CN103232971A - A method for B lymphocyte transformation by EB virus (Epstein-barr virus) for elite ice and snow athletes - Google Patents
A method for B lymphocyte transformation by EB virus (Epstein-barr virus) for elite ice and snow athletes Download PDFInfo
- Publication number
- CN103232971A CN103232971A CN2013101231329A CN201310123132A CN103232971A CN 103232971 A CN103232971 A CN 103232971A CN 2013101231329 A CN2013101231329 A CN 2013101231329A CN 201310123132 A CN201310123132 A CN 201310123132A CN 103232971 A CN103232971 A CN 103232971A
- Authority
- CN
- China
- Prior art keywords
- cell
- frozen
- elite
- immortalized
- virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a method for B lymphocyte transformation by EB virus (Epstein-barr virus) for elite ice and snow athletes. According to the present invention, athlete immortalized cell lines are successfully constructed by taking B lymphocytes from athlete blood, transforming by the EB virus, and culturing by using a suspension culture method; and various tests of biological characteristics of the constructed cell lines are performed, establishing a relatively complete system of test techniques for biological characteristics. The object of the present invention is to provide immortalized cell lines with a high cell conversion rate, unlimited cell proliferation of elite athletes. The immortalized cell lines of elite athletes related in the present invention may provide a large number of high-quality materials for life science researches such as medical science and cell and molecular biology, and can be permanently preserved as physical quality-related genetic resources of the most outstanding ice and snow athletes of all mankind. The contradiction exists in the prior art that experimental studies are carried out in a relay type while specimens are collected temporarily is solved, in order to effectively carry out continuous and long-lasting researches on a same research object.
Description
Technical field
The present invention relates to a kind of cultural method of outstanding winter sports person's immortal cell line, namely transform outstanding winter sports person's bone-marrow-derived lymphocyte by Epstein-Barr virus and set up the method for immortality cell strain, belong to sports science, human resource and human cell's biology cross discipline and field.
Background technology
Mankind's inheritance resources comprises the human genome DNA, and it is important genetic material.Transform the human peripheral blood B lymphocyte with Epstein-Barr virus (Epstein-Barr Virus), make it become a kind of lymphoblastoid strain that can divide continuously, forever survive.Cell strain (Cell strain) refers to the culture with special property or sign that obtains by back-and-forth method or clone forming method from primary culture or clone.And immortalization (immorlization) does not still have the clear and definite concept of generally acknowledging at present, is commonly considered as cultured cell in vitro and escapes from the propagation crisis through influence spontaneous or extraneous factor, thereby have the process of unlimited multiplication capacity.The probability of spontaneous immortalization is very little, and rodent zooblast is 10
-5～10
-6, the human cell is then rarer, less than 10
-12Therefore, scholars change exogenous immutalizing gene in people's purpose cell by technology such as gene transfections, with the incidence of increase immortalization, and then set up immortalized cell line.The permanent lymphoblastoid strain of Jian Liing like this, i.e. immortality cell strain.The complete genome of the individuality that blood sample is provided has originally been preserved in each immortality cell strain.
Part as mankind's inheritance resources; motion gene genetic resource is human valuable wealth; but because motion gene genetic resource exists only in the limitation of sportsmen's individuality; if outstanding sportsmen is not taked the gene preservation work of protectiveness; let alone retired back motor capacity and disappear gradually until death, beyond doubt the massive losses of China's sports.Therefore, also do not have the mankind before the development and use campaign gene genetic resource of more reasonable science, preserve effectively and it is carried out necessary research and just seem and be even more important.Motion gene genetic resource has close contacting with the exploitation of motor capacity, perfect along with the Human Genome Project, and increasing motion gene is found, and also will go deep into more the research of its function.Given this, it is a kind of convenient just to need, and efficient, stable method is preserved motion gene genetic resource.Because China lacks good method to the preservation of peripheral blood and cell sample, can't do further research, causes the waste of the great outstanding motion genetic resources of China.In recent years, it is to set up the better method of continuous passage, cell strain that proterties is stable that the external transduction of using virogene makes cell immortalityization (immortalization), and the cell source can infinitely be provided, and is conducive to motion gene Study on Mechanism.
Up to now, can represent the only a few colony of the world's top athletes ' body quality gene, just might become world champion.Mankind's inheritance resources is that the mankind carry out self exploring and the irreplaceable valuable source of research, and the foundation in outstanding winter sports person's immortality cell storehouse is exactly for the most outstanding sportsmen genetic resources relevant with body constitution in can the permanent preservation universe.Simultaneously also carry out the contradiction that exists in the temporary collection with sample for solving the relay-type of experimental study in the past, in order to effectively same research object is carried out lasting continuity research.
The cell in healthy tissues source can grow and divide under common condition of in vitro culture, but through behind the passage of limited number of time, will stop propagation, takes place old and feeble and dead.This has just limited the further application of cell culture technology.Sexual cell, (embryo) stem cell, part tumour cell and allogenic gene transform the ability that the immortalized cells that makes up then has unlimited division and propagation, are immortalized cells.Therefore, scholars propose to set up immortalized cell line, make the cell of vitro culture have unlimited multiplication capacity and iuntercellular indifference.The cell immortality technical development has had significant progress to today, Epstein-Barr virus, SV40 virus all are immortalized cells transforming virus commonly used, and as papilloma virus E6, hepatitis B virus (HBV) though be not the virus commonly used that makes cell immortality, they can make the multiplication capacity of some special cells strengthen.
Make in the method and technology of its immortalization at numerous conversion cell in vitro, often convenient to gather sample, virus transforms immortalized cells rate height, short Epstein-Barr virus of operational cycle transforms the peripheral blood B lymphocyte and sets up immortal cell line, for preserving elite's gene, research influences the optimum method of motor capacity gene.Be suitable for the cultivation of external extensiveization cell.Therefore setting up immortalized cell line is good to adopt Epstein-Barr virus to transform the peripheral blood B lymphocyte.
