Immunohistological study for the invasion and metastasis of oral malignant melanoma cell line (SSM-1)

口腔恶性黑色素瘤细胞系（SSM-1）侵袭和转移的免疫组织学研究

• 批准号: 09672075
• 负责人: MIZUTANI Naritaka
• 金额: $ 1.98万
• 依托单位: Kanagawa Dental College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672075/
• 关键词: maligrant melanoma; cell culture; metastasis; invasion; MMP-2; MMP-9; E-Cadherin; 侵潤

The invasive and metastatic ability are most important characteristics of oral malignant melanoma. In order to elucidate the factors contributory to the expression of invasiveness and metastasis of oral malignant melanoma, I used high melanin productive cell line(SSM-1) with three-dimensional type I collagen gel matrix. This method is usefull to obtain the condition of iv vivo. The results were folloewd :1. SSM-1 was confluent after 2-3weeks.2. The samples were fixed with 10% formalin, and embedded in paraffin. Paraffin sections were stained with H-E.The shape of SSM-1 were spindle and polygonal.3. I tried two method for an in vitro invasion model. (1) mixed the gel of human fibroblast with the colony of SSM-1 (2)after incubated human fibroblast in the fibroblast-embedded collagen gel matrix, overlaid SSM-1 on the gel matrix.4. The samples were fixed with 4% paraformaldehyde for immunohistochemical stain of anti-human E-cadherin antibody. The sections were immunostained for cadherin by SAB method. SSM-1 and the biopsy of primary lesion were not stained. This result suggest that the decrease of E-cadherin expression indicated the decrease of cell-cell adhesion.5. MMP-2 was detected with RT-PCR.6. SSM-1 and the biopsy of primary lesion were immunostained for MMP-2 MMP9 by SAB method.Because of melanocyto, I could not detect MMP-2,9 in primary lesion. In SSM-1, MMP-9 was not stained, but MMP-2 was stained in the fibroblast too.These results suggest that fibroblast is well correlated with the invasive and metastatic ability.

侵袭转移能力是口腔恶性黑色素瘤最重要的特征。为了阐明口腔恶性黑色素瘤侵袭和转移表达的影响因素，我使用了具有三维I型胶原凝胶基质的高黑色素生产细胞系（SSM-1）。该方法可用于体内给药条件的研究。结果如下：1. SSM-1在2- 3周后融合.将样品用10%福尔马林固定，并包埋在石蜡中。石蜡切片进行H-E染色，SSM-1呈梭形和椭圆形.我尝试了两种方法建立体外侵袭模型。(1)将人成纤维细胞在包埋成纤维细胞的胶原凝胶基质中孵育后，将人成纤维细胞的凝胶与SSM-1的殖民地混合（2），将SSM-1覆盖在凝胶基质上。标本经4%多聚甲醛固定后进行抗人E-钙粘蛋白抗体免疫组化染色。采用SAB法对切片进行钙粘蛋白免疫染色。SSM-1和原发灶活检组织均未染色。提示E-cadherin表达的降低提示细胞间粘附能力的降低. RT-PCR检测MMP-2。采用免疫组化SAB法对SSM-1和原发灶活检标本进行MMP-2、MMP-9的免疫组化染色，由于原发灶中存在黑素细胞，故不能检测到MMP-2、MMP-9。在SSM-1中，MMP-9不表达，而MMP-2在成纤维细胞中表达，提示成纤维细胞与肿瘤的侵袭转移能力密切相关。