CN103289953B - Fish leukocyte separation method - Google Patents
Fish leukocyte separation method Download PDFInfo
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- CN103289953B CN103289953B CN201310197729.8A CN201310197729A CN103289953B CN 103289953 B CN103289953 B CN 103289953B CN 201310197729 A CN201310197729 A CN 201310197729A CN 103289953 B CN103289953 B CN 103289953B
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Abstract
The invention provides a kind of Fish leukocyte separation method, the method is aseptically, and fish complete blood cell be inoculated into and do not cultivate containing in the Zooblast culture medium of animal serum, wherein complete blood cell inoculum density is 10
6-10
10individual/milliliter substratum, culture condition is the carbonic acid gas of the temperature of 15 DEG C-37 DEG C, the humidity of 90%-98% and 0-8%.White corpuscle without the need to additionally using the lysate of cellular segregation liquid or certain cell, but by the method that cell culture fluid and temperature combine, is successfully separated by the present invention, also without the need to using the protein ingredient of animal serum and so in substratum; The cell that the inventive method is separated, do not have the side effect that the chemical reagent of parting liquid and lysate and so on produces, cell purity is high, and active strong, security is good, and cost is low; In addition, the cell separated by the inventive method, when carrying out vitro culture, just can normal growth in low levels blood serum medium.
Description
Technical field
The present invention relates to the leukocytic separation method of a kind of animal, particularly a kind of Fish leukocyte separation method being applicable to vitro culture.
Background technology
White corpuscle has defence and immunologic function, cures, resists the invasion of cause of disease and play an important role to the immunology of disease at body injury.Study the immunologic mechanism of body, acquisition sufficient amount, the white corpuscle that purity is high and activity is strong are primary conditions, and the foundation of leukocytic vitro culture and stimulus modality is prerequisite.Existing white corpuscle separation method mainly contains two kinds: one adopts erythrocyte cracked liquid By Direct Pyrolysis red corpuscle and is separated white corpuscle, and another kind adopts lymph parting liquid carry out gradient centrifugation and be separated each cellular blood components.The white corpuscle purity separated by first method is high, but poor activity, be not suitable for carrying out vitro culture further, be therefore mainly used in leukocytic temporary detecting with the white corpuscle that the method is separated; The white corpuscle vigor be separated by second method is better, but component is more single, mostly only has lymphocyte, and is dropped with the granulocyte of red corpuscle similar density, and needs when cultivating in vitro to add relevant cofactor.
Summary of the invention
In order to overcome the deficiency that above-mentioned prior art exists, the invention provides a kind of brand-new Fish leukocyte separation method, the method uses and just white corpuscle successfully can be separated with red corpuscle containing the Zooblast culture medium of serum, and the isolated leukocyte activity of institute is high, is easy to vitro culture.
Technical scheme of the present invention is to provide a kind of Fish leukocyte separation method, and the method is aseptically, and fish complete blood cell be inoculated into and do not cultivate containing in the Zooblast culture medium of animal serum, wherein complete blood cell inoculum density is 10
6-10
10individual/milliliter substratum, culture condition is the carbonic acid gas of the temperature of 15 DEG C-37 DEG C, the humidity of 90%-98% and 0-8 %.
Described complete blood cell inoculum density is preferably 10
7~ 10
9individual/milliliter substratum, complete blood cell inoculum density is within the scope of this, and the net result difference that white corpuscle is separated is little.
Described culture condition is preferably the carbonic acid gas of the temperature of 25 DEG C-30 DEG C, the humidity of 92%-96% and 3-5 %.