The immortality cell strain that ER virus transforms the foundation of elite's peripheral blood B lymphocyte allows script have the human body cell in certain life-span to breed down incessantly, makes every field elite's gene be able to permanent preservation.Relevant individual gene not only can be forever preserved in the foundation of immortality cell strain, and owing to can be each motion of research group's gene structure difference, inquire into the Xiang Qun that respectively moves common ground and between difference, explore important inhibition motor capacity gene in each motion Xiang Qunzhong distribution characteristics and genetics meaning, set up a series of and endurance, strength, the item colony that speed is relevant is cell bank, and gene probe, many sports and scientific researches fields such as gene stimulant provide cell and DNA endlessly, will more and more be paid attention to so set up elite's immortality cell storehouse in Sports Scientific Research.
For the preservation of elite's cell, germiparity and activity all have higher requirement, at present, owing to be that exogenous virus, proto-oncogene etc. is led people's purpose cell, its gene integration is randomness, but unexpected change takes place, as loses the control of differentiating characteristic and cell cycle check point in physiological pathway in its expression product interference cell.Simultaneously, what obtain by transfections such as virus, proto-oncogenes is transformant but not normal cell, and its phenotype, caryogram etc. can change, and reduce contact inhibition forfeiture etc. as the serum dependency.And the present immortalized cell line of setting up not is all to be " infinite multiplication ", and what have has only tided over the M1 phase and prolonged cell survival etc.Therefore, be badly in need of now the standard that its method improvement makes elite's cell reach high cell transformation rate immortalization has been obtained good preservation and continues.
Summary of the invention
The elite's immortalized cell line that the purpose of this invention is to provide high cell transformation rate, high cell infinite multiplication.
Another object of the present invention is to provide the cultural method of above-mentioned immortalized cell line.
For achieving the above object, the present invention takes following technical scheme:
This elite's immortalized cell line has following feature: the B958 cell was being cultivated about 5 days, cell aggregation becomes the spherical cell mass of grape, the cell mass volume is bigger when watching under the inverted microscope, refractivity is good, the color of substratum also becomes yellow by original light red, but cell during confluent culture bottle bottom surface just equivalent divide bottle to go down to posterity to continue to cultivate, divide the cell density behind the bottle to reduce, cell is less cell mass or individual cells, and form remains unchanged round and full.
The cultural method of a kind of high conversion, infinite multiplication elite immortalized cell line, this method comprises the steps:
The preparation of 1EBV
1.1B958 cell recovery
(1) the B958 cell that in 40 ℃ of water-baths, thaws frozen;
The B958 cell that (2) will thaw moves to the 25cm that the full substratum of 6ml RPMI1640 is housed
2In the culturing bottle, place 37.5 ℃, 5%CO
2Cultivate in the incubator.
1.2B958 cell cultures
Treat that the substratum color becomes when isabelline, replenish the fresh culture of equivalent, continue to cultivate.
1.3B958 passage
When cell reaches about 3 * 10
5During/bottle, can go down to posterity.After cell fully shaken up, equivalent was sub-packed in 2 25cm
2In the culturing bottle, and in bottle, add the proper amount of fresh substratum, continue to cultivate.
1.4B958 cell cryopreservation
(1) 1d before the freeze-stored cell adds an amount of fresh culture in culturing bottle;
(2) collect the B958 cell in the 15ml centrifuge tube, the centrifugal 15min of 1500rpm removes supernatant under the normal temperature;
(3) use the frozen storing liquid re-suspended cell, adjusting cell density is 1 * 10
6～3 * 10
6Cell/ml is sub-packed in cell suspension in the frozen pipe with the 1ml/ pipe;
(4) earlier frozen pipe is placed 4 ℃ of refrigerator half an hour, put into then Virahol is housed the programmed cooling box in-80 ℃ of refrigerator overnight, drop into prolonged preservation in the liquid nitrogen container.
1.5EBV extract
(1) EBV solution is placed-80 ℃ of refrigerator 10min, 37 ℃ of water-bath freeze thawing then.So repeatable operation is 3 times.
(2) the centrifugal 15min of 1800rpm, 0.22 μ m membrane filtration, standby in-80 ℃ of preservations after the packing.
2 use EBV translational movement person bone-marrow-derived lymphocyte
2.1 blood specimen collection
(1) record blood sampling sportsmen personal information is corresponding with the numbering on the bloodletting tube;
(2) use the disposable bloodletting tube that has heparin sodium, extract sportsmen's venous blood 4ml/ people;
(3) the static placement 1～2d of blood sample normal temperature;
Annotate: if whole blood can not transform at that time at once, must do following processing: with blood sample 2500rpm at normal temperatures, centrifugal 15min, centrifugal back changes the tunica albuginea layer in the centrifuge tube that contains the full substratum of 3ml1640 with Pasteur's pipe, then centrifuge tube is put into 37.5 ℃ of incubators, be lying on the square plate.
2.2 cell immortalityization
(1) with the two anti-RPMI1640 basic mediums that add 1%, with sportsmen's hemodilution of gathering, blow even gently.
(2) lymphocyte separation medium places 37.5 ℃ of water-bath preheatings, during use its solution is shaken up.
(3) slowly pour the 4ml lymphocyte separation medium in the 15ml centrifuge tube that sportsmen's blood sample is housed, make the two interface clear, the centrifugal 10min of 3000rpm makes the blood layering;
(4) be divided into four layers in the centrifugal back centrifuge tube, the tunica albuginea layer that the one-tenth poplar on serum lower floor, red corpuscle upper strata is cotton-shaped is managed immigration with Pasteur the centrifugal 10min of 1000rpm is housed in the centrifuge tube of 6ml RPMI1640 substratum;
(5) after centrifugal supernatant liquor is discarded, add 10ml RPMI1640 substratum again, the centrifugal 10min of 1000rpm abandons supernatant liquor.After repeating once, abandoning supernatant is struck cell even stand-by;
(6) 2ml is contained full substratum, 1.4ml Epstein-Barr virus, the 0.4ml CyA of PHA and the white corpuscle of collecting and make mixed solution, altogether about 4ml;
(7) mixed solution is joined in 24 well culture plates every hole 1ml;
(8) 24 well culture plates are placed 37.5 ℃, 5%CO
2Cultivate in the incubator.
The cultivation of 3 sportsmen's immortalized cell lines and frozen
3.1 going down to posterity of cell
(1) immortalized cells is transferred to 25m from 24 orifice plates
2Culturing bottle in continue to cultivate;
(2) 3～4d carry out once half amount and change liquid, and each half amount is changed liquid and is passage one time.
