欧洲鳗鲡淋巴细胞转化试验条件的建立

Optimum conditions of lymphocyte proliferation of European eel

通过对培养时间、培养温度、刀豆蛋白A(ConA)质量浓度和鳗鲡血清质量分数4个参数的测定,确定欧洲鳗鲡(Anguilla anguilla)外周血淋巴细胞转化试验四甲基偶氮唑(MTT)法的实验条件。结果表明,在培养时间为42-90 h,培养温度分别为15.0℃、20.0℃、25.0℃和30.0℃时,细胞培养液中ConA质量浓度分别为0、10μg/mL、20μg/mL和30μg/mL,鳗鲡血清质量分数分别为0、5%、10%和15%时,淋巴细胞于20.0℃、含10μg/mL ConA,10%鳗鲡血清的RPMI1640细胞培养液中培养66 h后可获得最好的淋巴细胞转化效果。方差分析结果显示,4个因素中仅鳗鲡血清水平对淋巴细胞转化试验OD值具有极显著影响(P<0.01),而温度、时间和ConA对其影响均不显著(P>0.05)。本研究建立的鳗鲡外周血淋巴细胞转化试验条件为鳗鲡的细胞免疫建立了研究方法。

One of experiments to indicate the immune status of the fish is the in vitro culture of lymphocytes and mitogen which will cause series changes of metabolizability and morphology of the lymphocytes and then induce the lymphocytes transforming to the lymphoblasts. This experiment of lymphocyte proliferation can be used as an index of cell immunology level and has been used for determining T cells mediated immunology. Many researches on mammal and avian indicated that culture temperature,culture time, concentration of animal serum and ConA had significant effects on lymphocyte proliferation in vitro culture, but few studies on the suitable condition for the fish lymphocyte proliferation were reported. To study the optimum condition of the lymphocyte proliferation, we isolated the peripheral blood lymphocyte of European eel （Anguilla anguilla） and incubated it in the RPMI 1640 medium （containing 10% fetal bovine serum, FBS） under the different temperature, cultured time, eel serum and ConA concentrations. We expect the results of this study would provide an effective method for peripheral blood lymphocyte proliferation of eel and other fish, and an important reference for the further studies on the cell-mediated immunology of fish. In the experiment,European eel （150- 200 g） obtained from Tongan Farm of Xiamen in China were cultured in aquaria supplied with aerated water in dark environment at room temperature. The fish were fed with commercial pellete feed twice a day. Prior to the experiments the fish were acclimatized at 25 ℃ for aminimum of four weeks. When preparing blood lymphocyte, 5 mL blood aseptically released from eel caudal sinus with a syringe containing 0.5 mL heparin sodium. The blood samples were diluted with 5 mL RPMI-1640 medium containing 10% FBS, adding isometric lymphocytes separation medium （Ficoll 1.077, The Second Reagent Factory of Shanghai）, then, isolated by leucocytes for 20 min at centrifugation of 3 000 g. Collecting the interface of the centrifuged medium which consisted of over 90 % lymphocytes, then washing twice with RPMI1640. The viability of the cells was determined by the trypan blue exclusion test and quantified by a haemocytometer. The cell suspension was then adjusted to 1 × 10^7 cells·mL^-1. To culturing lymphocyte.s, the temperature was from 15 ℃ to 30 ℃ ,and the incubation time was from 42 h to 90 h;the eel serum concentration was from 5% to 15% ,and the ConA concentration was from 0 μg/mL to 30 μg/mL. At the four incubation temperatures （15 ℃,20 ℃,25 ℃,30 ℃ ）, each was designed with three incubation periods （42 h,66 h,and 90 h）,four eel serum concentrations （0% ,5%, 10%, 15% ） and four ConA concentrations （0 μg/mL, 10 μg/mL,20 μg/mL,30 μg/mL）. Then 48 culture mediums with different combinations of incubation time, eel serum and ConA concentration were made. The results of anova analysis of four factors indicated that only eel serum concentration had significant （P〈0.01） influence on the lymphocyte proliferation rate. Simultaneously, there were significant （P 〈 0.05） interaction effects between the three factors of incubation temperature, incubation time and eel serum concentration. Some researchers found out 10% serum of brook trout （ Salvelinus fontinalis ） could increase 30 to 100 times of OD value in lymphocyte proliferation,but the OD value can never doubled when eel serum concentration was increased from 5% to 15 %. In general, the temperature of lymphocyte proliferation of mammals is 37 ℃, and avian lymphocytes cultured in vitrio is over 39℃. Optimum temperature of fish lymphocytes cultured in vitro was hard to ascertain because fish are allotherms. The results of our study found out the best temperature for lymphocyte proliferation was 20 ℃. In addition, effect of ConA concentration was significantly influenced by other three factors, but 10 μg/mL was the best. To sum up, the optimum stimulation index of the experiment can be acquired after 90 h under the condition of 20.0 ℃ ,RPMI 1640 medium containing 10 μg/mL ConA and 10% eel serum. The results in this experiment acquired optimum conditions for cell-mediated immunity study of European eel （Anguilla anguilla ） and provided an important reference for the further studies on the cellmediated immunology of fish. [ Journal of Fishery Sciences of China, 2007,14 （3） ： 403 - 411 ]