CN110343658A - A kind of medical cell experiment process liquid configuration method and products thereof - Google Patents
A kind of medical cell experiment process liquid configuration method and products thereof Download PDFInfo
- Publication number
- CN110343658A CN110343658A CN201910700566.8A CN201910700566A CN110343658A CN 110343658 A CN110343658 A CN 110343658A CN 201910700566 A CN201910700566 A CN 201910700566A CN 110343658 A CN110343658 A CN 110343658A
- Authority
- CN
- China
- Prior art keywords
- solution
- concentration
- cell
- glucose
- configuration method
- 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
- 238000000034 method Methods 0.000 title claims abstract description 44
- 238000002474 experimental method Methods 0.000 title claims abstract description 27
- 239000007788 liquid Substances 0.000 title claims abstract description 25
- 230000008569 process Effects 0.000 title claims abstract description 16
- 239000000243 solution Substances 0.000 claims abstract description 75
- 239000002253 acid Substances 0.000 claims abstract description 31
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 30
- 235000005300 cardamomo Nutrition 0.000 claims abstract description 30
- 239000008103 glucose Substances 0.000 claims abstract description 30
- 239000012530 fluid Substances 0.000 claims abstract description 26
- TWJNQYPJQDRXPH-UHFFFAOYSA-N 2-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C#N TWJNQYPJQDRXPH-UHFFFAOYSA-N 0.000 claims abstract description 14
- 235000021360 Myristic acid Nutrition 0.000 claims abstract description 14
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000012360 testing method Methods 0.000 claims abstract description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 48
- 238000001816 cooling Methods 0.000 claims description 42
- 210000004027 cell Anatomy 0.000 claims description 40
- 240000002943 Elettaria cardamomum Species 0.000 claims description 29
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 22
- 239000011259 mixed solution Substances 0.000 claims description 16
- 102000004895 Lipoproteins Human genes 0.000 claims description 15
- 108090001030 Lipoproteins Proteins 0.000 claims description 15
- 239000007853 buffer solution Substances 0.000 claims description 13
- 150000001875 compounds Chemical class 0.000 claims description 12
- 239000007789 gas Substances 0.000 claims description 12
- 238000002156 mixing Methods 0.000 claims description 12
- 229910052786 argon Inorganic materials 0.000 claims description 11
- 230000001954 sterilising effect Effects 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 9
- 210000001789 adipocyte Anatomy 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 230000010355 oscillation Effects 0.000 claims description 6
- 230000005540 biological transmission Effects 0.000 claims description 5
- 230000004927 fusion Effects 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- NJPPVKZQTLUDBO-UHFFFAOYSA-N novaluron Chemical compound C1=C(Cl)C(OC(F)(F)C(OC(F)(F)F)F)=CC=C1NC(=O)NC(=O)C1=C(F)C=CC=C1F NJPPVKZQTLUDBO-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 238000011017 operating method Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000007306 turnover Effects 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 2
- 229910021641 deionized water Inorganic materials 0.000 claims description 2
- 238000002604 ultrasonography Methods 0.000 claims description 2
- 239000004744 fabric Substances 0.000 claims 1
- 150000001720 carbohydrates Chemical group 0.000 abstract description 7
- 206010012601 diabetes mellitus Diseases 0.000 abstract description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 abstract description 4
- 238000004090 dissolution Methods 0.000 abstract description 4
- 238000010790 dilution Methods 0.000 abstract description 3
- 239000012895 dilution Substances 0.000 abstract description 3
- 235000021588 free fatty acids Nutrition 0.000 abstract description 3
- 230000001575 pathological effect Effects 0.000 abstract description 3
- 244000222991 cardamomo Species 0.000 abstract 1
- 150000002772 monosaccharides Chemical class 0.000 abstract 1
- 238000013461 design Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 102000015735 Beta-catenin Human genes 0.000 description 4
- 108060000903 Beta-catenin Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000112 cooling gas Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 206010068150 Acoustic shock Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000016362 Catenins Human genes 0.000 description 1
- 108010067316 Catenins Proteins 0.000 description 1
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 1
- 206010021703 Indifference Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 208000017442 Retinal disease Diseases 0.000 description 1
- 206010038923 Retinopathy Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 238000002242 deionisation method Methods 0.000 description 1
- 208000033679 diabetic kidney disease Diseases 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 208000030172 endocrine system disease Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000011810 insulating material Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229910052755 nonmetal Inorganic materials 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 210000001626 skin fibroblast Anatomy 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0656—Adult fibroblasts
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/36—Lipids
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Rheumatology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
The present invention provides a kind of medical cell experiment process liquid configuration methods and products thereof, belong to medical cell and tests high saccharide ring border simulated solution high in fat, the cell experiment treatment fluid includes the myristic acid of 10-25mmol/L and the glucose of 1-5mol/L, for myristic acid as a kind of for simulating the environment high in fat of cell of free fatty acid, glucose is used to simulate the high saccharide ring border of cell as a kind of monosaccharide.Configuration method provided by the invention can effectively dissolve myristic acid and glucose, solve the technical issues of cardamom acid dissolution is unstable, solute is precipitated, the high sugared treatment fluid concentration high in fat configured is high and concentration is accurate, experimenter can be according to different cell experiments, dilution or directly using the cell treatment fluid to simulate the influence of high saccharide ring border high in fat to cell under various concentration, the pathological study for greatly facilitating diabetes works.
