CN101294145B - Process for the separation of brain synaptosome - Google Patents
Process for the separation of brain synaptosome Download PDFInfo
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- CN101294145B CN101294145B CN2007100398752A CN200710039875A CN101294145B CN 101294145 B CN101294145 B CN 101294145B CN 2007100398752 A CN2007100398752 A CN 2007100398752A CN 200710039875 A CN200710039875 A CN 200710039875A CN 101294145 B CN101294145 B CN 101294145B
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Abstract
The invention discloses a separation method of brain synaptosome, which comprises the following steps: (1) homogenizing brain tissue containing brain synaptosome in a 0.32M sucrose solution, directly adding into 1.2M sucrose solution, balance-centrifuging at least 20,000 g for 20 to 60 min, and sucking the lower 1.2M sucrose solution with a sucker; and (2) adding the supernate obtained in step (1) in 0.8M sucrose solution, balance-centrifuging at least 30,000 g for 20 to 60 min, and collecting the precipitate to obtain the refined brain synaptosome. Compared with prior art, the separation method has the advantages of simplified operation step and shortened separation time. The separated brain synaptosome has very stable protein content, and has the advantages of high accuracy, good repeatability, high efficiency and simple operation when being used for uptake test of monoamine neurotransmitters.
Description
Technical field
The present invention relates to a kind of separation method of brain synaptosome.
Background technology
Brain synaptosome is the part of expanding of the central nervous system nerve ending that obtains by cell separation technology.In the body neuron with neurogenous be connected with the signal transmission be cynapse realization by nerve ending, the cynapse tip contains neurotransmitter.When nerve impulse arrival nerve ending, the synaptosome release neurotransmitters acts on time one-level neuron, makes it to produce effect.Different nerve ending contains different neurotransmitters, disappears by it is acted on after neurotransmitters such as monoamine, L-glutamic acid, glycine discharge.Therefore brain synaptosome is one of significant points of physiology and pharmacology worker research, can be used for the research relevant with the picked-up activity of these neurotransmitters, for example can be used for the inhibitor and the anti-depressant pharmacological research of these neurotransmitters.Wherein, brain synaptosome is used for monoamine neurotransmitter, research as serotonin (5-HT), norepinephrine (NA) and Dopamine HCL (DA) picked-up effect, and, be internationally recognized, advanced research method as the screening model that acts on the antipsychotic drug of monoamine transmitters re-uptake link.
The inventor is once on the method for bibliographical information, the brain synaptosome separation method has been carried out research and improved (Dong Wenxin, Li Jianqi, Ni Xianglian, Huang Cheng is equal. the foundation and the application of brain synaptosome picked-up serotonin research method. and Chinese Journal of Pharmaceuticals .2001.31 (3): 118-120), its key step is: 1) rat broken end back is taken out brain rapidly, remove pia mater and vascular tissue down at cold condition (4 ℃ physiological saline), get pallium 2ml in the sucrose solution of 0.32M (30ml) after the homogenate, with 1500g equilibrium centrifugation (4 ℃) 10min, purpose is to remove big cell debris; 2) get supernatant liquor in 17000g equilibrium centrifugation (4 ℃) 30min once more, purpose be collect in this liquid institute in a organized way, carry out following gradient centrifugation, to separate brain synaptosome; 3) will precipitate subsequently that the centrifugal force with 38000g carries out gradient centrifugation 60min in the sucrose gradient centrifugation pipe be placed on 0.32M, 0.8M and 1.2M that suspends, carefully collect suspension between 0.8M and 1.2M interface with puncture needle, this suspension promptly contains brain synaptosome; 4) the brain synaptosome suspension of collecting is carried out centrifugal speed with 20000g (4 ℃) 30min once more, the gained throw out is the purified brain synaptosome.Protein content in the prepared synaptosome suspension of this method is stabilized in 5.6～6.8g/L, and (6.18 ± 0.36g/L, n=14), its between-group variation is 5.8%, at selected 300nmolL
-1The 3H-monoamine of/pipe is during as concentration of substrate, and the content of synaptosome is between 120-220 μ g albumen/pipe, and the effectiveness indifference of synaptosome picked-up monoamine does not need to carry out protein determination at every turn, has simplified experimental arrangement greatly; And can carry out simultaneously the re-uptake of 5-HT, NA and DA is studied.Adopt this method the representative medicine of some known monoamine re-uptake inhibitor to be studied the IC that records
50Value is very identical with bibliographical information, has the new drug (CN ZL02111934.1, US10/516,205) that very strong dual reuptake suppresses (5-HT, NA) effect but also filtered out.Yet this method operation steps is also more loaded down with trivial details, and it is bigger particularly carefully to collect the step operation easier of the suspension between 0.8M and 1.2M interface with puncture needle, if misoperation can cause the variation of synaptosome content; And disengaging time is also longer.
