CAJ | 학술논문

目的应用SYBR Green Ⅰ荧光定量PCR法检测外伤性视神经损伤后P53、bax和caspase 3基因mRNA表达的变化.方法应用液压颅脑损伤仪建立大鼠外伤性视神经损伤动物模型,伤后1、3、5、7、9、14、28d处死,以Trizol法提取新鲜视网膜组织的总RNA,以Oligo (dt) 18 为引物逆转录合成cDNA 并进行扩增,以T/A克隆法将纯化的目的片断与T/A克隆载体(pTZ57R/T)连接成重组质粒并转化入E.coli DH5α.采用碱裂解法提取重组质粒,经蓝白斑筛选、酶切、测序鉴定后,根据标准品建立标准曲线,由软件自动计算出待测样本中靶基因mRNA的含量,并以靶基因和内参GAPDH mRNA含量的比值作为评价靶基因表达水平的指标.结果由pTZ57R/T与目的基因所构建的标准曲线的线性关系良好、灵敏度高、特异性强、准确可靠.P53和bax均在视神经损伤后3d mRNA表达明显增加,5d时达到高峰,7d后开始下降;伤后5d caspase 3 mRNA表达明显增加,9d时达到高峰,14d后开始下降.三者表达水平与对照组相比,差异均有统计学意义(P＜0.05).结论促凋亡基因P53、bax和caspase 3在视神经损伤后视网膜神经节细胞(RGCs)的凋亡发生中起到重要作用.
목적 응용SYBR Green Ⅰ형광정량PCR법검측외상성시신경손상후P53、bax화caspase 3기인mRNA표체적변화.방법 응용액압로뇌손상의건립대서외상성시신경손상동물모형,상후1、3、5、7、9、14、28d처사,이Trizol법제취신선시망막조직적총RNA,이Oligo (dt) 18 위인물역전록합성cDNA 병진행확증,이T/A극륭법장순화적목적 편단여T/A극륭재체(pTZ57R/T)련접성중조질립병전화입E.coli DH5α.채용감렬해법제취중조질립,경람백반사선、매절、측서감정후,근거표준품건립표준곡선,유연건자동계산출대측양본중파기인mRNA적함량,병이파기인화내삼GAPDH mRNA함량적비치작위평개파기인표체수평적지표.결과 유pTZ57R/T여목적 기인소구건적표준곡선적선성관계량호、령민도고、특이성강、준학가고.P53화bax균재시신경손상후3d mRNA표체명현증가,5d시체도고봉,7d후개시하강;상후5d caspase 3 mRNA표체명현증가,9d시체도고봉,14d후개시하강.삼자표체수평여대조조상비,차이균유통계학의의(P＜0.05).결론 촉조망기인P53、bax화caspase 3재시신경손상후시망막신경절세포(RGCs)적조망발생중기도중요작용.
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 cDNA using 01igo(dt) 18 as primer and then amplified. The target fragments of bax, P53 and caspase 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 increase 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 caspase 3 between model group and control group (P ＜ 0. 05) . Conclusion The pro-apoptotic protein P53, bax and caspase 3 play an important role in RGCs apoptosis.