CN103992984A - Method for treating whole blood used for fish chromosome technique based on whole blood culture method - Google Patents
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Abstract
The invention provides a method for treating whole blood used for a fish chromosome technique based on a whole blood culture method. The method comprises the following steps: firstly, anesthetizing test fish, sampling blood, mixing the obtained blood with an ACD (Acid, Citrate and Dextrose) anticoagulant to obtain mixed blood; secondly, mixing the obtained mixed blood with a culture medium to obtain a mixed solution; thirdly, superposing the obtained mixed solution onto a Percoll separating medium, centrifuging and removing the upper-layer plasma, thereby obtaining lymphocytes at the junction of the plasma layer and the separating medium layer; fourthly, adding the obtained lymphocytes to the culture medium, mixing thoroughly and evenly, and centrifugally washing to obtain a cell precipitate; finally, transferring the cell precipitate to the culture medium and culturing at 20-30 DEG C for 72-96 hours. The processing method for preparing the whole blood for the fish chromosome technique based on the whole blood culture method is capable of successfully and effectively separating out the lymphocytes and eliminating red blood cell interference, and thus increasing the preparation success rate and laying a foundation for guaranteeing test results.
Description
Technical field
The invention belongs to Cytogenetic techniques field, be specifically related to a kind of method based on whole blood culture method treat fish chromosomoid technology whole blood used.
Background technology
Chromosome karyotype analysis is one of important content of fish cell genetics research, not only categorizing system, evolutionary relationship and the karyomit(e) evolutionary process tool significance to understanding fish, also can be fish germplasm research and population qualification provides cytogenetics foundation.What the making method of Fishes Chromosomes was the most frequently used at present has live body injection and cell culture method etc.Wherein, PHA live body injection, does not have in the situation of good equipment in the wild, and this method is particularly applicable, but this method requires to cut open and kill fish, is unfavorable for the preservation of fingerling, especially to rare fish with to plant fish not too suitable.Lymphocyte in fish peripheral blood is under the stimulation of certain PHA concentration, and division growth rapidly, is applicable to carrying out chromosomal research.Utilize micro-peripheral blood to carry out vitro culture and prepare karyomit(e) and can effectively address the above problem, different fish, film-making process, observational technique are all not quite similar.
Cell culture method is due to the difference of the aspects such as the phylogenetic features of fish, film-making season, PHA quality and operator's experience, and the quality and quantity of the metacinesis phase obtaining is extremely unstable.In order to reduce experimentation cost, chromosomal preparation is used domestic PHA conventionally, because of not purified, there is red corpuscle coagulability in various degree, especially some fingerling, red corpuscle rich content in blood, short solidifying effect to PHA is very sensitive, very easily causes the aggegation of culture sample, causes cultivating unsuccessfully; In addition, karyomit(e) prepare conventional antithrombotics heparin due to the purity height of its preparation with and shelf time varying length, its anti-freezing effect is not identical yet, amount may cause greatly haemolysis, suppress lymphocytic conversion and division, I haven't seen you for ages occurs to occur all can affecting even test failure of lymphocyte transformation efficiency by the membrane structure that scleroproein forms in blood coagulation or culture for amount.Therefore, the karyomit(e) preparation condition of every kind of fish maturation need to constantly be groped could set up with repetition test, and wherein, the processing of whole blood sample is most important.
Summary of the invention
The object of this invention is to provide a kind of method based on whole blood culture method treat fish chromosomoid technology whole blood used, the method can successfully effectively be isolated lymphocyte, gets rid of red corpuscle and disturbs, and improves and is prepared into power, for warranty test result lays the foundation.
Based on the method for whole blood culture method treat fish chromosomoid technology whole blood used, comprise the following steps:
Step 1, will test fish anesthesia, and blood sampling, mixes gained blood with ACD antithrombotics, obtain mixing blood;
Step 2, mixes step 1 gained mixing blood with the first substratum, obtain mixed solution;
The first substratum is 0.1% FCS-L-15 substratum;
Step 3, adds to step 2 gained mixed solution on the Percoll parting liquid of percent by volume 55～65, centrifugal, removes upper plasma, obtains plasma layer and the lymphocyte that separates liquid layer intersection;
Step 4, adds step 3 gained lymphocyte in the second substratum, fully mixes, and centrifuge washing, obtains cell precipitation thing;
Two substratum are 0.1% FCS-L-15 substratum;
Step 5, goes to step 4 gained cell precipitation thing in the 3rd substratum, cultivates 72～96h for 20～30 DEG C;
The 3rd substratum is L-15 substratum.