Description
Technical field
The present invention relates to medical cells to test high saccharide ring border simulated solution high in fat, and in particular to a kind of medical cell experimental station
Manage liquid configuration method and products thereof.
Background technique
2 patients with type Ⅰ DM (type 2 diabetes mellitus, T2DM) are endocrine system disease, 2 patients with type Ⅰ DM original names
Adult onset diabetes are, mostly falls ill after 35~40 years old, accounts for 90% or more diabetic.2 patients with type Ⅰ DM it is long-term
Occur, develop the generation that can lead to cardiovascular pathological changes, retinopathy and diabetic nephropathy etc..
In order to study the pathology and pathogenesis of 2 patients with type Ⅰ DM, scientific research personnel needs to simulate diabetic in vitro
Then internal hyperglycemia, the experimental situation of high free fatty acid study high saccharide ring border high in fat to different doctors by cell experiment again
Treat the influence and lesion of cell.Wherein, myristic acid is as common free fatty acid in human body and animal body, in adiposis patient, sugar
Urine patient's is widely present in vivo, is the excellent selection for simulating the environment high in fat of cell.
However, how myristic acid dissolves the technical problem for becoming the field in cell culture medium as a kind of solid.
In the prior art, without the dissolving method of pertinent literature record myristic acid, also without recording matching for high glucose and high fat cell treatment fluid
Details are set, scientific research personnel can only voluntarily grope, and only a few personnel use methanol, ethyl alcohol equal solvent is prepared, the disadvantage is that configuration
There is solid precipitation soon afterwards, lead to the concentration inaccuracy of cell treatment fluid, concentration inaccuracy will lead to experimental data and knot
By unreliable, cell model is not perfect.Also only a few personnel directly configure using bovine serum albumin(BSA), this configuration method is suitable
The case where closing low concentration, can not configure the cell treatment fluid higher than 10mmol/L, therefore can not simulate the diabetes of high concentration
Environment, usage scenario are limited.
Summary of the invention
In view of this, providing a kind of medical cell experiment it is an object of the invention in view of the problems of the existing technology
Treatment fluid configuration method and products thereof, this method can effectively dissolve myristic acid and glucose, solve cardamom acid dissolution it is unstable,
The technical issues of solute is precipitated, the high sugared treatment fluid concentration high in fat configured is high and concentration is accurate, and experimenter can be according to not
Same cell experiment, dilution or directly using the cell treatment fluid to simulate under various concentration high saccharide ring border high in fat to the shadow of cell
It rings, greatly facilitates the pathological study work of diabetes.
In order to achieve the object of the present invention, the present invention adopts the following technical scheme:
A kind of medical cell experiment process liquid configuration method, which is characterized in that successively include that stoste prepares to merge two with stoste
Step, in which: the stoste preparation process includes the BSA solution that compound concentration is 45-50g/100ml and compound concentration is 20-
The cardamom acid solution of 50mmol/L；The stoste fusion steps specifically include following operating method:
1) low-temperature mixed: using pipettor by 60-65 DEG C and cardamom acid solution that concentration is 20-50mmol/L is added to 80-85
DEG C and concentration be 45-50g/100ml BSA solution in and vibrate mixing, obtain mixed solution, the cardamom acid solution and BSA are molten
The mixed volume ratio of liquid is 1:1, by mixed solution fast cooling to -20 DEG C, hydrotropy 3-5min in the environment of -20 DEG C；
2) it adjusts back PH: adding the enriching hydrochloric acid of 12mol/L to mixed liquor and be uniformly mixed, be adjusted to the PH numerical value of mixed liquor
7.3-7.5 obtains high lipoprotein solution；
3) it adds glucose: high lipoprotein solution being moved into centrifuge tube to be placed in 30-35 DEG C of water-bath and is heated, high lipoprotein solution is heated to
At 30-35 DEG C, weighs glucose and be added in high lipoprotein solution and be put in the speed centrifugation 8- on centrifuge with 6000r/min
10min obtains that concentration of glucose is 1-5mol/L, cardamom acid concentration is 10- until glucose is dissolved completely in high lipoprotein solution
The high glucose and high fat cell experiment treatment fluid of 25mmol/L.
