摘要
背景与目的:基因治疗是生命科学研究的前沿领域,但目前其主要技术手段———基因转染及表达尚缺乏理想的无创性评价方法,影像学与分子生物学技术结合有可能解决这一问题,但这方面研究才刚刚开始。本研究利用酪氨酸酶基因作为报告基因,在体外细胞中进行实验,以探索MR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HepG2细胞,利用其合成大量黑色素而在T1WIMR影像上呈高信号的特性来反映基因表达的情况,同时应用Fontana染色法检测黑色素和RT-PCR检测cDNA片段的方法来进一步验证。结果:(1)Fontana染色法可检测到HepG2细胞内的黑色素颗粒。(2)采用RT-PCR方法可检测到转染的HepG2细胞中含酪氨酸酶基因的cDNA片段。(3)pcDNA3tyr质粒转染进入HepG2细胞并在其中表达生成黑色素,其数量能够被MR检测到,这种黑色素与在正常细胞中生成的黑色素一样在T1WIMR影像上呈高信号,且MR信号强度与转染质粒量成正相关。结论:MR能够检测到体外细胞内合成的黑色素,说明影像学与分子生物学技术结合可能成为一种评价体外细胞基因表达的方法。
BACKGROUND & OBJECTIVE:Gene therapy is the frontier of life science. There is no perfect method to evaluate gene expression without invasion at present. Medical imaging connecting with molecular biology might be helpful; however, the technology is just on the horizon. The authors conducted this study in vitro by transferring reporter tyrosinase gene into HepG2 cell to apply magnetic resonance imaging (MR) for evaluating gene expression. METHODS:The plasmid of pcDNA3tyr carried the full length cDNA of tyrosinase gene was transfected into HepG2 cell by lipofectin, its property of synthesizing melanin was used to produce high signal in T1WI MR image and then to evaluate gene expression. Further identification were performed with searching melanin granules by Fontana staining and searching cDNA of tyrosinase gene using reverse transcription polymerase chain reaction (RT PCR). RESULTS:(1)The melanin granules were found in HepG2 cell using Fontana staining. (2)The cDNA fragment of tyrosinase gene was detectable in transferred HepG2 cell by RT PCR. (3)Plasmid of pcDNA3tyr was transfected into HepG2 cell and synthesized a large amount of melanin in HepG2 cell; the synthetic melanin appeared high signal in T1WI MR imaging as same as natural melanin and was enough to be detected by MR. And further, the signal intensity was positively related to the amount of transferred plasmid. CONCLUSION:The fact that synthetic melanin of HepG2 cell can be detected by MR demonstrates that medical imaging connecting with molecular biology can be used to evaluate the result of gene expression in vitro.
出处
《癌症》
SCIE
CAS
CSCD
北大核心
2003年第2期156-159,共4页
Chinese Journal of Cancer
