双阴性T细胞促进Babl／C小鼠B16F10种植瘤生长的机制研究

摘要

目的：探讨双阴性 T 细胞（DNT）促进 B16F10黑色素瘤生长的可能机制。方法：以 B16F10－luc －G5细胞系接种 Babl／C 小鼠建立种植瘤模型，IVIS 监测生长动态。流式细胞仪检测外周血中 T 淋巴细胞的比例。流式分选正常小鼠的 DNT 及 CD4＋细胞。在体内，以从正常小鼠体内获得的外源性 DNT 干预种植瘤，IVIS 监测肿瘤的生长动态，病理切片分析肿瘤组织病变。在体外，建立混合培养系统，观察 DNT 对淋巴细胞的杀伤功能及分化的影响，以及对 CD4＋细胞杀伤功能的影响。结果：荷瘤鼠 DNT 含量高于正常鼠（P ＜0．05）；CD4＋细胞含量与肿瘤大小高度负相关（R2＝－0．99）。外源性 DNT 干预后种植瘤生长速度加快（P ＜0．05），病理显示肿瘤组织中淋巴细胞浸润较对照组少。体外实验中，DNT 抑制总淋巴细胞的杀伤功能；DNT细胞直接抑制 CD4＋细胞杀伤功能。结论：DNT 促进 B16F10种植瘤生长主要机制之一可能为 DNT 直接抑制CD4＋细胞杀伤功能，从而减少了 CD4＋细胞对肿瘤的杀伤作用。%Objective:To study the possible mechanism that DNT promotes the growth of B16F10 melanoma. Methods:Tumor -bearing mice were established by subcutaneous inoculation of B16F10 -luc -G5 cells.Flow cy-tometry was used to detect the composition changes of lymphocyte in peripheral blood from tumor -bearing mice and normal mice.DNT and CD4 + cells were separated by Aria Cell Sorter.DNT was injected in tumor -bearing mice and the effect on tumor growth was observed by IVIS in vivo and pathological changes.Mixture culture system (MLC)in vitro were established to reveal the mechanism of DNT negative -effect on tumor growth:DNT influence lymphocytes, killing function and differentiation,respectively were monitored by IVIS and flow cytometry after MLC.Effects of DNT direct influence on lymphoid killing function of CD4 + cells were detected by IVIS after MLC.Results:Luminescence between DNT group and tumor control group:there was no significant difference (P >0.05)of mean photon count [photon/(cm2 .ser.s)]on day 0,but significant difference (P <0.05)on day 3 ~9.Differentiation of T cells in pe-ripheral blood:In tumor control group,CD4 + increased first to peak on day 5 and then decreased,with a strong nega-tive correlation to tumor cells (bioluminescence)(R2 =-0.99).Contrast to normal mice,in tumor -bearing mice, DNT increased (P <0.05)but CD4 +CD25 +T decreased (P <0.05),there was strong correlation between two cells (R2 =-0.99).Also a strong correlation between number of CD8 + cells and DNT cells (R2 =-0.97)was also demonstrated in tumor control group.Pathology analysis of tumor:With less lymphocytes infiltration,the tumor was bigger in DNT effect group than in tumor control group.But in following day 5 ~9,the differences decreased gradual-ly.DNT influenced lymphoid killing function:)DNT influenced lymphoid killing function of total lymphocytes:24 ~72h,there was significant difference (P <0.05)of mean photon count and contents of total lymphocytes between any two groups of three.DNT also influenced lymphoid killing function of CD4 + cells.Conclusion:DNT down -regulate the immune responses and these may occur through the direct inhibition on CD4 + cells to promote the growth of mela-noma.