It preferably, further include ultrasonic mixing operation between low-temperature mixed and readjustment PH operation, the ultrasound mixing operation
It specifically includes: -20 DEG C of low-temperature mixed solution after hydrotropy being placed in 20-25 DEG C of environment, and use the super of 200-500W power
6-8min is swung in acoustic shock.
Preferably, it includes: by the PBS buffer solution in centrifuge tube that the compound concentration, which is the BSA solution of 45-50g/100ml,
It is placed in 60-65 DEG C of water-bath and heats, when PBS buffer solution is heated to 60-65 DEG C, weighs BSA and be added in PBS buffer solution and be put in
12-15min is centrifuged with the speed of 10000r/min on centrifuge, until BSA is dissolved completely in PBS buffer solution, obtaining concentration is 45-
The BSA solution of 50g/100ml, BSA solution is placed in 60-65 DEG C of water-bath and is kept the temperature.
Preferably, it includes: to weigh NaOH to be dissolved in deionization that the compound concentration, which is the cardamom acid solution of 20-50mmol/L,
Water is made into 0.15mol/L NaOH solution, then weighs myristic acid and be added in NaOH solution, be placed in 80-85 DEG C of water-bath 35-
The cardamom acid solution for being dissolved in NaOH solution is put on centrifuge and is centrifuged 3-5min with the speed of 10000r/min, until beans by 50min
Cool acid is dissolved completely in NaOH solution, obtains the cardamom acid solution that concentration is 20-50mmol/L, cardamom acid solution is placed in 80-85
DEG C water-bath in keep the temperature.
Preferably, it is described by mixed solution fast cooling to -20 DEG C include: using argon gas cooling device that mixed solution is fast
Speed is cooled to -20 DEG C, and the argon gas cooling device includes liquid argon tanks, controlled valve, tracheae and cooler, the switch valve
Door is mounted in the opening of liquid argon tanks, tracheae one end connection switch valve, and the other end of tracheae connects cooler, institute
Cooler is stated for being quickly cooled down test tube, centrifuge tube, beaker or pipettor.
Preferably, the cooler includes cooling ring, the adjusting on the handle and mounting knob for being mounted on cooling ring side
Valve, the cooling ring are the hollow cylinder of both ends open, and the inner surface of cooling ring is evenly arranged with cold air nozzle, the tune
The one end for saving valve connects intake interface, the other end connecting gas transmission pipe of control valve, and the intake interface connects the tracheae,
The hollow cavity of the appendix connection cooling ring can be realized 40-80 DEG C of mixed solution/min cooling speed by control valve
Degree.
Preferably, the control valve includes valve body, pressure spring, choked flow magnet, button, driving magnet and fixture nut,
In: the valve body is mounted on inside handle by fixture nut；The valve inner is provided with air cavity, and the both ends of air cavity are provided with
Attachment base, side attachment base connect intake interface, and other side attachment base connecting gas transmission pipe, the bottom of the air cavity offers bottom
Spring chamber, the bottom spring chamber is bottom-up to be disposed with pedestal, pressure spring and choked flow magnet, the choked flow magnet can be in spring chamber on
Lower movement, the valve body are provided with top guidance cavity in the position close to air cavity, are equipped with button in the top guidance cavity, institute
The bottom for stating button is equipped with driving magnet, and the button can move up and down in guidance cavity, the driving magnet and choked flow
Magnetic pole is identical adjacent to one another for magnet.
Preferably, it includes being mixed using turbula shaker or turnover oscillator oscillation that the oscillation, which mixes operation,.
It preferably, further include filter sterilization step, the filter sterilization step includes: to set high glucose and high fat cell treatment fluid
In on super-clean bench, is filtered using bacterial filter and use ultraviolet lamp sterilization.
A kind of cell experiment treatment fluid configuration method of the invention has the advantages that
1) the cell treatment fluid is instant liquid, is used after directly can using or dilute, and is specifically 1-5mol/ comprising concentration
The glucose and concentration of L is the myristic acid of 10-25mmol/L, can effectively simulate the fluid environment of diabetes high glucose and high fat, energy
It enough greatly facilitates scientific research occurrences in human life and carries out cell processing experiment.
