CN103160438A - Method using nanometer needle-shape materials to increase cell permeability - Google Patents
Method using nanometer needle-shape materials to increase cell permeability Download PDFInfo
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Abstract
The invention discloses a method using nanometer needle-shape materials to increase cell permeability. The method includes the steps of stirring mixed suspension of the nanometer needle-shape materials and cells, enabling the nanometer needle-shape materials to stab repairable small holes in cell membranes or cell walls, and achieving the purpose of increasing the cell permeability. The method has the advantages of being simple in operation, free from expensive precise instruments, capable of effectively enabling the cells to obtain permeability and ensuring cell viability, and suitable for increasing the cell permeability, and processed samples are not restricted by size.
Description
Technical field
The invention belongs to biological technical field, is about improving the method for cell permeability, more particularly, is about the mechanical force that produces by stirring, the acicular nanometer material to be produced on barrier cell can by the aperture of cell self-regeneration, increase cell permeability.
Background technology
Barrier cell by cytolemma and cell wall components is separated cell and outside atmosphere, limited the transhipment of polar molecule, hydrophilic molecule and biomacromolecule, the saturating property of reversible increase barrier cell is to make much to be difficult under normal circumstances be undertaken spreading prerequisite (Mazurkiewicz, the US6107095 of transhipment by barrier cell across the material of barrier cell transhipment by diffusion; Hapala, 1997, Crit.Rev.Biotechnol., 17,105).Usually can't carry out under normal circumstances estimating across the material of barrier cell transhipment the effect (Shimizu that cell permeability increases with ble (bleomycin) and NAD (Reduced nicotinamide-adenine dinucleotide) etc., 1996, Biol Phardm Bull, 19,1279).
Make material pass through the method for barrier cell all significant to pharmaceutical developments and biotechnology, therefore researcher has been done a large amount of research work in this field, and has developed many methods (being mainly that ECM is entered in cell) that increase cell permeability by physical or chemical treatment.
These methods that increase cell permeability can be divided three classes: biological method, chemical method and physical method.
Biological method can guarantee to greatest extent cell survival rate and the molecule that is transferred is had strict selectivity, but can only be used for limited species and limited molecular species (Hapala, 1997, Crit.Rev.Biotechnol., 17,105); The destruction of chemical method cell membrane is normally irreversible, can cause necrocytosis (Hapala, 1997, Crit.Rev.Biotechnol., 17,105), and gentle chemical method such as Ca+ coordinate heat shock (Mandel and Higa, 1992, Biotechnology, 24,198) the very low and complicated operation of efficient; At present most study is most widely used is Physical, physical method can be used for being permitted eurypalynous cell and also general operation fairly simple.The method that increases cell permeability by physics method mainly contains following several (Canham, US 6770480):
1. microinjection: directly material is expelled in cell, this method can effectively be controlled material and enter the quantity of cell and guarantee very high success ratio, but operator are required very high, can only process at every turn a cell (Canham, US 6770480; Soreq andSeidman, 1992, Methods Enzymol, 207,225).This method can't be used for small-sized cell (Ando and Anonymous, US 07479388).
2. laser boring: utilize laser radiation to make barrier cell produce damage, the material in solution is transported (Ando and Anonymous, US 07479388) by diffusion.But this technology efficient except the instrument of needs precision is also very low, can only act on a cell at every turn, even also can only act on the scope (Turovets of 10 * 25mm by scattering, 1993, Biotechniques, 15,1022), and can only act on the surface cell.Therefore this technology seldom is employed, but adopts more economical physical means.
3. particle gun: break through barrier cell with high velocity particle; the nucleic acid or other materials that are adsorbed on particle are sent into cell (Klein; 1987; Nature; 327; particle or the drop that 70), also can directly accelerate to be transported material bombard (Mazurkiewicz, US6107095) in cell.Particle bombardment also can make material spread transhipment (Sanford, US 5478744,2570) by barrier cell by the saturating property that increases barrier cell.This method for application extensively can be transported polytype material (Sanford, 1993, Methods Enzymol, 217,483), and can be used for the minicell such as E.coli (Smith, 1992, Journalof General Microbiology, 138,239).But these class methods need to have accurate instrument to control accurately the size of particle and speed to avoid cell killing that enough kinetic energy breakthrough barrier cell (Ginaven and Facciotti are arranged simultaneously, US 5457041), owing to directly could being broken through barrier cell by particle in the face of the cell of jet orifice so treatment effect is inhomogeneous, the cell quantity of processing is limited.
4. electroporation: make cytolemma produce aperture by high-voltage electric shock, the solution of the materials such as gene is transported (Rubinsky and Huang, US 07718409) by these apertures.This is the method for the non-property of a kind of efficient transhipment molecule, owing to being the mode transport molecule that spreads, transhipment is subjected to molecular chemistry property effect (being applicable to hydrophilic molecules, amphipathic molecule and macromole) fast and not, and can control cell survival rate (Sixou and Teissie by adjusting strength of electric field, electric field time length and number of shocks, 1993, BioelectrochBioener, 31,237).Electroporation is the method (Rubinsky and Huang, US 07718409) of at present the most frequently used increase cell permeability, and is used for gene therapy and research and drug transport (Gehl, 2003, Acta Physiol Scand, 177,437).But electroporation only has the perforation effect to the cell that is between electrode connecting line; The cell quantity that produces the each processing of high-intensity electric field (suspension is generally 0.5kV/cm (Golzio, 2002, Proc Natl Acad SciUSA, 99,1292)) due to needs is limited; Need to accurately control voltage waveform, electric shock intensity, electric shock frequency and treatment time in order to improve cell survival rate and piercing efficiency, thereby accurate instrument need to be arranged.
5. increase cell permeability by shearing force: increase cell permeability by the physical abuse to barrier cell.Major way have cell is mixed with granulated glass sphere after slight vibrations (Mcneil, 1989, Method Cell Biol, 29,153), cell is mixed rear concussion (Shimizu and Kawazoe, 1996, Biol Pharm Bull with the film destabilizing agent, 19,1279) or with cell pass through syringe pressure-vaccum (Clarke and Mcneil, 1992, J Cell Sci repeatedly, 102,533).These methods are simple to operate and do not need accurate instrument, and front two kinds of methods can also be used for processing simultaneously a large amount of samples, but the efficient of increase cell permeability is very low, cell survival rate is low and be only applicable to acellular wall and the larger zooblast of cell volume.
