CAJ | 학술논문

背景 N-甲基-N-亚硝基脲(MNU)诱导的大鼠光感受器细胞凋亡可用于研究视网膜变性类疾病,但视网膜变性类疾病早期基因水平的研究尚未见报道. 目的应用基因芯片技术研究MNU诱导的视网膜变性大鼠早期基因表达谱的变化.方法 6周龄雌性SD大鼠50只分为正常组20只、12h模型组20只和24 h模型组10只.模型组大鼠皮下注射MNU(40 mg/kg),正常组大鼠皮下注射等容量的生理盐水作为对照.分别于造模后12、12、24 h处死正常组和12h模型组、24 h模型组大鼠,大鼠右眼球进行常规视网膜组织病理学检查,正常组和12h模型组大鼠左眼的新鲜视网膜用基因芯片技术检测差异基因的表达,用荧光实时定量PCR(real-time PCR)法验证基因芯片技术检测出的差异表达率≥2.0的基因mRNA表达水平. 结果全层视网膜厚度测量表明,24 h模型组大鼠的厚度值明显低于正常组大鼠和12h模型组大鼠,差异均有统计学意义(t=9.926,P=0.002;t2.736,P=0.028).24 h模型组大鼠外核层厚度值为(26.58±2.90) μm,明显低于正常组的(38.11±1.01)μm和12h模型组的(35.07±3.03) μm,差异均有统计学意义(t=6.028,P=0.009;t=6.839,P=0.006),正常组与12h模型组间大鼠全层视网膜厚度和外核层厚度值比较差异均无统计学意义(全层厚度:t=1.541,P=0.324;外核层厚度:t=2.040,P=0.134).大鼠cDNA基因芯片技术检测结果表明,12h模型组大鼠全部17 000个基因的表达谱中涉及生物过程的基因为142个,涉及分子功能的基因为94个,排除重复基因共有74个基因,差异表达基因主要涉及丝裂原活化蛋白激酶(MAPK)通路、Toll样受体通路和细胞凋亡通路.对基因芯片技术检测的差异表达率≥2.0的基因进行的real-time PCR定量分析表明,CCL2、IL-1b、CCL3、c-fos、c-myc、p53和MMP3基因mRNA表达值与基因芯片技术检测的表达趋势一致. 结论 MNU诱导的视网膜变性早期有明显的基因表达改变.基因芯片检测的基因表达改变结果与real-time PCR定量分析结果一致. 揹景 N-甲基-N-亞硝基脲(MNU)誘導的大鼠光感受器細胞凋亡可用于研究視網膜變性類疾病,但視網膜變性類疾病早期基因水平的研究尚未見報道. 目的應用基因芯片技術研究MNU誘導的視網膜變性大鼠早期基因錶達譜的變化.方法 6週齡雌性SD大鼠50隻分為正常組20隻、12h模型組20隻和24 h模型組10隻.模型組大鼠皮下註射MNU(40 mg/kg),正常組大鼠皮下註射等容量的生理鹽水作為對照.分彆于造模後12、12、24 h處死正常組和12h模型組、24 h模型組大鼠,大鼠右眼毬進行常規視網膜組織病理學檢查,正常組和12h模型組大鼠左眼的新鮮視網膜用基因芯片技術檢測差異基因的錶達,用熒光實時定量PCR(real-time PCR)法驗證基因芯片技術檢測齣的差異錶達率≥2.0的基因mRNA錶達水平. 結果全層視網膜厚度測量錶明,24 h模型組大鼠的厚度值明顯低于正常組大鼠和12h模型組大鼠,差異均有統計學意義(t=9.926,P=0.002;t2.736,P=0.028).24 h模型組大鼠外覈層厚度值為(26.58±2.90) μm,明顯低于正常組的(38.11±1.01)μm和12h模型組的(35.07±3.03) μm,差異均有統計學意義(t=6.028,P=0.009;t=6.839,P=0.006),正常組與12h模型組間大鼠全層視網膜厚度和外覈層厚度值比較差異均無統計學意義(全層厚度:t=1.541,P=0.324;外覈層厚度:t=2.040,P=0.134).大鼠cDNA基因芯片技術檢測結果錶明,12h模型組大鼠全部17 000箇基因的錶達譜中涉及生物過程的基因為142箇,涉及分子功能的基因為94箇,排除重複基因共有74箇基因,差異錶達基因主要涉及絲裂原活化蛋白激酶(MAPK)通路、Toll樣受體通路和細胞凋亡通路.對基因芯片技術檢測的差異錶達率≥2.0的基因進行的real-time PCR定量分析錶明,CCL2、IL-1b、CCL3、c-fos、c-myc、p53和MMP3基因mRNA錶達值與基因芯片技術檢測的錶達趨勢一緻. 結論 MNU誘導的視網膜變性早期有明顯的基因錶達改變.基因芯片檢測的基因錶達改變結果與real-time PCR定量分析結果一緻.