Summary of the invention
The technical problem to be solved in the present invention provides that a kind of more above-mentioned existing method steps is simplified, the time shortens and protein content in the synaptosome suspension does not have separation method considerable change, improved brain synaptosome.
The inventor finds by many tests back, to have method steps now reconfigures, by control centrifugal speed and time, it is centrifugal that the stripped cerebral tissue (pallium) that will contain brain synaptosome at first places the sucrose solution of 0.32M and 1.2M to carry out, and at first removes precipitable big cell debris and sediment in the sucrose solution of 1.2M; The sucrose solution that then will contain the 0.32M of brain synaptosome places the sucrose solution recentrifuge of 0.8M, and in the pipe end, taking precipitate is the purified brain synaptosome with the direct centrifugation of synaptosome suspension that was positioned at 0.8M and 1.2M interface originally.
Therefore, technical scheme of the present invention is: a kind of separation method of brain synaptosome, it comprises the following steps:
1. will exsomatize brain cortical tissue in the sucrose solution of 0.32M after the homogenate, the sucrose solution that directly places 1.2M is inhaled the sucrose solution of the 1.2M of sub-cloud with 20000g equilibrium centrifugation at least after 20～60 minutes with suction pipe;
2. the supernatant liquor that 1. step is obtained place 0.8M sucrose solution so that the 30000g equilibrium centrifugation is after 20～60 minutes at least, taking precipitate is the purified brain synaptosome.
Wherein, centrifugal speed is fast more, and precipitation is more abundant in the identical time; In other words, for reaching same centrifugal effect, required time can shorten.Certainly, consider centrifugation apparatus commonly used and energy consumption, the centrifugal speed of step of the present invention in 1. is general preferred in 20000～40000g scope, and the centrifugal speed of step in 2. is preferably in 30000～40000g scope.
More preferably, the centrifugal speed of step in 1. is 20000g, and centrifugation time is 30 minutes.
The centrifugal speed of step in 2. is 30000g, and centrifugation time is 30 minutes.
With conventional, two centrifugation step of the present invention are carried out under low temperature (4 ℃) condition; As long as and the sucrose solution consumption of 0.32M can make the homogenate of stripped brain cortical tissue fully, the volume ratio with stripped brain cortical tissue is at least 10:1 usually, preferably is 15～20:1; As long as 1.2M or the consumption of the sucrose solution of 0.8M can with the clear boundary of the sucrose solution of 0.32M, and can admit wherein that throw out gets final product, the volume ratio of itself and stripped brain cortical tissue is at least 5:1 usually.
According to the present invention, said brain synaptosome derives from experimental animal usually, so the present invention is that example illustrates with test rat commonly used.
The step of separation method of the present invention, particularly centrifugation step changed for 2 steps into by 4 steps, had simplified schedule of operation; Centrifugation time can be shortened to about 60min by 130min, has shortened the whole experiment process of the daylong brain synaptosome picked-up of span monoamine experiment; Omitted operation easier big, to the synaptosome content influence bigger with the careful step of collecting the suspension that is positioned at 0.8M and 1.2M interface of puncture needle; The protein content of prepared synaptosome is highly stable, do not have than existing methods obviously and reduce, be stabilized in 4.5～5g/L, when meeting 3H-monoamine at selected 300nM/ pipe as concentration of substrate, the standard of the content of synaptosome between 120-220 μ g albumen/pipe, the synaptosome (synaptosome) that capacity is provided is for re-uptake required (as shown in table 1).
Table 1 is quantitative
3Under the H-5-HT concentration of substrate, and the actuating quantity of the required synaptosome of every pipe (n=4, x ± s)
Description of drawings
Fig. 1 absorbs brain synaptosome for fluoxetine
3The inhibiting dose-effect relationship figure of H-5-HT (n=4, x ± s).
Embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.
At first according to conventional methods, Wistar or SD rat (Shanghai Si Laike laboratory animal responsibility company limited) broken end back are taken out brain rapidly, remove pia mater and vascular tissue down at cold condition (4 ℃ physiological saline), get the test that pallium carries out the following example.
Wherein, the content of synaptosome comes quantitatively with protein content contained in every liter of solution in the synaptosome suspension (being suspended in the 1ml artificial cerebrospinal fluid), adopts the total protein test kit (biuret colorimetry) of Shanghai claim reagent company limited to carry out protein determination.Whizzer is (German Sigma company, 3K30 type high speed freezing centrifuge).
Embodiment 1
1. the 2ml of brain cortical tissue that will exsomatize after the homogenate, directly places the sucrose solution of 10ml, 1.2M after 30 minutes, to inhale the sucrose solution of the 1.2M of sub-cloud with the 20000g equilibrium centrifugation with suction pipe in the sucrose solution of 20ml, 0.32M;
2. the supernatant liquor that 1. step is obtained place 10ml, 0.8M sucrose solution with the 30000g equilibrium centrifugation after 30 minutes, taking precipitate is the purified brain synaptosome.
