人角质形成细胞与MV3人黑素瘤细胞体外构建人工皮肤黑素瘤组织模型

In vitro construction of an artificial skin model of melanoma with human keratinocytes and human MV3 melanoma cells

目的 用人角质形成细胞（KC）和MV3人黑素瘤细胞混合接种培养于去表皮的真皮（DED）,体外构建人工皮肤黑素瘤组织模型,探讨KC对黑素瘤侵袭性的影响.方法 用两步酶消化法处理小儿包皮,获得表皮细胞悬液,用KC无血清培养基培养并传代KC.用RPMI 1640培养基培养及传代MV3人黑素瘤细胞.分别取对数生长期KC、MV3人黑素瘤细胞制备细胞悬液.将KC、MV3人黑素瘤细胞悬液接种于DED表面（KC：MV3人黑素瘤细胞约为3：1）.采用液下培养和空气-液面培养相结合方式进行混合培养,2周后取出进行HE染色、S-100蛋白、HMB45及角蛋白免疫组化染色.结果 HE染色显示,MV3人黑素瘤细胞在DED表面形成条带状瘤团或瘤灶,KC间杂于瘤细胞间,未形成典型的表皮样结构;部分瘤细胞浸润到DED浅层,呈群聚性分布;瘤细胞侵入DED管腔内,呈环形附着于管壁上,部分瘤细胞穿过管壁进入周围真皮组织;在DED底面及侧面,瘤细胞呈单个细胞弥散状浸润分布.瘤灶S-100蛋白阳性表达,HMB45弱阳性表达,角蛋白阳性表达.结论 KC增强MV3人黑素瘤细胞在人工皮肤黑素瘤组织模型中的侵袭.

Objective To construct an artificial skin model of melanoma by mixed culture of human keratinocytes and MV3 melanoma cells on de-epidermized dermis （DED） in order to study the effect of keratinocytes on melanoma invasion. Methods Epidermal cell suspension was obtained by a two-step digestion method from the circumcised foreskin of a child, keratinocyte serum-free medium was applied to the culture and passage of keratinocytes. MV3 melanoma cells were cultured and passaged in RPMI 1640 medium. Log-phase keratinocytes and MV3 cells were mixed with a ratio of 3：1 and seeded onto the surface of DED followed by a liquid culture and air-liquid culture for a total of 2 weeks. Thereafter, the artificial tissue model was assessed by HE staining and immunohistochemical staining for S-100 protein, HM64S and keratin. Results HE staining showed that MV3 cells formed band-like tumor masses or foci on the surface of DED, with keratinocytes intermingling among the tumor cells, but no typical epidermis-like structure was observed. Some tumor cells infiltrated into the surface of DED and showed a cluster distribution; some tumor cells invaded the lumen of the DED, and attached to the luminal wall in a ring shape; some tumor cells penetrated through the wall into the surrounding dermal tissue. On the bottom and lateral side of DED, tumor cells were infiltrating dispersedly.The tumor loci stained positive for S-100 protein and keratin, and weakly positive for HMB45. Conclusion Keratinocytes enhance the invasion of MV3 melanoma cells into the skin tissue model of melanoma.