2) cell treatment fluid performance is stable, concentration is accurate, concentration is high, and solve that solid after reagent configures is precipitated asks
Topic, wherein cardamom acid concentration is that the 10-25mmol/L concentration can satisfy cell processing experiments several greatly absolutely.
3) the cell treatment fluid can long-term preservation, can be saved in the environment of -20 DEG C 12-14 months.
4) quick cooler based on processing mode design, can be fast for cooling test tube, centrifuge tube, beaker or pipettor
Prompt drop temperature, the speed of the adjustable cooling of one side on the other hand can be to stir in cooling, which can be placed on whirlpool
On the platform for revolving oscillator, reagent to be cooled down is wrapped, agitation while cools down.
Detailed description of the invention
Fig. 1 is the first configuration method flow diagram of the invention；
Fig. 2 is the second configuration method flow diagram of the invention；
Fig. 3 is the structural schematic diagram of cooling device in the present invention；
Fig. 4 is control valve closing structure schematic diagram in the present invention；
Fig. 5 is control valve Unclosing structure schematic diagram in the present invention；
Fig. 6 and Fig. 7 is that cell treatment fluid of the invention acts on β-catenin cell datagram.
In figure, 1- cooling ring, 2- handle, 3- control valve, 301- valve body, 302- air cavity, 303- attachment base, the bottom 304-
Spring chamber, 305- pedestal, 306- pressure spring, 307- choked flow magnet, the top 308- guidance cavity, 309- button, 310- driving magnet, 311-
Fixture nut, 4- cold air nozzle, 5- intake interface, 6- appendix.
Specific embodiment
The present invention may be implemented in different forms, be not limited to the examples mentioned in the following.Following implementation
Example only as the present invention difference towards and feature in representative.
As shown in Figure 1, a kind of medical cell experiment process liquid configuration method successively includes that stoste prepares and stoste fusion two
A step, in which:
It is 20-50mmol/L that BSA solution that compound concentration is 45-50g/100ml and compound concentration are needed in stoste preparation process
Cardamom acid solution.
Specifically, the BSA solution that compound concentration is 45-50g/100ml includes: to be placed in the PBS buffer solution in centrifuge tube
It is heated in 60-65 DEG C of water-bath, when PBS buffer solution is heated to 60-65 DEG C, weighs BSA and be added in PBS buffer solution and be put in centrifugation
12-15min is centrifuged with the speed of 10000r/min on machine, until BSA is dissolved completely in PBS buffer solution, obtaining concentration is 45-50g/
The BSA solution of 100ml, BSA solution is placed in 60-65 DEG C of water-bath and is kept the temperature.
It should be noted that BSA may be dissolved in PBS buffer solution soon under high speed centrifugation, at this time, it is desired nonetheless to
It is centrifuged 12-15min.Meanwhile directly centrifugation mixing is not heated, or rock or shake and be likely to that BSA is caused to form lump shaped crystalline
It is insoluble, therefore, to avoid shaking in heating process.
Specifically, the cardamom acid solution that compound concentration is 20-50mmol/L includes: to weigh NaOH to be dissolved in deionized water,
It is made into 0.15mol/L NaOH solution, then weighs myristic acid and is added in NaOH solution, is placed in 80-85 DEG C of water-bath 35-50min,
The cardamom acid solution for being dissolved in NaOH solution is put on centrifuge, 3-5min is centrifuged with the speed of 10000r/min, until myristic acid is complete
Fully dissolved obtains the cardamom acid solution that concentration is 20-50mmol/L, cardamom acid solution is placed in 80-85 DEG C of water in NaOH solution
It is kept the temperature in bath.
It should be noted that the process kept the temperature in water-bath is essential, if cooling, it is solid that white gels shape can be obtained
Body needs to be heated to clarifying in 80-85 DEG C again.
Stoste fusion steps specifically include following operating method:
1) low-temperature mixed: using pipettor by 60-65 DEG C and cardamom acid solution that concentration is 20-50mmol/L is added to 80-85
DEG C and concentration be in the BSA solution of 45-50g/100ml and to vibrate mixings, obtain mixed solution, cardamom acid solution and BSA solution
Mixed volume ratio is 1:1, by mixed solution fast cooling to -20 DEG C, hydrotropy 3-5min in the environment of -20 DEG C；
Specifically, it includes being mixed using turbula shaker or turnover oscillator oscillation that oscillation, which mixes operation,.Meanwhile fast cooling
It needs to reach the cooling rate of 40-80 DEG C/min to -20 DEG C, is quickly cooled down for mixing most important, otherwise later period solution meeting
There is cotton-shaped object.