Describedly be not made up of culture medium dry powder composition and supporting agent containing the Zooblast culture medium of animal serum, wherein the component of culture medium dry powder composition and content comprise Calcium Chloride Powder Anhydrous .2H2O 250.00-270.00 mg for often liter of substratum, iron nitrate .9H2O 0.05-0.15 mg, Repone K 300.00-500.00 mg, anhydrous magnesium sulfate 95.00-100.00 mg, sodium-chlor 6300.00-6500.00 mg, AMSP 108.00-110.00 mg, succinic acid 70.00-80.00 mg, Soduxin 90.00-110.00 mg, L-arginine hydrochloride 82.00-86.00 mg, L-hydrochloric acid Gelucystine 60.00-65.00 mg, glycine 20.00-40.00 mg, L-histidine monohydrochloride 40.00-44.00 mg, ILE 100.00-110.00 mg, L-Leu 100.00-110.00 mg, LYS 144.00-148.00 mg, METHIONINE 20.00-60.00 mg, L-Phe 64.00-68.00 mg, Serine 38.00-45.00 mg, L-threonine 90.00-100.00 mg, L-Trp 14.00-18.00 mg, TYR 70.00-74.00 mg, Valine 92.00-96.00 mg, D-VB5 calcium 2.00-6.00 mg, choline tartrate 7.00-7.40 mg, folic acid 2.00-6.00 mg, inositol 7.00-7.60 mg, niacinamide 2.00-6.00 mg, riboflavin 0.20-0.60 mg, thiamine hydrochloride 2.00-6.00 mg, pyridoxine hydrochloride 2.00-6.00 mg, glucose 800.00-3000.00 mg, Sodium.alpha.-ketopropionate 100.00-120.00 mg, phenol red 9.00-9.60 mg, sodium bicarbonate 3500.00-4000.00 mg.
The component of described culture medium dry powder composition and content are preferably often liter of substratum and comprise Calcium Chloride Powder Anhydrous .2H2O 255.00-265.00 mg, iron nitrate .9H2O 0.08-0.12 mg, Repone K 350.00-450.00 mg, anhydrous magnesium sulfate 96.00-98.00 mg, sodium-chlor 6350.00-6450.00 mg, AMSP 108.00-110.00 mg, succinic acid 72.00-78.00 mg, Soduxin 95.00-105.00 mg, L-arginine hydrochloride 83.00-85.00 mg, L-hydrochloric acid Gelucystine 62.00-64.00 mg, glycine 25.00-35.00 mg, L-histidine monohydrochloride 41.00-43.00 mg, ILE 101.00-109.00 mg, L-Leu 101.00-109.00 mg, LYS 145.00-147.00 mg, METHIONINE 205.00-55.00 mg, L-Phe 65.00-67.00 mg, Serine 40.00-44.00 mg, L-threonine 92.00-98.00 mg, L-Trp 15.00-17.00 mg, TYR 71.00-73.00 mg, Valine 93.00-95.00 mg, D-VB5 calcium 2.50-5.50 mg, choline tartrate 7.10-7.30 mg, folic acid 3.00-5.00 mg, inositol 7.10-7.50 mg, niacinamide 3.00-5.00 mg, riboflavin 0.30-0.50 mg, thiamine hydrochloride 3.00-5.00 mg, pyridoxine hydrochloride 3.00-5.00 mg, glucose 900.00-2000.00 mg, Sodium.alpha.-ketopropionate 105.00-115.00 mg, phenol red 9.10-9.50 mg, sodium bicarbonate 3600.00-3800.00 mg.
Not within the specific limits, the net result that its white corpuscle is separated is similar for each component concentration of culture medium dry powder composition in the above-mentioned Zooblast culture medium not containing animal serum.
One of described Zooblast culture medium constituent supporting agent be preferably in distilled water, deionized water, water for injection, redistilled water and ultrapure water one or more.
Zooblast culture medium dry powder composition is dispersed in supporting agent by the preparation method of described Zooblast culture medium, refilters degerming.
The dispersing method of described Zooblast culture medium dry powder composition in supporting agent can be able to be realized, as sonic oscillation, magnetic agitation etc. by the dispersing method that this area is conventional; Described filtration sterilization method is that bacteriological filtration film well known in the art carries out one or many filtration, and wherein the aperture of bacteriological filtration film is not more than 0.22 μm.
The all ingredients that the present invention uses can be purchased, the method preparation that this area also can be adopted conventional.For the reagent of cell cultures, not only purity requirement is high, and can not there is any material affecting cell normal physiological activity, therefore also needs the requirement meeting pharmaceutical products, preferred injection stage source chemicals.
