한우 T 및 B 림프구 분리 동정

Separation and identification of T and B lymphocyte subpobul in Korean native cattle

The purpose of the present study was to evaluate and standardize the techniques for identification of bovine lymphocyte subpopulations. Mononuclear cells(MNCs) were obtained from peripheral blood, prescapular lymph node, thymus and spleen of Korean native cattle by density gradient centrifugation, and monocytes were depleted by phagocytosis of carbonyl iron particles. Various methods for separatino of lymphocyte subpopulations have been modified and adapted for use in isolating and identifying the subpopulations of monocyte depleted MNCs. Separation of T lymphocytes was achieveed best by nylon wool column(NWC) method whereas separation of B lymphocytes was obtained best by EA- and EAC-rosette using Korean native goat erythrocytes as target binding cells. The former yielded 94.1±1.6% T lymphocytes and the later gave 88.3±1.0% B lymphocytes as evaluated by direct and indirect immunofluorescent- or alkaline phosphatase- and immunoperoxidase-staining with 2-aminoethyl isothiouronium bromide(Aet)- and dexran(Dex) treated erythrocytes, both Aet and Dex not only enhanced rosette formation but also made it easy to enumerate rosettes by increasing numbers of erythrocytes attacked on lymphocytes. Using erythrocytes treated with 0.1M Aet and 8% Dex in combination, the maximum rosettes were shown, and the rates of rosetting cells were not changed duting 5 to 24 hours incubation at 4℃. This method detedted 56.6±6.8% of peripheral blood MNC as T lymphocytes in female, and 65.8±6.3% in male. These rosetting cells were confirmed as T lymphocytes by immunoperoxidase staining with monoclonal antibody, BLT₁ to bovine T lymphocytes.