PHA—CD3AK细胞的体外诱导及其生物学特性初步研究

目的：研究PHA—CD3AK细胞的体外诱导方法及其生物学特性，并与LAK细胞进行比较。方法：分离人外周血单个核细胞（PBMC），采用植物血凝素（PHA）、单克隆抗体（anti—CD3McAb）和基因重组人白细胞介素2（rhIL-2）、共同诱导制备PHA—CD3AK细胞；应用流式法分析PHA—CD3AK细胞的免疫表型、乳酸脱氢酶法（LDH）检测PHA—CD3AK细胞的杀伤活性，并观察其形态；吉姆萨染色法观察其核型。结果：微量anti—CD3McAb（0．05μg／m1）辅以少量rhIL～2（300U／ml）和PHA（100μg／m1）共同培养，即能诱导和大量扩增PHA—CD3AK细胞，其扩增倍数显著高于LAK细胞，维持高扩增的时间也远较LAK细胞持久；当效靶细胞比为80：1时，PHA—CD3 AK细胞对体外肿瘤细胞（K562）杀伤的百分率为56．5％；免疫表型检测PHA—CD3 AK细胞中CD3^＋、CD4^＋、Cd8^＋细胞的比率分别为（86．5±5．89）％、（38．20±5．27）％、（42．63±3．50）％；核型为正常二倍体，染色体数目为46条。结论：PHA—CD3 AK细胞是以CD3^＋、CD4^＋、CD8^＋细胞为主的异质细胞群，并具有淋巴母细胞样特征，PHA—CD3AK细胞为正常二倍体细胞。PHA—CD3AK细胞是易于体外诱导、扩增能力强，体外存活时间长、杀瘤活性高的一种具有广阔应用前景的肿瘤过继免疫效应细胞。

A preliminary experimental study on inducement in vitro and biological features of PHA- CD3AK cells

Objective： To investigate the inducement method in vitro and the preliminary biological features of PHA- CD3AK cells in comparison with LAK cells. Methos： We adopted anti -CD3 monoclonal antibody （anti - CD3McAb） ,recombinant human interleukin- 2 （rhIL- 2 ） and phytohemaglutinin （PHA） to induce jointly human peripheral blood mononucleus cells （PBMCs）. FCM, LDH,and Giemsa＇s staining were used to assay the biological features of PHA - CD3AK cells, such as phenotype, killing activity and nuclear type. Results： PHA - CD3AK cells could be induced and proliferated by tracing anti -CD3mAb （0. 05μg/ml） plus rhIL- 2（300U/ml） and PHA （100μg/ml）. The proliferation multiples and time in vitro of high level proliferation multiple of PHA- CD3AK cells was longer than that of LAK cells. When the proportion between effector cells and target cells was 80 ： 1, PHA - CD3AK cells could kill 56.5% of K562 cells. The cell phenotype ratio with CD3^＋ , CD4^＋ , and CD8^＋ of PHA - CD3AK cells were（86.5±5.89）%, （33.20±5.27）% ,and （42.63 ±3.50）% ,respectively. Karyotype of PHA - CD3AK cell was a normal diploid. The chromosome number was 46. Conclusion： PHA - CD3AK cells are a group of heterogeneous cells with lymphoblast -like features. PHA -CD3 AK cells are normal liploid cells, and easily not only to be induced and cloned in vitro, but also to induce long time in vitro and to enhance killing activity. It might be a promising tumor immunotherapy cell.