2) it adjusts back PH: adding the enriching hydrochloric acid of 12mol/L to mixed liquor and be uniformly mixed, adjust the PH numerical value of mixed liquor
For 7.3-7.5, high lipoprotein solution is obtained；
It should be noted that due to using NaOH solution when dissolution, it is therefore desirable to adjust back PH.Solution is brown color after adjusting back PH
Sample is clarified, with former BSA solution character indifference, without any solid or cotton-shaped impurity, character is stablized.
3) it adds glucose: high lipoprotein solution being moved into centrifuge tube to be placed in 30-35 DEG C of water-bath and is heated, high lipoprotein solution adds
When heat is to 30-35 DEG C, weighs glucose and be added in high lipoprotein solution and be put on centrifuge 8- is centrifuged with the speed of 6000r/min
10min obtains that concentration of glucose is 1-5mol/L, cardamom acid concentration is 10- until glucose is dissolved completely in high lipoprotein solution
The high glucose and high fat cell experiment treatment fluid of 25mmol/L.
It should be noted that using the above method when small measurement, low concentration configure.When needs large dosage configuration, and
When glucose is 4-5mol/L, cardamom acid concentration is 18-25mmol/L, it is also necessary to be set between low-temperature mixed and readjustment PH operation
Ultrasonic mixing operation is set, ultrasonic mixing operation specifically includes: -20 DEG C of low-temperature mixed solution after hydrotropy are placed in 20-25 DEG C
Environment, and use the ultrasonic vibration 6-8min of 200-500W power.
The present invention includes liquid argon for the technological design of mixed solution fast cooling new argon gas cooler, the cooler
Gas tank, controlled valve, tracheae and cooler, controlled valve are mounted in the opening of liquid argon tanks, tracheae one end connection switch
Valve, the other end of tracheae connect cooler, and cooler is for being quickly cooled down test tube, centrifuge tube, beaker or pipettor.Such as Fig. 3
With shown in Fig. 4, cooler includes cooling ring 1, the control valve 3 on the handle 2 and mounting knob 2 for being mounted on 1 side of cooling ring,
Cooling ring 1 is the hollow cylinder of both ends open, and the inner surface of cooling ring 1 is evenly arranged with cold air nozzle 4, control valve 3
One end connects intake interface 5, the other end connecting gas transmission pipe 6 of control valve 3, and intake interface 5 connects tracheae, and appendix 6 is connected to
The hollow cavity of cooling ring 1 can be realized 40-80 DEG C of mixed solution/min cooling rate by control valve 3.Wherein, handle 2
Installation and adjustment valve 3 and various pipelines are used for for hollow structure.
It should be noted that control valve 3 can be ball valve or other common valves.In the present embodiment, for the ease of adjusting
Section uses new-type control valve 3, and the button 309 by pressing control valve 3 can be realized valve flow and adjust, a hand
Finger can fast implement.
Specifically, control valve 3 includes valve body 301, pressure spring 306, choked flow magnet 307, button 309,310 and of driving magnet
Fixture nut 311, in which: valve body 301 is mounted on inside handle 2 by fixture nut 311；Valve body 301 is internally provided with air cavity
302, the both ends of air cavity 302 are provided with attachment base 303, and side attachment base 303 connects intake interface 5, and other side attachment base 303 connects
Appendix 6 is connect, the bottom of air cavity 302 offers bottom spring chamber 304, and bottom spring chamber 304 is bottom-up to be disposed with pedestal 305, pressure
Spring 306 and choked flow magnet 307, choked flow magnet 307 can move up and down in bottom spring chamber 304, and valve body 301 is close to air cavity
302 position is provided with top guidance cavity 308, is equipped with button 309 in top guidance cavity 308, button 309 can pass through sliding slot
Slide block device is mounted in the guidance cavity 308 of top, and the bottom of button 309 is equipped with driving magnet 310, and button 309 can push up
It is moved up and down in portion's guidance cavity 308, magnetic pole is identical adjacent to one another for driving magnet 310 and choked flow magnet 307.Wherein, pedestal 305
The installation for being convenient for pressure spring 306 and choked flow magnet 307 in 301 bottom of valve body can be detachably arranged in by screw thread or buckle.