3.2 cell is frozen
(1) collecting cell suspension, the centrifugal 10min of 1000rpm abandons
6Clearly;
(2) add the frozen storing liquid of predetermined amount in the centrifuge tube, cell is beaten gently even, the adjustment cell density is 1 * 10/ml～3 * 10
6/ ml installs to the frozen storing liquid branch in the frozen pipe, every pipe 1ml;
(3) pre-freeze: with frozen pipe pack into the cooling box in, be put in 4 ℃ of 20～30min, place-70 ℃ then more than the pre-freeze 4h, propose frozen pipe and put into
(4) frozen: as to propose frozen pipe and put into the liquid nitrogen cabinet, namely finish cell cryopreservation.
Above-mentioned high transfection efficiency,, the cultural method of wireless propagation elite immortalized cell line, wherein, can carry out cell recovery by the cell that step (4) is frozen according to actual needs, concrete steps are: frozen pipe is taken out from liquid nitrogen insert in 42 ℃ of water-baths, after rocking 1min continuously, cell moved in the culturing bottle be added with nutrient solution (RPMI1640+10% foetal calf serum) piping and druming evenly, place and contain 37.5 ℃ of 5%CO
2Incubator in can continue the cultivation of going down to posterity after continue cultivating 10～12h.
Advantage of the present invention and benefit: draw materials easily (several milliliters of peripheral bloods), it is stable, less demanding to substratum that karyomit(e) is built during strain is simple, cultured continuously goes down to posterity in conversion, save time economical, can obtain chromosome specimen synchronously, can be used for experiments such as cell hybridization, can obtain inexhaustible, nexhaustible DNA material, sustainable for every research.
Embodiment
Used key instrument and reagent source among the embodiment:
(1) main instruments and equipment
DL-CJ-2N high-performance aseptic experiment platform: east, Chinese Harbin connection electronic technology development corporation, Ltd.
37.5 ℃ 5%C02 incubator: German Heraeus company
TDL-40B horizontal centrifuge: the smart grand experimental instrument and equipment of Chinese Shanghai company limited
AR1530/C electronic balance: U.S. OHAUS company
IX-71 inverted phase contrast microscope: Japanese Olympus company
Electric-heated thermostatic water bath, blood counting chamber, liquid nitrogen container: BeiJing, China 61 factories
Pall ultrapure water instrument: German Pall company
Electronic pipettor: German Eppendorff company
Ultralow Temperature Freezer: company of China's Haier
Culture plate: U.S. Corning company
Grads PCR instrument: U.S. Applied Biosystems
The ultrapure water instrument Millipore U.S.
Gel imaging system: U.S. AlpHa Innotech
Ultraviolet spectrophotometer: U.S. Ultrospec1100pro GE
Ultraviolet spectrophotometer: U.S. ND-1000Nanodrop
Desk type high speed refrigerated centrifuge: German Eppendorf
Flow cytometer: U.S. Backman
(2) main agents and compound method
Elite's immortalized cell line is cultivated agents useful for same
(1)1N?NaOH
Take by weighing 2g NaOH and be dissolved in the 50ml tri-distilled water, 0.22 μ m membrane filtration degerming, 4 ℃ of preservations are standby.
(2)1N?Hepes
Take by weighing 11.925g Hepes and be dissolved in the 50ml tri-distilled water, with NaOH adjusting pH7.0～7.2,0.22 μ m membrane filtration degerming of 1N, 4 ℃ of preservations are standby.
(3)0.2M?L-Glutamine
Take by weighing 1.46g L-Glutamine and be dissolved in the 50ml tri-distilled water, place 37 ℃ of water-bath heating for dissolving, 0.22 μ m membrane filtration degerming ,-20 ℃ of preservations are standby.
(4) the RPMI1640 basic medium is with RPMI1640 dry powder and the 1g NaHCO of a packing
3Be dissolved in an amount of tri-distilled water, be settled to 1000ml, transfer about pH7.0,0.22 μ m filtering with microporous membrane degerming, the packing of 500ml/ bottle, 4 ℃ of preservations are standby.
(5) the full substratum of RPMI1640: RPMI1640 basic medium+10%FBS+1% is two to be resisted+1%L-Glutamine, regulates pH7.0 with Hepes, 0.22 μ m filtering with microporous membrane degerming, and 4 ℃ of preservations are standby.
(6) frozen storing liquid 95%FBS+5%DMSO
(7) preparation of CyA: take by weighing 5mg CyA and be dissolved in 250ml RPMI1640 basic medium, 0.22 μ m membrane filtration degerming-20 ℃ preservation is standby.
(8) preparation of PHA: 10mg PHA is dissolved in the 5ml physiological saline, and-20 ℃ of preservations are standby.
(9) contain the full substratum of RPMI1640 of PHA: RPMI1640 basic medium+20%FBS+1% two anti-+ 1%L-Glutamine+0.1%PHA, regulate PH7.0 with Hepes, 0.22 μ m filtering with microporous membrane degerming, 4 ℃ of preservations are standby.
(10) B958 primary cell strain
The microorganism detection agents useful for same:
(11) soybean Tryptones: 3g soybean Tryptones, add the 100ml tri-distilled water, transfer pH7.3, autoclaving 15～20min divides to install in the Glass tubing of 15mm * 150mm.
(12) wort: the 2g wort adds the 100ml tri-distilled water, transfers pH3.0～4.0, and autoclaving 15～20min divides to install in the Glass tubing of 15mm * 150mm.
(13) DAPI dye liquor: tri-distilled water preparation 1mg/ml DAPI storage liquid, the PBS dilution is 0.5～1mg/ml.
(14) citric acid of mounting liquid: 22.2ml0.1M, the Na of 27.8ml0.2M
2HPO
4Be dissolved in the 50ml glycerine, NaOH regulates pH5.5, is stored in 4 ℃.
(15) stationary liquid: Glacial acetic acid and methyl alcohol are with 1: 3 ratio of volume ratio (matching while using).
The chromosome specimen agents useful for same
(16) colchicine
1. stock solution: the 10mg colchicine is dissolved in the 10ml tri-distilled water.
2. working fluid: tri-distilled water is 100 μ g/ml with 10 times of stock solution dilutions to final concentration.
(17) hypotonic KCI solution
0.5g KCI be dissolved in the 100ml tri-distilled water.