As the further improvement of foregoing invention, the volumetric ratio of mixing blood and substratum in described step 2 is 1:1～1:2.
As the further improvement of foregoing invention, in described step 3, the volumetric ratio of mixed solution and Percoll parting liquid is 1:1～3:1.
As the further improvement of foregoing invention, in described step 3, centrifugal condition is 350g, 18～25 DEG C of centrifugal 15～30min.
As the further improvement of foregoing invention, the volume ratio of described step 4 medium size lymphocyte and the second substratum is 1:4～1:5.
As the further improvement of foregoing invention, in described step 4, centrifugal condition is 4 DEG C, the centrifugal 10min of 140g.
Substratum in each step can carry out routine according to the correlation technique knowledge of this area to be selected, and " first, second, third " of the present invention do not form the restriction whether identical to the kind of substratum, just for describing the use order of substratum.
It is a lot of that the lymphocytic factor of a large amount of fish is obtained in impact, evolution degree of the collection of enough peripheral bloods, centrifugal force, centrifugation time, envrionment temperature, individual difference, blood viscosity, fish etc. all can affect lymphocyte quantity and the purity of separation, but the cellular segregation liquid of selecting best in quality concentration is to affect the key factor that lymphocyte effectively separates.The Percoll using in the present invention is a kind of silica gel particle that is surrounded by V-Pyrol RC, can not penetrate microbial film, to cell toxicological harmless; (< 20mo sm/kg H is forced down in infiltration
2o), viscosity little, can form the density up to 1.3g/ml; In addition, its diffusion constant is low, and the gradient forming is very stable, can be at low centrifugal force (200-1000g) in several minutes to reaching satisfied separating effect in tens of minutes while adopting preformed density gradient.Separate the parting liquid of the lymphocyte requirement use different mass concentration in different genera peripheral blood, lymphocyte from peripheral blood of fish mass concentration is between 1.085～1.075g/ml, configure the centrifugate of 55%～65% density according to the concentration of Percoll, in the time of Percoll concentration < 55% or > 65%, cannot collect lymphocyte; Envrionment temperature is controlled at 18～25 DEG C simultaneously, can ensure the separating effect of the survival rate of cell and the cellular segregation liquid of maximum; Blood can reduce blood self stickiness and erythrocytic gathering through a certain proportion of dilution, improves lymphocyte harvest yield.
The treatment process of preparing the whole blood of Fishes Chromosomes technology based on whole blood culture method provided by the invention, can successfully effectively isolate lymphocyte, gets rid of red corpuscle and disturbs, and improves and is prepared into power, for warranty test result lays the foundation.
Brief description of the drawings
Fig. 1 in step 2 is superimposed to mixed solution the design sketch on Percoll parting liquid, and wherein 1 is mixed solution, and 2 is Percoll parting liquid;
Fig. 2 is mixed solution and the design sketch of Percoll parting liquid after centrifugal in step 2, wherein 1 blood plasma for dilution, and 2 is lymphocyte, and 3 is Percoll parting liquid, and 4 is granulocyte, and 5 is red corpuscle;
Fig. 3 is the chromosomal light microscopic figure of the prepared swamp eel of embodiment 1 (amplifying 400 times);
Fig. 4 does not process the chromosomal light microscopic figure of prepared swamp eel (amplifying 400 times) through whole blood;
From the contrast of Fig. 3 and Fig. 4 can find out swamp eel chromosome specimen red corpuscle that embodiment 1 obtains disturb little, number is complete, good dispersion, form are clear;
Fig. 5 is the chromosomal light microscopic figure of the prepared tilapia of embodiment 2 (amplifying 400 times);
Fig. 6 does not process the chromosomal light microscopic figure of prepared tilapia (amplifying 400 times) through whole blood;
From the contrast of Fig. 5 and Fig. 6 can find out tilapia chromosome specimen red corpuscle that embodiment 2 obtains disturb little, number is complete, good dispersion, form are clear.
