CN101451120A - Abalone blood cell anticoagulation protecting agent and preparation method thereof - Google Patents
Abalone blood cell anticoagulation protecting agent and preparation method thereof Download PDFInfo
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- CN101451120A CN101451120A CNA2008100725392A CN200810072539A CN101451120A CN 101451120 A CN101451120 A CN 101451120A CN A2008100725392 A CNA2008100725392 A CN A2008100725392A CN 200810072539 A CN200810072539 A CN 200810072539A CN 101451120 A CN101451120 A CN 101451120A
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Abstract
The invention discloses an abalone blood cell anticoagulant protective agent and a preparation method thereof, and relates to an anticoagulant protective agent. The invention provides an abalone blood cell anticoagulant protective agent for maintaining the activity of isolated abalone blood cells within longer time and a preparation method thereof. The abalone blood cell anticoagulant protective agent comprises the following raw materials by mass percentage: 1.95 to 2.08 percent of glucose, 0.78 to 0.8 percent of sodium citrate (2H2O), 0.063 to 0.107 percent of citric acid, 0.37 to 0.57 percent of sodium chloride, 0.21 to 0.24 percent of hydroxyethyl piperazinyl ethyl thiosulfonic acid, and 100 percent of double distilled water, wherein the pH value is between 6.0 and 6.4. The abalone blood cell anticoagulant protective agent is obtained by dissolving the glucose, the sodium citrate (2H2O), the sodium chloride and the hydroxyethyl piperazinyl ethyl thiosulfonic acid into the double distilled water, stirring and dissolving the mixture, using the citric acid to adjust the pH value to be between 6.0 and 6.4, and performing high-temperature sterilization.
Description
Technical field
The present invention relates to a kind of anticoagulation protecting agent, especially relate to a kind of abalone blood cell anticoagulation protecting agent and preparation method thereof.
Background technology
Bao is the important sea farming kind of China, and is nutritious, the economic worth height; But its poor growth requires the very aquaculture water of cleaning.Bao disease problem is more and more serious in recent years, seeks effective means and promotes the Bao healthy aquaculture to become the task of top priority.Bao is similar to other shellfishes, and the defence of invading cause of disease is realized by the phagolysis of hemocyte to external microorganism that mainly therefore the research to abalone blood cell is to explore its immune important ring.Shellfish hemocytes such as Bao have the characteristics of a highly significant, and under the situation that does not add antithrombotics, in a single day hemocyte exsomatizes, and is a large amount of aggegations in the several minutes.And being suspended in the crustaceans once reported and the abalone blood cell mass mortality in 1 hour in the shellfish antithrombotics, these characteristics have seriously hindered the exploration to its function.Therefore exploitation can the reasonable time in prevent aggegation and keep the Bao blood antithrombotics of cytoactive to be further to study the immune basis of Bao Zhizhi shellfish.
The antithrombotics that often is applied at present the abalone blood cell manipulation in vitro is sterilization seawater (Sterilized seawater, SS), phosphate buffered saline buffer (PBS) (Zhang Feng, Li Guangyou. the influence [J] that several heavy metal species produce the thin active oxygen of haliotis discus hannai Ino blood. marine environmental sciences, 2005,24 (1): 32-34).Many pieces of documents show SS and PBS and the suspension of the abalone blood cell that is not suitable for exsomatizing and maintenance activity, and test of many times shows also that within 1h stripped abalone blood cell over half in SS and PBS aggegation takes place.The investigator of many shellfish hemocytes all attempts to add various compositions and obtains mammalian blood cell-preservation liquid (modified Alserver ' the s solution that improves in mammalian blood cell-preservation liquid (Alserver ' s solution), MAS) (Lebel, J.M., Giard, W., Favrel, P.and Boucaud-Camou, E.:Effects of different vertebrate growth factorson primary cultures of hemocytes from the gastropod mollusc, Haliotis tuberculata.Biol Cell 86 (1996) 67-72).The hemocyte (1h) though the MAS antithrombotics can of short duration preservation wart Bao (H.tuberculata) exsomatizes, and be applied to the in vitro conservation (Xue of flat oyster hemocyte, Q.G., Renault, T.and Chilmonczyk, S.:Flow cytometricassessment of haemocyte sub-populations in the European flat oyster, Ostrea edulis, but it is to anti-freezing effect of wart Bao and oyster hemocyte clear and definite report haemolymph.Fish Shellfish Immunol 11 (2001) 557-567).