(18) stationary liquid 2.
Glacial acetic acid: the ratio preparation of methyl alcohol (v/v)=1: 3.
(19) Giemsa staining fluid:
1. stoste: 1g Giemsa is dyed powder and 20ml glycerine is poured mortar into, in mortar, be ground to till the no particle.Glycerol adding is mended to 66ml, methyl alcohol 66ml.After mixed solution is placed on 56 ℃ of insulation can 2h, place brown bottle lucifuge, 4 ℃ of storages, minimum needs can use after one week.
2. working fluid: with PBS with Giemsa stoste dilution (PBS:Giemsa is 9: 1) matching while using.
Liposome transfection cell agents useful for same:
(20) plasmid DNA (DsRed, pEYFP-N1 and pEGFP-N3), liposome Lipofectamine does not contain the MEM nutrient solution of serum, contains the MEM nutrient solution of serum, the PBS of pH7.4.
The Isozyme Analysis agents useful for same:
(21) 0.9% (mass ratio) NaCl-0.06mmol EDTA solution: take by weighing 0.9g NaCI and 0.0223g EDTA respectively and be dissolved in the 100ml tri-distilled water and get final product.
(22) protein extract: with namely getting protein extract in 5ml TritonX-100 adding 75ml0.9% (mass ratio) the NaCl-0.06mmol EDTA solution, it is stored in 4 ℃ of refrigerators preserve.TritonX-100: 0.9% (mass ratio) NaCl-0.06mmolEDTA solution=1: 15.
(23) 40% (mass volume ratio) sucrose solution: 40g sucrose is dissolved in the 100ml water.
(24) preparation of sol solution
A liquid: 1mol/L HCI48.0ml, Tris36.6g, TEMED0.23ml transfers to pH8.9 with concentrated hydrochloric acid
B liquid: Acr40.0g, Bis2g
C liquid: ammonium persulphate 0.14g (now with the current)
D liquid: 1mol/L HCI48.0ml, Tris5.98g, TEMED0.46ml transfers to pH6.7 with concentrated hydrochloric acid
E liquid: Acr10.0g, Bis2.5g
F liquid: riboflavin 4.0mg
G liquid: sucrose 40.0g
(25) polyacrylamide gel electrophoresis separation gel and concentrated glue see Table 1
Table 1
(26) serum lactic dehydrogenase staining fluid and malate dehydrogenase (malic acid dehydrogenase) staining fluid see Table 2
Table 2
Cell cycle is detected agents useful for same
(27) PI dye liquor preparation (100ml): PI5mg, RNase2mg, 1.0%Triton X-1000.25ml, physiological saline 65ml, Citric Acid are received 100mg, and adding distil water is to 100ml, adjust pH 7.2-7.6, and with brown bottle packing, 4 ℃ keep in Dark Place.
Apoptosis detects agents useful for same:
(28) the two transfection reagent boxes of Annexin V-FITC/PI apoptosis: shellfish is won biological Chinese Shanghai
The structure of a kind of high conversion of embodiment, wireless propagation elite's immortalized cell line
(1) former foster the using of being commissioned to train adds two anti-RPMI1640 basic mediums of 1%, with sportsmen's hemodilution of gathering, blows even gently, in the 15ml centrifuge tube that sportsmen's blood sample is housed, slowly pour the 4ml lymphocyte separation medium into, make the two interface clear, the centrifugal 10min of 3000rpm makes the blood layering; Be divided into four layers in the centrifuge tube of centrifugal back, the tunica albuginea layer that the one-tenth poplar on serum lower floor, red corpuscle upper strata is cotton-shaped is managed immigration with Pasteur the centrifugal 10min of 1000rpm is housed in the centrifuge tube of 6ml RPMI1640 substratum; After centrifugal supernatant liquor is discarded, add 10ml RPMI1640 substratum again, the centrifugal 10min of 1000rpm abandons supernatant liquor.After repeating once, abandoning supernatant is struck cell even stand-by; 2ml is contained full substratum, 1.4ml Epstein-Barr virus, the 0.4ml CyA of PHA and the white corpuscle of collecting is made mixed solution, altogether about 4ml; Mixed solution is joined in 24 well culture plates, and every hole 1ml places 37.5 ℃ with 24 well culture plates, 5%CO
2Cultivate in the incubator.
(2) immortalized cells that goes down to posterity of cell is transferred to 25m from 24 orifice plates
2Culturing bottle in continue to cultivate; 3～4d carries out once half amount and changes liquid, and each half amount is changed liquid and is passage one time.
(3) the frozen collecting cell suspension of cell, the centrifugal 10min of 1000rpm abandons supernatant, adds the frozen storing liquid of predetermined amount in the centrifuge tube, cell is beaten gently even, and the adjustment cell density is 1 * 10/ml～3 * 10
6/ ml installs to the frozen storing liquid branch in the frozen pipe, every pipe 1ml, with frozen pipe pack into the cooling box in, be put in 4 ℃ of 20～30min, place-70 ℃ then more than the pre-freeze 4h, propose frozen pipe and put into the liquid nitrogen cabinet, namely finish cell cryopreservation.
(4) cell recovery: frozen pipe is taken out from liquid nitrogen, inserts rapidly in 42 ℃ of water-baths, rock 1 fen kind continuously after, seeing surplusly has little ice group to become the big h of soybean grain to put into Bechtop; Cell moved in the culturing bottle be added with nutrient solution (the superfine foetal calf serum of RPMI1640+10%) piping and druming gently evenly, place and contain 37.5 ℃ of 5%CO
2CO
2Can continue the cultivation of going down to posterity after continuing in the incubator to cultivate 10～12h.
The detection of embodiment 2 elite's immortalized cell line biological characteristicses
Elite's immortalized cell line that embodiment 1 is cultivated carries out the biological characteristics inspection, and method and conclusion are as follows.
One, morphological observation
Method: cell carries out routine examination to cell in the vitro culture process, and whether observation of cell growth conditions, nutrient solution colour-change situation and cell pollute.