Embodiment
Embodiment 1
Whole blood culture method is prepared the karyomit(e) of swamp eel
After whole blood processing: MS-222 anesthesia, test fish is with after cotton ball soaked in alcohol sterilization afterbody body surface, in super clean bench, after anus, 2mL blood is adopted at 1cm place, lay down after syringe needle, blood is proceeded to (syringe and ACD shift to an earlier date precooling) in the aseptic centrifuge tube that contains appropriate ACD antithrombotics, the gentle blood that fully mixes; After mixed blood mixes with 0.1% FCS-L-15 substratum dilution such as head for precooling such as grade, slowly join in the centrifuge tube of the Pereol1 parting liquid that has filled 2mL 60% as shown in Figure 1, room temperature, horizontal rotor, 350g, centrifugal 15～30min, obtains plasma layer and the lymphocyte that separates liquid layer intersection, as shown in Figure 2; Lymphocyte is placed in to aseptic centrifuge tube, adds 0.1% FCS-L-15 substratum of 4 times of volumes, after fully mixing, under 4 DEG C, the centrifugal 10 min conditions of 140g, wash 2 times; Cell precipitation thing goes in 5mL L-15 substratum, after shaking up gently, builds lid, cultivates 72～96 h for 28 DEG C.
ACD antithrombotics formula: citric acid 0.48g, Trisodium Citrate 1.32g, glucose 1.47g, add water to 100mL, for subsequent use after sterilizing.
0.1% FCS-L-15 culture medium prescription: 0.1mL FCS(foetal calf serum), 1mL S/P(mycillin mixed solution, 100 times of working fluids), add L-15 substratum to 100mL, for subsequent use, use front 4 DEG C of precoolings.
60% Percoll parting liquid formula: 6 mL Percoll stostes, 1mL HBSS(10 times working fluid, containing Yihong), 3mL sterilized water mixes.
L-15 culture medium prescription: 1% S/P, 5% FCS and PHA, pH:6.8～7.2.
By the lymphocyte that processing obtains through whole blood through pre-treatment and collection, hypotonic and fixing, drip after sheet and dyeing, obtain swamp eel chromosome specimen, microscopy is according to as shown in Figure 3.From the contrast of Fig. 3 and Fig. 4 can find out the present embodiment obtained red corpuscle disturb little, number is complete, good dispersion, form swamp eel chromosome specimen clearly.
Embodiment 2
Whole blood culture method is prepared the karyomit(e) of tilapia
After whole blood processing: MS-222 anesthesia, test fish is with after cotton ball soaked in alcohol sterilization afterbody body surface, in super clean bench, adopt 2mL blood at lateral line scales place, lay down after syringe needle, blood is proceeded to (syringe and ACD shift to an earlier date precooling) in the aseptic centrifuge tube that contains appropriate ACD antithrombotics, the gentle blood that fully mixes; After mixed blood mixes with 0.1% FCS-L-15 substratum dilution such as head for precooling such as grade, slowly join in the centrifuge tube of Pereol1 parting liquid that has filled 2mL 60%, as shown in Figure 1, room temperature, horizontal rotor, 350g, centrifugal 15～30min, obtain plasma layer and the lymphocyte that separates liquid layer intersection, as shown in Figure 2; Lymphocyte is placed in to aseptic centrifuge tube, adds 0.1% FCS-L-15 substratum of 5 times of volumes, after fully mixing, at 4 DEG C, under the centrifugal 10 min conditions of 140g, wash 2 times.Cell precipitation thing goes in 5mL L-15 substratum, after shaking up gently, builds lid, cultivates 72～96 h for 28 DEG C.
ACD antithrombotics formula: citric acid 0.48g, Trisodium Citrate 1.32g, glucose 1.47g, add water to 100mL, for subsequent use after sterilizing.
0.1% FCS-L-15 culture medium prescription: 0.1mL FCS(foetal calf serum), 1mL S/P(mycillin mixed solution, 100 times of working fluids), add L-15 substratum to 100mL, for subsequent use, use front 4 DEG C of precoolings.
60% Percoll parting liquid formula: 6 mL Percoll stostes, 1mL HBSS(10 times working fluid, containing Yihong), 3mL sterilized water mixes.
L-15 culture medium prescription: 1% S/P, 5% FCS and PHA, pH:6.8～7.2.
