酪氨酸酶基因在HEK293细胞表达的MRI评价

目的:以酪氨酸酶基因作为报告基因转染HEK293细胞, 利用其合成大量黑色素而能被MRI检测的特性来反映基因表达的情况, 以探索磁共振成像(MRI)评价体外细胞基因表达的方法。方法:以脂质体将含酪氨酸酶基因完全cDNA的pcDNA3tyr质粒转染到HEK293细胞, 以MRIT₁WI、T₁WI/SPIR、T₂WI序列扫描转染细胞, 观察表达的黑色素的MRI信号。应用Fontana染色检测黑色素的合成, RT-PCR检测酪氨酸酶基因的cDNA片段, 以进一步验证酪氨酸酶基因的转染与表达。结果:(1)pcDNA3tyr质粒转染进入HEK293细胞并在其中表达生成黑色素, 转染5μg、10μg、20μg质粒的10⁶个细胞内生成的黑色素能够被MRI检测到并在MRIT₁WI、T₁WI/SPIR、T₂WI检查呈高信号, MRI信号强度与转染质粒量成正相关。(2)Fontana染色法检测到HEK293细胞内的黑色素颗粒;(3)采用RT-PCR方法检测到转染的HEK293细胞含酪氨酸酶基因的cDNA片段。结论:MRI能够检测到HEK293细胞内由外源基因表达合成的黑色素, 说明影像学与分子生物学技术结合可以评价体外细胞基因表达的情况。

Evaluation of tyrosinase gene's expression in HEK293 cells by magnetic resonance imaging

AIM:Tyrosinase gene was transfected into HEK293 cell as a reporter gene, it's property of synthesizing melanin, which can be examined by magnetic resonance imaging(MRI), is used to evaluate the tyrosinase gene's expression. The aim of this study was to search a way to evaluate the results of gene expression by MRI in vitro.METHODS:The plasmid of pcDNA3tyr which carried the full-length cDNA of tyrosinase gene was transfected into HEK293 cell by lipofectin. To observe the MRI signals of expressed melanin, the transfected cells were scanned by MRI sequences of T₁WI, T₁WI/SPIR and T₂WI. On the other hand, fontana stain was used to search for melanin granules in transfected cells, RT-PCR method was used to search for cDNA of tyrosinase gene.RESULTS:(1) Plasmids of pcDNA3tyr could be transfected into HEK293 cells and could synthesize a large amount of melanin. The synthetic melanins of 10⁶ cells, which had been transfected 5μg, 10μg, 20μg plasmids of pcDNA3tyr separately, were all sufficient to be detected by MRI and appeared high signal in MRI T₁WI、T₁WI/SPIR、T₂WI sequences. The signal intensities of MRI imaging were related to the amounts of transfected plasmids positively. (2) The melanin granules could be found in HEK293 cells by Fontana stain. (3) The cDNA fragment of tyrosinase gene could be detected in transfected HEK293 cells by RT-PCR.CONCLUSION:The fact that MRI could detect the synthet ic melanin of HEK293 cells, which controlled by expression of exogenous gene, demonstrates that medical imaging connecting with molecular biology technology can evaluate the result of gene expression in vitro.