铁缺乏致聋相关基因表达谱的实验研究

Changes in gene expression profile in Wistar rats with iron hearing loss: an experimental study

目的应用基因表达谱芯片研究铁缺乏耳聋Wistar大鼠耳蜗膜性结构基因表达的改变。方法选用体重28～45 g的健康、无耳疾、ABR阈值正常的幼龄Wistar大鼠23只,随机分成两组。正常大鼠对照组12只,喂以标准鼠饲料12周;缺铁耳聋大鼠组11只,缺铁饲料喂养12周;将包含了4096条大鼠的靶基因用点样仪点在特制玻片上制备成表达谱芯片,取缺铁耳聋组和正常对照组的耳蜗膜性组织mRNA,通过逆转录方法将Cy3和Cy5两种荧光分别标记到两种组织的cDNA上,制成cDNA探针,并与表达谱芯片进行杂交后扫描。用RT-PCR方法验证部分差异表达基因。结果缺铁耳聋组与正常对照组比较,筛选出差异表达的基因896条,其中上调基因447条,下调基因449条。肌动蛋白基因及其相关调节蛋白基因的表达发生了改变。其中肌动蛋白基因arpc1b,actc1,actb,actal都表现为下调,肌动蛋白的抑制蛋白基因profilinⅡ上调。细胞骨架蛋白tubulin、myosin和热休克蛋白HSP下调。选取5条差异表达基因BC072490(Ctsb)、CF111145(Actb)、NM013146(Cald1)、AB011679(Tubb5)、BC063175 (Ctsl)用RT-PCR验证,RT-PCR结果与基因芯片结果基本一致。结论铁缺乏通过影响耳蜗膜性结构中多种基因的表达,包括肌动蛋白基因及其相关调节蛋白基因的表达从而导致感音神经性聋。

Objective To explore changes in gene expression in the inner ear of Wistar rats with iron deficiency hearing loss. Methods Twenty-three normal hearing Wistar rats were randomized into 2 groups. Group A rats （n= 12） were fed with normal dietary for 12 weeks. Group B rats （n = 11） were fed with iron deficiency diet for 12 weeks to make iron deficiency hearing loss models. Two cDNA probes labelled with Cy3 and Cy5 fluorescence dyes were prepared from mRNA of the cochlear of Group B/A and were hybridized with cDNA microarray of target gene expression profile which were scanned for fluorescent intensity. The mixed probes were hybridized with two pieces of BioDoor 4096 double dot rat whole gene chip. Changes in gene expression were further confirmed by RT-PCR and were analyzed. Results Comparison analysis revealed that the expression of 447 genes was up-regulated and 449 genes were down-regulated in the cochlea of iron deficiency hearing loss rats. In the differentially expressed genes,actin-related genes such as arpclb,actcl, actb ＆ actal were down-regulated. Actin-control gene profilin Ⅱ was up-regulated. Tubulin, myosin and heat shock protein were down-regulated. Conclusions The expression of many genes, such as actin-related genes, may change in the cochlea of iron deficiency Wistar rats and cause hearing loss.