6. endocytosis: this is a kind of method that physics combines with biology, will be by transhipment material and coprecipitation of calcium phosphate, and coprecipitated product can enter cell (Graham and van der Eb, 1973, Virology, 52,456 by endocytosis; Wigler, 1979, Cell, 16,777).Also material can be coated in and make material enter cell (Mizuguchi, 1996, Br J Cancer, 73,472) in liposome.These class methods can be processed a large amount of cells simultaneously, but success ratio is very low, are only applicable to the cell of acellular wall.
Research finds that the acicular nanometer material can pierce through cell by force of sliding friction when the mixed solution of solid surface coating needle-like nano material and cell, and this phenomenon is called as Yoshida effect (Yoshida, 2007, Recent Pat Biotechnol, 1,194).This discovery is used to Plasmid Transformation and its principle has been carried out inference (Tan Haidong, 2010, biotechnology journal Chinese Journal of Biotechnology, 26,1379; Yoshida, 2008, ApplMicrobiol Biotechnol, 80,813; Tan, 2010, InternationalJournal of Molecular Sciences, 11,4962), but the force of sliding friction of generation Yoshida effect can only make this discovery can't act on simultaneously a large amount of cells in the solid surface generation, and single job only obtains plasmids less than 250 cells, and operation the time need to be coated on solid surface to cell, increased schedule of operation.
Process a large amount of cells when the method for above-described increase cell permeability all can't efficient economy, until the people such as Rojas-Chapana have developed the method (Giersig that increases cell permeability with multi-walled carbon nano-tubes, DE102004063150), the method is by the suspension liquid of microwave irradiation cell and carbon nanotube, make the carbon nanotube tip charged, the electrostatic force promotion needle-like carbon nanotube of carbon nanotube and cell surface produces aperture and realizes substance transportation (Rojas-Chapana on barrier cell, 2005, Lab on a Chip 5,536).The method is higher and can effectively guarantee cell survival rate with the method phase specific efficiency that increases cell permeability by shearing force; No longer be confined to the suspension surface owing to carrying out in cell suspension, can process simultaneously the cell in the certain volume suspension, improved processing efficiency.But excessive microwave radiation can cause the cell mortality must strictly control treatment time and content of carbon nanotubes, and microwave radiation can weaken gradually when propagating in liquid and causes treatment effect inhomogeneous, therefore sample size will be controlled in certain volume, and sample volume also is subject to the restriction of microwave processing equipment.
Summary of the invention
The present invention relates to a kind of method that improves cell permeability, more particularly, is by the mechanical force that stirring produces, the acicular nanometer material to be produced on barrier cell can by the aperture of cell self-regeneration, increase cell permeability.
Be the experiment above-mentioned purpose, the technical solution used in the present invention is:
A kind of method of utilizing the acicular nanometer material to increase cell permeability,
Use physical method to pierce through cytolemma or cell walls and cytolemma, form the aperture of diameter 0.02-0.12 μ m at cell surface, the cell permeability that produces 0-94% increases efficient.
Described physical method is under liquid environment, add the acicular nanometer material in liquid, the mechanical force that produces by stirring makes the acicular nanometer material pierce through cytolemma or cell walls and cytolemma, produces aperture on cytolemma or cell walls and cytolemma, makes cell obtain saturating property;
The aperture that the acicular nanometer material produces on barrier cell can be by cytothesis, thereby guarantees the survival rate of cell; Increase efficient with cell permeability and improve, cell survival rate reduces.
The acicular nanometer material refers to diameter at 0.02-0.12 μ m, the material of length between 0.34-2.30 μ m, or the surface is with the material of the spicule that meets above-mentioned specification; The acicular nanometer material mixes with the state of cell with suspension.
Described acicular nanometer material is sepiolite.
Standard conditions: cell density is 1OD, stirring time to be that 1min, impact strength are 3000rpm, and sepiolite concentration is 0.5%, and not outer glycerol adding changes viscosity.Carry out following checking:
In suspension, the acicular nanometer material concentration is higher, and property increase efficient is higher thoroughly; Cell density be 1OD, stirring time be 1min, when impact strength is 3000rpm, when adding the concentration of acicular nanometer material sepiolite to change between 0.25%-0.75% (mass ratio) in mixed solution, property increase efficient changes between 81%-94% thoroughly, and cell survival rate changes between 80%-69%;
In suspension, cell concn is higher, and property increase efficient is higher thoroughly, but property increase efficient is less thoroughly; Sepiolite concentration is 0.5%, the stirring time is 1min, when impact strength is 3000rpm, when in mixed solution, cell density changed between 0.5OD-2OD, property increase efficient changed between 95%-88% thoroughly, cell survival rate changes between 73%-78%.
Suspension viscosity is higher, and property increase efficient is lower thoroughly; Suspension viscosity can change by adding glycerine, and viscosity improves to increase the system glycerol concentration and realizes; Sepiolite concentration is 0.5%, cell density be 1OD, stirring time be 1min, when impact strength is 3000rpm, suspension viscosity is 54 * 10
-3Pas-65 * 10
-3When changing between Pas, property increase efficient changes between 91%-83% thoroughly, and cell survival rate changes between 74%-77%.
The mechanical force that described stirring produces is enough to make the acicular nanometer material pierce through the mechanical force of barrier cell by the container generation that suspension is equipped with in direct stirring suspension itself or concussion.
By stirring the generation of direct stirring suspension own when enough making the acicular nanometer material pierce through the mechanical force of barrier cell, agitation strength is larger, and property increase efficient is higher thoroughly;
Sepiolite concentration is 0.5%, cell density is 1OD, stirring time when being 2min, stirs suspension intensity when changing between 800rpm-400rpm, and property increase efficient changes between 28%-38% thoroughly, and cell survival rate changes between 98%-89%.
When the container that suspension is housed by concussion produced and enough to make the acicular nanometer material pierce through the mechanical force of barrier cell, impact strength was larger, and property increase efficient is higher thoroughly;
Sepiolite concentration is 0.5%, cell density is 1OD, stirring time when being 1min, and when impact strength changed between 1000rpm-2000rpm, property increase efficient changed between 63%-90% thoroughly, and cell survival rate changes between 84%-73%.
