Detect of P53,bax and caspase 3 genes expression after optic nerve injury in rats with SYBR green I fluorescence quantitative PCR

Abstract

Objective Previous study showed that the histopathological basis of visual function damage caused by optical nerve injury is apoptosis of retinal ganglion cells（RGCs）. This procedure is regulated by P53 ,bax and caspase 3 genes. Present study aimed to observe the expression of bax,P53 and caspase 3 mRNA in RGCs after traumatic optic nerve damage in the rats by SYBR green I fluorescence quantitative PCR method. Methods The animal model of optic nerve injury was established in the right eyes of 56 adult Wistar rats by a fluid percussion brain injury device （FPI）. Animal were killed on days 1,3,5,7,9,14,28 days separately after injury. Other 16 Wistar rats were divided into normal control group and sham operation group. The total RNA was isolated from rat fresh retina tissue by Trizol method and was treated by reverse transcription to eDNA using Oligo（dt） 18 as primer and then amplified. The target fragments of bax,P53 and easpase 3 cDNA were linked with carrier pTZ57 R/T to construct recombined plasmids which were transformated to E. coli DH5α by T/A clone method. Recombined plasmids were extracted with alkaline lysis method and the plasmids were selected in white colonies by ampicillin screening,EcoR I restrictive enzyme analysis, and their specificity was evaluated using DNA sequencing. The standard curves were created by plasmid DNA and the precise expression level of target genes in samples were determined using software. The results were expressed as the ratios of target gene mRNA to GAPDH mRNA. Results The standard curve drawn by pTZ57R/T and target gene presented a good linear tendency with the higher sensitivity and specificity. The expression of P53 and bax mRNA began to inerease on the third day after the injury of optic nerve and peaked on the fifth day and started to decline on the seventh day. The expression of caspase 3 mRNA increased from the fifth day through the ninth days after injury and declined on the fourteenth day. The significant differences were found in the expression of P53, bax and easpase 3 between model group and control group （P 〈 0.05 ）. Conclusion The pro-apoptotie protein P53 ,bax and easpase 3 play an important role in RGCs apoptosis. Key words: SYBR green I method; fluorescence quantitative polymerase chain reaction; T/A clone method; traumatic optic nerve injury; P53; bax; caspase 3;