高效分离培养肿瘤浸润淋巴细胞方法的建立

Establishment of an Effective Way for Separating and Culturing Tumor Infiltrating Lymphocytes

提高肿瘤浸润淋巴细胞（ＴＩＬ）的增殖能力和杀伤活性，是其临床应用前需解决的关键问题。为此，作者对ＴＩＬ的分离和培养条件进行了探讨，建立了以机械分散法，０．０５％胶原酶和０．００３％ＤＮＡ酶室温消化６小时加冷消化１８小时的酶消化法和７５％与１００％Ｆｉｃｏｌｌ非连续密度梯度离心法相结合的分离纯化ＴＩＬ方法。分离出的ＴＩＬ在体外条件培养基中多数能扩增到１０９以上的应用标准，ＮＫ、ＬＡＫ活性分别可达５８．３±１１．７％和４８．６±１０．６％，其中发挥主要作用的是ＣＤ８Ｔ细胞。因此，本法不失为一种有效的分离培养ＴＩＬ的方法。它的建立为应用ＴＩＬ治疗恶性肿瘤奠定了良好的实验基础。

The key to preparing TILs for clinicalapplication is to enhance its capacity of proliferationand cvtotoxicity,For this purpose we have rnade astudy of methodology and established an effective wayconsisting of 3 processes to separate and culture TILs.It included:(1) mechanical splitting; (2) digestingwith 0. 05%collagenase Ⅱ and 0. 003%DNAase Ⅰmixture in room temperature for 6 hrs and 18hrs incool , and(3) centrifuging with 75%and 100%Ficollnon-continuous grade density,The separated TILswere suspended in conditional culture medium for pro-liferation. Most of them reached the amount of 109 atthe end of culture,which was essential for clinicaltherapy. Their NK and LAKactivities were 56.3±11.7%and 48.6%±10.6%respectively,in which theCD5 T cells played an important role,The resultssuggest this a perfect way for separating and culturingTILs.