CN109777777A - A kind of 4-phenylbutyrate solution and preparation method thereof mitigating er stress - Google Patents
A kind of 4-phenylbutyrate solution and preparation method thereof mitigating er stress Download PDFInfo
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- CN109777777A CN109777777A CN201910097643.5A CN201910097643A CN109777777A CN 109777777 A CN109777777 A CN 109777777A CN 201910097643 A CN201910097643 A CN 201910097643A CN 109777777 A CN109777777 A CN 109777777A
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Abstract
The present invention provides a kind of 4-phenylbutyrate solution and preparation method thereof for mitigating er stress, belong to solvent preparation technique field.Described method includes following steps: 1) mixing 4-phenylbutyrate with serum-free DME/F-12 culture medium, by obtained blend in 48~52 DEG C of water-bath 8~12min of mesoscale eddies, obtain pretreatment fluid；2) pretreatment fluid of the step 1) is stood into 22~26h, is mitigated the 4-phenylbutyrate solution of er stress.The present invention makes solution alkaline, damage of the acidic materials to cell is balanced, so that the 4-phenylbutyrate solution for the mitigation er stress being prepared be enable to reduce the toxicity to cell by the way that the DME/F-12 culture medium containing 4-phenylbutyrate to be placed in air.
Description
Technical field
The invention belongs to solvent preparation technique fields, and in particular to a kind of 4-phenylbutyrate solution for mitigating er stress
And preparation method thereof.
Background technique
Endoplasmic reticulum is a kind of important film property desmacyte device in cell, is primarily involved in the conjunction of protein, lipid and carbohydrate
At with metabolism, regulation calcium ion storage and release.It respectively organizes in human body in organ cell, endoplasmic reticulum is the richest in liver cell
It is rich.It can lead to endoplasmic reticulum stable state when physiology or pathological factor (nutritional deficiency, acid poisoning, virus infection etc.) occurs in body
It is destroyed.And endoplasmic reticulum stable state is once destroyed, calcium ion level cataclysm, misfolded protein in endoplasmic reticulum and unfolded
Protein accumulation can induce er stress (endoplasmicreticulum stress, ERS), unfolded egg then occurs
White reaction and stress reaction.Non-adhesion inhibition index is the important link for mediating ERS, continues strong ERS meeting active cell
Apoptotic signal access.Many diseases are related with er stress, as cardiovascular disease, respiratory disease, virus hepatitis,
Autoimmune disease etc..It inquires into er stress disease molecules mechanism associated therewith and has become new challenge in recent years.
The inducer of induction er stress includes thapsigargin, tunicamycin, oleic acid, stearic acid, palmitinic acid etc. at present.
4-phenylbutyrate (PBA) can be used as a kind of molecular chaperones, reverses mistake displacement or the misaggregation of protein molecule, helps it
Normal space structure is established, to mitigate the burden of endoplasmic reticulum, inhibit er stress induction of signal, mitigates er stress
Caused tissue damage.The use of endoplasmic reticulum inducer and PBA can more sufficiently inquire into the molecular mechanism of er stress,
But the method for preparing PBA solution in the prior art is so that 4-phenylbutyrate is obtained PBA solution using water-bath, but this solution is in acid
Property, there is apparent cytotoxic effect to cell, pH value is adjusted to by cell culture optimal pH using sodium bicarbonate or sodium hydroxide
After value, still there is apparent damaging action to cell.
Summary of the invention
In view of this, the present invention provides a kind of 4-phenylbutyrate solution and preparation method thereof for mitigating er stress,
The 4-phenylbutyrate solution that preparation method provided by the invention enables preparation to obtain effectively mitigates the toxic action to cell.
To solve the above-mentioned problems, the present invention provides following technical schemes:
The present invention provides a kind of preparation methods of 4-phenylbutyrate solution for mitigating er stress, including walk as follows
It is rapid:
1) 4-phenylbutyrate is mixed with serum-free DME/F-12 culture medium, the water by obtained blend at 48~52 DEG C
8~12min of mesoscale eddies is bathed, pretreatment fluid is obtained；
2) pretreatment fluid of the step 1) is stood into 22~26h, the 4-phenylbutyrate for being mitigated er stress is molten
Liquid.
