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

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。

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

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.