CN107988140A - A kind of abalone blood cell anticoagulation protecting agent and preparation method thereof - Google Patents
A kind of abalone blood cell anticoagulation protecting agent and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of abalone blood cell anticoagulation protecting agent and preparation method thereof, the anticoagulation protecting agent consists of the following compositions：2.45~2.55g of sodium chloride, EDTA0.9~1.1g, two ethyl sulfonic acid 0.22~0.24g and 0.1mM PBS 100ml of Isosorbide-5-Nitrae piperazine.Abalone blood cell anticoagulation protecting agent using the present invention can ensure that aggegation does not occur and maintains higher survival rate in a long time for abalone haemocyte; aggegation is agglomerating in the short time after solving the problems, such as abalone haemocyte in vitro, lays the foundation for research abalone non-specific immunity.
Description
Technical field
The present invention relates to blood cell anticoagulation protecting agent technical field, and in particular to a kind of abalone blood cell anticoagulation protecting agent and
Its preparation method.
Background technology
For abalone due to full of nutrition, economic value is high, becomes the important sea-farming kind in China.In recent years, abalone is supported
Grow and perplex by disease, yield is significantly affected.Research for abalone non-specific immunity becomes solution abalone disease
One of key element of evil problem.Abalone mainly realizes basic defence by phagocytosis of the haemocyte to pathogenic microorganism
Function, therefore the research to abalone blood cell is to explore the important step of nospecific immunity.It is being not added with the situation of anticoagulation protecting agent
Under, abalone haemocyte is once in vitro, i.e. a large amount of aggegations in a few minutes.Even if the abalone haemocyte for adding some anticoagulation protecting agents exists
In short time also can mortality, this feature, which seriously hinders our and carries out biochemical, immune and molecule to abalone haemocyte, gives birth to
Research in terms of thing.Therefore, exploitation can prevent abalone hemagglutination and keep the anti-of cytoactive within a certain period of time
Solidifying protective agent is the basis for studying abalone non-specific immunity.
The anticoagulation protecting agent applied to abalone haemocyte has sterilizing seawater, phosphate buffer (PBS), Tris salt to delay at present
The anticoagulation protecting agent of fliud flushing (TBS), the anticoagulation protecting agent of Mammalian blood cells and other shellfish (oyster and scallop) haemocytes
Deng.In application effect, sterilizing seawater, PBS and TBS are not appropriate for, and a large amount of aggegations will occur in a short time for abalone haemocyte,
And the phagocytic activity of haemocyte is significantly suppressed.Liquid is preserved using the Mammalian blood cells of improvement
(modifiedAlserver ' s solution, MAS), i.e. MAS anticoagulation protecting agents, of short duration can preserve wart Bao and oyster blood is thin
Born of the same parents, but its anticoagulant effect is not known.Good anticoagulant effect can be obtained using oyster and scallop anticoagulation protecting agent, but it is anti-
The solidifying time cannot last long.
The content of the invention
It is an object of the invention to provide a kind of abalone blood cell anticoagulation protecting agent, to solve abalone blood in the prior art
After cells ex vivo in the short time the problem of a large amount of aggegations.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of abalone blood cell anticoagulation protecting agent, consists of the following compositions：2.45~2.55g of sodium chloride, EDTA 0.9~
1.1g, two ethyl sulfonic acid 0.22~0.24g and 0.1mM PBS 100ml of Isosorbide-5-Nitrae-piperazine.
Preferably, the anticoagulation protecting agent consists of the following compositions：Sodium chloride 2.5g, EDTA 1g, Isosorbide-5-Nitrae-piperazine diethyl sulphur
Sour 0.23g and 0.1mM PBS 100ml.