It should be noted that being based on magnet since magnetic pole is identical adjacent to one another for driving magnet 310 and choked flow magnet 307
The principle of homopolar-repulsion.Firstly, turning on the switch valve, secondly in the case where button 309 is not by external force, choked flow magnet 307 exists
Under the action of bottom pressure spring 306, bounces and cooling air-flow is hindered to enter in cooling ring 1, at this time cooling gas flow very little, cooling velocity
Generally, in the case that button 309 is by external force, driving magnet 310 is moved down, due to homopolar-repulsion, at this point, choked flow magnet 307
The elastic force of pressure spring 306 is overcome to move down, at this point, the drag reduction of air-flow, cooling gas flow become larger.In conclusion passing through control
The dynamics of system pressing just can be realized the adjusting of cooling velocity.Specifically, choked flow magnet 307, bottom pressure spring 306 and driving magnet
310 size and interaction force needs to design in advance, to guarantee that control valve 3 in the initial state for normally off, uses
The structure eliminates traditional sealing structure, which is intended merely to hinder air-flow, for air cavity 302 and choked flow magnet 307 it
Between whether be fully sealed it is of less demanding.
It needs to illustrate again, cooling ring 1 and handle 2 can be made of non-metal heat-insulating material.307 He of choked flow magnet
The outer surface of driving magnet 310 is designed for arc transition, reduces frictional force, avoids stuck in moving process, 307 He of choked flow magnet
Driving magnet 310 can also use cylinder design, and when using cylinder design, the diameter of choked flow magnet 307 should be slightly less than air cavity 302
Internal diameter, the topology view do not provide.
Finally, it should be noted that further including filter sterilization step, filter sterilization step includes: will be at high glucose and high fat cell
Reason liquid is placed on super-clean bench, is filtered using bacterial filter and is used ultraviolet lamp sterilization.
β-catenin cell is handled based on the cell treatment fluid, research high glucose and high fat expresses shadow to skin fibroblasts
Loud experimental study, it is specific to detect the fibroblast β-after different disposal using western blot western blot method
Catenin protein expression situation, if Fig. 6 and Fig. 7 is β-catenin after the high myristic acid effect different time of high glucose joint
The variation of protein expression level compared with the control group, * P < 0.05.Wherein, it dilutes the cell treatment fluid to be tested, in experiment
Concentration of glucose is 40mmol/L, cardamom acid concentration is 300 μm of ol/L, and 8h is shown to β-catenin protein expression
Depression effect, it was demonstrated that the validity of the cell treatment fluid.
Configuration method provided by the invention can effectively dissolve myristic acid and glucose, solve cardamom acid dissolution it is unstable,
The technical issues of solute is precipitated, the high sugared treatment fluid concentration high in fat configured is high and concentration is accurate, and experimenter can be according to not
Same cell experiment, dilution or directly using the cell treatment fluid to simulate under various concentration high saccharide ring border high in fat to the shadow of cell
It rings, greatly facilitates the pathological study work of diabetes.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Claims (10)
1. a kind of medical cell experiment process liquid configuration method, which is characterized in that successively include that stoste prepares and stoste fusion two
A step, in which:
The stoste preparation process includes the BSA solution that compound concentration is 45-50g/100ml and compound concentration is 20-50mmol/
The cardamom acid solution of L；
The stoste fusion steps include following operating method:
1) low-temperature mixed: using pipettor by 60-65 DEG C and cardamom acid solution that concentration is 20-50mmol/L is added to 80-85
DEG C and concentration be 45-50g/100ml BSA solution in and vibrate mixing, obtain mixed solution, the cardamom acid solution and BSA are molten
The mixed volume ratio of liquid is 1:1, by mixed solution fast cooling to -20 DEG C, hydrotropy 3-5min in the environment of -20 DEG C；
2) it adjusts back PH: adding the enriching hydrochloric acid of 12mol/L to mixed liquor and be uniformly mixed, be adjusted to the PH numerical value of mixed liquor
7.3-7.5 obtains high lipoprotein solution；
3) it adds glucose: high lipoprotein solution being moved into centrifuge tube to be placed in 30-35 DEG C of water-bath and is heated, high lipoprotein solution is heated to
At 30-35 DEG C, weighs glucose and be added in high lipoprotein solution and be put in the speed centrifugation 8- on centrifuge with 6000r/min
10min obtains that concentration of glucose is 1-5mol/L, cardamom acid concentration is 10- until glucose is dissolved completely in high lipoprotein solution
The high glucose and high fat cell experiment treatment fluid of 25mmol/L.