배경 N-갑기-N-아초기뇨(MNU)유도적대서광감수기세포조망가용우연구시망막변성류질병,단시망막변성류질병조기기인수평적연구상미견보도. 목적 응용기인심편기술연구MNU유도적시망막변성대서조기기인표체보적변화.방법 6주령자성SD대서50지분위정상조20지、12h모형조20지화24 h모형조10지.모형조대서피하주사MNU(40 mg/kg),정상조대서피하주사등용량적생리염수작위대조.분별우조모후12、12、24 h처사정상조화12h모형조、24 h모형조대서,대서우안구진행상규시망막조직병이학검사,정상조화12h모형조대서좌안적신선시망막용기인심편기술검측차이기인적표체,용형광실시정량PCR(real-time PCR)법험증기인심편기술검측출적차이표체솔≥2.0적기인mRNA표체수평. 결과 전층시망막후도측량표명,24 h모형조대서적후도치명현저우정상조대서화12h모형조대서,차이균유통계학의의(t=9.926,P=0.002;t2.736,P=0.028).24 h모형조대서외핵층후도치위(26.58±2.90) μm,명현저우정상조적(38.11±1.01)μm화12h모형조적(35.07±3.03) μm,차이균유통계학의의(t=6.028,P=0.009;t=6.839,P=0.006),정상조여12h모형조간대서전층시망막후도화외핵층후도치비교차이균무통계학의의(전층후도:t=1.541,P=0.324;외핵층후도:t=2.040,P=0.134).대서cDNA기인심편기술검측결과표명,12h모형조대서전부17 000개기인적표체보중섭급생물과정적기인위142개,섭급분자공능적기인위94개,배제중복기인공유74개기인,차이표체기인주요섭급사렬원활화단백격매(MAPK)통로、Toll양수체통로화세포조망통로.대기인심편기술검측적차이표체솔≥2.0적기인진행적real-time PCR정량분석표명,CCL2、IL-1b、CCL3、c-fos、c-myc、p53화MMP3기인mRNA표체치여기인심편기술검측적표체추세일치. 결론 MNU유도적시망막변성조기유명현적기인표체개변.기인심편검측적기인표체개변결과여real-time PCR정량분석결과일치.
Background The rat model of N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell apoptosis is often used to study retinal degeneration.But the changes in the gene expression patterm in retinal degeneration in rats have not been reported.Objective This study was undertaken to investigate regulation of gene expression in the retina of MNU-induced retinal degeneration in rats by performing microarray analysis of retinal RNA.Methods Fifty 6-week-old SD rats were numbered and randomized into the normal group and the model group.The retinal degeneration model was established by a single hypodermic injection of 40 mg/kg of MNU,and the rats in the normal group received equivalent volume of physiological saline in the same way.The rats were sacrificed 12 hours or 24 hours after injection.Retinal sections from the right eyes were prepared for the measurement of the retinal thickness by histopathological examination,and retinas from the left eyes were used to confirm the differential gene expression as detected by microarray (normal group and 12 hours model group).Genes exhibiting changes in expression by ≥2.0 folds were further confirmed using real-time PCR.Results The whole thickness of the retina declined in the rats from the 24 hours model group compared to the normal group and 12 hours model group (t =9.926,P=0.002;t=2.736,P=0.028).The thickness of the outer nuclear layer was (26.58±2.90) μm in the 24 hours model group,showing a significant decrease in comparison with (38.11 ± 1.01) μm in the normal group and (35.07t3.03) μm in the 12 hours model group (t=6.028,P=0.009;t=6.839,P=0.006).However,there was no significant difference in retinal thickness between the normal group and the 12 hours model group (whole thickness:t=1.541,P=0.324;outer nuclear layer thickness:t=2.040,P=0.134).Microarray analysis of the rat genes showed that out of 17 000 genes,142 genes involved in biological process and 94 genes involved in molecular functions were differentially expressed,where most of them participate in the mitogen activated protein kinase signaling pathway,Tolllike receptor signaling pathway and apoptosis pathway.Real-time PCR analysis demonstrated that the expression of CCL2,IL-1b,CCL3,c-fos,c-myc,p53 and MMP3 were consistently up-regulated,conforming with the results from microarray analysis.Conclusions The changes in gene expression pattern appear in the early stage of MNUinduced retinal degeneration.These microarray results provided clues to understanding the molecular pathways underlying photoreceptor degeneration and indicating the directions for future studies.