Cell is single dispersion or less cell bulk, and at the bottom of the cell density, the microscopically refractivity is strong.Cultivated about 3 days, single lymphocyte or less lymphocyte are rolled into a ball just can assemble and are grown into bigger lymphocyte group, and cell density can reach 70%～80%; At the bottom of the visible fine sand saccharoid of the visual inspection confluent culture bottle, one shake and to float gently, as smog; The color of substratum becomes yellow by original pink colour, and pH value does not change substantially; There is the phenomenon of " long root ", " becoming mildewed ", " intermuscular needling fork " in microscopically visible cell group, proves that cell mass grows fine.
Two, microorganism detection
1, bacterium, fungi detect
Method:
(1) get the freeze-stored cell of total freeze-stored cell ampoule 0.5%, be dissolved in respectively in the 8ml nonreactive substratum, behind the mixing with cell suspension with the centrifugal 20min of 2000g, and repeat twice, to eliminate antibiotic influence.Be resuspended in again in the nutrient solution of 2ml antibiotic-free.
(2) cell is inoculated in Trypsin beans peptone and the malt extract medium with 0.5ml, places the incubator of 37 ℃ and 26 ℃ to cultivate for two weeks respectively.
(3) establishing contrast is:
A. positive control: subtilis and Candida albicans bacterium are inoculated into respectively in Trypsin beans peptone and the malt extract medium to be cultivated, and places the incubator of 37 ℃ and 26 ℃ to cultivate for two weeks.
B. negative control: Trypsin beans peptone substratum and malt extract medium place the incubator of 37 ℃ and 26 ℃ to cultivate for two weeks respectively.
Conclusion: the soybean Tryptones nutrient solution and the wort nutrient solution that detect cell visual inspection inoculation elite immortalized cell line, all present limpid transparence, place microscopically to observe in test tube, except the rounded transparence bright spot of zooblast swims in the nutrient solution, do not have other foreign matter.In the whole process of proof and cell recovery frozen in cell cultures, cell is not by bacterium, fungal contamination.
2, detection of mycoplasma
Method:
(1) sample: immortalized cells is made cell suspension, and adjusting cell concn is 1 * 10
5Individual/ml.
(2) cultivate: tested cell goes down to posterity to cultivate in not containing antibiotic nutrient solution and (is not less than 3 times) more than 3 times, goes down to posterity the last time to cultivate to breed the interim bright nutrient solution that renews, and continues to cultivate 2～3d.
(3) inoculation: the negative control ware adds cell nutrient solution RPMI16402ml; Add sample 2ml to be checked in the ware to be checked, treat from bottle, to take out before the cover plate culturing cell converges (converge fully as cell, influence is to the observation of mycoplasma).
(4) rinsing: the cell cover plate is placed the dish ware, use the PBS rinsing, cold wind dries up.
(5) fixing: soak cover glass with stationary liquid, behind the fixed cell 10min, repeating step (4).
(6) dyeing: will prepare the Hoechst33258 dye liquor and be added drop-wise on the cell that fixes, 30min dyes under the room temperature.
(7) rinsing: embathe cover glass 3 times after the dyeing with PBS, each 3～5min, cold wind dries up.
(8) film-making a: PBS who contains 1% glycerine is added to cell surface after the dyeing, will has the cover glass of cell to face down and cover on the slide glass.
(9) observe: with 100 * to 400 * fluorescence microscope, open light source 10min after, excite with 330～380nm purple fluorescence, whether observation of cell nuclear is outer has the fluorescence of blue-fluorescence point or thread point to produce.
Conclusion:
The Hoechst33258 fluorescence dye can see through the complete cell of cell membrane, embeds in the nucleus DNA, makes it to send bright blue-fluorescence.Also contain DNA in the mycoplasma, also can be painted, under the fluorescence microscopy Electronic Speculum, observe and can see blue-fluorescence.Therefore, in positive control, except the nucleus position has the blue-fluorescence, around reaching, cell surface also can see blue point-like or thread fluorescence.And at cell surface mycoplasma DNA fluorescent dye particle and thread point are arranged simultaneously, detected result shows: after the film-making of vitro culture elite's immortalized cell line, under inverted fluorescence microscope, purple fluorescence with 380nm excites, can find that visible nucleus surface is smooth sample in the whole visual field, nucleus is round shape or ellipticity, and the DNA in the nucleus sends blue-fluorescence, and cell peripheral does not have thread or the particulate state point.
Three, the drafting of immortalized cell line cell growth curve
Method:
(1) gets P10 for cell, the conventional method collecting cell;
(2) counting, adjusting cell density is 5.0 * 10
4Cell/ml is inoculated in 24 orifice plates, every hole liquid feeding 1ml (the cell generation is P11 after the reclosing kind);
(3) 3 holes of picked at random every day are collected, are counted, every hole counting 3 times, determination of trypan blue staining cell motility rate, 8d continuously;
(4) with the incubation time be X-coordinate, cell count is ordinate zou, draws growth curve.
Conclusion:
Observe and count P10 for the propagation situation of immortality cell cultured continuously 8d and draw long curve.P10 has not been ordinary cells for the growth curve of immortality cell as can be seen, " S " type curve of growth.After the latent period of cell experience 3d, enter logarithmic phase; At the logarithmic growth after date of experience 1d, an extremely short plateau 1d appears, directly enter the logarithmic phase of molecular marker for increased proliferation then again, the paracme of having skipped cell.With the growth cycle of ordinary cells great difference is arranged, but meet the propagation characteristics of immortality cell.
Four, immortalized cell line nucleus type analysis
(1) cell of cultivating exponential phase of growth is gone down to posterity at 1: 2 after, place 37.5 ℃, 5%CO
2Cultivate 48h in the incubator of saturated humidity;
(2) add colchicine in culturing cell, making its final concentration is 0.8 μ g/ml, puts incubator and continues to cultivate 1.5h;
(3) collect the interior cell suspension of culturing bottle with the 10ml centrifuge tube, the centrifugal 10min of 2500rpm abandons supernatant;
(4) add the KCI hypotonic solution 8ml of the 0.075mol/L of 37.5 ℃ of preheatings, immediately with suction pipe piping and druming, evenly after, water-bath 30min in 37.5 ℃ of water-baths;
(5) add methyl alcohol-Glacial acetic acid stationary liquid (volume ratio 3: 1) 1ml, piping and druming makes cell suspension even gently, and room temperature is transferred 5min and fixed;
(6) the centrifugal 10min of 2000rpm, abandon supernatant after, add methyl alcohol-Glacial acetic acid stationary liquid 8ml, room temperature is transferred 30min, and is centrifugal, remove supernatant, repeats to fix 1 time;
(7) add a small amount of stationary liquid and make cell suspension, get 2 cell suspensions and drip sheet, dry or dry naturally for 37.5 ℃;
(8) Giemsa-PBS mixed solution dyeing (volume ratio 1: 9) is washed out mixed solution gently with clear water behind the 30min, by the time use the copal gum mounting after dripping the sheet drying, in order to observe.
