CN105055467A - Styrax and cell bath solution mixture and application thereof in inhibition of inward current of Kir2.1 - Google Patents
Styrax and cell bath solution mixture and application thereof in inhibition of inward current of Kir2.1 Download PDFInfo
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- 239000004870 Styrax Substances 0.000 title claims abstract description 87
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- 101000944277 Homo sapiens Inward rectifier potassium channel 2 Proteins 0.000 title claims abstract description 67
- 102100033114 Inward rectifier potassium channel 2 Human genes 0.000 title claims abstract description 65
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
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- AQKDBFWJOPNOKZ-UHFFFAOYSA-N Celastrol Natural products CC12CCC3(C)C4CC(C)(C(O)=O)CCC4(C)CCC3(C)C2=CC=C2C1=CC(=O)C(=O)C2C AQKDBFWJOPNOKZ-UHFFFAOYSA-N 0.000 description 1
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- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical group [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- XFDVJGKSQRUEEM-UHFFFAOYSA-N N-(2,6-Dichloro-4-(((2-chloroethyl)methylamino)methyl)phenyl)-4,5-dihydro-1H-imidazol-2-amine Chemical compound ClC1=CC(CN(CCCl)C)=CC(Cl)=C1NC1=NCCN1 XFDVJGKSQRUEEM-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a styrax and cell bath solution mixture. The styrax and cell bath solution mixture comprises styrax and a cell bath solution in the volume ratio being 1: (10-500), wherein the pH of the cell bath solution is 7.2-7.4, and the osmotic pressure is adjusted by sucrose to be 300 mOsm/L. An application method of the styrax and cell bath solution mixture is as follows: the mixture is used for incubating cells transfected with Kir2.1 channel protein and effectively inhibiting inward current in a Kir2.1 channel. The inhibition rate of the styrax and cell bath solution mixture on the inward current of the Kir2.1 reaches 98%, the half effective inhibition concentration is 0.0113, and one way for developing an effective treating drug for short QT syndrome 3 is provided.
Description
Technical field
The present invention is the screening belonging to inward rectifier potassium channels Kir2.1 regulator, is specifically related to Styrax and body lotion mixed liquor to the suppression of Kir2.1 passage inward electric current.
Background technology
Ion channel is can only the duct property albumen of penetrating a certain or certain several ion on cell membrane, in maintenance transmembrane potential, carries out intracellular signaling and plays very important effect.Inward rectifyimg potassium channel Kir2.1 is the one of potassium-channel, and it allows potassium ion this duct penetrating.When there is magnesium ion or polyamines ion in cell, potassium ion more easily flows into cell and not easily flows out cell, is therefore called " inward rectifier potassium channels ".The unconventionality expression of Kir2.1 passage can cause a lot of sudden death type heart diseases, such as Anderson syndrome, short QT3 syndrome, Barre syndrome etc., and its symptom is all relevant with arrhythmia.These diseases are all because the gene mutation in some site of Kir2.1 to such an extent as to its curent change cause.So the regulator finding Kir2.1 channel current is significant for these diseases for the treatment of.
The laboratory facilities of screening ion channel modulators are generally the method for fluorescence and the method for Patch-clamp techniques electric current.The method of fluorescence utilizes to infiltrate vehicles cells---fluorescent probe in HEKC (HEK293T)---FluxOR.This probe can significantly strengthen in conjunction with the fluorescence sent after exciting by 460-490 nanometer visible laser after thallium ion, and when Kir2.1 passage is opened, thallium ion can replace potassium ion to enter cell by this passage, and then causes the enhancing of fluorescence intensity.On the contrary, if add the inhibitor of Kir2.1, then Kir2.1 passage is suppressed, is also namely in closed condition, and thallium ion cannot enter cell, then fluorescence intensity cannot strengthen.The advantage of this method is that efficiency is high, but cannot be quantitative.Patch-clamp can record the size of the electric current on cell membrane, and this method efficiency is low, but can be quantitative.Two kinds of methods combining get up, and just can carry out the screening that ion channel regulates reagent fast and effectively.
