摘要
肿瘤组织经机械剪切和酶消化分离肿瘤浸润淋巴细胞(TIL)。TIL在含1000U/ml重组人白细胞介素-2(rhIL-2)的培养液中培养6d后,植入含1000U/ml rhIL-2的软琼脂半固体培养基中培养,7d后将形成的克隆转移到含rhIL-2的培养液中培养。^(51)Cr释放法测定结果表明,约30％的克隆对NK敏感的K562细胞和对NK不敏感的H7402肝癌细胞有细胞毒性,为具有杀瘤活性的TIL杀伤克隆(TIL-K)。60％以上TIL-K克隆在含IL-2的培养液中可持续地增殖2～3个月,其细胞数可扩增至10~8～10~9,并始终保持对肿瘤细胞的细胞毒性。TIL-K克隆的表型为CD3^+、CD4^-、CD8^+、CD16^-,有T细胞抗原受体β链基因的表达,说明其属T细胞系统。采用半固体-液体两步培养法可获取大量高纯度具有广谱杀瘤活性的TIL,本研究有助于TIL的深入研究和临床应用。
A method for cloning and large-scale expansion of human tumor-infiltrating lymphocytes(TIL) with wide spectrum of cytotoxicity against tumor cells was described. TILs were obtained by combination of mechanical release and enzymatic disaggregation from human solid tumors. After incubation with 1 000 U /ml rhIL-2 for 6 days, TILs were plated in RPMI-1640 medium containing 0.3% agar and 1 000 U/ml rhIL-2 and incubated for 7 days. The clones formed in the agar culture were transferred into 96-well flat bottom culture plates and then to 24-well culture plates for expansion. About 30% clones showed cytotoxicity against either NK-sensitive human myeloid leukemia cell line K562 or NK-resistant human hepatoma cell line H7402 in a 4 hour 51Cr release assay. More than 60% clones could expand up to 108cells with good maintenance of cytotoxicity over a period of 2 to 3 months in liquid culture containing 5% human AB serum and 1 000 U/ml rhIL-2. Cell surface phenotyping by flow cytometry showed that the cloned TIL expressed CD3 and CD8 antigens. Northern blot analysis demonstrated T-cell receptor β chain gene expression in these clones. Consequently, cloned TIL belongs to a subset of T cell lineage.
出处
《中国应用生理学杂志》
CAS
CSCD
1992年第3期209-213,共5页
Chinese Journal of Applied Physiology
关键词
肿瘤
浸润
淋巴细胞
克隆化
tumor-infiltrating lymphocytes
interleukin-2
T cell clone
cytotoxicity