By the lymphocyte that processing obtains through whole blood through pre-treatment and collection, hypotonic and fixing, drip after sheet and dyeing, obtain tilapia chromosome specimen, microscopy is according to as shown in Figure 5, from the contrast of Fig. 5 and Fig. 6 can find out this experiment obtained red corpuscle disturb little, number is complete, good dispersion, form tilapia chromosome specimen clearly.
Embodiment 3
Whole blood culture method is prepared the karyomit(e) of jian carp
After whole blood processing: MS-222 anesthesia, test fish is with after cotton ball soaked in alcohol sterilization afterbody body surface, in super clean bench, adopt 2mL blood at lateral line scales place, lay down after syringe needle, blood is proceeded to (syringe and ACD shift to an earlier date precooling) in the aseptic centrifuge tube that contains appropriate ACD antithrombotics, the gentle blood that fully mixes; After mixed blood and 4mL precooling 0.1% FCS-L-15 substratum dilution mix, slowly join in the centrifuge tube of Pereol1 parting liquid that has filled 6mL 55%, as shown in Figure 1, room temperature, horizontal rotor, 350g, centrifugal 15～30min, obtain plasma layer and the lymphocyte that separates liquid layer intersection, as shown in Figure 2; Lymphocyte is placed in to aseptic centrifuge tube, adds 0.1% FCS-L-15 substratum of 4 times of volumes, after fully mixing, at 4 DEG C, under the centrifugal 10 min conditions of 140g, wash 2 times.Cell precipitation thing goes in 5mL L-15 substratum, after shaking up gently, builds lid, cultivates 72～96 h for 28 DEG C.
ACD antithrombotics formula: citric acid 0.48g, Trisodium Citrate 1.32g, glucose 1.47g, add water to 100mL, for subsequent use after sterilizing.
0.1% FCS-L-15 culture medium prescription: 0.1mL FCS(foetal calf serum), 1mL S/P(mycillin mixed solution, 100 times of working fluids), add L-15 substratum to 100mL, for subsequent use, use front 4 DEG C of precoolings.
55% Percoll parting liquid formula: 5.5 mL Percoll stostes, 1mL HBSS(10 times working fluid, containing Yihong), 3.5mL sterilized water mixes.
L-15 culture medium prescription: 1% S/P, 5% FCS and PHA, pH:6.8～7.2.
By the lymphocyte that processing obtains through whole blood through pre-treatment and collection, hypotonic and fixing, drip after sheet and dyeing, obtain jian carp chromosome specimen, that the jian carp chromosome specimen red corpuscle that the present embodiment obtains disturbs is little, number is complete, good dispersion, form are clear.
Embodiment 4
Whole blood culture method is prepared the karyomit(e) of cabrilla
After whole blood processing: MS-222 anesthesia, test fish is with after cotton ball soaked in alcohol sterilization afterbody body surface, in super clean bench, adopt 2mL blood at lateral line scales place, lay down after syringe needle, blood is proceeded to (syringe and ACD shift to an earlier date precooling) in the aseptic centrifuge tube that contains appropriate ACD antithrombotics, the gentle blood that fully mixes; After mixed blood and 4mL precooling 0.1% FCS-L-15 substratum dilution mix, slowly join in the centrifuge tube of Pereol1 parting liquid that has filled 2mL 65%, as shown in Figure 1, room temperature, horizontal rotor, 350g, centrifugal 15～30min, obtain plasma layer and the lymphocyte that separates liquid layer intersection, as shown in Figure 2; Lymphocyte is placed in to aseptic centrifuge tube, adds 0.1% FCS-L-15 substratum of 4 times of volumes, after fully mixing, at 4 DEG C, under the centrifugal 10 min conditions of 140g, wash 2 times.Cell precipitation thing goes in 5mL L-15 substratum, after shaking up gently, builds lid, cultivates 72～96 h for 28 DEG C.
ACD antithrombotics formula: citric acid 0.48g, Trisodium Citrate 1.32g, glucose 1.47g, add water to 100mL, for subsequent use after sterilizing.
0.1% FCS-L-15 culture medium prescription: 0.1mL FCS(foetal calf serum), 1mL S/P(mycillin mixed solution, 100 times of working fluids), add L-15 substratum to 100mL, for subsequent use, use front 4 DEG C of precoolings.