Chinese herbal medicine is China's rarity of 5,000 years, and its advantage is a lot of.Search according to " Chinese medicine voluminous dictionary " accordingly and can treat ARR relevant Chinese herbal medicine, find Chinese medicine Styrax Be very effective in the adjustment rhythm of the heart.Styrax is a kind of oily liquids, and the process of resin that the trunk for plant Styrax tree oozes out is refining to be formed, and is a kind of conventional Chinese crude drug.
Summary of the invention
Technical problem to be solved by this invention is when suppressing reagent to lack for current Kir2.1 potassium current, there is provided the mixture of a kind of Styrax and cell body lotion, mixture can play very strong inhibitory action to the inward electric current of the Kir2.1 passage being expressed in HEK293T cell.Under Laser Scanning Confocal Microscope, Styrax and cell body lotion hatch the HEK293T cell of expressing K ir2.1 channel protein, then the fluorescence that in HEK293T cell, FluxOR sends is unchanged.Utilize the mixture of Styrax and cell body lotion to hatch the HEK293T cell of transfection Kir2.1, then the inward electric current of Kir2.1 that Patch-clamp techniques arrives disappears.The two demonstrates Styrax and cell body lotion plays inhibitory action to Kir2.1 passage inward electric current, is the inhibitor of Kir2.1.When re perfusion is without the cell body lotion of Styrax, then Kir2.1 channel current is restored, and this proves that Styrax and cell body lotion do not destroy the structure of Kir2.1 channel protein.
Technical scheme of the present invention is:
A kind of Styrax and cell bath mixture, its composition comprises Styrax and cell body lotion, and volume ratio is Styrax: cell body lotion=1:10 ~ 500;
The pH value of described cell body lotion is 7.2 ~ 7.4, and osmotic pressure is 290 ~ 300mOsm/L.
The application process of described Styrax and cell bath mixture, utilize this mixture to hatch cell that transfection has Kir2.1 channel protein, effectively suppresses Kir2.1 passage inward electric current.
The application process of described Styrax and cell bath mixture, specifically comprises the following steps:
(1) cell body lotion is prepared;
(2) Styrax is mixed with cell body lotion shake up, and with the membrane filtration of 0.22 ~ 0.44 micron, the liquid obtained is stand-by; Wherein, volume ratio is Styrax: cell body lotion=1:500 ~ 1:10;
(3) liquid utilizing step (2) to obtain hatches the HEK293T cell of transfection Kir2.1 plasmid, and inhibitor Styrax and HEK293T cell are coexisted.
Described cell body lotion is preferably cell body lotion a, cell body lotion b, cell body lotion c, cell body lotion d, cell body lotion e or cell body lotion f; Wherein,
The composition of described cell body lotion a comprises: the test kit FluxOR of (MOLECULARPROBES) of Life Technologies, Inc. of the U.S.
tMpreparation of reagents in PotassiumIonChannelAssay forms, obtain every 10mL cell body lotion comprise the HEPES200uL of 1M, the reagent B of 1mL, the reagent D of 100uL, the deionized water of 8.7mL, and regulate its pH value to be 7.4 with NaOH;
The composition of described cell body lotion b comprises: the MgCl of the KCl of 116.88mM, 5mM
2, the KH of the EDTA of 2mM, 2.83mM
2pO
4, the K of 7.17mM
2hPO
4, solvent is ultra-pure water, and KOH regulates pH value of solution to be 7.3, and osmotic pressure is 300mOsm/L;
The composition of described cell body lotion c or comprise: concentration is respectively the KCl of 140mM, the K-EGTA of the K-HEPES of 10mM, 1mM, and solvent is ultra-pure water, and KOH regulates pH value of solution to be 7.3, and osmotic pressure is 300mOsm/L;
The composition of described cell body lotion d comprises: concentration is respectively the KCl of 110mM, the KH of 10mM
2pO
4, the K of the HEPES of the EDTA of 2mM, 5mM, 1.9mM
2aTP, solvent is ultra-pure water, and KOH regulates pH value of solution to be 7.3, and osmotic pressure is 300mOsm/L;
The composition of described cell body lotion e or comprise: concentration is respectively the KCl of 123mM, the K of 5mM
2the K of EDTA, 7.2mM
2hPO
4, the KH of 8mM
2pO
4, solvent is ultra-pure water, and KOH regulates pH value of solution to be 7.3, and osmotic pressure is 300mOsm/L;
The composition of described cell body lotion f comprises: concentration is respectively the KCl of 120mM, the K of 4mM
2the K of EDTA, 7.2mM
2hPO
4, the KH of 2.8mM
2pO
4, solvent is ultra-pure water, and KOH regulates pH value of solution to be 7.3, and osmotic pressure is 300mOsm/L.
