酸性α-醋酸萘酯酶染色法在鳜细胞免疫水平检测中的应用研究

研究了酸性α-醋酸萘酯酶染色法在鳜细胞免疫水平检测中应用的可行性。用不同比重的淋巴细胞分离液对鳜外周血中的淋巴细胞进行分离,结果表明比重为1.080的分离液可最大程度地将鳜外周血中的淋巴细胞分离出来。用酸性α-醋酸萘酯酶染色法对分离出的淋巴细胞进行染色,结果显示最佳的染色条件为在pH6.0~6.5的孵育液中37℃孵育90 m in,然后2%甲基绿37℃复染10 m in,酸性α-醋酸萘酯酶阳性细胞胞核被染成绿色而胞浆被染成红色,阴性细胞胞核和胞浆都被绿染。对正常未免疫鳜与嗜水气单胞菌脂多糖和鳜病毒主要衣壳蛋白免疫一周后的鳜外周血淋巴细胞染色计数,结果酸性α-醋酸萘酯酶阳性细胞所占的比例分别为15%~18%、33%~40%和26%~29%,方差分析表明免疫组与对照组阳性率差异极显著,说明α-醋酸萘酯酶染色法可用于鳜细胞免疫水平的检测。

Application of Acid A-naphtyl Acetate Esterase Staining Method in Siniperca chuatsi Cell Immune Evaluation

The feasibility of using acid A-naphtyl acetate esterase(ANAE)staining method to evaluate the cellular immunity level in immunized Siniperca chausti was tested in this paper.Ficoll-Uragafin gradient solution with different density was used to isolate lymphocytes from peripheral blood of Siniperca chautsi and the solution with a density of 1.080 showed the best result in isolation.ANAE was used to stain the isolated lymphocytes and it indicated that the optimal staining condition was at 37 ℃ incubation for 90 min in ANAE followed by staining at 37 ℃ for 10 min in 2% methyl green solution.At 7 days after immunization the lymphocytes from immunized Siniperca chausti with Aeromonas hydrophila LPS and Siniperca Chausti virus major capsid protein(MCP)were isolated and stained with ANAE.The results showed that the percentage of ANAE positive lymphocytes in LPS and MCP immunized groups were 33%~40% and 26%~29%,respectively,while in the control group the result was 15%~18%,which was significantly less than either of the immunized groups statistically analyzed by One-way ANOVA(P<0.01).