2. medical cell experiment process liquid configuration method according to claim 1, which is characterized in that in low-temperature mixed and return
Adjusting between PH operation further includes ultrasonic mixing operation, and the ultrasound mixing operation specifically includes: -20 DEG C of low temperature after hydrotropy are mixed
The environment that solution is placed in 20-25 DEG C is closed, and uses the ultrasonic vibration 6-8min of 200-500W power.
3. medical cell experiment process liquid configuration method according to claim 1, which is characterized in that the compound concentration is
The BSA solution of 45-50g/100ml includes:
PBS buffer solution in centrifuge tube is placed in 60-65 DEG C of water-bath and is heated, when PBS buffer solution is heated to 60-65 DEG C, is weighed
BSA, which is added in PBS buffer solution and is put on centrifuge, is centrifuged 12-15min with the speed of 10000r/min, until BSA is completely dissolved
In PBS buffer solution, the BSA solution that concentration is 45-50g/100ml is obtained, BSA solution is placed in 60-65 DEG C of water-bath and is kept the temperature.
4. medical cell experiment process liquid configuration method according to claim 1, which is characterized in that the compound concentration is
The cardamom acid solution of 20-50mmol/L includes:
Weigh NaOH and be dissolved in deionized water, be made into 0.15mol/L NaOH solution, then weigh myristic acid be added NaOH solution in,
It is placed in 80-85 DEG C of water-bath 35-50min, the cardamom acid solution for being dissolved in NaOH solution is put on centrifuge with 10000r/min
Speed be centrifuged 3-5min, until myristic acid is dissolved completely in NaOH solution, obtain the cardamom acid solution that concentration is 20-50mmol/L,
Cardamom acid solution is placed in 80-85 DEG C of water-bath and is kept the temperature.
5. medical cell experiment process liquid configuration method according to claim 1, which is characterized in that described by mixed solution
Fast cooling includes: to -20 DEG C
Using argon gas cooling device by mixed solution fast cooling to -20 DEG C, the argon gas cooling device include liquid argon tanks,
Controlled valve, tracheae and cooler, the controlled valve are mounted in the opening of liquid argon tanks, and tracheae one end connection is opened
Closing valve, the other end of tracheae connect cooler, and the cooler is for being quickly cooled down test tube, centrifuge tube, beaker or pipettor.
6. medical cell experiment process liquid configuration method according to claim 5, which is characterized in that the cooler includes
Cooling ring, the control valve being mounted on the handle and mounting knob of cooling ring side, the cooling ring are the sky of both ends open
Heart cylindrical body, the inner surface of cooling ring are evenly arranged with cold air nozzle, and one end of the control valve connects intake interface, adjusts
The other end connecting gas transmission pipe of valve, the intake interface connect the tracheae, and the appendix is connected to the hollow cavity of cooling ring,
It can be realized 40-80 DEG C of mixed solution/min cooling rate by control valve.
7. medical cell experiment process liquid configuration method according to claim 6, which is characterized in that the control valve packet
Include valve body, pressure spring, choked flow magnet, button, driving magnet and fixture nut, in which: the valve body is mounted on by fixture nut
Inside handle；The valve inner is provided with air cavity, and the both ends of air cavity are provided with attachment base, and side attachment base connects air inlet connecting
Mouthful, other side attachment base connecting gas transmission pipe, the bottom of the air cavity offers bottom spring chamber, the bottom-up cloth of bottom spring chamber
It is equipped with pedestal, pressure spring and choked flow magnet, the choked flow magnet can move up and down in the spring chamber of bottom, and the valve body is close to gas
The position of chamber is provided with top guidance cavity, is equipped with button in the top guidance cavity, the bottom of the button is equipped with driving
Magnet, the button can move up and down in the guidance cavity of top, the driving magnet and choked flow magnet magnetic pole adjacent to one another
It is identical.
8. medical cell experiment process liquid configuration method according to claim 1, which is characterized in that the oscillation mixes behaviour
Make to include mixing using turbula shaker or turnover oscillator oscillation.
9. medical cell experiment process liquid configuration method according to claim 1, which is characterized in that further include filter sterilization
Step, the filter sterilization step include:
High glucose and high fat cell treatment fluid is placed on super-clean bench, is filtered using bacterial filter and uses ultraviolet lamp sterilization.