The invention has the beneficial effects as follows: the mixture of Styrax and cell body lotion effectively inhibits the inward electric current of Kir2.1, suppression ratio is close to a hundred per cent.
Up to now, the inhibitor of Kir2.1 passage has ML133, tamoxifen, chloroethylclonidine, wherein, only have ML133 (N-(4-Methoxybenzyl)-1-(naphthalen-1-yl) methanamine) inhibition obvious, its half effective inhibition concentration only has 290nM.But it is a kind of micromolecule of chemosynthesis, it still needs checking to the side effect of human body.Although the celastrol in addition in discovery Chinese herbal medicine micromolecule and gamlogic acid also have inhibitory action to Kir2.1 passage inward electric current, can not suppress completely, DeGrain.
Styrax is from natural storesin, little to human body side effect, " Chinese medicine voluminous dictionary " second edition 1509 pages, " Styrax gavage; the myocardial ischemia ability under mice normal pressure can be improved, quivering incidence rate in the mice room significantly reducing chloroform induction, improves coronary flow ".The present invention reaches 98% by the mixing material of itself and cell body lotion to Kir2.1 inward electric current suppression ratio, and half effective inhibition concentration is that this effective medicine for the short QT3 syndrome of development of 0.0113. opens a fan gate.
Accompanying drawing explanation
Fig. 1 is the fluorescence photo of HEK293T cell under Laser Scanning Confocal Microscope of transfection Kir2.1 in embodiment 1, wherein:
Figure 1A is the HEK293T cell of transfection Kir2.1, adds the fluorescence photo before thallium ion;
Figure 1B is the HEK293T cell of transfection Kir2.1, adds the fluorescence photo after thallium ion;
Fig. 1 C, after the HEK293T cell of transfection Kir2.1 is hatched under Styrax and cell body lotion, adds the fluorescence photo before thallium ion;
Fig. 1 D, after the HEK293T cell of transfection Kir2.1 is hatched under Styrax and cell body lotion, adds the fluorescence photo after thallium ion.
Fig. 2 is in embodiment 1, the representative curve that individual cells change in fluorescence compares.
Fig. 3 is in embodiment 1, when hatching the HEK293T cell of single transfection Kir2.1 passage under not containing and contain the cell body lotion mixed liquor situation of Styrax, according to Laser Scanning Confocal Microscope record, the change ratio bar graphs adding the fluorescence intensity of the cell before and after thallium ion obtained, wherein, I
maxfor adding the fluorescence intensity after thallium ion, I
minfor adding the fluorescence intensity before thallium ion, n=5.
Fig. 4 is in embodiment 2, and by the change of the electric current on the HEK293T cell of transfection Kir2.1 that is recorded to external schema in patch-clamp with transmembrane potential, abscissa is voltage, and vertical coordinate is electric current.Wherein:
Fig. 4 A, the Kir2.1 curent change of non-perfusion Styrax;
Fig. 4 B, Styrax and cell body lotion volume ratio are the curent change of the Kir2.1 after the mixed liquor perfusion of 1:20;
Fig. 4 C, utilizes cell body lotion to wash out the curent change of the Kir2.1 after Styrax.