White corpuscle separation method of the present invention can be separated the freshwater fish white corpuscles such as swamp eel white corpuscle, leucocytes of grass carp and crucian, and the white corpuscle after separation can carry out vitro culture.
Advantage of the present invention is: the invention provides a kind of brand-new Fish leukocyte separation method, without the need to additionally using the lysate of cellular segregation liquid or certain cell, but by method that cell culture fluid and temperature combine, white corpuscle is successfully separated, also without the need to using the protein ingredient of animal serum and so in substratum; The cell that the inventive method is separated, do not have the side effect that the chemical reagent of parting liquid and lysate and so on produces, cell purity is high, and active strong, security is good, and cost is low; In addition, the cell separated by the inventive method, when carrying out vitro culture, just can normal growth in low levels blood serum medium, the cell be separated by the inventive method, different batches difference is little.Therefore this invention simplifies the isolation technique of Fish leukocyte, cost-saving, improve white corpuscle vigor.
Accompanying drawing explanation
Fig. 1 is the white corpuscle photo that the white corpuscle separation method of embodiment 1 is separated;
Fig. 2 is the white corpuscle photo that the white corpuscle separation method of embodiment 2 is separated;
Fig. 3 is the white corpuscle photo that the white corpuscle separation method of comparative example 1 is separated;
Fig. 4 is the white corpuscle photo that the white corpuscle separation method of comparative example 2 is separated;
Fig. 5 is the picture that white corpuscle that the white corpuscle separation method of embodiment 1 is separated is cultivated 72 hours;
Fig. 6 is the picture that white corpuscle that the white corpuscle separation method of embodiment 2 is separated is cultivated 72 hours;
Fig. 7 is the picture that white corpuscle that the white corpuscle separation method of comparative example 1 is separated is cultivated 72 hours;
Fig. 8 is the picture that white corpuscle that the white corpuscle separation method of comparative example 2 is separated is cultivated 72 hours.
Embodiment
Now in conjunction with specific embodiments, technology contents of the present invention is further described.
This embodiment of embodiment 1(is for illustration of white corpuscle separation method of the present invention)
The present embodiment used medium not containing any animal serum, each component of described culture medium dry powder composition and content as shown in table 1:
Table 1
In table 1 and following table, the unit mg/L of each component of culture medium dry powder composition refers to, with by the volume of this culture medium dry powder composition and the final liquid nutrient medium of carrier mixing gained for benchmark, be equivalent to often liter of final liquid nutrient medium, the milligram number of the solid matter that needs add when preparing substratum.
The concrete preparation method of the Zooblast culture medium that the present invention is used is by the consumption of each component of the solid medium dry powder composite shown in table 1 except sodium bicarbonate according to 1 liter of final liquid nutrient medium, after the mixing of ten thousand/balance one accurate weighing, add 900 milliliters of ultrapure waters again, uniform dispersion is obtained after stirring 2 hours with magnetic stirring apparatus at 25 DEG C, then add 3.7 grams of sodium bicarbonates and its pH value is adjusted to 7.0,1000 milliliters are settled to ultrapure water, bacteriological filtration membrane filtration with 0.22 μm after constant volume is degerming, and 4 DEG C save backup.
Leukocytic concrete separation method is aseptically, with pipettor, above-mentioned for 5ml nutrient solution is transferred to 25cm
2in Tissue Culture Flask, inoculate fresh swamp eel complete blood cell, inoculum density is 2 × 10
7individual cell/ml substratum; Then under 25 DEG C of temperature condition, 95% humidity condition and 5% carbon dioxide conditions, cultivation 3-7 days is carried out, record the time that unit odd number red corpuscle and leukocytic relative proportion and red corpuscle all disappear under inverted microscope, Rui Shi Giemsa staining detects the cell composition of institute's culturing cell.Rui Shi Giemsa staining the results are shown in Figure 1, and Fig. 5 is shown in by the cell cultures photo of 72 hours.As seen from Figure 1, at 25 DEG C, cultivate the complete blood cell of 72 hours, primarily of lymphocyte, monocyte, granulocyte composition, there is no red corpuscle.As seen from Figure 5, by the complete blood cell of cultivating after 72 hours, red corpuscle all disappears, and remaining white corpuscle is in good condition.
