利用GFP示踪细胞内源性P53活性检测DNA损伤

GFP Reporting Endogenous P53 Transcriptional Activation as a Detector for DNA Damage

ＤＮＡ损伤的检测对预防癌症和遗传病等非常重要。采用分子克隆技术，将报告基因—绿色荧光蛋白（ＧＦＰ）置于ＳＶ４０基本启动子调控下，构建成对照载体ｐＳＶ－ＧＦＰ。在ＳＶ４０基本启动子上游插入寡核苷酸Ｐ５３ＲＥ，构建成示踪载体ｐ５３ＲＥ－ＧＦＰ。转染ＮＩＨ３Ｔ３细胞，以ＧＦＰ示踪细胞内源性Ｐ５３的转录激活活性。紫外线照射或Ｈ２Ｏ２处理转化细胞使ＤＮＡ损伤，诱导细胞内源性Ｐ５３的表达。用激光扫描共聚焦成像系统（ＬＳＣＩＳ）对细胞进行红、绿、蓝三色光融合成像，并测定ＧＦＰ经４８８ｎｍ激发后发出的绿色荧光光密度，验证ＧＦＰ示踪Ｐ５３的特异性。ｐ５３ＲＥ－ＧＦＰ转化细胞３Ｔ３－ＲＥＧ经紫外线照射或Ｈ２Ｏ２处理后，ＧＦＰ的表达增高，处理后１ｈｒ光密度即达到最高水平，随后逐渐降低。血清“饥饿”—非ＤＮＡ损伤处理的３Ｔ３－ＲＥＧ细胞，以及经紫外和Ｈ２Ｏ２处理的对照载体ｐＳＶ－ＧＦＰ转化细胞３Ｔ３－ＳＶＧ，ＧＦＰ的表达无明显增强。实验表明：ＧＦＰ示踪内源性Ｐ５３转录激活活性用于检测ＤＮＡ损伤有很高的灵敏度和特异性，适宜推广应用。

Indentifying and measuring DNA damage are important in hazard assessment. Reporter gene, green fluorescent protein(GFP), with P53 response element(P53RE) fusion gene was constructed and transfected NIH 3T3 cells. Transformed cells were treated with ultraviolet and H 2O 2 to make DNA damage and induce endogenous P53 expression. The GFP expression and the intensity of green fluorescence in cells were measured with Laser Scanning Confocal Imaging System(LSCIS). The fluorescence intensity increased rapidly aftern ultraviolet and H 2O 2 treatment and reached the maxism 1 hr later, then decreased slowly. The fluoorescent intensity of cells treated with fatal calf serum(FCS)drawing, a non DNA damage treatment, increased slightly 4hr later, as well as cells transformed with control vector pSV-GFP after ultraviolet treatment. The results suggest that GFP as a roporter of P53 transcriptinal activation is a sensitive and specific detector for DNA damage.