微波治疗后坏死肿瘤组织致敏的树突状细胞介导肿瘤免疫治疗

目的 探讨微波凝固治疗（MCT）后坏死肿瘤组织能否致敏树突状细胞（DC）,致敏DC是否具有特异性抗肿瘤作用。方法 对BALB／c小鼠皮下肿瘤结节进行MCT治疗后,取出坏死的组织碎块研磨过滤,滤液与体外培养的小鼠骨髓来源的DC共同孵育获得致敏DC。观察DC的形态并检测其表型。3↑H-TdR掺人法测定DC刺激T细胞反应性增殖和^51Cr释放测定法检测DC诱导特异性CTL的细胞毒作用,以及体内对小鼠皮下肿瘤的抑制情况。最后再检测致敏DC治疗后的荷瘤鼠脾细胞悬液提取的T细胞在体内外的杀伤特异性。结果 与MCT治疗后坏死CT-26肿瘤组织滤液共同培养的DC具有典型的致敏DC形态特征和表型。致敏DC和未致敏DC诱导T细胞增殖强度在不同DC／T比下分别为：DC／T比1510时,80±10 vs 10±5;DC／T比1：20时,58±7 vs 9±4,两两比较差异均有统计学意义（均P〈0．01）。致敏DC和未致敏DC诱导的CTL在不同E／T比下对CT-26肿瘤细胞杀伤率分别为：E／T比10时,13．6±2．5 vs 1．1±0．4;E／T比20时,27．5±4．4 vs 1．4±0．4;E／T比50时,51．2±8．1 vs 1．4±0．5,两两比较差异均有统计学意义（P〈0．01）。二者诱导的CTL在不同E／T比下对MIP／NIM淋巴瘤细胞杀伤率分别为：E／T比10时,2．61±0．64 vs 0．87 ±0．15;E／T比20时,5．22±0．65VS2．18±0．41;E／T比50时,6．09 ± 0．83 vs 3．91±0．51,两两比较差异均无统计学意义（均P〉0．01）。体内实验,皮下移植瘤大小在致敏DC组、未致敏DC组和生理盐水对照组间随小鼠存活时间延长越来越明显,至40d时3组肿瘤平均重量分别为4．5g±1．1g,6．9g±1．6g,9．0g±1．5g,组间差异有统计学意义（均P〈0．01）。致敏DC治疗后的荷瘤鼠脾细胞悬液内T细胞在体内外也均显示了对CT-26肿瘤的杀伤作用。结论 作为一种全细胞抗原,MCT治疗后的肿瘤坏死组织能够致敏DC,致敏DC在体内外均具有明显的特异性抗小鼠CT-26肿瘤作用。

Immunotherapy against tumor with dendritic cell sensitized by necrotic tumor tissue after microwave coagulation therapy

OBJECTIVE: To evaluate if the necrotic tumor tissue treated with microwave coagulation therapy (MCT) can sensitize dendritic cell (DCs), and if the sensitized DCs have the specific inhibit effect on tumor cell. METHODS: CT-26 tumor nodules planted percutaneously in BALB/c mice were treated with MCT. The treated tumor tissue was levigated and filtrates and the filtrate obtained was incubated with the DCs from marrow of the BALB/c mice to get sensitized DCs. The shape of the DCs was observed by the microscopy and the electron microscopy respectively and the immune phenotype was detected by flow cytometer. The ability of the DCs to induce proliferation of T cells was measured by (3)H-TdR incorporation assay. The cytotoxicity of CTL activated by the DCs was measured by (51)Cr release assay. The inhibition of the DCs on the growth of CT-26 tumor in the mice was observed. RESULTS: The DCs cultured with the filter from necrotic tissue of CT-26 tumor had the typical shape and immune phenotype of the sensitized DCs. The intensity of the sensitized DCs and the naive DC to stimulate T lymphocyte proliferation at the DC/T of 1:10 was 80 +/- 10 vs 10 +/- 5 (P < 0.01); and at the DC/T of 1:20 was 58 +/- 7 vs 9 +/- 3 (P < 0.01). The killing effect of specific CTLs on CT-26 cancer cells in the sensitized DCs and the naive DCs group were 13.6 +/- 2.5 vs 1.1 +/- 0.4 (P < 0.01) at the E/T of 10:1, 27.5 +/- 4.4 vs 1.4 +/- 0.4 (P < 0.01) at E/T 20:1, and 51.2 +/- 8.1 vs 1.4 +/- 0.5 (P < 0.01) at the E/T of 50:1 respectively. The specific CTL's killing effect on MIP/ND4 lymphoma cells in the sensitized DCs and the naive DCs group were 2.61 +/- 0.64 vs 0.87 +/- 0.15 (P > 0.01) at the E/T of 10:1, 5.22 +/- 0.65 vs 2.18 +/- 0.41 (P > 0.01)at the E/T of 20:1, and 6.09 +/- 0.83 vs 3.91 +/- 0.51 (P > 0.01) at the E/T of 50:1 respectively. The difference of the volumes of the tumor implanted percutaneously in the BALB/c mice became more and more obviously with the prolongation of mice survival times in the sensitized DC group, the naive DC group and the control group. The weight of the tumor at the fortieth day were 4.5 g +/- 1.1 g, 6.9 g +/- 1.6 g, 9.0 g +/- 1.5 g in the three groups respectively (P < 0.01). Finally the special killing effect of the T cells which extracted from the spleen of the mice whose tumor had been treated by the sensitized DCs in vivo and in vitro were observed. CONCLUSION: As an whole cell antigen, the necrotic CT-26 tumor tissue treated with MCT can sensitize DCs, and the sensitized DCs have the specific inhibitory effect on CT-26 tumor cells both in vivo and in vitro.