Embodiment 2 (this embodiment is for illustration of white corpuscle separation method of the present invention)
The present embodiment used medium is not containing any animal serum, and each component of described culture medium dry powder composition and content are with table 1.
The preparation method of the Zooblast culture medium used by the present invention is as embodiment 1, and leukocytic concrete separation method is aseptically, with pipettor, above-mentioned for 5ml nutrient solution is transferred to 25cm
2in Tissue Culture Flask, inoculate fresh swamp eel complete blood cell, inoculum density is 2 × 10
7cell/ml; Then under 37 DEG C of temperature condition, 95% humidity condition and 5% carbon dioxide conditions, cultivation 2-4 days is carried out, record the time that unit odd number red corpuscle and leukocytic relative proportion and red corpuscle all disappear under inverted microscope, Rui Shi Giemsa staining detects the cell composition of institute's culturing cell.Rui Shi Giemsa staining the results are shown in Figure 2, and Fig. 6 is shown in by the cell cultures photo of 72 hours.As seen from Figure 2, the complete blood cell after cultivating 72 hours at 37 DEG C, primarily of granulocyte, lymphocyte, monocyte composition, does not have red corpuscle.As seen from Figure 6, at 37 DEG C by the complete blood cell of cultivating after 72 hours, red corpuscle all disappears, and remaining white corpuscle is in good condition.
Below see two groups of contrast experiment's examples, they are for illustration of existing white corpuscle separation method.
Comparative example 1
Erythrocyte splitting fluid component is as shown in the table:
Aseptically, with pipettor, above-mentioned for 2ml lysate is transferred in 10ml centrifuge tube, then the fresh finless eel blood of 2ml is added, after mixing, room temperature leaves standstill 10 minutes, after solution is transparent, 25 DEG C of temperature condition, rotating speed under being 1500rpm condition centrifugal 5 minutes, abandon supernatant, after phosphoric acid buffer is resuspended by cell precipitation, rotating speed is under 1500rpm condition centrifugal 5 minutes, abandon supernatant, with embodiment 1 same medium re-suspended cell used, under embodiment 1 same culture conditions, cultivate washing rear portion pipettor transfer to 25cm
2in Tissue Culture Flask, with the substratum identical with embodiment 1, cultivate by under the identical condition of embodiment 1, do Rui Shi Giemsa staining simultaneously and detect cell composition, cultured cells observation of cell form taking pictures under inverted microscope.Hemocyte composition Rui Shi Giemsa staining after erythrocyte splitting the results are shown in Figure 3.State after hemocyte after erythrocyte splitting cultivates 72 hours is shown in Fig. 7.As seen from Figure 3, after conventional cracking process removing red corpuscle, remaining cell forms primarily of primarily of macrolymphocyte, small lymphocyte, monocyte, granulocyte, and its composition forms identical with the white corpuscle separated with the present invention.As seen from Figure 7, with the white corpuscle after erythrocyte cracked liquid process, cultivate in non-additive situation, by cultured cells, most cells is dead after 72 hours.
Comparative example 2
Fish lymphocyte separation medium is purchased from Tianjin Hao ocean biological products company limited.Aseptically, with pipettor, above-mentioned for 2ml lymph parting liquid is transferred in 10ml centrifuge tube, then add the fresh finless eel blood of 2ml along tube wall, 25 DEG C of temperature condition, rotating speed under being 2000rpm condition centrifugal 15 minutes, after getting the cell phosphoric acid buffer washing of buffy coat, transfer to 25cm
2in Tissue Culture Flask, with the identical substratum of embodiment 1, cultivate by under the identical condition of embodiment 1, do Rui Shi Giemsa staining simultaneously and detect cell composition.Observe culturing cell form under inverted microscope and take pictures.The cell Rui Shi Giemsa staining that lymph parting liquid is separated the results are shown in Figure 4.The cell cultures state of 72 hours is shown in Fig. 8, as seen from Figure 4, with Conventional density gradients centrifugation lymph confluent monolayer cells out, primarily of macrolymphocyte, small lymphocyte composition.As seen from Figure 8, the lymphocyte separated by density gradient centrifugation with lymph parting liquid, is cultivated in non-additive situation, by cultured cells after 72 hours most cells start death.
