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目的构建稳定过表达人表皮生长因子受体2(HER2/ErbB2)基因的黑素瘤细胞株,研究其对黑素瘤细胞的增殖、侵袭和迁移的影响。方法将pCMV3-ErbB2(人源)重组质粒通过脂质体法转染至黑素瘤B16细胞株,用潮霉素B筛选出耐药阳性克隆。实时定量PCR(qRT-PCR)检测各单克隆ErbB2 mRNA相对表达量,免疫荧光技术检测ErbB2蛋白的表达。MTT法检测转染后细胞增殖能力,TranswellTM实验检测细胞侵袭能力,划痕实验检测细胞迁移能力。结果实时定量PCR检测结果表明,通过脂质体法成功构建过表达HER2的B16细胞株,其HER2的表达显著高于空载体对照细胞。TranswellTM等实验显示HER2过表达增强黑素瘤细胞增殖、侵袭和迁移的能力。结论过表达HER2促进黑素瘤细胞的增殖、侵袭和迁移。
Objective To construct a melanoma cell line stably overexpressing human epidermal growth factor receptor 2 (HER2 / ErbB2) gene and study its effect on the proliferation, invasion and migration of melanoma cells. Methods The recombinant plasmid pCMV3-ErbB2 (human) was transfected into melanoma B16 cell line by lipofectamine and the positive clone was screened with hygromycin B. Real-time quantitative PCR (qRT-PCR) was used to detect the relative expression of ErbB2 mRNA, and the expression of ErbB2 protein was detected by immunofluorescence assay. Cell proliferation was detected by MTT assay. TranswellTM assay was used to detect cell invasion. Scratch assay was used to detect cell migration. Results The results of real-time quantitative PCR showed that the HER2 expression in HER2-positive cells was significantly higher than that in empty vector cells by liposome method. TranswellTM and other experiments show that HER2 overexpression enhances the ability of melanoma cells to proliferate, invade and migrate. Conclusion Overexpression of HER2 can promote the proliferation, invasion and migration of melanoma cells.