Preferably, the mass volume ratio of 4-phenylbutyrate and serum-free DME/F-12 culture medium is in the step 1)
32.8mg:20mL。
Preferably, the time of the step 1) mesoscale eddies is 10min.
Preferably, it is carried out under super-clean bench ultraviolet light irradiation condition when being stood in the step 2).
It preferably, further include filtration sterilization after being stood in the step 2).
Preferably, the method for the filtration sterilization is film filtration sterilization, and the aperture of the film is 0.22 μm.
Preferably, the time stood in the step 2) is for 24 hours.
The present invention provides a kind of 4-phenylbutyrates for the mitigation er stress that above scheme the method is prepared
Solution.
Preferably, when it is described mitigate er stress 4-phenylbutyrate solution be diluted when, use mass concentration for
15%~20% fetal calf serum culture medium is diluted.
Compared with prior art, technical solution provided by the invention has the advantage that
The present invention provides a kind of preparation methods of 4-phenylbutyrate solution for mitigating er stress, including walk as follows
It is rapid: 1) to mix 4-phenylbutyrate with serum-free DME/F-12 culture medium, by obtained blend in 48~52 DEG C of water-bath
Be vortexed 8~12min, obtains pretreatment fluid；2) pretreatment fluid of the step 1) is stood into 22~26h, is mitigated endoplasmic reticulum
Stress 4-phenylbutyrate solution.The DME/F-12 culture medium containing 4-phenylbutyrate is placed in air in the present invention, is made molten
Liquid alkalization balances damage of the acidic materials to cell, to make the 4-phenylbutyrate of mitigation er stress being prepared
Solution can reduce the toxicity to cell.Meanwhile the 4-phenylbutyrate solution for mitigating er stress that the present invention is prepared
It is stability, reproducible and easy to operate.
Detailed description of the invention
Fig. 1 be it is pre- intervene HepG2 cell after Western Blot detect GRP78, CHOP result figure, wherein from left to right four
Band is followed successively by the 4- phenyl for giving DME/F-12 culture medium containing 10% fetal calf serum, the tunicamycin of 1 μ g/mL, 2mmol/L
Butyric acid solution, 2mmol/L 4-phenylbutyrate solution+tunicamycin in predry prognosis testing result band.
Specific embodiment
The present invention provides a kind of preparation methods of 4-phenylbutyrate solution for mitigating er stress, including walk as follows
It is rapid:
1) 4-phenylbutyrate is mixed with serum-free DME/F-12 culture medium, the water by obtained blend at 48~52 DEG C
8~12min of mesoscale eddies is bathed, pretreatment fluid is obtained；
2) pretreatment fluid of the step 1) is stood into 22~26h, the 4-phenylbutyrate for being mitigated er stress is molten
Liquid.
The present invention mixes 4-phenylbutyrate with serum-free DME/F-12 culture medium, by obtained blend at 48~52 DEG C
Water-bath 8~12min of mesoscale eddies, obtain pretreatment fluid.In the present invention, the 4-phenylbutyrate and serum-free DME/F-12 are trained
The mass volume ratio for supporting base is preferably 32.8mg:20mL.Serum-free DME/F-12 culture medium is the basis culture of HepG2 culture
On the one hand base, in the present invention, the serum-free DME/F-12 culture medium can cultivate cell, on the other hand dissolve as solvent
4-phenylbutyrate.The present invention is not particularly limited the source of the serum-free DME/F-12 culture medium and 4-phenylbutyrate, adopts
With this field conventional products.DME/F-12 culture medium described in the embodiment of the present invention is bought in HyClone company；The 4-
Phenylbutyric acid is bought from sigma company.
In the present invention, the temperature of the water-bath is 48~52 DEG C, preferably 50 DEG C.In the present invention, the vortex
Time is 8~12min, preferably 10min.In the present invention, by obtained blend 48~52 DEG C water-bath mesoscale eddies 8~
12min can be such that 4-phenylbutyrate sufficiently dissolves.Blend is red before dissolution, because containing phenol in DME/F-12 culture medium
It is red, therefore after 4-phenylbutyrate dissolution, become orange pretreatment fluid.