(9) observe under the mirror, microscopically is observed and 50～100 of the statistics of taking a picture are sprawled intact karyomit(e) metacinesis phase, utilizes the PHotoshop imgae processing software to clip and paste, sort.By Denver meeting and Levan canonical measure and calculate chromosomal relative length, centromere index, and definite kinetochore type.Calculate as follows:
Relative length refers to individual chromosome length and the ratio that comprises the haploid chromosomes length overall of X (or Y) karyomit(e), represents with percentage.
Relative length=each chromosome length/(haploid chromosomes+X chromosome total length) * 100% centromere index refers to that galianconism accounts for the ratio of this chromosome length, represents with percentage.It determines centric relative position.
Centromere index=(disconnected arm lengths/this karyomit(e) length overall) * 100% is pressed the criteria for classifying of Levan (1964), and the arm ratio index is at kinetochore karyomit(e) (M) between 1.0～1.7, in the middle part of the title of centromere index between 50.0%～37.5%; The arm ratio index between 1.7～3.0 the silk
The title submedian centromere karyomit(e) (SM) of grain index between 37.5%～25.0%; The arm ratio index between 3.0～7.0, centromere index claims inferior telocentric chromosome (ST) between 25.0%～12.5%; Arm ratio index＞7.0, centromere index are telocentric chromosome (T) between 12.5%～0.0%.
Conclusion:
Find out from sportsmen's immortalized cells karyotype, sportsmen's immortality cell chromosome number is 2n=46, macrochromosome length is successively decreased obviously, be easy to distinguish, the microchromosome number is difficult for distinguishing, show that according to the observation statistics to sportsmen's immortality cell metacinesis phase cell chromosome male sex sportsmen's immortality cell caryogram is 46, XY; Women sport person's immortality cell caryogram is 46, XX.According to chromosomal form and size, in 23 pairs of euchromosomes of male sex sportsmen's immortality cell, 6 pairs 12 is metacentric chromosome, 11 pairs of 22 submetacentric chromosomes, and 5 pairs 10 is inferior telocentric chromosome.X chromosome is greater than the 8th pair, less than the 7th pair submetacentric chromosome; Y is minimum kinetochore, inferior end karyomit(e); In 23 pairs of euchromosomes of women sport person's immortality cell, 6 pairs 12 is metacentric chromosome, 11 pairs of 22 submetacentric chromosomes, and 5 pairs 10 is inferior telocentric chromosome.X chromosome is greater than the 8th pair, less than the 7th pair submetacentric chromosome.Press the Denver meeting, chromosome number, form to karyotype is arranged immortality cell with Denver meeting standard basically identical, illustrate that above-mentioned index does not have significant difference after cell immortalityization.
Five, the expression study of three kinds of external source fluorescence protein genes in immortalized cell line
(1) collecting cell, picking growth is vigorous, the form good cell is with PBS (pH7.4) or do not contain twice of the nutrient solution rinsing of serum.Be inoculated into during 24 holes pull, density is 2 * 10
4/ hole;
(2) 2ug plasmid (pEGFP-N3, pEYFP-N1, three kinds of fluorescence protein genes of pDsRedl-N1) is diluted incubated at room 15min respectively with 50 μ l serum-free mediums;
(3) dilute 6 μ l Lipofectamine2000 (liposome 2000), incubated at room 15min with 50 μ l serum-free mediums;
(4) with the plasmid DNA of dilution and the Lipofectamine2000 mixing of dilution, incubated at room 45min;
(5) remove nutrient solution in the Tissue Culture Plate, slowly drip plasmid DNA/Lipofectamine2000 transfection composite, mixing gently;
(6) 37 ℃, 5%CO
2Cultivate 6～8h in the incubator, change full nutrient solution;
(7) 37 ℃, continue in the 5%C02 incubator to cultivate, observing and recording pEGFP-N3, pEYFP-N1, three kinds of fluorescence protein genes of pDsRedl-N1 expression in immortalized cells under the exciting light of 24h usefulness laser scanning co-focusing microscope at specific wavelength after the transfection, be standard to amplify 100 times field of microscope, count 5 fields of microscope and express the cell count of fluorescence, ask its mean value, estimate transfection efficiency with this;
(8) cross the fluorescence intensity that observation is respectively organized in the positive cell under high power lens and determine the distribution of fluorescin in positive cell.
Conclusion:
Immortalized cells has been carried out the transfection of pEGFP-N3, pEYFP-N1, pDsRedl-N1 three kinds of fluorescence protein genes, test-results shows, the transfection efficiency of immortalized cells is very high by about 85%, and the external source fluorescence protein gene can be expressed in whole cell, proved flushing of immortalized cells, ability with expression alien gene can be used as the transgenic research material, also can be used as following Vectors in Gene Therapy.
Six, immortalized cell line Isozyme Analysis
1, protein extraction
(1) collect immortalized cells, the centrifugal 10min of 1000r/mim abandons supernatant;
(2) with 2ml PBS re-suspended cell, piping and druming is cleaned gently, and the centrifugal 10min of 1000r/mim abandons supernatant;
(3) add protein extract, the piping and druming cell;
(4) the centrifugal 2min of 12000r/mim collects supernatant, and packing is stand-by.
2, discontinuous gradient polyacrylamide gel electrophoresis
(1) making sheet is lived the sheet glass three side sealing with 2% agarose solution, prevents leakage;
(2) make separation gel and concentrated glue;
(3) in the sheet glass of making, add separation gel to 3/4 of cumulative volume, place slightly one transfer to after the meeting in the incubator gelling is solid;
(4) continue to add concentrated glue at the separation gel that solidifies sheet glass is filled, insert comb, be positioned over shady and cool place and solidify;
(5) go up sample, electrophoresis (first 220V electrophoresis 1h; 50V electrophoresis 16h then);
(6) dye glue, take a picture.
