食管癌引流淋巴结细胞体外培养及其抑瘤作用的观察

The cell growth and cytotoxic activity to antilogous tumor cells of esophageal tumor draining lymph node cells

目的观察食管癌引流淋巴结(TDLN)细胞体外培养结果及其抑瘤作用。方法术中切取食管癌患者的TDLN进行培养,分为3组:A组,培养基中添加IL-2;B组,添加IL-2+IL-4+粒—巨噬细胞集落刺激因子(GM-CSF);C组,添加IL-2+IL-4+GM-CSF+自身食管癌细胞抗原。于培养第1、7、14、21天行细胞计数;采用流式细胞技术测定TDLN细胞的CD3+、CD4+、CD8+、CD5+6、CD8+3;MTT法测定各组TDLN细胞和淋巴因子激活杀伤(LAK)细胞对自体瘤细胞的抑瘤率。结果每1.0 g淋巴结组织培养第7、14、21天收获细胞数三组间差异不显著;A组第14天细胞明显多于第7天和第21天(P<0.01),第7天和第21天之间无明显差异;B组、C组结果类似。三组间CD4+、CD8+、CD8+3细胞数相比,P<0.05。未经培养的TDLN细胞、LAK细胞和各培养组TDLN细胞对自体肿瘤细胞的杀伤率有显著差异(P<0.01)。结论TDLN细胞通过培养可以获得大量成熟树突状细胞和T细胞;培养基中添加GM-CSF和IL-4的TDLN培养后对自身肿瘤细胞的杀伤率最高。

Objectives To investigate the cell growth and the cytotoxie activity to antilogous tumor cells of esophageal tumor draining lymph node （TDLN） cells. Methods Lymph nodes from esophageal tumor drainage region were removed during operation. TDLN eell was suspended in RPMI 1640 medium containing IL-2 （Group A） , IL-2 ＋ IL-4 ＋ GM-CSF （ Group B）, IL-2 ＋ IL-4 ＋ GM-CSF ＋ Antilogous tumor antigen （ Group C） in etdture flask. The cell number was eounted at 1st d, 7th d, 14th d, and 21st d. The ratio ofCD3＋, CD8＋, CD4＋, CD56＋ and CD83＋ cells were analyzed by flow cytom- etry （FCM） ; The TDLN cells not cultured（ Control Group） ,the LAK ceils, the TDLN ceils of group A, B, C were com- pared for their cytotoxic activity to antilogous tumor cells using mononuclear cell direct cytotoxicity assay （ MTT）. Results The difference of the numbers of cells that obtained from 1.0 g of lymphatic tissue between the three groups was not significant. At 14th d, the number of cells was higher than at 7th d and 21st d （ P 〈0.01 ）. The ratios of CD4＋ ,CD8＋ ,CD83＋ between the three groups are significantly different （P 〈 0.05 ）. There were great difference between the four groups （P 〈 0. 01） on the cytotoxic ratio to antilogous tumor cells. Conclusion It is possible to obtain substantial matured DC and T cells in vitro culture of TDLN cells. The cells have strong capacity to kill antilogous tumor cells.