@article{oai:u-ryukyu.repo.nii.ac.jp:02002042, author = {小倉, 剛 and 杉本, 哲朗 and 野口, 規子 and 川島, 由次 and 寺尾, 恵治 and Ogura, Go and Sugimoto, Tetsuro and Noguchi, Noriko and Kawashima, Yoshitsugu and Terao, Keiji}, issue = {3}, journal = {霊長類研究, Primate research}, note = {サル類の末梢血リンパ球サブセットを解析する際のリンパ球分離法の妥当性を明らかにする目的で,Ficollを用いた比重遠心法,染色前後に溶血処理をおこなう2種類の全血法,dextran処理と比重遠心法との併用法の計4法を用いてアカゲザルのリンパ球分離を行い,末梢血リンパ球サブセットレベル,赤血球の残存率およびリンパ球回収率を比較した。得られた結果は以下のとおりである。\n1.4種の主要リンパ球サブセットレベルには比重遠心法と溶血後に染色する全血法Aとの間に差は認められなかった。このことから,ヒトで報告されている比重遠心操作の過程での特定のサブセットの消失は生じないと判断され,比重1.077の分離剤を用いた比重遠心法はアカゲザルに適応可能と考えられる。\n2.染色後に溶血処理を行う全血法BではLeu-lla抗体で検出されるCD16抗原陽性細胞がほとんど検出されなかった。原因として,染色時に血漿中のIgGによるFcγレセプターのブロックが生じた可能性が考えられ,Leu-lla抗体を用いる場合は,抗体反応前に血球を洗浄する必要がある。\n3.Dextran処理と比重遠心の併用法はリンパ球分画への赤血球の混在を効果的に防止するが,リンパ球の回収率は低下した。しかしながら,特定のサブセットが低下することがないことから,アカゲザルのリンパ球分離法としては有効な方法である。\n4.NH_4Clを基剤とする溶血剤を用いた溶血処理は,蒸留水による溶血処理に比べてリンパ球回収率が高く,安定して良好な溶血が行える処置と判断される。, Determination of peripheral lymphocyte subset levels becomes important method for analyzing immune function in nonhuman primates. However, It has been well-known that the separation of peripheral lymphocytes by density gradient centrifuge is relatively difficult in nonhuman primates, because the gravity of red blood cells (RBC) varies among individuals. The purpose of this paper is to compare the major lymphocyte subset levels, the recovery rate of lymphocyte and the contamination of RBC in lymphocyte fraction among four different methods of lymphocyte separation, Method-1: conventional density gradient centrifuge using Ficoll, Method-2: density gradient centrifuge after sedimentation of RBC using 5% dextran, Method-3: whole blood method A; hemolysis before staining lymphocyte, and Method-4: whole blood method B; hemolysis after staining lymphocytes. The lymphocyte recovery rate as well as RBC contamination were also compared between two different hemolysis methods, 1) hemolysis with distilled water and 2) hemolysis with NH_4Cl solution. The results obtained are as follows.\n1. There was no difference in lymphocyte subset levels between Method-1 and Method-3, indicating that loss of specific lymphocyte subset(s) did not occur during density gradient centrifuge in method-1. The density gradient centrifugal method using Ficoll (d=1.077) was able to seem to adapt for rhesus monkeys.\n2. CD16-positive lymphocytes could not be detected by method-4. It might be necessary to wash cells to remove IgG in sample before staining with anti-CD16, Leu-lla monoclonal antibody in the case of applying Method-4 to analyzing CD16-positive NK cells.\n3. Method-2 was effective to remove RBC from lymphocyte fraction, but the lymphocyte recovery rate was significantly lower as compared to Method-1.\n4. The lymphocyte recovery rate was significantly higher when contaminated RBC were removed by NH_4Cl solution as compared when RBC were removed by distilled water., 論文}, pages = {361--368}, title = {アカゲザルのリンパ球サブセット解析におけるリンパ球分離法の比較検討}, volume = {15} }