The invention also discloses the preparation method of above-mentioned abalone blood cell anticoagulation protecting agent, specifically comprise the following steps：
(1) take 0.1mM PBS buffer to pour into beaker, then 3-5 points of solid sodium chloride powder stirring is added into beaker
Clock, until sodium chloride all dissolves, then, addition two ethyl sulfonic acid powder of Isosorbide-5-Nitrae-piperazine, which is sufficiently stirred, dissolves to obtain mixed solution A；
(2) and then mixed solution A is put into 38-40 DEG C of water-bath and heated, be slow added into EDTA, stirred when adding
Mix, until EDTA is completely dissolved to obtain mixed solution B；
(3) mixed solution B is gone to and magnetic stirrer 0.5-1h is used under room temperature state, each component is fully dissolved into
In PBS buffer；
(4) superclean bench opens ultraviolet-sterilization function 30-35min, then on superclean bench that mixed solution B is first
It is put into 0.45um sterile filters filtering and impurity removings in the blue lid bottle of sterilizing, then filtered liquid in blue lid bottle with 0.22um sterile filters
Abalone blood cell anticoagulation protecting agent (being denoted as AP) that is degerming, then obtaining.
Preferably, in the preparation method of above-mentioned abalone blood cell anticoagulation protecting agent, water-bath heating temperature in the step (2)
Spend for 40 DEG C.
Preferably, in the preparation method of above-mentioned abalone blood cell anticoagulation protecting agent, magnetic stirring apparatus stirs in the step (3)
It is 0.5h to mix the time.
Above-mentioned abalone blood cell anticoagulation protecting agent can be stored in spare in the blue lid bottle of sterilizing at 4 DEG C or under room temperature.
The method of the present invention has the following advantages that：
Obtained abalone blood cell anticoagulation protecting agent can be protected in 4 DEG C of preservations, abalone blood cell anticoagulation using the present invention
Shield agent can ensure that abalone haemocyte maintains higher survival rate in a long time.Established for research abalone non-specific immunity
Fixed basis.
Brief description of the drawings
Fig. 1 is that (no anticoagulation protecting agent group is for aggegation situation after the in vitro 1.5h of abalone haemocyte in each anticoagulation protecting agent
Control group)；
Fig. 2 is the aggegation situation in each anticoagulation protecting agent after the in vitro 3h of abalone haemocyte；
Fig. 3 is the aggegation situation in each anticoagulation protecting agent after the in vitro 4h of abalone haemocyte；
Fig. 4 is the aggegation situation in each anticoagulation protecting agent after the in vitro 5h of abalone haemocyte；
In figure, arrow meaning is cell agglutination phenomenon.
Embodiment
The present invention will be further explained by specific embodiment below, it being understood, however, that can be with each
Kind form realizes the present invention without should be limited by embodiments set forth here.Conversely, there is provided these embodiments are to be able to
The present invention is best understood from, and the scope of the present invention can be completely communicated to those skilled in the art.
"comprising" or " comprising " as mentioned in working as in specification in the whole text and claim are an open language, therefore should
It is construed to " including but not limited to ".To implement the better embodiment of the present invention, so description is specification subsequent descriptions
For the purpose of the rule of specification, the scope of the present invention is not limited to.Protection scope of the present invention is when regarding appended power
Profit is required subject to institute's defender.
Embodiment 1
A kind of abalone blood cell anticoagulation protecting agent, consists of the following compositions：Sodium chloride 2.5g, EDTA1g, Isosorbide-5-Nitrae-piperazine two
Ethyl sulfonic acid 0.23g and 0.1mM PBS 100ml.
The preparation method of above-mentioned abalone blood cell anticoagulation protecting agent is specially：
(1) take 100ml 0.1mM PBS buffer to be put into beaker, then 2.5g solid sodium chloride powder is added into beaker
Stirring 3 minutes, until sodium chloride all dissolvings, then adds 0.23g1, two ethyl sulfonic acid powder of 4- piperazines is sufficiently stirred dissolving
Obtain mixed solution A；
(2) and then mixed solution A is put into 40 DEG C of water-baths and heated, be slow added into EDTA 1g, stirred when adding
Mix, until EDTA is completely dissolved to obtain mixed solution B；
(3) mixed solution B is gone to and magnetic stirrer 0.5h is used under room temperature state, each component is fully dissolved into
In PBS buffer；
(4) superclean bench opens ultraviolet-sterilization function 30min, then first uses mixed solution B on superclean bench
0.45um sterile filters filtering and impurity removings are put into the blue lid bottle of sterilizing, then are crossed liquid in blue lid bottle with 0.22um sterile filters and filtered out
Bacterium, the abalone blood cell anticoagulation protecting agent then obtained.
Embodiment 2
A kind of abalone blood cell anticoagulation protecting agent, consists of the following compositions：Sodium chloride 2.55g, EDTA1.1g, Isosorbide-5-Nitrae-piperazine
Two ethyl sulfonic acid 0.24g and 0.1mM PBS 100ml.
The preparation method of above-mentioned abalone blood cell anticoagulation protecting agent is specially：
(1) take 100ml 0.1mM PBS buffer to be put into beaker, then 2.55g solid chlorine sodium powders are added into beaker
End stirring 4 minutes, until sodium chloride all dissolves, then, addition 0.24g1, two ethyl sulfonic acid powder of 4- piperazines is sufficiently stirred dissolving
Obtain mixed solution A；
(2) and then mixed solution A is put into 39 DEG C of water-baths and heated, be slow added into 1.1g EDTA, stirred when adding
Mix, until EDTA is completely dissolved to obtain mixed solution B；
(3) mixed solution B is gone to and magnetic stirrer 45min is used under room temperature state, each component is fully dissolved into
In PBS buffer；
(4) superclean bench opens ultraviolet-sterilization function 32min, then first uses mixed solution B on superclean bench
0.45um sterile filters filtering and impurity removings are put into the blue lid bottle of sterilizing, then are crossed liquid in blue lid bottle with 0.22um sterile filters and filtered out
Bacterium, the abalone blood cell anticoagulation protecting agent then obtained.
Embodiment 3
A kind of abalone blood cell anticoagulation protecting agent, consists of the following compositions：Sodium chloride 2.45g, EDTA 0.9g, Isosorbide-5-Nitrae-piperazine
Two ethyl sulfonic acid 0.22g and 0.1mM PBS 100ml of piperazine.
The preparation method of above-mentioned abalone blood cell anticoagulation protecting agent is specially：
(1) take 100ml 0.1mM PBS buffer to be put into beaker, then 2.45g solid chlorine sodium powders are added into beaker
End stirring 5 minutes, until sodium chloride all dissolves, then, addition 0.22g Isosorbide-5-Nitraes-two ethyl sulfonic acid powder of piperazine is sufficiently stirred molten
Solve mixed solution A；
(2) mixed solution A is put into 38 DEG C of water-baths and heated, be slow added into 0.9g EDTA, stir while adding,
Until EDTA is completely dissolved to obtain mixed solution B；
(3) mixed solution B is gone to and magnetic stirrer 1h is used under room temperature state, each component is fully dissolved into PBS
In buffer solution；
(4) superclean bench opens ultraviolet-sterilization function 35min, then first uses mixed solution B on superclean bench
0.45um sterile filters filtering and impurity removings are put into the blue lid bottle of sterilizing, then are crossed liquid in blue lid bottle with 0.22um sterile filters and filtered out
Bacterium, the abalone blood cell anticoagulation protecting agent then obtained.
In order to further prove beneficial effects of the present invention, applicant has also carried out following experiment：
First, the anticoagulating active contrast of different anticoagulation protecting agents
No. 1 anticoagulation protecting agent formula (anticoagulation protecting agent formula of the invention, be denoted as AP)：Sodium chloride 2.5g, 0.1mM PBS
100ml, EDTA 1g, two ethyl sulfonic acid 0.23g of Isosorbide-5-Nitrae-piperazine；
No. 2 anticoagulation protecting agent formulas：Sodium chloride 2.5g, 0.1mM PBS 100ml, EDTA 1g；
No. 3 anticoagulation protecting agent formulas：Sodium chloride 2.5g, 0.1mM PBS 100ml, EDTA 1g, sodium oxalate 0.15g.
Abalone haemocyte is pressed 1 from different anticoagulation protecting agents (1,2, No. 3 anticoagulation protecting agent) first：1 volume mixture, room temperature
The aggegation situation of haemocyte is shown in Table 1 and Fig. 1 after holding 1.5h, is added in this contrast and does not add the pure Bao blood of anticoagulation protecting agent thin
Born of the same parents are as control.
Aggegation situation of the 1 abalone haemocyte of table in different anticoagulation protecting agents
Note：Aggegation group's number refers in 20 visuals field of random observation that be averaged each visual field cell mass number.
Micro- Microscopic observation, the abalone haemocyte for being not added with anticoagulation protecting agent occur aggegation, multiple cell agglutination groups occur, and
The cell agglutination number of each aggegation group is more than 20 cells.Abalone haemocyte is presented in AP and is uniformly distributed, in random observation
Haemocyte group number is less than 2 in 20 visuals field, and the blood cell count of each cell mass is less than 3, and anti-freezing collection effect is best.No. 2
The effect of anticoagulation protecting agent is taken second place, and hemagglutination phenomenon generally occurs in No. 3 anticoagulation protecting agents.
Continue on the basis of above-mentioned experiment to add 1,2, No. 3 anticoagulation protecting agent abalone haemocyte anti-freezing situation into
Row observation.At ambient temperature, respectively to keep 3h, 4h, 5h after haemocyte No. 1, No. 2, No. 3 anticoagulation protecting agent aggegation
Situation is contrasted, and specific comparative result is shown in Table 2 and Fig. 2-4.
Condensation situation of the 2 different time sections abalone haemocyte of table in different anticoagulation protecting agents
Figure it is seen that 3 it is small when after haemocyte in AP still be evenly distributed, haemocyte has becoming for aggegation in the visual field
Gesture, the cell number of aggegation only have 3-4.Haemocyte skewness in No. 2 anticoagulation protecting agents, haemocyte has bright in the visual field
There is cotton-shaped impurity phenomenon more than 5 in the phenomenon of aobvious aggegation, the cell number of aggegation.Haemocyte is in No. 3 anticoagulation protecting agents
Skewness, haemocyte has the phenomenon of obvious aggegation in the visual field, and the cell number of aggegation is more than 10.
From figure 3, it can be seen that 4 it is small when after haemocyte distribution uniform in AP, there is a small amount of cell agglutination group to go out in the visual field
It is existing, but the cell number of aggegation is no more than 4, and agglutination phenomenon is not serious.Haemocyte skewness in No. 2 anticoagulation protecting agents
It is even, there is obvious cell agglutination group in the visual field, and the cell number of aggegation is more than 8.Haemocyte divides in No. 3 anticoagulation protecting agents
Cloth is extremely uneven, and cell agglutination group is more in the visual field, and the cell number of aggegation is more than 20.
From fig. 4, it can be seen that 5 it is small when after haemocyte be distributed in AP more uneven, have a small amount of cell agglutination group in the visual field
Occur, but the cell number of aggegation is no more than 6, and agglutination phenomenon is not serious.As seen from the figure, 5 it is small when after haemocyte it is anti-at No. 2
Skewness in protective agent is coagulated, has substantial amounts of cell agglutination group to occur in the visual field, agglutination phenomenon is more serious.5 it is small when after blood it is thin
Born of the same parents' distributed pole in No. 3 anticoagulation protecting agents is uneven, and cell agglutination group is more in the visual field, and the more (＞ 22 of the cell number of aggegation
It is a), agglutination phenomenon is serious.
Test proves that at different time sections (0-Sh), AP anticoagulation protecting agents are either maintaining cellular morphology, still
Prevent from being all substantially better than anticoagulation protecting agent No. 2 and No. 3 in terms of cell agglutination, be ideal choosing in research abalone experiment
Select.
2nd, different anticoagulation protecting agent haemocyte death rate contrasts
The measure of the haemocyte death rate is carried out using flow cytometer.Concrete operation step is as follows：Take out 500ul abalone blood
(after adding different anticoagulation protecting agents) is each to add PI dye liquor 10ul (final concentration 20ug/ml) into new 2ml sterile centrifugation tubes, 20 DEG C
10min is incubated in darkroom.20 DEG C, 400g centrifuges 5min to remove undyed PI reagents.It is resuspended with 600ul sterilizing seawater thin
Born of the same parents, are put into 5ml streaming pipes.With Flow cytometry 30 seconds.There is fluorescence signal under the conditions of 630nm, use flow cytometer
The one-parameter histogram of FL2 channels, distinguishes dead cell and living cells.
The cell mortality that abalone blood cell measures after being mixed in equal volume with various anticoagulation protecting agents the results are shown in Table 3.
The death rate of 3 haemocyte of table in each anticoagulation protecting agent
From table 3 it is observed that the haemocyte in AP shows the relatively low death rate in 1.5-5h, and No. 2 and No. 3
The death rate of anti-coagulants is higher than AP.Take the anticoagulant effect of table 1 and 2 into consideration, AP anticoagulation protecting agent effects of the invention are more preferable.
Although above with general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (5)
1. a kind of abalone blood cell anticoagulation protecting agent, it is characterised in that the anticoagulation protecting agent consists of the following compositions：Sodium chloride
2.45~2.55g, 0.9~1.1g of EDTA, two ethyl sulfonic acid 0.22~0.24g and 0.1mM PBS 100ml of Isosorbide-5-Nitrae-piperazine.
2. abalone blood cell anticoagulation protecting agent according to claim 1, it is characterised in that the anticoagulation protecting agent is by following
Component forms：Sodium chloride 2.5g, EDTA 1g, two ethyl sulfonic acid 0.23g and 0.1mM PBS 100ml of Isosorbide-5-Nitrae-piperazine.
3. a kind of preparation method of abalone blood cell anticoagulation protecting agent as claimed in claim 1 or 2, it is characterised in that described
Preparation method includes the following steps：
(1) 0.1mM PBS buffer is poured into beaker, then solid sodium chloride powder stirring 3-5 minutes is added into beaker, directly
All dissolved to sodium chloride, then add after two ethyl sulfonic acid powder of Isosorbide-5-Nitrae-piperazine is sufficiently stirred dissolving and obtain mixed solution A；
(2) mixed solution A is put into 38-40 DEG C of water-bath and heated, be slow added into EDTA, stirred while adding, until
EDTA obtains mixed solution B after being completely dissolved；
(3) mixed solution B is gone to and magnetic stirrer 0.5-1h is used under room temperature state, each component is fully dissolved into PBS
In buffer solution；
(4) superclean bench opens ultraviolet-sterilization function 30-35min, then first uses mixed solution B on superclean bench
0.45um sterile filters filtering and impurity removings are put into the blue lid bottle of sterilizing, then are crossed liquid in blue lid bottle with 0.22um sterile filters and filtered out
Bacterium, finally obtains abalone blood cell anticoagulation protecting agent.
4. the preparation method of abalone blood cell anticoagulation protecting agent according to claim 3, it is characterised in that the step (2)
Middle water-bath heating-up temperature is 40 DEG C.
5. the preparation method of abalone blood cell anticoagulation protecting agent according to claim 3, it is characterised in that the step (3)
The middle magnetic stirrer time is 0.5h.
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