Fig. 5 is in embodiment 3, Styrax and the suppression percentage ratio of cell body lotion mixed liquor to Kir2.1 electric current and the variation relation of Styrax concentration, namely amount effect curve.Abscissa is Styrax and body lotion volume ratio, and vertical coordinate is the percentage ratio suppressing Kir2.1.IC
50＝0.0113。
Detailed description of the invention
The Styrax that the present invention relates to is from Chinese food medicine identification research institute (kind numbering 120931, lot number 120931-201003)
Embodiment 1
Adopt the representative experimental results that FluxOR fluorescent probe method record Styrax and cell body lotion mixed liquor suppress Kir2.1 inward electric current.
FluxOR is a kind of fluorescent probe, after exciting, can send the green fluorescence of 525nm wavelength after thallium ion combines with it by 460-490 nanometer visible laser.Thallium ion is monovalent cation, can pass through Kir2.1 passage as potassium ion.Reagent used by the present embodiment and FluxOR all come from commercially available test kit, and test kit is from the FluxOR of the MOLECULARPROBES of Life Technologies, Inc. of the U.S.
tMpotassiumIonChannelAssay, article No.: F10016.
The cell body lotion that the present embodiment uses is primarily of the test kit FluxOR of Life Technologies, Inc. of the U.S. (MOLECULARPROBES)
tMpreparation of reagents in PotassiumIonChannelAssay forms, and is specially:
NaOH is utilized to regulate its pH value to be 7.4.(wherein, " reagent B " and " reagent D " are respectively the reagent of fixing composition in this test kit, carry out, obtain above-mentioned cell body lotion during preparation according to the explanation in test kit description.In the present embodiment, the follow-up reagent related in test kit uses in like manner.)
Styrax with cell bath mixture compound method is: by Styrax with cell body lotion according to volume ratio: Styrax: cell body lotion=1:20 mixes and shakes up, and with the membrane filtration of 0.22 micron.
Kir2.1 plasmid transfection is as follows to the method for HEK293T cell: HEK293T plating cells is in 24 orifice plates containing 0.13-0.17mm thickness creep plate, after 37 degree of 5% carbon dioxide aseptic culture case cultivates 24 hours, density reaches 40%, utilizes liposome method to carry out transfection Kir2.1 plasmid.Wherein liposome is from the X-tremeGENEHPDNATransfectionReagent of Roche Holding Ag of the U.S..Liposome: Kir2.1 plasmid: subtract blood serum medium (Opti-MEMI, Life Technologies, Inc. of the U.S.)=1.5 microlitres: 0.5 sodium gram: 50 microlitres.Above-mentioned condition continued cultivation after 24 hours, obtained the HEK293T cell of expressing Kir2.1 passage.
First utilize fluorescence probe method to verify HEK293T successful expression Kir2.1 channel protein, specific experiment step is according to FluxOR
tMpotassiumIonChannelAssay description:
1, remove the culture medium in a hole in 24 orifice plates, then, add 400uL sample-loading buffer; The composition of described sample-loading buffer and proportioning are that (reagent A, reagent B, reagent C, reagent D are from test kit FluxOR
tMpotassiumIonChannelAssay):
2, room temperature lucifuge hatches 60 minutes, removes sample-loading buffer, then adds 400uL cell body lotion.
3, utilize German Lycra SP5 laser scanning co-focusing microscope record fluorescence photo, wherein excitation wavelength is 488nm, wavelength of transmitted light 520-540nm.The number of microscope continuous sweep photo is 100, and adjacent photo is spaced apart 0 second.Be scanned up at the beginning of the tenth photo, in the cell body lotion of step 2, add irritation cell body lotion 80uL, wherein measure being formulated as of cell body lotion:
Add intracellular Fluorescence probe before and after irritation cell body lotion send the change of fluorescence intensity to compare, choose arbitrarily the fluorescence photo before adding irritation cell body lotion (for ease of comparing, here the 8th is chosen, fluorescence photo Figure 1A) and after adding irritation cell body lotion is (for ease of comparing, here the 20 is chosen, Figure 1B).Relatively Figure 1A and Figure 1B, finds that the fluorescence intensity that the cell in Figure 1B sends significantly strengthens, demonstrates thallium ion and enter HEK293T cell by Kir2.1 passage, namely proves HEK293T cell Successful transfection Kir2.1 passage.