10. according to claim 1 to cell treatment fluid prepared by method described in 9 any one.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910700566.8A CN110343658A (en) | 2019-07-31 | 2019-07-31 | A kind of medical cell experiment process liquid configuration method and products thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910700566.8A CN110343658A (en) | 2019-07-31 | 2019-07-31 | A kind of medical cell experiment process liquid configuration method and products thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110343658A true CN110343658A (en) | 2019-10-18 |
Family
ID=68183439
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910700566.8A Pending CN110343658A (en) | 2019-07-31 | 2019-07-31 | A kind of medical cell experiment process liquid configuration method and products thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110343658A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR920012935U (en) * | 1990-12-29 | 1992-07-25 | 포항종합제철 주식회사 | Rapid cooling system using adiabatic low pressure tank |
| CN101041550A (en) * | 2006-12-28 | 2007-09-26 | 北京交通大学 | Method and device for improving MCVD deposition efficiency and quality of low-temperature refrigeration |
| CN103853203A (en) * | 2014-03-19 | 2014-06-11 | 中国科学院高能物理研究所 | Cooling device |
| CN109182247A (en) * | 2018-09-04 | 2019-01-11 | 江苏省家禽科学研究所 | A kind of Embryo liver cell Isolation and culture and steatosis method for establishing model |
| CN208727424U (en) * | 2018-08-30 | 2019-04-12 | 西安鲲创科技发展有限公司 | A kind of high lipocyte additive configuration equipment |
-
2019
- 2019-07-31 CN CN201910700566.8A patent/CN110343658A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR920012935U (en) * | 1990-12-29 | 1992-07-25 | 포항종합제철 주식회사 | Rapid cooling system using adiabatic low pressure tank |
| CN101041550A (en) * | 2006-12-28 | 2007-09-26 | 北京交通大学 | Method and device for improving MCVD deposition efficiency and quality of low-temperature refrigeration |
| CN103853203A (en) * | 2014-03-19 | 2014-06-11 | 中国科学院高能物理研究所 | Cooling device |
| CN208727424U (en) * | 2018-08-30 | 2019-04-12 | 西安鲲创科技发展有限公司 | A kind of high lipocyte additive configuration equipment |
| CN109182247A (en) * | 2018-09-04 | 2019-01-11 | 江苏省家禽科学研究所 | A kind of Embryo liver cell Isolation and culture and steatosis method for establishing model |
Non-Patent Citations (1)
| Title |
|---|
| 主父中印等: ""高葡萄糖高游离脂肪酸对皮肤成纤维细胞β一连环蛋白表达的影响"", 《中华实验外科杂志》, vol. 31, no. 1, pages 57 - 60 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106124429B (en) | A kind of Bionic digestion measuring method of feed digestible carbohydrate total amount | |
| JP2001504692A (en) | Cell culture incubator | |
| CN104851346B (en) | The in-vitro simulated system of modularization animal alimentary canal and its human intestine's analogy method | |
| CN207717742U (en) | A kind of simulation slaking apparatus of food allergen | |
| CN109439517A (en) | Dot hybridization reaction unit and its application and immune blotting detection method | |
| CN110343658A (en) | A kind of medical cell experiment process liquid configuration method and products thereof | |
| CN111435134A (en) | Gastrointestinal dynamic simulation device | |
| CN109385374A (en) | A kind of minicell culture apparatus and its inside cavity environment control method | |
| CN113744610A (en) | Simple in-vitro dynamic simulation digestion device and use method | |
| CN117393158A (en) | AI prediction method for drug-induced acute kidney injury based on kidney organoid model | |
| WO2016081810A1 (en) | System for the concomitant assessment of drug dissolution, absorption and permeation and methods of using the same | |
| CN103245754B (en) | Improved radix sophorae flavescentis thin-layer chromatography identification method | |
| CN209894783U (en) | Gastrointestinal dynamic simulation device | |
| CN112284865A (en) | Slide pretreatment system | |
| CN209513389U (en) | Fecal specimens processing unit | |
| CN208933384U (en) | A kind of culture tube | |
| CN104569419B (en) | A kind of chemiluminescence immunoassay test kit detecting human blood GLP-1 total content | |
| CN201459143U (en) | Anterior segment eye perfusion system | |
| CN113413170B (en) | Urine collecting device | |
| CN205353089U (en) | Detection apparatus for clenobuterol hydrochloride | |
| CN207803317U (en) | A kind of constant current mouse liver ex vivo perfusion system | |
| CN208937601U (en) | Fully-automatic multi-channel urinates Iodine assay system | |
| CN108709987B (en) | Urine conventional and sediment microscopic examination electronic imaging device | |
| CN208540657U (en) | The storage device for avoiding cell or blood from precipitating | |
| CN220357713U (en) | Human digestive system simulator based on intestinal canal |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |