摘要
目的研究pcDNA3.0-tyr在正常人成纤维母细胞株(GM0639细胞)中的表达。方法pcDNA3.0-tyr真核表达质粒扩增、纯化后经测序、酶切鉴定;电穿孔法将pcDNA3.0-tyr转染GM细胞,RT-PCR检测;测定490nm吸光度值,细胞爬片Fontana染色,证实酪氨酸酶基因表达及其表达水平。结果测序及酶切结果提示pcDNA3.0-tyr质粒含有酪氨酸酶全长cDNA片段;转染pcDNA3.0-tyr的细胞的PCR产物长度与预期长度一致,而转染pcDNA3.0空载体的细胞PCR产物无此条带;转染pcDNA3.0-tyr的GM细胞的A490显著高于转染空载体的细胞及未转染的GM细胞(P<0.001);转染pcDNA3.0-tyr的GM细胞Fontana染色于胞浆内见棕褐色银沉着颗粒,而转染pcDNA3.0空载体以及未转染的细胞缺乏此颗粒。结论pcDNA3.0-tyr可体外转染GM0639细胞并成功表达,为后续研究奠定基础。
Objective To study the gene expression of pcDNA3.0-tyr in GM cells （GM0639） from the skin of the normal subject in vitro. Methods pcDNA3.0-tyr eukaryotic expression plasmids were multiplied and purified, and its integrity was determined by means of sequencing and restriction analysis. The plasmids were transfected into GM cells by electroporation. Then reverse transcription PCR was performed in transfected cells. The tyrosinase gene expression and its levels in transfected cells were confirmed by measuring absorption spectrum at 490 nm and staining with the Fontana stain. Results The result of sequencing and restriction analysis confirmed that the eukurytoic expression vector induced contains the full-length human tyrosinase cDNA. Reverse transcription PCR product of transfected cells showed a band that coincided with that of reverse primers, whereas the non-transfected cells don＇ t show this band. The value of A490 in transfected cells is greater than that in mocktransfected and non-transfected cells （P 〈 0. 001）. Fontana stain showed that the brownish color granules in pcDNA3.0-tyr transfected cells, which represented induced melanin, and the absence of these in non-transfected cells. Conclusion The plasmids of pcDNA3.0-tyr can be transfected into human GM0639 cells and synthesize melanin.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2005年第6期955-958,共4页
Suzhou University Journal of Medical Science
基金
江苏省135工程医学重点人才基金(RC2003096)