When stirring suspension, the time of stirring continuously is longer, and property increase efficient is higher thoroughly;
Sepiolite concentration is 0.5%, cell density is 1OD, when impact strength is 3000rpm, when the concussion time changed between 1min-5min, property increase efficient changed between 93%-96% thoroughly, cell survival rate changes between 74%-54%.
The cell permeability verification method: the present invention uses NAD (nadide) or these the two kinds material checking cell permeabilities that can't pass through the transportation of free diffusing cross-film of ble (bleomycin) to increase effect.The transhipment of NAD can detect by HPLC the variation of NAD quantity in cell, and the transhipment of ble can detect the lethal effect of cell by ble.Wherein NAD is available from the prosperous Bioisystech Co., Ltd (production code member DH207) of Beijing ancient cooking vessel state, and ble is available from Invitrogen company (production code member R250-01).
Saturating property increase efficiency rating method: find in the invention process process, when adding the NAD of different concns, absorbed ble and dead cell proportion when changing by increasing property between 0-95%, therefore this ratio always is directly proportional to the NAD transfer amount, has absorbed ble by increasing property and saturating property efficient that dead cell proportion can the whole cell colony of quantitative response obtains.Therefore in the present invention, saturating property increase definitions of efficiency is added the colony number of ble after for (placing the colony number that adds ble after the colony number that adds ble after 30min-stirrings at once)/placement 30min.Wherein (place the colony number that horse back after the colony number that adds ble after 30min-stirrings adds ble) and reflected reversible increase the cell count of saturating property, i.e. survivaling cell; Place the colony number that adds ble after the 30min cell count of saturating property that reflected the cell count of acquisition property not and reversible increase.
The survival rate that has increased the cell of saturating property is defined as (placing the colony number that adds ble after the colony number that adds ble after 30min-stirrings at once)/(agitation treatment does not directly add the colony number of ble-rear horse back of stirrings and adds the colony number of ble)).In above formula (agitation treatment does not directly add the colony number that adds ble after the colony number of ble-stirring at once) reflected the cell count that has been increased saturating property.
In the present invention, saturating property increase efficient and cell survival rate are processed the rear plate count results by ble and are calculated acquisition.
Thereby the present invention stir mode that mixed solution produces mechanical force including, but not limited to: the concussion of whirlpool concussion instrument is processed, 200rpm wave and culture, magnetic stirring apparatus drive that in mixed solution, magneton stirs in shaking table.Wherein whirlpool concussion instrument uses KMC-1300V Korea S Vision whirlpool concussion instrument, and rotating speed can be regulated between 0-3000rpm; Shaking table uses the full temperature vibrator of east, the Harbin connection electronic technology HZQ-Q of development corporation, Ltd., and rotating speed can be regulated between 40-350rpm; Magnetic stirring apparatus uses regularly bidirectional electronic constant temperature blender with magnetic force of the bank head beneficial friend of Jintan City laboratory apparatus JB-4 of factory, and rotating speed can be regulated between 0-200rpm.
The acicular nanometer material that the present invention adopts including, but not limited to: available from the sepiolite (production code member 58945) of German KremerPigmente company limited.Sepiolite is a kind of hydrosilicate, and molecular formula is H
4Mg
2Si
3O
10, outward appearance is off-white powder.Its particle is the fiber of mean length 2 μ m, mean diameter 20nm, wherein is no more than particle more than 5%, 90% less than 5 μ m greater than the particle of 10 μ m.Between 0.02-0.12 μ m, length is (Yoshida, 2007, Recent PatBiotechnol, 1,194) between 0.34-2.30 μ m according to its diameter of scanning electron microscope analysis.Therefore use sepiolite at the hole diameter that produces on cytolemma or cell walls and cytolemma between 0.02-0.12 μ m.
The cell type that the present invention investigates is including, but not limited to the Saccharomyces cerevisiae BY4741 (S.cerevisiae) of yeast belong in the Escherichia coli BW25113 (E.coli) of Escherichia and eukaryote in prokaryotic organism.
The present invention gets the mixed solution of different viscosity by the glycerin obtained that adds different concns in PBS, corresponding viscosity coefficient reference literature (Liu Zhuqin, 2005, Yanan University's journal (natural science edition) Journal of Yanan University (Natural Science Edition), 24,58), wherein 50% glycerine viscosity is 54 * 10
-3Pas, 60% glycerine viscosity is 58 * 10
-3Pas, 70% glycerine viscosity is 65 * 10
-3Pas.
The invention has the beneficial effects as follows:
Method provided by the invention is not strict with equipment, as long as can stir mixed solution thereby to produce mechanical force just passable; The acicular nanometer material that method provided by the invention is selected easily obtains, uses safety, reasonable price; Method provided by the invention does not rely on cell type, size, shape, quantity and whether cell walls is arranged; Method provided by the invention makes the acicular nanometer material pierce through barrier cell by the mechanical force that stirs the suspension generation increases cell permeability, thereby the processing sample is not to be subjected to volume restrictions; The intensity that in method provided by the invention, cell permeability increases can be adjusted by adjustment stirring velocity, adjustment nano material additional proportion; Method provided by the invention can directly be used in culturing process, does not need to interrupt culturing process or sample is taken out from culture vessel; Method provided by the invention can guarantee cell survival rate effectively, does not affect Growth of Cells.
Description of drawings
Figure .1 is that the acicular nanometer material increases the E.coli cell permeability with the suspension that mixes of E.coli through stir process, makes under normal circumstances and can not transport by free diffusing across the NAD of barrier cell transhipment.Fig. 1 bend shape post represents mixing the NAD that adds different concns in suspension but not stirring of acicular nanometer material and E.coli cell, so NAD can't carry out transporting across barrier cell; Solid post represent acicular nanometer material and E.coli cell mix the NAD that adds different concns in suspension after stir, NAD can spread with concentration gradient.
What Fig. 2 represented acicular nanometer material and E.coli cell mixes suspension through the amount of the NAD that transports by diffusion after stir process, the difference of NAD content in the thalline of NAD content and standing sample in the thalline of the sample that stirs under each NAD concentration in each point corresponding diagram 1 in figure.Fig. 2 illustrates that NAD transports by diffusion.
Scheme .3 for to add NAD in the different time, the variation of the amount of NAD in the E.coli thalline.
