半滑舌鳎外周血T淋巴细胞的分离、鉴定及TCRβ基因免疫应答分析

ISOLATION AND IDENTIFICATION OF T-LYMPHOCYTES FROM PERIPHERAL BLOOD AND IMMUNE RESPONSE ANALYSIS OF TCRβ CHAIN GENE IN CYNOGLOSSUS SEMILAEVIS

为开展半滑舌鳎(Cynoglossus semilaevis)免疫学研究提供细胞平台,利用密度梯度离心法分离半滑舌鳎外周血淋巴细胞,采用短期细胞培养法分离悬浮淋巴细胞,悬浮淋巴细胞在含有0.3μg/m L的PHA的DMEM完全培养基,于24℃条件下可连续培养3—4d左右,采用自制的尼龙毛柱可将悬浮淋巴细胞中的非黏附淋巴细胞和黏附淋巴细胞成功分离;利用流式细胞仪结合特异抗体检测对非黏附淋巴细胞和黏附淋巴细胞进行鉴定,结果表明,非黏附细胞与鼠抗人FTIC-CD3单克隆抗体特异结合,为T样淋巴细胞;黏附细胞和鼠抗人FTICCD19单抗特异结合,为B样淋巴细胞。T细胞表面抗原受体TCRβ基因可特异性的在非黏膜细胞中表达,而在黏附细胞中不表达,证明分离获得的非黏膜细胞为T淋巴细胞,采用q RT-PCR(Quantitative Real-Time PCR)方法检测TCRβ基因表达,结果表明,TCRβ基因在半滑舌鳎肝、脾、头肾、后肾、小肠、胃、血液、鳃、皮肤、肌肉、心脏、脑、卵巢组织中均有表达,其中在肠、胃、脾、头肾中表达量较高;鳗弧菌感染后TCRβ基因在肝、脾、鳃中呈现明显的上调表达,且表达峰值出现在感染后72—96h,表明TCRβ基因在获得性免疫应答中起重要作用。

The density gradient method was used to isolate lymphocytes from peripheral blood of Cynoglossus semilaevis, and the isolated lymphocytes were cultured in complete DMEM medium（with 0.3 μg/m L PHA） for 3—4 days.Peripheral blood lymphocytes were separated to non-adherent lymphocytes and adherent lymphocytes using self-made nylon hair column. Non-adherent lymphocytes specifically bind with mouse anti-human FTIC-CD3 monoclonal antibody, while adherent lymphocytes specifically bind to anti-human FTIC-CD19 monoclonal antibody. TCRβ specifically expressed on non-adherent lymphocytes. Taken together, non-adherent lymphocytes were confirmed as T lymphocytes. The results of q RT-PCR showed that TCRβ was expressed in all detected 13 tissues with high expression level in the intestine, stomach, spleen, head kidney and the lowest level in muscle. The expression of TCRβ gene was significantly induced in the liver, spleen and gill with peak level at 72—96 h after V. Anguillarum infection. These results indicate that TCRβ gene plays an important role in adaptive cellular immune response.