The content of synaptosome is 4.8g albumen/L in the synaptosome suspension.
Embodiment 2
1. the 2ml of brain cortical tissue that will exsomatize after the homogenate, directly places the sucrose solution of 10ml, 1.2M after 20 minutes, to inhale the sucrose solution of the 1.2M of sub-cloud with the 40000g equilibrium centrifugation with suction pipe in the sucrose solution of 20ml, 0.32M;
2. the supernatant liquor that 1. step is obtained place 10ml, 0.8M sucrose solution with the 30000g equilibrium centrifugation after 60 minutes, taking precipitate is the purified brain synaptosome.
The content of synaptosome is 4.9g albumen/L in the synaptosome suspension.
Embodiment 3
1. the 2ml of brain cortical tissue that will exsomatize after the homogenate, directly places the sucrose solution of 10ml, 1.2M after 60 minutes, to inhale the sucrose solution of the 1.2M of sub-cloud with the 20000g equilibrium centrifugation with suction pipe in the sucrose solution of 30ml, 0.32M;
2. the supernatant liquor that 1. step is obtained place 10ml, 0.8M sucrose solution with the 40000g equilibrium centrifugation after 20 minutes, taking precipitate is the purified brain synaptosome.
The content of synaptosome is 4.6g albumen/L in the synaptosome suspension.
Embodiment 4
1. the 2ml of brain cortical tissue that will exsomatize after the homogenate, directly places the sucrose solution of 10ml, 1.2M after 40 minutes, to inhale the sucrose solution of the 1.2M of sub-cloud with the 30000g equilibrium centrifugation with suction pipe in the sucrose solution of 20ml, 0.32M;
2. the supernatant liquor that 1. step is obtained place 10ml, 0.8M sucrose solution with the 40000g equilibrium centrifugation after 40 minutes, taking precipitate is the purified brain synaptosome.
The content of synaptosome is 5.0g albumen/L in the synaptosome suspension.
Application Example 1
Adopt the inventive method to prepare brain synaptosome, and carry out fluoxetine brain synaptosome picked-up 5-HT inhibition test (testing sequence is with reference to prior art such as CN ZL02111934.1).Add the 1.0mlTris-Krebs damping fluid in the test tube earlier, add 20 μ l synaptosome suspensions (brain synaptosome of embodiment 1 suspends with artificial cerebrospinal fluid) subsequently, the fluoxetine 10 μ l of the adding 0.1mM that continues mix, and temperature is bathed 5min in 37 ℃ of water-baths.Add again 10 μ l substrates (
3H-5-HT), mixing, 37 ℃ of temperature are bathed 5min.Test tube is put into 4 ℃ of frozen water termination reactions rapidly, vacuum filtration, and wash 2 times.Take off filter membrane, 60-70 ℃ of oven dry put into scintillation vial with filter membrane, adds the toluene scintillation solution, in β-liquid flashing counting device counting.The clean intake of synaptosome: 37 ℃ cpm value (initiatively re-uptake) deducts 0 ℃ cpm value (non-specific aggregation), and its dose-effect relationship figure as shown in Figure 1.
The IC of the representative medicine fluoxetine of the 5-HT reuptake inhibitor that records
50Value is 0.04 μ molL
-1/ pipe is with very identical (the 0.049 μ molL of bibliographical information
-1).
Claims (3)
1. the separation method of a brain synaptosome, it comprises the following steps:
1. will exsomatize brain cortical tissue in the sucrose solution of 0.32M after the homogenate, the sucrose solution that directly places 1.2M is inhaled the sucrose solution of the 1.2M of sub-cloud with 20000g equilibrium centrifugation at least after 20～60 minutes with suction pipe;
2. the supernatant liquor that 1. step is obtained place 0.8M sucrose solution so that the 30000g equilibrium centrifugation is after 20～60 minutes at least, taking precipitate is the purified brain synaptosome;
Wherein, the centrifugal speed of described step in 1. is 20000～40000g, and the centrifugal speed of step in 2. is 30000～40000g.
2. separation method as claimed in claim 1 is characterized in that the centrifugal speed during step 1. is 20000g, and centrifugation time is 30 minutes.
3. separation method as claimed in claim 1 is characterized in that the centrifugal speed during step 2. is 30000g, and centrifugation time is 30 minutes.
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| KR20230148118A (en) | 2022-04-15 | 2023-10-24 | 재단법인대구경북과학기술원 | Method for purification and enrichment of lipid-associated proteins for mass spectrometry analysis of lipid-associated proteins in biological samples |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20230148118A (en) | 2022-04-15 | 2023-10-24 | 재단법인대구경북과학기술원 | Method for purification and enrichment of lipid-associated proteins for mass spectrometry analysis of lipid-associated proteins in biological samples |
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