65% Percoll parting liquid formula: 6.5 mL Percoll stostes, 1mL HBSS(10 times working fluid, containing Yihong), 2.5mL sterilized water mixes.
L-15 culture medium prescription: 1% S/P, 5% FCS and PHA, pH:6.8～7.2.
By the lymphocyte that processing obtains through whole blood through pre-treatment and collection, hypotonic and fixing, drip after sheet and dyeing, obtain cabrilla chromosome specimen, that the cabrilla chromosome specimen red corpuscle that the present embodiment obtains disturbs is little, number is complete, good dispersion, form are clear.
Claims (6)
1. the method based on whole blood culture method treat fish chromosomoid technology whole blood used, comprises the following steps:
Step 1, will test fish anesthesia, and blood sampling, mixes gained blood with ACD antithrombotics, obtain mixing blood;
Step 2, mixes step 1 gained mixing blood with the first substratum, obtain mixed solution;
The first substratum is 0.1% FCS-L-15 substratum;
Step 3, adds to step 2 gained mixed solution on the Percoll parting liquid of percent by volume 55～65, centrifugal, removes upper plasma, obtains plasma layer and the lymphocyte that separates liquid layer intersection;
Step 4, adds step 3 gained lymphocyte in the second substratum, fully mixes, and centrifuge washing, obtains cell precipitation thing;
The second substratum is 0.1% FCS-L-15 substratum;
Step 5, goes to step 4 gained cell precipitation thing in the 3rd substratum, cultivates 72～96h for 20～30 DEG C;
The 3rd substratum is L-15 substratum.
2. the method based on whole blood culture method treat fish chromosomoid technology whole blood used according to claim 1, is characterized in that: the volumetric ratio of mixing blood and the first substratum in described step 2 is 1:1～1:2.
3. the method based on whole blood culture method treat fish chromosomoid technology whole blood used according to claim 1, is characterized in that: in described step 3, the volumetric ratio of mixed solution and Percoll parting liquid is 1:1～3:1.
4. the method based on whole blood culture method treat fish chromosomoid technology whole blood used according to claim 1, is characterized in that: in described step 3, centrifugal condition is 350g, 18～25 DEG C of centrifugal 15～30 min.
5. the method based on whole blood culture method treat fish chromosomoid technology whole blood used according to claim 1, is characterized in that: the volume ratio of described step 4 medium size lymphocyte and the second substratum is 1:4～1:5.
6. the method based on whole blood culture method treat fish chromosomoid technology whole blood used according to claim 1, is characterized in that: in described step 4, centrifugal condition is 4 DEG C, centrifugal 10 min of 140g.
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| CN105255827A (en) * | 2015-12-02 | 2016-01-20 | 中国海洋大学 | Culture medium for flounder paralichthys lymphocyte proliferation |
| CN105779389A (en) * | 2016-05-18 | 2016-07-20 | 中国水产科学研究院淡水渔业研究中心 | Megalobrama amblycephala peripheral blood leucocyte separation and primary culture method |
| CN106434551A (en) * | 2016-08-31 | 2017-02-22 | 青岛大学 | Culturing method of fish lymphocyte |
| CN111110385A (en) * | 2019-12-31 | 2020-05-08 | 南京普恩瑞生物科技有限公司 | Construction method of human tumor xenograft model |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105255827A (en) * | 2015-12-02 | 2016-01-20 | 中国海洋大学 | Culture medium for flounder paralichthys lymphocyte proliferation |
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| CN105779389A (en) * | 2016-05-18 | 2016-07-20 | 中国水产科学研究院淡水渔业研究中心 | Megalobrama amblycephala peripheral blood leucocyte separation and primary culture method |
| CN105779389B (en) * | 2016-05-18 | 2019-06-28 | 中国水产科学研究院淡水渔业研究中心 | A kind of separation of megalobrama amblycephala peripheral white blood cells and primary culture method |
| CN106434551A (en) * | 2016-08-31 | 2017-02-22 | 青岛大学 | Culturing method of fish lymphocyte |
| CN111110385A (en) * | 2019-12-31 | 2020-05-08 | 南京普恩瑞生物科技有限公司 | Construction method of human tumor xenograft model |
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