Summary of the invention
The objective of the invention is at existing shellfish blood cell anticoagulation collection and the technical deficiency of preservation, a kind of active abalone blood cell anticoagulation protecting agent of abalone blood cell and preparation method thereof that exsomatizes that keeps in a long time is provided.
Technical scheme of the present invention be adopt generally keep add on citrate buffer, glucose and the sodium chloride solution basis that Premeabilisation of cells presses hydroxyethyl piperazine second thiosulfonic acid (2-[4-(2-Hydroxyethyl)-1-piperazinyl] ethanesulfonic acid, HEPES) this composition (this composition first Application is in the cultivation of shellfish hemocyte) with efficient shock absorption.
The raw material of abalone blood cell anticoagulation protecting agent of the present invention is formed and is glucose 1.95～2.08 by the content of mass ratio; Trisodium Citrate (2H2O) 0.78～0.8; citric acid 0.063～0.107; sodium-chlor 0.37～0.57; hydroxyethyl piperazine second thiosulfonic acid 0.21～0.24; distilled water 100, pH6.0～6.4.
The raw material of abalone blood cell anticoagulation protecting agent of the present invention is formed and is preferably glucose 2.05, Trisodium Citrate (2H2O) 0.8, citric acid 0.08, sodium-chlor 0.42, hydroxyethyl piperazine second thiosulfonic acid 0.23, distilled water 100, pH6.1 by the content of mass ratio.
The preparation method of abalone blood cell anticoagulation protecting agent of the present invention may further comprise the steps:
With glucose, Trisodium Citrate (2H
2O), sodium-chlor and hydroxyethyl piperazine second thiosulfonic acid be dissolved in the distilled water, stirring and dissolving is transferred pH to 6.0～6.4 with citric acid, high-temperature sterilization, abalone blood cell anticoagulation protecting agent.
The temperature that stirs is preferably 45 ℃, and the concentration of citric acid preferably is 10% by mass percentage, and the temperature of high-temperature sterilization is preferably 112.6 ℃, and the time of high-temperature sterilization is preferably 30min.
Prepared abalone blood cell anticoagulation protecting agent can use for 2 weeks 4 ℃ of preservations.Abalone blood cell anticoagulation protecting agent can guarantee that abalone blood cell keeps higher survival rate in 12h; if need the activity of keeping of abalone blood cell longer time; can be with abalone blood cell anticoagulation protecting agent and the general nutrient solution RPMI1640 of cell mixed with 1: 8, the antithrombotics of gained (being designated as RH) can guarantee more than the abalone blood cell survival 2d.
Embodiment
Following examples will the present invention is further illustrated.
Other marine invertebrate blood cell anticoagulation protecting agent effects that below provide abalone blood cell anticoagulation protecting agent of the present invention (being designated as HA) and heterogeneity compare.
Several different blood cell anticoagulation protecting agent sterilization seawater (Sterilized seawater shown in the table 1; SS), PBS phosphate buffered saline buffer, blood red born of the same parents' nutrient solution RPMI1640, (the DA antithrombotics is the antithrombotics of Chinese prawn to the DA antithrombotics; document sees reference: the war literary composition is refined; Zhang Lifeng; Zhang Zhi etc., the discriminating [J] of development of Chinese prawn hemocyte monoclonal antibody and prawn hemocyte type.The hi-tech communication, 2001,25 (6): 19-22) different fully with the composition of described abalone blood cell anticoagulation protecting agent.All add ethylenediamine tetraacetic acid (EDTA) (EDTA) in DA and the MAS composition, but do not add hydroxyethyl piperazine second thiosulfonic acid, other composition is identical and content and pH differ greatly.Concrete prescription is: sodium-chlor 0.82g, and glucose 1.98g, Trisodium Citrate 0.88g, citric acid 0.55g, ethylenediamine tetraacetic acid (EDTA) 0.37g is dissolved in the 100ml distilled water pH5.6.MAS is the antithrombotics (Xue of oyster, Q.G., Renault, T.and Chilmonczyk, S.:Flow cytometric assessment of haemocytesub-populations in the European flat oyster, Ostreaedulis, haemolymph.Fish Shellfish Immunol 11 (2001) 557-567), concrete prescription is 20.80g/l glucose, 8.00g/l Trisodium Citrate, 3.36g/l ethylenediamine tetraacetic acid (EDTA), 22.50g/l sodium-chlor, 10% citric acid is transferred pH to 7.5.The result shows:
1. abalone blood cell anticoagulation protecting agent (HA) has strong anti-freezing collection activity
Table 1 has showed that 1: 1 volume ratio of stripped Haliotis diversicolor hemocyte is suspended in the various antithrombotics, after room temperature keeps 1.5h, and the hemagglutination situation.Microscopically is observed, and abalone blood cell presents uniform distribution in HA and RH, and hemocyte group number average is less than 5 in 20 visuals field of random observation, and the blood cell count of each cell mass is less than 10, and anti-freezing collection effect is best.Next is MAS, and the agglutination phenomenon of hemocyte is general in SS, PBS, DA and RPMI-1640.HA obviously is better than other antithrombotics (table 1) on anticoagulating active.
The aggegation situation of table 1 Haliotis diversicolor hemocyte 1.5h in various antithrombotics
Annotate: aggegation group number is meant average each visual field cell mass number in 20 visuals field of random observation.
2. abalone blood cell anticoagulation protecting agent (HA) is preserved the high viability of cell
Table 2 has showed that 1: 1 volume ratio of stripped Haliotis diversicolor hemocyte is suspended in the mortality ratio in the various antithrombotics.Hemocyte among SS, PBS and the MAS all shows very high mortality ratio, and other 4 kinds of antithrombotics all have good survival rate.Take the anti-freezing effect of table 2 into consideration, the comprehensive action that HA of the present invention and RH possess anti-freezing and preserve abalone blood cell.
The mortality ratio (%) of Haliotis diversicolor hemocyte in the various antithrombotics of table 2
3. the system component content of abalone blood cell anticoagulation protecting agent (HA) changes the anti-freezing protection effect to abalone blood cell
The key point of abalone blood cell anticoagulation protecting agent (HA) is to add HEPES and controlled contents, strict control pH value and restriction adding sodium salt, below illustrates one by one with regard to above-mentioned 3 aspects.
1)HEPES
As shown in Tables 3 and 4; the anti-freezing collection of solution and protection effect were best when HEPES content was 0.23～0.24g among the 100ml HA; content is that the effect of 0.21g still can; when HEPES content during less than 0.19g; the anti-freezing collection effect of HA descends to some extent; surpass the 2.5g abalone blood cell and be suspended among the HA that mortality ratio significantly increases behind the 12h, agglutination phenomenon is serious.
The aggegation situation of Haliotis diversicolor hemocyte 1.5hr among the HA of the different HEPES content of table 3
The mortality ratio (%) of Haliotis diversicolor hemocyte among the HA of the different HEPES content of table 4
2) pH value
Shown in table 5 and 6, the pH value is that the anti-freezing protection effect of 6.1 o'clock HA is best, and the pH value is that 6.0～6.4 o'clock effects are fair, and the mortality ratio that pH is lower than 6.0 o'clock abalone blood cells has improved greatly, and it is serious that pH is higher than the aggegation of 6.6 cells.
The aggegation situation of table 5 Haliotis diversicolor hemocyte 1.5h in the HA of different pH
The mortality ratio of Haliotis diversicolor hemocyte (%) among the HA of the different pH of table 6
3) NaCl content
Shown in table 7 and 8, NaCl content hangs down the then easy aggegation of abalone blood cell, and high then mortality ratio hemocyte increases greatly.The NaCl that contains 3.7g-5.7g with 100ml is advisable.
The aggegation situation of table 7 Haliotis diversicolor hemocyte 1.5h in the HA of different N aCl content
The mortality ratio (%) of Haliotis diversicolor hemocyte among the HA of table 8 different N aCl content
4.HA anti-freezing protection effect to other shellfish and Young Crab
Shown in table 9 and 10, HA and RH also have good anti-freezing collection and preservation effect for the hemocyte of shellfishes such as east wind spiral shell and clam and mud crab.HA of the present invention and RH have good anti-freezing collection, high survival and versatility, are expected to become the general antithrombotics of shellfish hemocyte.
The different marine invertebrate hemocytes of table 9 aggegation situation behind the suspension 1.5h in HA
The mortality ratio (%) of the different marine invertebrate hemocytes of table 10 in HA
Claims (6)
1. abalone blood cell anticoagulation protecting agent is characterized in that its raw material is formed and is glucose 1.95～2.08 by the content of mass ratio, Trisodium Citrate (2H
2O) 0.78～0.8, citric acid 0.063～0.107, sodium-chlor 0.37～0.57, hydroxyethyl piperazine second thiosulfonic acid 0.21～0.24, distilled water 100, pH6.0～6.4.
2. abalone blood cell anticoagulation protecting agent as claimed in claim 1 is characterized in that its raw material is formed and is glucose 2.05 by the content of mass ratio, Trisodium Citrate (2H
2O) 0.8, citric acid 0.08, sodium-chlor 0.42, hydroxyethyl piperazine second thiosulfonic acid 0.23, distilled water 100, pH6.1.
3. the preparation method of abalone blood cell anticoagulation protecting agent as claimed in claim 1 is characterized in that may further comprise the steps:
With glucose, Trisodium Citrate (2H
2O), sodium-chlor and hydroxyethyl piperazine second thiosulfonic acid be dissolved in the distilled water, stirring and dissolving is transferred pH to 6.0～6.4 with citric acid, high-temperature sterilization, abalone blood cell anticoagulation protecting agent.
4. the preparation method of abalone blood cell anticoagulation protecting agent as claimed in claim 3 is characterized in that the temperature that stirs is 45 ℃.
5. the preparation method of abalone blood cell anticoagulation protecting agent as claimed in claim 3, the concentration that it is characterized in that citric acid is 10% by mass percentage.
6. the preparation method of abalone blood cell anticoagulation protecting agent as claimed in claim 3, the temperature that it is characterized in that high-temperature sterilization is 112.6 ℃, the time of high-temperature sterilization is 30min.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101975850A (en) * | 2010-09-13 | 2011-02-16 | 南京卡博生物科技有限公司 | Diluent for blood cell analyzer |
| CN102125543A (en) * | 2011-02-28 | 2011-07-20 | 王啸龙 | Heparin-free anticoagulant infection inhibitor for hemodialysis and preparation method thereof |
| CN102318837A (en) * | 2011-08-23 | 2012-01-18 | 安徽宝迪肉类食品有限公司 | Compound anticoagulant of animal blood |
| CN107988140A (en) * | 2018-01-25 | 2018-05-04 | 鲁东大学 | A kind of abalone blood cell anticoagulation protecting agent and preparation method thereof |
| CN108396008A (en) * | 2018-01-25 | 2018-08-14 | 鲁东大学 | A kind of abalone haemocyte processing method |
| CN117717066A (en) * | 2024-02-04 | 2024-03-19 | 广东海洋大学 | Embryo preservation solution, cryopreservation method thereof and application of embryo preservation solution in cryopreservation of Babylonia embryos |
-
2008
- 2008-12-31 CN CN2008100725392A patent/CN101451120B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101975850A (en) * | 2010-09-13 | 2011-02-16 | 南京卡博生物科技有限公司 | Diluent for blood cell analyzer |
| CN102125543A (en) * | 2011-02-28 | 2011-07-20 | 王啸龙 | Heparin-free anticoagulant infection inhibitor for hemodialysis and preparation method thereof |
| CN102318837A (en) * | 2011-08-23 | 2012-01-18 | 安徽宝迪肉类食品有限公司 | Compound anticoagulant of animal blood |
| CN107988140A (en) * | 2018-01-25 | 2018-05-04 | 鲁东大学 | A kind of abalone blood cell anticoagulation protecting agent and preparation method thereof |
| CN108396008A (en) * | 2018-01-25 | 2018-08-14 | 鲁东大学 | A kind of abalone haemocyte processing method |
| CN107988140B (en) * | 2018-01-25 | 2021-02-02 | 鲁东大学 | Abalone blood cell anticoagulant protective agent and preparation method thereof |
| CN117717066A (en) * | 2024-02-04 | 2024-03-19 | 广东海洋大学 | Embryo preservation solution, cryopreservation method thereof and application of embryo preservation solution in cryopreservation of Babylonia embryos |
| CN117717066B (en) * | 2024-02-04 | 2024-04-26 | 广东海洋大学 | Embryo preservation solution, cryopreservation method thereof and application of embryo preservation solution in cryopreservation of Babylonia embryos |
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