水牛外周血单核巨噬细胞的培养与鉴定

Identification and Culture of Buffalo Peripheral Blood Mononuclear Macrophage

本试验旨在为阐明水牛感染大片吸虫的免疫机理提供单核巨噬细胞材料。经过密度梯度离心分离,贴壁法培养水牛外周血单核巨噬细胞,以MTT法检测培养4、24、48、72、96、120、144、168、192h时贴壁单核巨噬细胞的相对存活数量,结果显示贴壁细胞体外培养72、96、120h时,细胞数量相对稳定,组间差异不显著(P<0.05)。贴壁72h的细胞通过倒置显微镜、瑞氏染色观察,结果显示其具备单核巨噬细胞的典型形态特征;酸性磷酸酶、非特异性酯酶染色阳性率分别为(72.8±0.5)%和(84.6±0.4)%,显示其具备单核巨噬细胞酶化学特性;贴壁细胞表达单核巨噬细胞特异性表面抗原MHCⅡ;墨汁的吞噬率为(87.5±0.6)%,表明该细胞具有单核巨噬细胞的吞噬功能。结果表明贴壁法分离,5%BSA的DMEM培养水牛外周血单核巨噬细胞,培养72h贴壁细胞具备单核巨噬细胞特征,在细胞数量相对稳定期(72、96、120h)可以进行相应的试验研究。

Buffalo is susceptible animals to Fasciola gigantic.To clarify the mechanism of immune,mononuclear macrophage cells have a very important role.In this experiment,buffalo peripheral blood mononuclear cells were separated by centrifugation,cultured in plates and then got adherent monouclear cells.The number of the adherent monouclear cells in culture was measured by MTT at the point 4,24,48,72,96,120,144,168,192 h.The results showed that the number of the adherent monouclear cells was relatively stable and no significant difference between groups（P0.05） at culture in 72,96,120 h in vitro.Adherent cells cultured 72 h identificate by inverted microscope.Wright＇s staining indicated that those cells had the cell morphology of mononuclear macrophage cell.Acid phosphatase（positive rate 72.8%±0.5%）,non-specific esterase（positive rates 84.6%±0.4%） had the cell cytochemical characteristics.The immunocytochemistry for the the adherent cells found that they display typical functional membrane protein MHC Ⅱ.Phagocytosis（positive rates 87.5%±0.6%） was measured by Chinese ink.The result indicated the adherent cells retain the phagocytic activities.The results showed that adherent cells from buffalo blood culture in 72 h have mononuclear macrophage characteristics and could used to the future study at culture in 72,96,120 h in vitro.