Conclusion:
The zymogram band feature of serum lactic dehydrogenase, malate dehydrogenase shows that the band mobility of four individualities is identical, and content and activity do not have significant difference yet, so can be used as the discriminating foundation, proves not to be subjected to crossed contamination between immortalized cells.
Seven, immortalized cell line cell cycle analysis
(1) the preparation single cell suspension is moved into immortalized cells in the centrifuge tube 1200rpm, centrifugal 8min with the 2ml transfer pipet;
(2) supernatant is outwelled, add 3ml PBS, clean cell with suction pipe piping and druming, make cell suspension, 1000rpm, centrifugal 8min;
(3) abandon supernatant, add precooling 70% dehydrated alcohol, 4 ℃ are spent the night;
(4) the centrifugal ethanol of abandoning adds 3ml PBS re-suspended cell 5min;
(5) 400 eye mesh screens filter 1 time, the centrifugal PBS that discards;
(6) add 1ml PI dye liquor, 4 ℃, lucifuge are hatched 30min;
(7) go up machine testing;
(8) by using the data of SPSS18.0 to analyze.
P8, P17, P20 are carried out the cell cycle for immortalized cells detect, detected result shows as a result, and P8 is that 80.9%, P17 and P20 are respectively 64.7% and 62% for the immortality cell G0/G1 phase for the immortality cell G0/G1 phase; The cell quantity of G0/G1 phase successively decreases along with the increase of cell generation.And division stage (S+G2/M) cell quantity be followed successively by from high to low: P20, P17, P8; The data presentation of gained, along with the increase of passage number of times, the multiplication capacity of immortalized cells is in rising trend, and the immortalized cells of high generation is molecular marker for increased proliferation still, meets the growing multiplication rule of immortalized cells.The immortalized cell line stable performance of vitro culture is described
Eight, the immortalized cell line apoptosis detects
Method:
(1) the preparation single cell suspension is moved into immortalized cells in the centrifuge tube 1200rpm, centrifugal 8min with the 2ml transfer pipet;
(2) collect (1～5) * 10 with PBS washed cell twice (the centrifugal 8min of 1000rpm)
5Cell;
(3) each sample adds the Binding Buffer suspension cell of 500 μ L;
(4) behind the adding 5 μ l AnnexinV-FITC mixings, add 5 μ l Propidium Iodide, mixing; Positive controls adds FITC and PI respectively;
(5) room temperature, lucifuge, reaction 5～10min;
(6) in 1h, carry out the flow cytometry detection by quantitative;
(7) by using the data of SPSS18.0 to analyze.
Conclusion:
P9, P18 are carried out the detection of apoptosis rate for immortality cell, the detected result data presentation, the apoptosis rate in P9 generation is 3.5%, P18 for the apoptosis rate of cell is 2.8%.ANOVA showed significant, apoptosis rate difference between the two generation cells is significantly (P＞0.05) not, illustrate that immortalized cells along with the increase of cell generation tangible phenomena of apoptosis does not take place, the immortalized cells of high generation still has higher motility rate, grows fine.
Above presentation of results the elite's immortalization system of cultivating can provide permanent experiment material for researchs such as human genetics, molecular biology.
Claims (3)
1. elite's immortalized cell line of one kind high cell transformation rate, high cell infinite multiplication.
2. method is according to claim 1, and the cultural method of elite's immortalized cell line of a kind of high cell transformation rate, high cell infinite multiplication is characterized in that this method comprises the steps:
2.1 blood specimen collection
(1) record blood sampling sportsmen personal information is corresponding with the numbering on the bloodletting tube;
(2) use the disposable bloodletting tube that has heparin sodium, extract sportsmen's venous blood 4ml/ people;
(3) the static placement 1～2d of blood sample normal temperature;
Annotate: if whole blood can not transform at that time at once, must do following processing: with blood sample 2500rpm at normal temperatures, centrifugal 15min, centrifugal back changes the tunica albuginea layer in the centrifuge tube that contains 3ml 1640 full substratum with Pasteur's pipe, then centrifuge tube is put into 37.5 ℃ of incubators, placed on the square plate.
2.2 cell immortalityization
(1) with the two anti-RPMI1640 basic mediums that add 1%, with sportsmen's hemodilution of gathering, blow even gently.
(2) lymphocyte separation medium places 37.5 ℃ of water-bath preheatings, during use its solution is shaken up.
(3) slowly pour the 4ml lymphocyte separation medium in the 15ml centrifuge tube that sportsmen's blood sample is housed, make the two interface clear, the centrifugal 10min of 3000rpm makes the blood layering;
(4) be divided into four layers in the centrifugal back centrifuge tube, the tunica albuginea layer that the one-tenth poplar on serum lower floor, red corpuscle upper strata is cotton-shaped is managed immigration with Pasteur the centrifugal 10min of 1000rpm is housed in the centrifuge tube of 6ml RPMI1640 substratum;
(5) after centrifugal supernatant liquor is discarded, add 10ml RPMI1640 substratum again, the centrifugal 10min of 1000rpm abandons supernatant liquor.After repeating once, abandoning supernatant is struck cell even stand-by;
(6) 2ml is contained full substratum, 1.4ml Epstein-Barr virus, the 0.4ml CyA of PHA and the white corpuscle of collecting and make mixed solution, altogether about 4ml;
(7) mixed solution is joined in 24 well culture plates every hole 1ml;
(8) 24 well culture plates are placed 37.5 ℃, 5%CO
2Cultivate in the incubator.
3. the cultivation of sportsmen's immortalized cell line and frozen
3.1 going down to posterity of cell
(1) immortalized cells is transferred to 25m from 24 orifice plates
2Culturing bottle in continue to cultivate;
(2) 3～4d carry out once half amount and change liquid, and each half amount is changed liquid and is passage one time.
3.2 cell is frozen
(1) collecting cell suspension, the centrifugal 10min of 1000rpm abandons supernatant;
(2) add the frozen storing liquid of predetermined amount in the centrifuge tube, cell is beaten gently even, adjusting cell density is 1 * 10
6/ ml～3 * 10
6/ ml installs to the frozen storing liquid branch in the frozen pipe, every pipe 1ml;
(3) pre-freeze: with frozen pipe pack into the cooling box in, be put in 4 ℃ of 20～30min, place then more than one 70 ℃ of pre-freeze 4h, propose frozen pipe and put into
(4) frozen: as to propose frozen pipe and put into the liquid nitrogen cabinet, namely finish cell cryopreservation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101231329A CN103232971A (en) | 2013-04-11 | 2013-04-11 | A method for B lymphocyte transformation by EB virus (Epstein-barr virus) for elite ice and snow athletes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2013101231329A CN103232971A (en) | 2013-04-11 | 2013-04-11 | A method for B lymphocyte transformation by EB virus (Epstein-barr virus) for elite ice and snow athletes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103232971A true CN103232971A (en) | 2013-08-07 |
Family
ID=48881040
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2013101231329A Pending CN103232971A (en) | 2013-04-11 | 2013-04-11 | A method for B lymphocyte transformation by EB virus (Epstein-barr virus) for elite ice and snow athletes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103232971A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293836A (en) * | 2014-06-24 | 2015-01-21 | 长沙赢润生物技术有限公司 | Making method of immune complex adsorption cells |
| CN104894050A (en) * | 2015-05-08 | 2015-09-09 | 西南大学 | Cryopreservation resuscitation method for silkworm embryonic cells infected with nosema bombycis |
| CN106520696A (en) * | 2016-11-11 | 2017-03-22 | 广西壮族自治区水产科学研究院 | Method for establishing and identifying immortalization tilapia mossambica macrophage system |
| CN109897822A (en) * | 2017-12-11 | 2019-06-18 | 复旦大学 | The foundation and application of lineup's immortalization bone-marrow-derived lymphocyte system |
| CN115181726A (en) * | 2022-03-15 | 2022-10-14 | 武汉百翼生物科技有限公司 | Construction method of lymphocyte immortalization |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2398783A (en) * | 2003-02-26 | 2004-09-01 | Antonio Lanzavecchia | A method for producing immortalised human B memory lymphocytes |
-
2013
- 2013-04-11 CN CN2013101231329A patent/CN103232971A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2398783A (en) * | 2003-02-26 | 2004-09-01 | Antonio Lanzavecchia | A method for producing immortalised human B memory lymphocytes |
Non-Patent Citations (2)
| Title |
|---|
| 刘宇: "我国优秀单板U型场地滑雪运动员永生细胞库的建立与保存", 《中国优秀硕士学位论文全文数据库-社会科学Ⅱ辑》, no. 11, 24 October 2011 (2011-10-24) * |
| 裴丽君: "EB病毒转化人类外周血B-淋巴细胞建立永生细胞系", 《中国生育健康杂志》, vol. 14, no. 3, 30 June 2003 (2003-06-30), pages 186 - 188 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293836A (en) * | 2014-06-24 | 2015-01-21 | 长沙赢润生物技术有限公司 | Making method of immune complex adsorption cells |
| CN104894050A (en) * | 2015-05-08 | 2015-09-09 | 西南大学 | Cryopreservation resuscitation method for silkworm embryonic cells infected with nosema bombycis |
| CN106520696A (en) * | 2016-11-11 | 2017-03-22 | 广西壮族自治区水产科学研究院 | Method for establishing and identifying immortalization tilapia mossambica macrophage system |
| CN109897822A (en) * | 2017-12-11 | 2019-06-18 | 复旦大学 | The foundation and application of lineup's immortalization bone-marrow-derived lymphocyte system |
| CN115181726A (en) * | 2022-03-15 | 2022-10-14 | 武汉百翼生物科技有限公司 | Construction method of lymphocyte immortalization |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Coss | Mitosis in Chlamydomonas reinhardtii basal bodies and the mitotic apparatus | |
| CN107236704B (en) | From the method for placenta separating mesenchymal stem cell and the digestive enzyme compositions used | |
| CN103232971A (en) | A method for B lymphocyte transformation by EB virus (Epstein-barr virus) for elite ice and snow athletes | |
| CN107299082A (en) | Placenta interstitial cell and the method for being trained mescenchymal stem cell are separated from tissue | |
| CN104164403A (en) | Method for extracting and culturing adipose-derived stem cells | |
| CN102586184A (en) | Method for establishing placental mesenchyme stem cell library | |
| CN103667176A (en) | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof | |
| CN103966162A (en) | Novel menstrual blood-derived mesenchymal stem cell separation method | |
| CN104694464A (en) | Clinical dental pulp stem cell and preparation method thereof | |
| CN103849602B (en) | A kind of bull testis clone and establishment method thereof and application | |
| CN109294974A (en) | A kind of hybridized prussian carp myeloid tissue cell line and its construction method are applied with it | |
| CN104531608A (en) | Anguilla anguilla liver cell line and construction method and application thereof | |
| CN104726401A (en) | Method for improving success rate of umbilical cord blood mesenchymal stem cell culture by using placental mesenchymal stem cells | |
| CN104818241B (en) | Epinephelus lanceolatus fish-skin skin tissue cell line and its construction method | |
| CN104152403A (en) | Method for establishing goose embryo epithelial cell line and established goose embryo epithelial cell line | |
| CN104480068A (en) | Method of in-vitro amplification and purification culture of mesenchymal stem cells | |
| Mazalova et al. | Quantification of DNA content in freshwater microalgae using flow cytometry: a modified protocol for selected green microalgae | |
| CN104488852A (en) | Frozen stock solution for storing mammary epithelial cells of milk goat and freeze-saving method | |
| CN101012450B (en) | Mount Wuzhi Pig ear rim tissue fibroblast series and culturing method thereof | |
| RU2348689C1 (en) | Constant sso-2 cell line of parenchymic organ pool of siberian sturgeon acipenser baeri | |
| CN111759864B (en) | Application of amniotic fluid stem cells in preparation of medicine for treating lupus nephritis | |
| CN110192552B (en) | Application of glycylglycine conditioned medium in mesenchymal stem cell cryopreservation and preparation of commercial cryopreservation protective agent | |
| CN100506976C (en) | Ear margin tissue fibroblast line of Minxian county black fur sheep and its culture process | |
| Polyanskaya et al. | Sizes of bacterial cells in soils determined by cascade filtration technique | |
| Miazzi et al. | Handling of the biotrophic pathogen Uncinula necator (Schw.) Burr. under laboratory conditions and observations on its mating system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130807 |