/L; Altheine acid is 450-530mg/L, and the L-halfcystine is 200-260mg/L, and L-glutaminate is 570-620mg/L, and the L-Histidine is 480-520mg/L; The L-Isoleucine is 480-520mg/L, and the L-leucine is 220-270mg/L, and L-Methionin is 120-170mg/L, and the L-methionine(Met) is 120-170mg/L; The L-phenylalanine(Phe) is 230-280mg/L, and the L-Serine is 360-420mg/L, and the L-Threonine is 550-610mg/L, and the L-tryptophane is 36-43mg/L; L-tyrosine is 530-620mg/L, and the L-Xie Ansuan is 160-220mg/L, and choline chloride 60 is 1.7-2.2mg/L, and the D-VA is 1.8-2.3mg/L; Folic acid is 1.7-2.2mg/L, and vitamin PP is 1.6-2.2mg/L, and Y factor is 1.8-2.3mg/L, and vitamin G is 0.18-0.22mg/L; Thiamine monophosphate chloride is 1.9-2.1mg/L, and i-Inositol is 3.6-4.3mg/L, and calcium chloride is 250-300mg/L; Magnesium chloride is 170-198mg/L, and sal epsom is 180-200mg/L, and Repone K is 770-810mg/L; Potassium primary phosphate is 110-130mg/L, and sodium-chlor is 19500-20100mg/L, and SODIUM PHOSPHATE, MONOBASIC is 370-390mg/L; Sodium.alpha.-ketopropionate is 1050-1120mg/L, and D-galactose is 1650-1900mg/L, and phenol red is 18-21mg/L.
3. the method for preparing claim 1 or 2 described nutrient solutions; It is characterized in that may further comprise the steps: it is in the ultrapure water of 18.2 Ω that each component is added 0.8 liter of resistivity; Osmotic pressure is allocated to 750-800mOsm/kg, potential of hydrogen is transferred to 7.0-7.2, using resistivity is that the ultrapure water of 18.2 Ω transfers to 1 liter with the TV of nutrient solution; Abundant mixing, 4 ℃ of preservations behind the filtration sterilization; Add before using that to account for volume percent be 10% foetal calf serum.
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| CN101603026A (en) * | 2009-06-19 | 2009-12-16 | 华东理工大学 | Be suitable for the animal origin-free low-protein culture medium of zooblast products production |
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| CN101603026A (en) * | 2009-06-19 | 2009-12-16 | 华东理工大学 | Be suitable for the animal origin-free low-protein culture medium of zooblast products production |
Non-Patent Citations (3)
| Title |
|---|
| SIGMA-ALDRICH INC: "L-15 MEDIUM LEIBOVITZ，With L-glutamine,Product Number L4386,product information sheet", 《HTTP://WWW.SIGMAALDRICH.COM/ETC/MEDIALIB/DOCS/SIGMA/PRODUCT_INFORMATION_SHEET/1/L4386PIS.PAR.0001.FILE.TMP/L4386PIS.PDF》, 31 May 2007 (2007-05-31), pages 1 * |
| 石松林 等: "文昌鱼鳃组织原代培养", 《海洋科学》, vol. 33, no. 3, 31 December 2009 (2009-12-31), pages 44 - 48 * |
| 黄晓明 等: "文昌鱼（Branchiostoma belcheri）离体细胞原代培养初步研究", 《厦门大学学报（ 自然科学版）》, vol. 45, 31 December 2006 (2006-12-31), pages 181 - 183 * |
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Application publication date: 20121031 |