卵巢上皮性癌肿瘤浸润淋巴细胞分离、培养及生物学特性的研究

A study on the isolation,culture and biological characters of tumor infiltrating lymphocytes in ovarian epithelial carcinoma.

目的：分离培养卵巢上皮性癌肿瘤浸润淋巴细胞（ＴＩＬ），探讨其生物学特性。方法：用酶法或机械法消化肿瘤组织后，在有ＩＬ２、ＰＨＡ或ＯＫＴ３ 条件下培养。ＴＩＬ表型分析采用间接免疫荧光法，细胞毒活性测定采用ＭＴＴ检测法。结果：分离培养２０ 份卵巢癌ＴＩＬ，１８ 份ＴＩＬ全部活化，另２份污染，最大扩增５２３倍。初分离的卵巢癌ＴＩＬ处于功能抑制状态（抑制期５～１５ｄ），对Ｋ５６２ 、Ｒａｊｉ和自体瘤细胞的细胞毒活性均很低，经体外培养活化增殖后，细胞毒活性明显增加，以特异性杀伤自体瘤细胞的能力增加最为明显。表型分析表明，ＴＩＬ体外培养过程中，ＣＤ３ 、ＣＤ８、ＣＤ２５（ＩＬ２Ｒ）和ＨＬＡＤＲ的表达率均明显上升。结论：卵巢癌ＴＩＬ体外培养后特异性杀伤自体瘤细胞的能力明显增加，可用于生物学治疗。

Objectives:To isolate,culture and explore the biological characters of tumor infiltrating lymphocytes(TIL)in ovarian epithelial carcinoma(OEC).Methods:Tumors were digested enzymatically or mechanically,then cultured in the presence of IL 2,PHA and OKT3 mAb.The changes in phenotype and cytotoxicity of TIL in OEC were examined using indirect immunofluorescence method and MTT method separately.Results:Eighteen samples of TIL in OEC were activated (2 samples contaminated) and proliferated after supressive phase which lasted for 5-15 days.During supressive time the proliferative ability and cytotoxicity were poor.The most expanding time was 532.OKT3 mAb+IL-2 could induce higher expanding times of TIL.The cytotoxicity against K562,Raji and autologous tumor cells increased while TIL were cultured in vitro,and the increase of cytotoxicity against autologous tumor cells was most significant in TIL.Phenotypic analyses showed the expression of CD 3,CD 8,CD 25 and HLA DR increased in the process of cultivation,correlated to the cytotoxicity.Conclusion:TIL in OEC has strongly autologous tumor cytotoxicity and is potentially useful in clinical biological adoptive therapy.