树突状细胞与黑色素瘤细胞体外共培养体系对小鼠黑色素瘤形成的影响

Effect of co-culturing dendritic cells with melanoma cells on formation of melanoma in mice

目的探讨树突状细胞(DC)与黑色素瘤细胞(B16)体外共培养后对小鼠黑色素瘤形成的影响。方法提取小鼠骨髓细胞,分化DC,培养至第6天,将细胞分为4组,脂多糖(LPS)组、聚肌胞[Poly(IC)]组、Melan-A抗原肽(Melan-A)组分别加入DC佐剂LPS(终浓度100 ng/m L)、Poly(IC)(终浓度20 ng/m L)、Melan-A抗原肽(终浓度5μmol/L)进行处理,未处理组未进行干预。采用流式细胞术检测DC成熟状态。B16细胞与培养第6天的DC共培养2 d。取C57BL/6J小鼠皮下注射B16细胞(8只),DC+B16细胞(8只),LPS(8只)、poly(IC)(8只)、Melan-A抗原肽激活(8只)的DC+B16细胞,接种B16细胞1周再接种DC+B16细胞(5只),对照组(3只)不接种细胞。于接种第14天取部分小鼠处死,取脾脏,镜下观察其组织形态;接种第28天测算肿瘤体积。结果 LPS组、Poly(IC)组、Melan-A组成熟DC多于未处理组。接种DC+B16细胞的小鼠未见肿瘤生长;接种第28天,与接种B16细胞的小鼠比较,接种LPS、poly(IC)、Melan-A抗原肽激活的DC+B16细胞及接种B16细胞1周再接种DC+B16细胞的小鼠肿瘤体积减小(P均<0.05)。与对照组比较,第14天接种DC+B16细胞小鼠脾脏滤泡结构无明显变化,而接种B16细胞及LPS、poly(IC)、Melan-A抗原肽激活的DC+B16细胞小鼠,脾脏滤泡结构增加。结论DC与B16共培养可抑制小鼠黑色素瘤的形成。

Objective To investigate the effect of co-culturing dendritic cells（ DC） with melanoma cells（ B16） on growth of melanoma in mice. Methods We extracted the bone marrow cells. Mouse DC were cultured for 6 days and then were divided into 4 groups： lipopolysaccharide（ LPS） group,Poly（ IC） group,and Melan-A group and the control group.Cells in the LPS group,Poly（ IC） group,Melan-A group were incubated with LPS（ LPS with final concentration of 100 ng / m L）,poly-inosinic-cytidylic acid [Poly（ IC） with final concentration of 20 ng / m L] and Melan-A peptide（ with final concentration of 5 μmol / L）,respectively. The control group was not treated. Flow cytometry was used to study the maturation and activation state of DC. B16-F10（ B16） melanoma cells were co-cultured with DC for 2 days and then were used for inoculation of mice. For melanoma model,C57 BL /6J mice were inoculated subcutaneously. Some mice were killed at 14 days after inoculation and their spleens were taken to study the structural changes by histochemistry. Tumor size was measured at 28 days after inoculation. Results More mature DC were produced after LPS,Poly（ IC） or Melan-A peptide treatment as compared with that of the control group（ P 0. 05）. There was no tumor growth after DC ＋ B16 cell inoculation.Compared with the mice inoculated with B16 cells only,the tumor volume was smaller in the mice inoculated with LPS,Poly（ IC） or Melan-A peptide-activated DC ＋ B16 cells or the mice inoculated with B16 cells one week later followed by another inoculation of DC ＋ B16 cells（ all P 0. 05）. Compared with the control group,there was no structural change of the spleen follicles in the mice inoculated with DC ＋ B16,but the number of spleen follicles was increased in the mice inoculated with B16 or with LPS,Poly（ IC）,or Melan-A peptide-activated DC ＋ B16 cells. Conclusion DC co-cultured with B16 can inhibit the growth of melanoma in mice.