After obtaining pretreatment fluid, the pretreatment fluid is stood 22~26h by the present invention, is mitigated the 4- of er stress
Phenylbutyric acid solution.In the present invention, the time of the standing is preferably for 24 hours.DME/F-12 culture medium is placed in air can be with
It is converted into alkalinity, in the present invention, the DME/F-12 culture medium containing 4-phenylbutyrate is placed in air, makes PBA solution alkali
Property, to balance damage of the acidic materials to cell, to keep the 4-phenylbutyrate for the mitigation er stress being prepared molten
Liquid can reduce the toxicity to cell.
In the present invention, the standing preferably carries out under super-clean bench ultraviolet light irradiation condition.In the present invention, ultra-clean
Carrying out standing under irradiation condition under platform ultraviolet light can be sterilized, and prevent from polluting.During standing, pretreatment fluid alkalinity
Change, orange pretreatment fluid becomes the 4-phenylbutyrate solution of red mitigation er stress.
The present invention is after standing, and it is also preferable to include filtration sterilizations.In the present invention, the method for the filtration sterilization is preferably
Film filtration sterilization；The aperture of the film is preferably 0.22 μm.
The present invention provides a kind of 4-phenylbutyrates for the mitigation er stress that above scheme the method is prepared
Solution.
In the present invention, when the 4-phenylbutyrate solution for mitigating er stress is diluted, it is preferred to use matter
The fetal calf serum culture medium that amount concentration is 15%~20% is diluted, more preferably 16.67% fetal calf serum culture medium.
The present invention provides a kind of 4-phenylbutyrates for the mitigation er stress that above scheme the method is prepared
Solution.In the present invention, the 4-phenylbutyrate solution for mitigating er stress is applicable to the induction of induction endoplasmic reticulum
In reaction, mitigate that endoplasmic reticulum generates when being induced stress, and it is smaller to the murder by poisoning of cell.In the present invention, the mitigation
When the 4-phenylbutyrate solution of er stress is in the induced reaction for being applied to induction endoplasmic reticulum, the mitigation er stress
The concentration of 4-phenylbutyrate solution be preferably 1.5~2.5mmol/L, more preferably 2mmol/L.
In order to further illustrate the present invention, technical solution provided by the invention is retouched in detail below with reference to embodiment
It states, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
32.8mg 4-phenylbutyrate is weighed, is added in the serum-free DME/F-12 culture medium of 20mL, in 52 DEG C of water-bath
Mesoscale eddies 8min becomes orange, obtains pretreatment fluid.Pretreatment fluid is stood into 22h under super-clean bench ultraviolet irradiation, becomes red
Color, then the filter degerming for being 0.22 μm with filter sizes, are mitigated the 4-phenylbutyrate solution of er stress.
Embodiment 2
32.8mg 4-phenylbutyrate is weighed, is added in the serum-free DME/F-12 culture medium of 20mL, in 48 DEG C of water-bath
Mesoscale eddies 12min becomes orange, obtains pretreatment fluid.Pretreatment fluid is stood into 26h in super-clean bench ultraviolet irradiation, becomes red,
The filter degerming for being again 0.22 μm with filter sizes, is mitigated the 4-phenylbutyrate solution of er stress.
Embodiment 3
32.8mg 4-phenylbutyrate is weighed, is added in the serum-free DME/F-12 culture medium of 20mL, in 50 DEG C of water-bath
Mesoscale eddies 10min becomes orange, obtains pretreatment fluid.Pretreatment fluid is stood for 24 hours in super-clean bench ultraviolet irradiation, becomes red,
The filter degerming for being again 0.22 μm with filter sizes, is mitigated the 4-phenylbutyrate solution of er stress.
Embodiment 4
4-phenylbutyrate solution mass concentration by the mitigation er stress that embodiment 3 is prepared is 16.67%
Fetal calf serum culture medium be diluted to 2mmol/L, give the HepG2 cell in logarithmic phase to culture medium respectively containing 10% tire ox
The DME/F-12 culture medium of serum, the 4- that concentration is the tunicamycin of 1 μ g/mL, the 4-phenylbutyrate solution of 2mmol/L, 2mmol/L
In the tunicamycin of+1 μ g/mL of phenylbutyric acid solution, cellular morphology is observed respectively after intervening 1d in advance respectively, it is specific to observe result such as table
Shown in 1.And cell protein is extracted, detection GRP78, CHOP are carried out using WesternBlot, concrete outcome is as shown in Figure 1, by scheming
1 as can be seen that individually give 4-phenylbutyrate effect HepG2 cell, the expression and negative control group of GRP78 and CHOP
Indifference shows 4-phenylbutyrate independent role without obvious cytotoxicity；Give (tunicamycin) TM intervene HepG2 after it is visible
GRP78, CHOP are significantly raised, and show that TM can cause apparent er stress；And intervene TM induction in advance with 4-phenylbutyrate
Er stress, it is seen that GRP78, CHOP more individually give TM and intervene obvious downward, show that 4-phenylbutyrate predry prognosis can
Mitigate the er stress of endoplasmic reticulum inducer induction.In triplicate, test result is identical, shows offer of the present invention for the above test
4-phenylbutyrate stability of solution and reproducible.
