人原发性小肠恶性黑色素瘤裸鼠原位移植高转移模型的建立

Establishment of a highly-metastatic model of human primary melanoma of the small intestine orthotopically transplanted in the small intestine of nude mice

目的建立人原发性小肠恶性黑色素瘤裸小鼠原位移植高转移模型。方法将手术切除的人原发性小肠恶性黑色素瘤原发灶和肝转移灶新鲜瘤组织块分别植入裸鼠小肠黏膜层内，观察原位移植的成瘤率、移植瘤的侵袭性和转移率，并进行形态学、流式细胞分析和染色体核型分析。结果人小肠恶性黑色素瘤原发灶和肝转移灶新鲜组织均移植成功，建成人原发性小肠（原发灶）恶性黑色素瘤裸鼠原位移植高转移模型（HSIM-0602）和人原发性小肠（肝转移灶）恶性黑色素瘤裸鼠原位移植肝转移模型（HSIM-0603）。HSIM-0602和HSIM-0603模型分别传至21代和23代，共移植裸鼠227只，其肿瘤移植生长率和液氮冻存复苏成活率均为100％。HSIM-0602模型肝转移率为65．7％，肺转移率为84．8％，淋巴结转移率为63.8％。HSIM-0603模型肝转移率为100％，肺转移率为46．7％，淋巴结转移率为71．3％。移植瘤组织病理学为小肠高度恶性黑色素瘤。免疫组织化学显示，S-100蛋白和HMB-45均为阳性表达。电镜下，瘤细胞浆内可见大量的黑色素小体，也可见黑色素复合体。HSIM-0602模型移植瘤细胞DNA指数为1．59±0．07，HSIM-0603模型移植瘤细胞DNA指数为1．71±0．12，均为异倍体。染色体核型分析显示，HSIM-0602模型移植瘤细胞染色体数为55～57条，HSIM-0603模型移植瘤细胞染色体数为57～59条。结论HSIM-0602和HSIM-0603模型是成功的人原发性小肠恶性黑色素瘤裸鼠原位移植自发性高转移模型，完整地模拟了人原发性小肠恶性黑色素瘤患者的自然临床病理过程，为研究原发性小肠恶性黑色素瘤转移生物学和抗转移治疗提供了理想的动物模型。

Objective To provide an useful animal model for exploring metastatic biology and anti- metastatic therapy of primary malignant melanoma of the small intestine. Methods A 49-year old male patient with malignant melanoma was treated by surgery, and the primary tumor in the small intestine and a metastatic tumor in the liver were removed. The diagnosis of malignant melanoma was confirmed by histopathology. Fresh melanoma tissue fragments taken from the primary intestinal tumor and hepatic metastatic tumor were orthotopically implanted into the mucosal layer of small intestine in nude mice, respectively. The tumor growth rate, invasion and metastasis of the transplanted tumors were observed. Light and electron microscopy, immunophenotype analysis, flow cytometry and karyotype analysis were carried out. Results Fragments of the primary and liver metastatic malignant melanoma were successfully implanted in nude mice. After continuous passages in nude mice, an highly-metastatic model of human primary malignant melanoma of the small intestine （from the primary ＇lesion） in nude mice （termed HSIM- 0602） and a liver metastatic model of human primary malignant melanoma of the small intestine （ originally from the liver metastatic lesion ） in nude mice （termed HSIM-0603 ） were successfully estabIished. Histological examination of the transplanted tumors revealed a high-grade melanoma of the small intestine. Immunohistochemical stainings of S-100 protein and HMB45 were positive. Many scattered melanosomes and melanin complex were seen in the cytoplasm of tumor cells. Chromosomal modal number was between 55 and 59. DNA index （DI） was 1.59-1.71, representing a heteroploid. The HSIM-0602 and HSIM-0603 tumor models had been maintained for 21 and 23 passages in nude mice, respectively. 227 nude mice were used for transplantation. Both the growth rate after transplantation and resuscitation rate from liquid nitrogen cryopreservation were 100%. The HSIM-0602 model exhibited 84. 8% lung metastasis, 65.7% liver metastasis and 63.8% lymph node metastasis. However, HSIM-0603 displayed 100% liver metastasis, 46. 7% lung metastasis and 71.3% lymph node metastasis. The transplanted tumors actively and invasively grew in the small intestine of nude mice and showed hematogenous and lymphatic metastases. Conclusion To our knowledge it is the first time that two strains of spontaneous highly-metastatic nude-mouse model of human primary malignant melanoma of the small intestine have been successfully established in our department. The models are very closely mimic the natural clinicopathologic course of primary small intestinal melanoma in humans and provide ideal animal models for the researches on metastasis biology and anti-metastatic experimental therapy of malignant melanoma of the small intestine.