应用肝移植供体脾淋巴细胞预防受体鼠肝癌复发的研究

目的 探讨肝癌肝移植后应用活化供体脾脏来源的淋巴细胞,对受体实施过继性免疫治疗以预防肝癌复发的可行性.方法 以培养黏附法体外分离培养Wistar大鼠骨髓树突状细胞(DCs)前体细胞,经诱导后与Wistar大鼠肝癌细胞CBRH-7919裂解物抗原共同孵育,形成负载癌抗原的DCs.以此DCs分别诱导受体(Wistar大鼠)、供体(SD大鼠)来源的脾淋巴细胞作为两个实验组C(受体活化组)、D(供体活化组),以未经抗原诱导的受、供体脾淋巴细胞作为对照组A(受体对照组)、B(供体对照组).对比活化、非活化以及供体来源、受体来源的脾淋巴细胞在体外对受体肿瘤细胞的杀伤活性.以上述不同来源和处理的脾淋巴细胞对SD→Wistar大鼠肝移植后肿瘤复发模型行过继性免疫治疗,以盐水治疗作为对照组E,每组6只大鼠,观察各组免疫治疗对于肿瘤浸润淋巴细胞、受体肝脏成瘤率和供肝的排斥反应的影响.结果 DCs诱导脾细胞过程中,培养上清液γ干扰素(IFN-γ)分泌C、D组较A、B组有明显的升高.C、D组对肝癌细胞的杀伤活性较A、B组显著增强,D组较C组显著增强.D组脾细胞回输体内后,在移植后大鼠体内观察到了肿瘤浸润淋巴细胞增多,肿瘤组织坏死和成瘤率下降.免疫治疗前后供肝未见严重排斥反应发生.结论 活化的供体脾细胞较受体脾细胞有更强的肿瘤杀伤效果.应用供体脾淋巴细胞对肝移植受体进行过继性免疫治疗可以在不增加对移植肝排斥反应的同时,为预防肝癌肝移植术后复发、延长生存提供一种可能的方法.

Prevention of post-transplantation recurrence of hepatocellular carcinoma in rats with donor spleen lymphocytes infusion

Objective To explore the feasibility of using activated donor spleen lymphocytes to prevent liver cancer recurrence after liver transplantation.Methods The precursors of dendritic cells (DCs) were isolated from bone marrow of Wistar rats,and were then cultured and pulsed with CBRH7919 tumor cell antigens prepared with freeze-thawed tumor cell lysates. Spleen cells from donor (SD)/recipient (Wistar) rats were cocultured with these DCs respectively to induce CTL and to serve as the two experimental groups,C and D.Unactivited blank donor/recipient spleen cells were maintained to survive using low dose of rrIL-2.These served as the two control groups,A and B.Supernatant IFN-γ release were determined by ELISA,and the cytotoxicity of CTL was assayed by LDH release test.A post-transplantation HCC recurrence rat model was randomly assigned into five groups,6 rats each.Four groups were treated with adoptive lymphocyte infusion which came from the corresponding groups in vitro,and another group E was a saline infusion group to serve as a blank control.Tumorgenecity,tumor infiltrating-lymphocyte,and liver graft rejection were compared between the individual groups.Results The supernatant IFN-γ release level during DCs activation in group C and D were much higher than group A and B.Spleen cells in group C and D manifested much higher cytotoxicity to tumor cells than group A and B.The cytotoxicity of group D was significantly higher than group C.Aggregation of lymphocytes was detected within and near the tumor tissue with necrosis of tumor cells,and a decrease in tumorgenecity was observed in group D but without any evidence of liver graft rejection.Conclusion Activated donor spleen cells were more effective in preventing liver cancer recurrence after liver transplantation when compared with recipient spleen cells,and there was no impairment to the liver graft.