Step proves that Styrax and cell bath mixture play inhibitory action to Kir2.1 passage inward electric current below:
4, reselect this another hole of 24 orifice plate, repeat step 1 ~ 3, the 400uL Styrax wherein added in step 2 and cell body lotion volume ratio are the mixture of 1:20, then hatch 30 minutes.Add intracellular Fluorescence probe before and after irritation cell body lotion send the change of fluorescence intensity to compare, still choose the before adding irritation cell body lotion the 8th photo (Fig. 1 C) here for comparing with the result of step 3 and add the 20 photo (Fig. 1 D) after irritation cell body lotion.Can find out in figure that the fluorescence intensity that HEK293T cell sends does not have significant change.Demonstrate thallium ion and do not enter by Kir2.1 passage the change that cell causes fluorescence intensity, also just demonstrate when Styrax and cell body lotion volume ratio are 1:20 and successfully inhibit Kir2.1 passage inward electric current.
5, because HEK293T cell is entered in the transfection of Kir2.1 channel protein certain efficiency, so the successful cell of transfection Kir2.1 channel protein (being also change in fluorescence obviously cell) chosen respectively by obtain from step 3 and 4 100 HEK293T cell fluorescence photos, make its fluorescence curve over time, obtain Fig. 2.Cell body lotion fluorescence intensity as seen from the figure containing Styrax does not become substantially.Prove that Styrax and cell bath mixture inhibit the electric current of Kir2.1 passage.
6, in order to further illustrate the inhibitory action of Styrax to Kir2.1 passage, the 20 that obtains from step 3 and 4 and the 8th fluorescence photo, to the fluorescence intensity of the successful HEK293T cell of same transfection Kir2.1 according to formula I
20-I
8/ I
20(I
8and I
20expression the 8th and the 20th this cell fluorescence intensity respectively) do ratio, (wherein, in figure, " Styrax (-) representative is containing the cell body lotion of Styrax to obtain Fig. 3; Styrax (+) representative is containing the cell body lotion of Styrax.Fig. 4 is same).Being 2.321 without Styrax person ratio, is 0.017 containing Styrax person ratio.When proving that Styrax and cell body lotion are 1:20, well suppress the electric current of Kir2.1 passage.
When utilizing the method for FluxOR fluorescent probe to demonstrate Styrax and cell body lotion volume ratio for 1:20, effectively can suppress the inward electric current of Kir2.1 passage.Styrax is a kind of inhibitor of Kir2.1 passage.
Embodiment 2
Patch-clamp method of testing record Styrax and cell body lotion mixed liquor is adopted to suppress and the representative experimental results recovered Kir2.1 inward electric current:
In cell body lotion and glass microelectrode, formula of liquid is as follows: KCl116.88mM, MgCl
25mM, EDTA2mM, KH
2pO
42.83mM, K
2hPO
47.17mM, solvent is ultra-pure water, and KOH regulates pH to be 7.3, and cell body lotion osmotic pressure is 300mOsm/L.Liquid osmotic pressure in glass microelectrode is regulated to be 295mOsm/L.
The HEK293T cell preparation method of transfection Kir2.1 channel protein is with embodiment 1.
Styrax and cell body lotion compound method: by Styrax with cell body lotion according to volume ratio: Styrax: cell body lotion=1:20 mixes and shakes up, and with the membrane filtration of 0.22 micron.
Utilize patch clamp amplifier to record the electric current of Kir2.1 passage, model is the EPC10 of German HEKA company, its logging mode: inside outwards.Obtain Kir2.1 passage typicality inward electric current (Fig. 4).Wherein Fig. 4 A, B, C hatches HEK293T cell, the exemplary currents of the Kir2.1 passage obtained with the cell body lotion not containing Styrax, the cell body lotion containing Styrax, the cell body lotion not containing Styrax successively successively for same sealing-in.