Fig. 3 illustrates that the acicular nanometer material content is 0.5%, and when the whirlpool oscillator was processed 1min, the aperture that the acicular nanometer material forms on the E.coli barrier cell needed 20min to repair.
Fig. 4 be the acicular nanometer material with the S.cerevisiae cell mix suspension through the amount of the NAD that transports by diffusion after stir process, the difference of NAD content in the thalline of NAD content and standing sample in the thalline of the sample that stirs under each NAD concentration of each point correspondence in figure.Fig. 4 illustrates that NAD transports by diffusion.
Fig. 5 is that sepiolite is to the affects on the growth of E.coli in the M9 substratum.Trigonometric curve represents to add in 10mL M9 substratum 37 ℃ of growth curves of cultivating E.coli after 200 μ L water, adds 37 ℃ of growth curves of cultivating E.coli after 200 μ L 2.5% sterilization sepiolites in square curve representation 10mL M9 substratum.
Embodiment
Following examples help to understand this patent, but are not limited to content of the present invention.
Embodiment 1:
Cultivate E.coli to logarithmic phase in 37 ℃ of 100mL LB, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=10.
Get the above-mentioned bacterium liquid of 5mL and add the NAD (0,50mM, 0.1M, 0.125M, 0.15M) of 100 μ L different concns and the slight mixing of sepiolite of 500 μ L mass concentrations 5%.Every kind of NAD concentration is divided and is installed in 6 1.5mL centrifuge tubes, every pipe 900 μ L, and wherein three effective whirlpools concussion instrument 3000rpm stir 1min, and other three pipes do not stir.Six arms of every kind of NAD concentration are placed 20min at 30 ℃.Whizzer 4000rpm is centrifugal, and 5min removes supernatant, resuspended with 900 μ L PBS after, whizzer 4000rpm is centrifugal, and 5min removes supernatant, resuspended with 900 μ L PBS after, whizzer 13200rpm is centrifugal, and 5min removes supernatant.Add 37 ℃ of cracking 30min of 800 μ L N,O-Diacetylmuramidases, add 400 μ L chloroforms to process 1min with whirlpool concussion instrument 3000rpm, the centrifugal 10min of whizzer 13200rpm transfers to 2mL pipe, freeze-drying with supernatant.Add 200 μ L 50mM ammonium acetates in the freeze-drying sample, detect NAD concentration (Sporty, Kab ir et al.2008) with HPLC, thereby obtain the amount of NAD in thalline.
Embodiment the results are shown in Figure 1 and Fig. 2.According to Fig. 1, when when processing sample, the outer NAD concentration of born of the same parents was lower than 2mM, the sample of stir process compares stirred sample not because NAD oozes out NAD concentration was low; When processing sample, the outer NAD concentration of born of the same parents was higher than 2.5mM, the sample of stir process compares stirred sample not because NAD enters cell NAD concentration was high, illustrates that NAD spreads according to concentration gradient.According to Fig. 2, the amount of the inside and outside larger NAD of NAD concentration difference of cell transhipment before barrier cell is repaired is more, illustrates that the diffusion of NAD meets the faster rule of the larger velocity of diffusion of concentration difference.
The mixed solution that sepiolite and cell are stirred in embodiment 1 explanation can increase cell permeability effectively, make can not transmembrane transport molecule spread transhipment.
Embodiment 2:
Cultivate E.coli to logarithmic phase in 37 ℃ of 100mL LB, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=10.
Get the slight mixing of sepiolite that the above-mentioned bacterium liquid of 15mL adds 1.5mL mass concentration 5%.Divide to install in 15 1.5mL centrifuge tubes, every pipe 1mL, wherein three pipes add the NAD of 20 μ L concentration 0.1M, and 15 centrifuge tubes stir 1min with whirlpool concussion instrument 3000rpm.Do not add after stirring and get the NAD that three pipes add 20 μ L concentration 0.1M immediately in the pipe of NAD, 15 centrifuge tubes are 30 ℃ of placements, and interval 10min, 20min, 30min get the NAD that pipe that three pipes do not add NAD adds 20 μ L concentration 0.1M respectively.
Place 20min at 30 ℃ immediately after often adding by all means NAD.Whizzer 4000rpm is centrifugal, and 5min removes supernatant, resuspended with 900 μ L PBS after, whizzer 4000rpm is centrifugal, and 5min removes supernatant, resuspended with 900 μ L PBS after, whizzer 13200rpm is centrifugal, and 5min removes supernatant.Add 37 ℃ of cracking 30min of 800 μ L N,O-Diacetylmuramidases, add 400 μ L chloroforms to process 1min with whirlpool concussion instrument 3000rpm, the centrifugal 10min of 13200rpm transfers to 2mL pipe, freeze-drying with supernatant.Add 200 μ L 50mM ammonium acetates in the freeze-drying sample, detect NAD concentration with HPLC, thereby obtain the amount of NAD in thalline.
Embodiment the results are shown in Figure 3.According to Fig. 3, adding immediately NAD after adding NAD before stirring and stirring compares, the amount of NAD in thalline is more or less the same, illustrate that in the method that this patent mentions, NAD mainly transports by diffusion, do not need to be adsorbed in advance on sepiolite, therefore the method mentioned of this patent be applicable to transport all can be in solution the material of free diffusing, this material does not need to be adsorbed by sepiolite.According to Fig. 3, the aperture that the acicular nanometer material forms on the E.coli barrier cell is recoverable, and in repair process, the transport speed of NAD is more and more slower, and barrier cell can be completed reparation in 20min in the time of 30 ℃.
If process intensity greater than the reparation speed of barrier cell, cell permeability is excessive causes necrocytosis thereby the long meeting of continuous processing time causes, therefore, when needs are processed cell by higher processing intensity, can guarantee cell survival rate by intermittently processing cell.
The mixed solution that sepiolite and cell are stirred in embodiment 2 explanations can reversibly increase cell permeability, and the aperture that the acicular nanometer material forms on barrier cell is recoverable, along with the reduction of the saturating property of repair cell.
Embodiment 3:
Cultivate S.cerevisiae to logarithmic phase in 30 ℃ of 100mL YEPD, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=10.
