MR评价酪氨酸酶基因在人胚胎肾293细胞表达的实验研究  

Experiment study of tyrosinase gene′s expression in HEK293 cell by MR

作　　者：元建鹏[1] 梁碧玲[1] 钟镜联[1] 谢榜昆[1] 张卫东[1] 张琳[1] 

机构地区：[1]中山大学附属第二医院放射科,广州510120

出　　处：《中华放射学杂志》2004年第6期605-610,共6页Chinese Journal of Radiology

基　　金：广东省医学科学技术基金资助项目 (项目编号 :A2 0 0 3 2 14 );广东省自然科学基金 (A3 170 9)

摘　　要：目的　以酪氨酸酶基因作为报告基因转染人胚胎肾 (HEK) 2 93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HEK2 93细胞 ,以MRT1W、T1W/SPIR(selectivepartialinversionrecovery)、T2 W序列扫描转染细胞 ,观察表达的黑色素的MR信号。应用Fontana染色和电子显微镜检测黑色素的合成 ,逆转录聚合酶链式反应 (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HEK2 93细胞并在其中表达生成黑色素 ,转染 5、10、2 0 μg质粒的 10 6个细胞内生成的黑色素能够被MRI检测到并在MRT1WI、T1WI/SPIR、T2 WI呈高信号 ,转染质粒量越大 ,MRI信号强度越高。 6 2 5× 10 4个转染有 2 0 μg质粒的HEK2 93细胞仍可检测到高信号。 (2 )Fontana染色法检测到HEK2 93细胞内的黑色素颗粒 ;(3)电子显微镜观察到在HEK2 93细胞胞质内有较多的黑色素颗粒及黑色素前体 ;(4 )采用RT PCR方法检测到转染的HEK2 93细胞含酪氨酸酶基因的cDNA片段。结论　MRI能够检测到HEK2 93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?Objective To transfect the tyrosinase gene into HEK293 cell as a reporter gene, and to evaluate the tyrosinase gene′s expression by using MRI based on the gene′s property of synthetizing large amount of melanin, and to search a way for evaluating the results of gene expression by MR in vitro.Methods The plasmid of pcDNA3tyr which carried the full-length cDNA of tyrosinase gene was transfected into HEK293 cell by lipofectin, and MR signals of expressed melanin was observed by scanning the transfected cells with MR sequences of T 1WI, T 1WI/SPIR, and T 2WI.Fontana stain and electric microscopy were used to search for melanin granules in transfected cells, and 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 synthetize a large amount of melanin in them.The synthetic melanin in 106 cells, which had been transfected with 5 μg, 10 μg, and 20 μg plasmids of pcDNA3tyr separately, were all sufficient to be detected by MR and appeared as high signal on MR T 1WI, T 1WI/SPIR, and T 2WI sequences.The more the amounts of transfected plasmids, the higher the signal intensities of MR imaging.On the other hand, 6.25×10 4 cells with 20 μg-plasmid of pcDNA3tyr transfection could also be detected by MR; (2) The melanin granules could be found in HEK293 cells in Fontana stain; (3) The melanin granules and their front bodies could be found in intracytoplasm of HEK293 cell by electric microscopy.(4)The cDNA fragment of tyrosinase gene could be detected in transfected HEK293 cells by RT-PCR.Conclusion The fact that MR could detect the synthetic melanin in HEK293 cells controlled by expression of exogenous gene demonstrated that medical imaging combined with molecular biology technology could evaluate the result of gene expression in vitro, and it also indicated that medical imaging could play an important role in the evaluation of gene therapy following the development of technology.

关 键 词：MR评价 酪氨酸酶基因 人胚胎肾 293细胞 表达 实验研究 基因治疗 

分 类 号：R445.2[医药卫生—影像医学与核医学]