Claims (5)
1. Fish leukocyte separation method, is characterized in that: the method is aseptically, and fish complete blood cell be inoculated into and do not cultivate containing in the Zooblast culture medium of animal serum, wherein complete blood cell inoculum density is 10
6-10
10individual/milliliter substratum, culture condition be 15 DEG C-37 DEG C temperature, the humidity of 90%-98%, the carbonic acid gas of 0-8 % and 3-7 days incubation time, describedly be not made up of culture medium dry powder composition and supporting agent containing the Zooblast culture medium of animal serum, wherein the component of culture medium dry powder composition and content comprise CaCl for often liter of substratum
2.2H
2o 250.00-270.00 mg, iron nitrate .9H
2o 0.05-0.15 mg, Repone K 300.00-500.00 mg, anhydrous magnesium sulfate 95.00-100.00 mg, sodium-chlor 6300.00-6500.00 mg, AMSP 108.00-110.00 mg, succinic acid 70.00-80.00 mg, Soduxin 90.00-110.00 mg, L-arginine hydrochloride 82.00-86.00 mg, L-hydrochloric acid Gelucystine 60.00-65.00 mg, glycine 20.00-40.00 mg, L-histidine monohydrochloride 40.00-44.00 mg, ILE 100.00-110.00 mg, L-Leu 100.00-110.00 mg, LYS 144.00-148.00 mg, METHIONINE 20.00-60.00 mg, L-Phe 64.00-68.00 mg, Serine 38.00-45.00 mg, L-threonine 90.00-100.00 mg, L-Trp 14.00-18.00 mg, TYR 70.00-74.00 mg, Valine 92.00-96.00 mg, D-VB5 calcium 2.00-6.00 mg, choline tartrate 7.00-7.40 mg, folic acid 2.00-6.00 mg, inositol 7.00-7.60 mg, niacinamide 2.00-6.00 mg, riboflavin 0.20-0.60 mg, thiamine hydrochloride 2.00-6.00 mg, pyridoxine hydrochloride 2.00-6.00 mg, glucose 800.00-3000.00 mg, Sodium.alpha.-ketopropionate 100.00-120.00 mg, phenol red 9.00-9.60 mg, sodium bicarbonate 3500.00-4000.00 mg.
2. Fish leukocyte separation method according to claim 1, is characterized in that: described complete blood cell inoculum density is 10
7~ 10
9individual/milliliter substratum.
3. Fish leukocyte separation method according to claim 2, is characterized in that: described culture condition is the carbonic acid gas of the temperature of 25 DEG C-30 DEG C, the humidity of 92%-96% and 3-5 %.
4. the Fish leukocyte separation method according to claim 1 or 2 or 3, is characterized in that: the supporting agent in described Zooblast culture medium constituent is one or more in distilled water, deionized water, water for injection, redistilled water and ultrapure water.
5. Fish leukocyte separation method according to claim 4, is characterized in that: Zooblast culture medium dry powder composition is dispersed in supporting agent by the preparation method of described Zooblast culture medium, refilters degerming.
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Non-Patent Citations (3)
| Title |
|---|
| 丰培金,张俊辉等.鲤鱼外周血白细胞的分离和体外培养.《中国兽医学报》.2004,第24卷(第4期), * |
| 厉广坤,鄢采芹等.无小牛血清外周血淋巴细胞培养液的研究.《中国优生与遗传杂志》.1999,第7卷(第2期), * |
| 曹丽萍,殷国俊等.香菇多糖对鲤鱼离体培养免疫细胞的活性调节作用.《农业生物技术学报》.2008,第16卷(第4期), * |
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