半相合基因骨髓细胞移植治疗小鼠肾癌的实验研究

目的探讨半相合基因骨髓细胞移植的嵌合体水平对移植物抗肿瘤效应（Graft-Versus-Tumor effect,GVT）、移植物抗宿主病（Graft-Versus-Host Disease,GVHD）的影响及其机理。方法 25只8-12周龄BALB/C小鼠随机分为3组：混合型嵌合体组（A组10只）、完全型嵌合体组（B组10只）、对照组（C组5只）。A、B组小鼠行直线加速器全身照射,剂量分别为4Gy、8Gy,照射后4-6h内经尾静脉注射入CB6F1代小鼠的骨髓细胞（5×106/只）,C组无处理。A、B、C三组均给予Renca肾癌细胞腋窝下注射,每只2.6×106个肾癌细胞。观察肿瘤生长速率,流式细胞仪测定A、B组小鼠移植后不同时间的嵌合体水平。荷瘤32d时断颈处死小鼠,剥离肿瘤称重;留取血浆,ELISA（Enzyme-Linked Immunosorbent Assay）方法测定血浆白细胞介素-2（Interleukin-2,IL-2）,干扰素-γ（Interferon Gamma,IFN-γ）浓度;取肿瘤组织做成石蜡切片后行HE染色,测定肿瘤坏死面积比;留取小鼠的皮肤、肝脏及小肠用于制备石蜡切片,并行HE染色,观察GVHD。结果血液白细胞中供者源细胞比例：A组移植2周后形成混合嵌合体并达高峰,3-4周时下降至10%以下。B组小鼠移植后14d形成完全供者型嵌合体（供体细胞〉90%）,28d后仍达90%以上。与C组和A组相比,B组的肿瘤生长速度明显减慢,肿瘤重量显著减小（P〈0.01）,抑瘤率高达52%,IL-2、IFN-γ水平显著升高（P〈0.01）,肿瘤组织坏死面积比显著升高（P〈0.01）。病理学检查未见肝、小肠及皮肤出现GVHD明显变化。结论完全型嵌合体水平的半相合基因骨髓细胞移植能够激发小鼠系统性的抗肿瘤免疫反应,抑制肿瘤生长,其机理可能是肿瘤坏死增加。

Treatmen of kidney cancer in mice with haploidentical bone maraaow cell transplantation

Objective To study the effect of chimerism level on graft-versus-tumor（GVT） and graft-versus-host disease（GVHD） after haploidentical bone marrow cell transplantation and its mechanism.Methods Twenty-five 8-12 weeks old BALB/c mice were randomly divided control group（group C,n=5）,mixed chimerism group（group A,n=10）,full donor chimerism group（group B,n=10）.Mice in group C were only inoculated with Renca cells（2.6×106）.Mice in group A were irradiated at the dose of 4Gy,infused with bone marrow cells（5×106） from CB6F1 mice on day 1,and inoculated with Renca cells（2.6×106） on day 8.Mice in group B were were irradiated at the dose of 8Gy,infused with bone marrow cells（5×106） from CB6F1 mice on day 1,and inoculated with Renca cells（2.6×106） on day 8.Tumor growth was monitored every 4 days.Chimerism levels in groups A and B were measured with a FACScan cytometer on days 14,21,and 28 after haploidentical bone marrow cell transplantation.Tumor-bearing mice were killed 32 days by decapitation with the tumor isolated and weighed.Interleukin-2（IL-2） and interferon gamma（IFN-γ） levels in plasm were measured by ELISA.Liver,skin,and intestine,were cut into sections which were stained with HE to observe GVHD,Tumor tissues to observe area ratio of tumor necrosis.Results The mixed chimera level reached its peak in groups A and B 2 weeks after haploidentical bone marrow cell transplantation,and decreased to less than 10% in group A 3-4 weeks after haploidentical bone marrow cell transplantation but remained over 90% in group B 4 weeks after haploidentical bone marrow cell transplantation.The tumor growth was significantly slower and the tumor weight was notably less in the group B than in groups A and C（P0.01）.The tumor suppressing rate was 52% for group B.The levels of IL-2,IFN-γ and the area ratio of tumor necrosis were significantly higher in group B than in groups A and C（P0.01）.No significant change occurred in GVHD of liver,intestine and skin.Conclusion Haploidentical allogeneic bone marrow cell transplantation at full donor chimerism level may stimulate the systemic GVT immunity and inhibit tumor growth in mice by increasing tumor necrosis.