Get the above-mentioned bacterium liquid of 5mL and add the NAD (0,25mM, 50mM, 75mM, 0.1M, 0.125M) of 100 μ L different concns and the slight mixing of sepiolite of 500 μ L mass concentrations 5%.Every kind of NAD concentration is divided and is installed in 6 1.5mL centrifuge tubes, every pipe 900 μ L, and wherein three effective whirlpools concussion instrument 3000rpm stir 1min, and other three pipes do not stir.Six arms of every kind of NAD concentration are placed 20min at 30 ℃.Whizzer 4000rpm is centrifugal, and 5min removes supernatant, resuspended with 900 μ L PBS after, whizzer 4000rpm is centrifugal, and 5min removes supernatant, resuspended with 900 μ L PBS after, whizzer 13200rpm is centrifugal, and 5min removes supernatant.Add 100mg granulated glass sphere cell crushing instrument smudge cells, add 400 μ L chloroforms to process 1min with whirlpool concussion instrument 3000rpm, the centrifugal 10min of whizzer 13200rpm transfers to 2mL pipe, freeze-drying with supernatant.Add 200 μ L 50mM ammonium acetates in the freeze-drying sample, detect NAD concentration with HPLC, thereby obtain the amount of NAD in thalline.
Embodiment the results are shown in Figure 4.According to Fig. 4, when when processing sample, the outer NAD concentration of born of the same parents was lower than 1mM, the sample of stir process compares stirred sample not because NAD oozes out NAD concentration was low; When processing sample, the outer NAD concentration of born of the same parents was higher than 1.5mM, the sample of stir process compares stirred sample not because NAD enters cell NAD concentration was high, illustrates that NAD spreads according to concentration gradient; The amount of the inside and outside larger NAD of NAD concentration difference of cell transhipment before barrier cell is repaired is more, illustrates that the diffusion of NAD meets the faster rule of the larger velocity of diffusion of concentration difference.
The mixed solution that sepiolite and cell are stirred in embodiment 3 explanations can increase eukaryotic property effectively.
Embodiment 4:
Cultivate E.coli to logarithmic phase in 37 ℃ of 100mL LB, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=10.
In 15 50mL centrifuge tubes, get the sepiolite that the above-mentioned bacterium liquid of 1mL adds 4mL water and 5mL mass concentration 1%, add the slight mixing of NAD of 0.2mL 0.125M.Wherein 12 effective whirlpool concussion instrument 3000rpm stir different time (0.5min, 1min, 2min, 5min), and other 3 pipes do not stir.
Every arm is got 90 μ L mixed solutions and is added 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.Every arm is got 90 μ L mixed solutions again after 30 ℃ of placement 30min, adds 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.Sample dilutes 1000 times after 4 ℃ of standing 30min, get 50 μ L coating less salt LB (NaCl 0.5%) flat boards, enumeration after incubated overnight, the viable count that obtain viable count that agitation treatment not directly adds ble, adds ble after stirring at once with place the viable count that adds ble after 30min, every kind of sample is surveyed and is averaged for three times.
In 15 arms, remaining sample is placed 20min at 30 ℃.Whizzer 4000rpm is centrifugal, and 5min removes supernatant, resuspended with 900 μ L PBS after, whizzer 4000rpm is centrifugal, and 5min removes supernatant, resuspended with 900 μ L PBS after, whizzer 13200rpm is centrifugal, and 5min removes supernatant.Add 37 ℃ of cracking 30min of 800 μ L N,O-Diacetylmuramidases, add 400 μ L chloroforms to process 1min with whirlpool concussion instrument 3000rpm, the centrifugal 10min of whizzer 13200rpm transfers to 2mL pipe, freeze-drying with supernatant.Add 200 μ L 50mM ammonium acetates in the freeze-drying sample, detect NAD concentration with HPLC, thereby obtain the amount of NAD in thalline.
During nonoscillatory, it is 0, NAD transfer amount 0 that corresponding cell permeability increases efficient; When the concussion time was 0.5min, it was 78%, NAD transfer amount 8.1nmol that corresponding cell permeability increases efficient; When the concussion time was 1min, it was 89%, NAD transfer amount 9.2nmol that corresponding cell permeability increases efficient; When the concussion time was 2min, it was 92%, NAD transfer amount 9.4nmol that corresponding cell permeability increases efficient; When the concussion time was 5min, it was 95%, NAD transfer amount 9.4nmol that corresponding cell permeability increases efficient.
When embodiment 4 illustrated that adding NAD concentration is 2.5mM, when cell permeability increase efficient changed between 0-95%, cell permeability increased efficient and is directly proportional to the NAD transfer amount.
Embodiment 5:
Cultivate E.coli to logarithmic phase in 37 ℃ of 100mL LB, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=10.
In 15 50mL centrifuge tubes, get the sepiolite that the above-mentioned bacterium liquid of 1mL adds 4mL water and 5mL mass concentration 1%, add the slight mixing of NAD of 0.2mL 0.15M.Wherein 12 effective whirlpool concussion instrument 3000rpm stir different time (0.5min, 1min, 2min, 5min), and other 3 pipes do not stir.
Every arm is got 90 μ L mixed solutions and is added 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.Every arm is got 90 μ L mixed solutions again after 30 ℃ of placement 30min, adds 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.Sample dilutes 1000 times after 4 ℃ of standing 30min, get 50 μ L coating less salt LB (NaCl 0.5%) flat boards, enumeration after incubated overnight, the viable count that obtain viable count that agitation treatment not directly adds ble, adds ble after stirring at once with place the viable count that adds ble after 30min, every kind of sample is surveyed and is averaged for three times.
In 15 arms, remaining sample is placed 20min at 30 ℃.Whizzer 4000rpm is centrifugal, and 5min removes supernatant, resuspended with 900 μ L PBS after, whizzer 4000rpm is centrifugal, and 5min removes supernatant, resuspended with 900 μ L PBS after, whizzer 13200rpm is centrifugal, and 5min removes supernatant.Add 37 ℃ of cracking 30min of 800 μ L N,O-Diacetylmuramidases, add 400 μ L chloroforms to process 1min with whirlpool concussion instrument 3000rpm, the centrifugal 10min of whizzer 13200rpm transfers to 2mL pipe, freeze-drying with supernatant.Add 200 μ L 50mM ammonium acetates in the freeze-drying sample, detect NAD concentration with HPLC, thereby obtain the amount of NAD in thalline.