The form of 1 different dry prognosis cell of table
As can be seen from Table 1, after being intervened using 4-phenylbutyrate solution provided by the invention, cellular morphology with do not intervene
HepG2 cell is similar, and round, growth of uniting, refractivity is good, and without obvious apoptosis, test result shows: 4- provided by the invention
Small toxicity of the phenylbutyric acid solution to cell.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of preparation method for the 4-phenylbutyrate solution for mitigating er stress, which comprises the steps of:
1) 4-phenylbutyrate is mixed with serum-free DME/F-12 culture medium, by obtained blend in 48~52 DEG C of water-bath
Be vortexed 8~12min, obtains pretreatment fluid；
2) pretreatment fluid of the step 1) is stood into 22~26h, is mitigated the 4-phenylbutyrate solution of er stress.
2. preparation method according to claim 1, which is characterized in that 4-phenylbutyrate and serum-free in the step 1)
The mass volume ratio of DME/F-12 culture medium is 32.8mg:20mL.
3. preparation method according to claim 1 or 2, which is characterized in that the time of the step 1) mesoscale eddies is
10min。
4. preparation method according to claim 1, which is characterized in that in super-clean bench ultraviolet light when being stood in the step 2)
It is carried out under irradiation condition.
5. preparation method according to claim 1, which is characterized in that further include filtering after being stood in the step 2)
Degerming.
6. preparation method according to claim 5, which is characterized in that the method for the filtration sterilization is film filtration sterilization,
The aperture of the film is 0.22 μm.
7. preparation method according to claim 1 or 5, which is characterized in that the time stood in the step 2) is for 24 hours.
8. the 4-phenylbutyrate solution for the mitigation er stress that claim 1~7 any one the method is prepared.
9. the 4-phenylbutyrate solution according to claim 8 for mitigating er stress, which is characterized in that when the mitigation
When the 4-phenylbutyrate solution of er stress is diluted, use mass concentration for 15%~20% fetal calf serum culture medium
It is diluted.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111329852A (en) * | 2020-04-14 | 2020-06-26 | 温州医科大学 | Application of 4-phenylbutyrate derivative in preparation of medicine for treating cerebral ischemia-reperfusion injury |
| WO2022191296A1 (en) * | 2021-03-10 | 2022-09-15 | 昭和電工マテリアルズ株式会社 | Medium composition, method for producing cell culture, and cell suspension |
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| CN101272763A (en) * | 2005-07-01 | 2008-09-24 | 纳文塔有限责任公司 | Process for preparation of liquid dosage form containing sodium 4-phenylbutyrate |
| CN103266144A (en) * | 2013-05-06 | 2013-08-28 | 中国药科大学 | Novel system for transferring biocatalytic reaction substrate based on high-molecular material |
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2019
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| CN101272763A (en) * | 2005-07-01 | 2008-09-24 | 纳文塔有限责任公司 | Process for preparation of liquid dosage form containing sodium 4-phenylbutyrate |
| CN103266144A (en) * | 2013-05-06 | 2013-08-28 | 中国药科大学 | Novel system for transferring biocatalytic reaction substrate based on high-molecular material |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111329852A (en) * | 2020-04-14 | 2020-06-26 | 温州医科大学 | Application of 4-phenylbutyrate derivative in preparation of medicine for treating cerebral ischemia-reperfusion injury |
| WO2022191296A1 (en) * | 2021-03-10 | 2022-09-15 | 昭和電工マテリアルズ株式会社 | Medium composition, method for producing cell culture, and cell suspension |
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