Can be found by Fig. 4 A, B, after cell body lotion is replaced by Styrax and cell body lotion mixed liquor, electric current is obviously suppressed, and suppresses ratio 98%.The fluorescence results that result and Laser Scanning Confocal Microscope detect is consistent.In addition, after the cell body lotion containing Styrax is replaced by the cell body lotion without Styrax, the inward electric current of Kir2.1 passage recovers again, this demonstrate that Styrax does not destroy and expresses Kir2.1 protein function on HEK293T cell.
Embodiment 3
Adopt the representative experimental results that the different Styrax of patch-clamp method of testing record and cell body lotion volume ratio suppress Kir2.1 inward electric current:
HEK293T cell prepares and transfection, cell body lotion, formula of liquid, patch-clamp arrange pattern with embodiment 2 in glass microelectrode.
Styrax and cell body lotion compound method: Styrax is respectively Styrax with cell body lotion according to volume ratio: cell body lotion=0.002,0.01,0.014,0.022,0.033,0.05 mixes and shakes up, and with the membrane filtration of 0.22 micron.
IC
50be worth for certain inhibitor suppresses passage maximum current half required dosage, it reflects that this kind of reagent suppresses the ability of passage inward electric current.IC
50value is provided by its amount effect curve, and amount effect curve is the change curve of passage inward electric current size with inhibitor concentration.Patch-clamp, to the same sealing-in of HEK293T cell, records the Kir2.1 electric current under different Styrax concentration.Under getting different Styrax concentration, the Kir2.1 inward electric current of-80mV, draws the amount effect curve (Fig. 5, n=6) that Styrax suppresses Kir2.1.Wherein IC
50be 0.0113, when also namely Styrax and cell body lotion volume ratio are 0.0113, Kir2.1 can be suppressed at the inward electric current of-80mV half.
Embodiment 4-5
Other steps are with embodiment 2 and 3, and difference is that the K-EGTA of the K-HEPES of the KCl that cell body lotion comprises 140mM, 10mM, 1mM, KOH regulate pH value of solution to be 7.3, and osmotic pressure is 300mOsm/L.Its patch-clamp test effect is with embodiment 2 and 3.
Embodiment 6-7
Other steps are with embodiment 2 and 3, and difference is the KH of the KCl that cell body lotion comprises 110mM, 10mM
2pO
4, the K of the HEPES of the EDTA of 2mM, 5mM, 1.9mM
2aTP, KOH regulate pH value of solution to be 7.3, and osmotic pressure is 300mOsm/L.Its patch-clamp test effect is with embodiment 2 and 3.
Embodiment 8-9
Other steps are with embodiment 2 and 3, and difference is the K of the KCl that cell body lotion comprises 123mM, 5mM
2the K of EDTA, 7.2mM
2hPO
4, the KH of 8mM
2pO
4, KOH regulates pH value of solution to be 7.3, and osmotic pressure is 300mOsm/L.Its patch-clamp test effect is with embodiment 2 and 3.
Embodiment 10-11
Other steps are with embodiment 2 and 3, and difference is the K of the KCl that cell body lotion comprises 120mM, 4mM
2the K of EDTA, 7.2mM
2hPO
4, the KH of 2.8mM
2pO4, KOH regulate pH value of solution to be 7.3, and osmotic pressure is 300mOsm/L.Its patch-clamp test effect is with embodiment 2 and 3.
In embodiment 1-11, after utilizing the cell body lotion containing Styrax to have the HEK293T cell incubation of Kir2.1 channel protein to expression, thallium ion and potassium ion effectively can be stoped to enter cell, namely can suppress the inward electric current of Kir2.1 passage.Demonstrate the inhibitor that Styrax is Kir2.1 passage inward electric current.And the cell body lotion containing Styrax can not destroy the structure of Kir2.1 channel protein.
Unaccomplished matter of the present invention is known technology.
Claims (4)
1. Styrax and a cell bath mixture, it is characterized by its composition and comprise Styrax and cell body lotion, volume ratio is Styrax: cell body lotion=1:10 ~ 500;
The pH value of described cell body lotion is 7.2 ~ 7.4, uses sucrose to regulate osmotic pressure to be 300mOsm/L.