During nonoscillatory, it is 0, NAD transfer amount 0 that corresponding cell permeability increases efficient; When the concussion time was 0.5min, it was 81%, NAD transfer amount 12.4nmol that corresponding cell permeability increases efficient; When the concussion time was 1min, it was 91%, NAD transfer amount 13.6nmol that corresponding cell permeability increases efficient; When the concussion time was 2min, it was 94%, NAD transfer amount 13.9nmol that corresponding cell permeability increases efficient; When the concussion time was 5min, it was 96%, NAD transfer amount 13.7nmol that corresponding cell permeability increases efficient.
When embodiment 5 explanation cell permeabilities increase efficient changed between 0-94%, cell permeability increased efficient and is directly proportional to the NAD transfer amount.Further improve along with cell permeability increases efficient, the damage increase of the unrepairable that is caused by the increase of saturating property causes the NAD transfer amount to descend.
In conjunction with the embodiments 4, the transfer amount of different concns NAD all is directly proportional to born of the same parents' saturating property increase efficient, so cell permeability increases efficient and can be used as the standard of estimating cell permeability.
Embodiment 6:
Cultivate E.coli to logarithmic phase in 37 ℃ of 100mL LB, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=10.
In the 10mL round-bottomed flask, get the sepiolite that the above-mentioned bacterium liquid of 1mL adds 4mL water and 5mL mass concentration 1%: a. and get 90 μ L mixed solutions and add 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.B. get the 5mL mixed solution in the 10mL round-bottomed flask, add the long magneton of 1cm, stir (stirring intensity is 800rpm, 600rpm, 400rpm) with the magnetic stirring apparatus different rotating speeds and stir sample 2min, get 90 μ L mixed solutions and add 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.In c.b, remaining sample after 30 ℃ of placement 30min, is got 90 μ L mixed solutions and is added 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.
Sample dilutes 1000 times after 4 ℃ of standing 30min, get 50 μ L coating less salt LB (NaCl 0.5%) flat boards, enumeration after incubated overnight, the viable count that obtain viable count that agitation treatment not directly adds ble, adds ble after stirring at once with place the viable count that adds ble after 30min, every kind of sample is surveyed and is averaged for three times.
When stirring intensity was 800rpm, it was 38% that corresponding cell permeability increases efficient, and cell survival rate is 89%; When stirring intensity was 600rpm, it was 33% that corresponding cell permeability increases efficient, and cell survival rate is 91%; When stirring intensity was 400rpm, it was 28% that corresponding cell permeability increases efficient, and cell survival rate is 98%.
When embodiment 6 presentation of results enough made the acicular nanometer material pierce through the mechanical force of barrier cell by stirring the suspension generation, agitation strength was larger, and property increase efficient is higher thoroughly.
Embodiment 7:
Cultivate E.coli to logarithmic phase in 37 ℃ of 100mL LB, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=10.
In the 1.5mL centrifuge tube, get the sepiolite that the 100 above-mentioned bacterium liquid of μ L add 400 μ L water and 500 μ L mass concentrations 1%: a. and get 90 μ L mixed solutions and add 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.B. get the 1mL mixed solution in the 1.5mL centrifuge tube, stir sample 1min with the whirlpool concussion different impact strengths of instrument (3000rpm, 2000rpm, 1000rpm), get 90 μ L mixed solutions and add 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.In c.b, remaining sample after 30 ℃ of placement 30min, is got 90 μ L mixed solutions and is added 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.
Sample dilutes 1000 times after 4 ℃ of standing 30min, get 50 μ L coating less salt LB (NaCl 0.5%) flat boards, enumeration after incubated overnight, the viable count that obtain viable count that agitation treatment not directly adds ble, adds ble after stirring at once with place the viable count that adds ble after 30min, every kind of sample is surveyed and is averaged for three times.
When impact strength was 3000rpm, it was 90% that corresponding cell permeability increases efficient, and cell survival rate is 73%; When impact strength was 2000rpm, it was 83% that corresponding cell permeability increases efficient, and cell survival rate is 75%; When impact strength was 1000rpm, it was 63% that corresponding cell permeability increases efficient, and cell survival rate is 84%.
When the container that embodiment 7 presentation of results are equipped with suspension by concussion produced and enough to make the acicular nanometer material pierce through the mechanical force of barrier cell, impact strength was larger, and property increase efficient is higher thoroughly.
Embodiment 8:
Cultivate E.coli to logarithmic phase in 37 ℃ of 100mL LB, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=10.
In the 1.5mL centrifuge tube, get the sepiolite that the 100 above-mentioned bacterium liquid of μ L add 400 μ L water and 500 μ L mass concentrations 1%: a. and get 90 μ L mixed solutions and add 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.B. get the 1mL mixed solution in the 1.5mL centrifuge tube, 3000rpm stirs different time (1min, 2min, 5min) with whirlpool concussion instrument, gets 90 μ L mixed solutions and adds 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.In c.b, remaining sample after 30 ℃ of placement 30min, is got 90 μ L mixed solutions and is added 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.
Sample dilutes 1000 times after 4 ℃ of standing 30min, get 50 μ L coating less salt LB (NaCl 0.5%) flat boards, enumeration after incubated overnight, the viable count that obtain viable count that agitation treatment not directly adds ble, adds ble after stirring at once with place the viable count that adds ble after 30min, every kind of sample is surveyed and is averaged for three times.
When the concussion time was 1min, it was 93% that corresponding cell permeability increases efficient, and cell survival rate is 74%; When the concussion time was 2min, it was 95% that corresponding cell permeability increases efficient, and cell survival rate is 68%; When the concussion time was 5min, it was 96% that corresponding cell permeability increases efficient, and cell survival rate is 54%.
When embodiment 8 presentation of results stirred suspension, the time of stirring continuously was longer, and property increase efficient is higher thoroughly.
Embodiment 9:
Cultivate E.coli to logarithmic phase in 37 ℃ of 100mL LB, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=10.
In the 1.5mL centrifuge tube, get the sepiolite that the 100 above-mentioned bacterium liquid of μ L add 400 μ L water and 500 μ L different mass concentration (0.5%, 1%, 1.5%): a. and get 90 μ L mixed solutions and add 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.B. get the 1mL mixed solution in the 1.5mL centrifuge tube, 3000rpm stirs sample 1min with whirlpool concussion instrument, gets 90 μ L mixed solutions and adds 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.In c.b, remaining sample after 30 ℃ of placement 30min, is got 90 μ L mixed solutions and is added 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.
Sample dilutes 1000 times after 4 ℃ of standing 30min, get 50 μ L coating less salt LB (NaCl 0.5%) flat boards, enumeration after incubated overnight, the viable count that obtain viable count that agitation treatment not directly adds ble, adds ble after stirring at once with place the viable count that adds ble after 30min, every kind of sample is surveyed and is averaged for three times.
When in mixed solution, sepiolite concentration was 0.25%, it was 81% that corresponding cell permeability increases efficient, and cell survival rate is 80%; When in mixed solution, sepiolite concentration was 0.5%, it was 92% that corresponding cell permeability increases efficient, and cell survival rate is 72%; When in mixed solution, sepiolite concentration was 0.75%, it was 94% that corresponding cell permeability increases efficient, and cell survival rate is 69%.
In embodiment 9 presentation of results suspension, the acicular nanometer material concentration is higher, and property increase efficient is higher thoroughly.
Embodiment 10:
Cultivate E.coli to logarithmic phase in 37 ℃ of 100mL LB, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=20.Be diluted to OD with PBS
600=5,10.
In the 1.5mL centrifuge tube, get 100 μ L OD
600=5,10,20 bacterium liquid adds the sepiolite of 400 μ L water and 500 μ L mass concentrations 1%: a. to get 90 μ L mixed solutions and adds 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.B. get the 1mL mixed solution in the 1.5mL centrifuge tube, 3000rpm stirs sample 1min with whirlpool concussion instrument, gets 90 μ L mixed solutions and adds 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.In c.b, remaining sample after 30 ℃ of placement 30min, is got 90 μ L mixed solutions and is added 10 μ L100mg/mL bleomycins, 4 ℃ of standing 30min.
Sample dilutes 1000 times after 4 ℃ of standing 30min, get 100 μ L coating less salt LB (NaCl 0.5%) flat boards, enumeration after incubated overnight, the viable count that obtain viable count that agitation treatment not directly adds ble, adds ble after stirring at once with place the viable count that adds ble after 30min, every kind of sample is surveyed and is averaged for three times.
Cell density is OD in mixed solution
600=0.5 o'clock, it was 95% that corresponding cell permeability increases efficient, and cell survival rate is 73%; Cell density is OD in mixed solution
600=1 o'clock, it was 93% that corresponding cell permeability increases efficient, and cell survival rate is 73%; Cell density is OD in mixed solution
600=2 o'clock, it was 88% that corresponding cell permeability increases efficient, and cell survival rate is 78%.
In embodiment 10 presentation of results suspension, cell concn is higher, and the cell quantity of property increase is higher thoroughly, but property increase efficient is less thoroughly.
Embodiment 11:
Cultivate E.coli to logarithmic phase in 37 ℃ of 100mL LB, collect thalline and wash twice with PBS, use the resuspended thalline of PBS to OD
600=10.
In the 1.5mL centrifuge tube, getting the 100 above-mentioned bacterium liquid of μ L adds the glycerine (volume by volume concentration is respectively 62.5%, 75%, 87.5%) of PBS preparation of 800 μ L different concns and the sepiolite of 100 μ L mass concentrations 5%: a. to get 90 μ L mixed solutions to add 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.B. get the 1mL mixed solution in the 1.5mL centrifuge tube, 3000rpm stirs sample 1min with whirlpool concussion instrument, gets 90 μ L mixed solutions and adds 10 μ L100mg/mL bleomycins, 4 ℃ of standing 30min.In c.b, remaining sample after 30 ℃ of placement 30min, is got 90 μ L mixed solutions and is added 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min.
Sample dilutes 1000 times after 4 ℃ of standing 30min, get 50 μ L coating less salt LB (NaCl 0.5%) flat boards, enumeration after incubated overnight, the viable count that obtain viable count that agitation treatment not directly adds ble, adds ble after stirring at once with place the viable count that adds ble after 30min, every kind of sample is surveyed and is averaged for three times.
When glycerol concentration in mixed solution was 50%, it was 91% that corresponding cell permeability increases efficient, and cell survival rate is 74%; When glycerol concentration in mixed solution was 60%, it was 89% that corresponding cell permeability increases efficient, and cell survival rate is 76%; When glycerol concentration in mixed solution was 70%, it was 83% that corresponding cell permeability increases efficient, and cell survival rate is 77%.
Embodiment 11 presentation of results suspension viscosities are higher, and property increase efficient is lower thoroughly.
Embodiment 12:
Add the E.coli of 400 μ L logarithmic phases in 40mL M9 substratum, assign in four 50mL pipes, every pipe 9mL bacterium liquid, wherein two pipes add 500 μ L water, other two pipes add 500 μ L 5% sterilization sepiolites, 200rpm wave and culture in 37 ℃ of shaking tables detects cell density and changes, two duct ligations definite growth curve of really averaging.
Embodiment the results are shown in Figure 5.According to Fig. 5, sepiolite can slow down the growth of stirring cultured cells but impact is very little; Make cell accelerate to enter decline phase the stationary phase that can shorten cell, but impact effect is very little.
Embodiment 12 explanation sepiolites can be used for cell liquid culturing process, and sepiolite affects Growth of Cells hardly under normal culture condition.
Embodiment 13:
Add E.coli and the 2mL mass concentration 5% sterilization sepiolite of 400 μ L logarithmic phases in 40mL M9 substratum, be distributed into four pipes, every pipe 10mL bacterium liquid, 200rpm wave and culture in 37 ℃ of shaking tables, respectively at 10min, 30min, 1h takes out a pipe during 8h.
Get 90 μ L bacterium liquid and add 10 μ L 100mg/mL bleomycins, 4 ℃ of standing 30min; All the other samples are got 90 μ L mixed solutions and are added 10 μ L100mg/mL bleomycins, 4 ℃ of standing 30min after 30 ℃ of placement 30min.Sample dilutes 100 times (1000 times of diluted samples during 8h) after 4 ℃ of standing 30min, get 50 μ L coating less salt LB (NaCl 0.5%) flat boards, enumeration after incubated overnight, obtain to add after the rear horse back of sampling adds the viable count of ble and places 30min the viable count of ble, every kind of sample is surveyed and is averaged for three times.Owing to determining
When cultivating 10min with shaking table, it is 31% that corresponding cell permeability increases efficient; When cultivating 30min with shaking table, it is 79% that corresponding cell permeability increases efficient; When cultivating 1h with shaking table, it is 59% that corresponding cell permeability increases efficient; When cultivating 8h with shaking table, it is 32% that corresponding cell permeability increases efficient.
Embodiment 13 explanation sepiolites can increase cell permeability in cell liquid culturing process, cell permeability increases Efficiency Decreasing when cell growth rate is advanced the speed higher than cell permeability.
The present invention is by the mix suspension of stirring acicular nanometer material with cell, and the acicular nanometer material stings out recoverable aperture on cytolemma or cell walls and cytolemma, reach the purpose that increases cell permeability.The advantage of the method is, and sample simple to operate, that process is not subjected to volume restrictions, need not expensive accurate instrument, can makes efficiently cell obtain saturating property and guarantee cell survival rate.Be applicable to increase cell permeability.
Claims (10)
1. method of utilizing the acicular nanometer material to increase cell permeability is characterized in that:
Use physical method to pierce through cytolemma or cell walls and cytolemma, form the aperture of diameter 0.02-0.12 μ m at cell surface, the cell permeability that produces 28-96% increases efficient.
2. method according to claim 1 is characterized in that:
Described physical method is under liquid environment, add the acicular nanometer material in liquid, the mechanical force that produces by stirring makes the acicular nanometer material pierce through cytolemma or cell walls and cytolemma, produces aperture on cytolemma or cell walls and cytolemma, makes cell obtain saturating property;
The aperture that the acicular nanometer material produces on barrier cell can be by cytothesis, thereby guarantees the survival rate of cell; Increase efficient with cell permeability and improve, cell survival rate reduces.
3. method according to claim 2, it is characterized in that: the acicular nanometer material refers to diameter at 0.02-0.12 μ m, the material of length between 0.34-2.30 μ m, or the surface is with the material of the spicule that meets above-mentioned specification;
The acicular nanometer material mixes with the state of cell with suspension.
4. according to claim 2 or 3 described methods, it is characterized in that: described acicular nanometer material is sepiolite; The particle of sepiolite more than 90% be less than 5 μ m, particle mean length 2 μ m, mean diameter 20nm.
5. method according to claim 4 is characterized in that:
In suspension, the acicular nanometer material concentration is higher, and property increase efficient is higher thoroughly; Cell density be 1OD, stirring time be 1min, when impact strength is 3000rpm, when adding the concentration of acicular nanometer material sepiolite to change between 0.25%-0.75% (mass ratio) in mixed solution, property increase efficient changes between 81%-94% thoroughly, and cell survival rate changes between 80%-69%;
In suspension, cell concn is higher, and property increase efficient is higher thoroughly, but property increase efficient is less thoroughly; Sepiolite concentration is 0.5%, the stirring time is 1min, when impact strength is 3000rpm, when in mixed solution, cell density changed between 0.5OD-2OD, property increase efficient changed between 95%-88% thoroughly, cell survival rate changes between 73%-78%.
6. method according to claim 4, it is characterized in that: suspension viscosity is higher, and property increase efficient is lower thoroughly; Suspension viscosity can change by adding glycerine, and viscosity improves to increase the system glycerol concentration and realizes;
Sepiolite concentration is 0.5%, cell density be 1OD, stirring time be 1min, when impact strength is 3000rpm, suspension viscosity is 54 * 10
-3Pas-65 * 10
-3When changing between Pas, property increase efficient changes between 91%-83% thoroughly, and cell survival rate changes between 74%-77%.
7. method according to claim 2 is characterized in that: the mechanical force that described stirring produces is enough to make the acicular nanometer material pierce through the mechanical force of barrier cell by the container generation that suspension is equipped with in direct stirring suspension itself or concussion.
8. method according to claim 7 is characterized in that: by stirring the generation of direct stirring suspension own when enough making the acicular nanometer material pierce through the mechanical force of barrier cell, agitation strength is larger, and property increase efficient is higher thoroughly;
Sepiolite concentration is 0.5%, cell density is 1OD, stirring time when being 2min, stirs suspension intensity when changing between 800rpm-400rpm, and property increase efficient changes between 28%-38% thoroughly, and cell survival rate changes between 98%-89%.
9. method according to claim 7 is characterized in that: when the container that suspension is housed by concussion produced and enough makes the acicular nanometer material pierce through the mechanical force of barrier cell, impact strength was larger, and property increase efficient is higher thoroughly;
Sepiolite concentration is 0.5%, cell density is 1OD, stirring time when being 1min, and when impact strength changed between 1000rpm-2000rpm, property increase efficient changed between 63%-90% thoroughly, and cell survival rate changes between 84%-73%.
10. method according to claim 7 is characterized in that: when stirring suspension, the time of stirring continuously is longer, and property increase efficient is higher thoroughly;
Sepiolite concentration is 0.5%, cell density is 1OD, when impact strength is 3000rpm, when the concussion time changed between 1min-5min, property increase efficient changed between 93%-96% thoroughly, cell survival rate changes between 74%-54%.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010101461A1 (en) * | 2009-03-03 | 2010-09-10 | Technische Universiteit Eindhoven | Device and method for treating cells |
| CN101991851A (en) * | 2009-08-24 | 2011-03-30 | 中国医学科学院药物研究所 | Ultrasound microbubble agent for treating tumors by low-intensity focused ultrasound and preparation method thereof |
-
2011
- 2011-12-19 CN CN2011104279725A patent/CN103160438A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010101461A1 (en) * | 2009-03-03 | 2010-09-10 | Technische Universiteit Eindhoven | Device and method for treating cells |
| CN101991851A (en) * | 2009-08-24 | 2011-03-30 | 中国医学科学院药物研究所 | Ultrasound microbubble agent for treating tumors by low-intensity focused ultrasound and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 谭海东等: "基于矿石纳米材料的DNA转化的机理初探及方法改进", 《生物工程学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110108905A (en) * | 2019-05-22 | 2019-08-09 | 长春理工大学 | A kind of nervous cell membrane potential and neuron membrane repair behavioral value method and device |
| CN110108905B (en) * | 2019-05-22 | 2021-08-06 | 长春理工大学 | Method and device for detecting membrane potential and nerve cell membrane repairing behaviors of nerve cells |
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