2. Styrax as claimed in claim 1 and cell bath mixture, is characterized by described cell body lotion and be preferably cell body lotion a, cell body lotion b, cell body lotion c, cell body lotion d, cell body lotion e or cell body lotion f; Wherein,
The composition of described cell body lotion a comprises: the test kit FluxOR of (MOLECULARPROBES) of Life Technologies, Inc. of the U.S.
tMpreparation of reagents in PotassiumIonChannelAssay forms, obtain every 10mL cell body lotion comprise the HEPES200uL of 1M, the reagent B of 1mL, the reagent D of 100uL, the deionized water of 8.7mL, and regulate its pH value to be 7.4 with NaOH;
The composition of described cell body lotion b comprises: the MgCl of the KCl of 116.88mM, 5mM
2, the KH of the EDTA of 2mM, 2.83mM
2pO
4, the K of 7.17mM
2hPO
4, solvent is ultra-pure water, and KOH regulates pH value of solution to be 7.3, and sucrose regulates osmotic pressure to be 300mOsm/L;
The composition of described cell body lotion c comprises: concentration is respectively the KCl of 140mM, the K-EGTA of the K-HEPES of 10mM, 1mM, and solvent is ultra-pure water, and KOH regulates pH value of solution to be 7.3, and sucrose regulates osmotic pressure to be 300mOsm/L;
The composition of described cell body lotion d comprises: concentration is respectively the KCl of 110mM, the KH of 10mM
2pO
4, the K of the HEPES of the EDTA of 2mM, 5mM, 1.9mM
2aTP, solvent is ultra-pure water, and KOH regulates pH value of solution to be 7.3, and sucrose regulates osmotic pressure to be 300mOsm/L;
The composition of described cell body lotion e comprises: concentration is respectively the KCl of 123mM, the K of 5mM
2the K of EDTA, 7.2mM
2hPO
4, the KH of 8mM
2pO
4, solvent is ultra-pure water, and KOH regulates pH value of solution to be 7.3, and sucrose regulates osmotic pressure to be 300mOsm/L;
The composition of described cell body lotion f comprises: concentration is respectively the KCl of 120mM, the K of 4mM
2the K of EDTA, 7.2mM
2hPO
4, the KH of 2.8mM
2pO
4, solvent is ultra-pure water, and KOH regulates pH value of solution to be 7.3, and sucrose regulates osmotic pressure to be 300mOsm/L.
3. the application process of Styrax as claimed in claim 1 and cell bath mixture, is characterized by utilize this mixture to hatch HEK293T cell that transfection has Kir2.1 channel protein, effectively suppression Kir2.1 passage inward electric current.
4. the application process of Styrax as claimed in claim 3 and cell bath mixture, is characterized by the application process of described Styrax and cell bath mixture, specifically comprises the following steps:
(1) cell body lotion is prepared;
(2) Styrax is mixed with cell body lotion shake up, and with the membrane filtration of 0.22 ~ 0.44 micron, the liquid obtained is stand-by; Wherein, volume ratio is Styrax: cell body lotion=1:500 ~ 1:10;
(3) liquid utilizing step (2) to obtain hatches the HEK293T cell of transfection Kir2.1 plasmid.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101234169A (en) * | 2007-02-01 | 2008-08-06 | 河北以岭医药研究院有限公司 | Application of Chinese medicine composition in preparing medicament for adjusting cardiac muscle cell potassium ion channel |
| WO2012118172A1 (en) * | 2011-03-03 | 2012-09-07 | 国立大学法人熊本大学 | Drug for improving central functions during pain |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101234169A (en) * | 2007-02-01 | 2008-08-06 | 河北以岭医药研究院有限公司 | Application of Chinese medicine composition in preparing medicament for adjusting cardiac muscle cell potassium ion channel |
| WO2012118172A1 (en) * | 2011-03-03 | 2012-09-07 | 国立大学法人熊本大学 | Drug for improving central functions during pain |
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| 李涛等: "Kir2.1与钾通道病", 《医学综述》 * |
| 王峰等: "苏合香化学成分研究", 《中国实验方剂学杂志》 * |
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