兔眼慢性高眼压模型视网膜损害相关基因的克隆及筛选

目的　构建慢性高眼压条件下兔眼视网膜组织差异表达基因的消减cDNA文库并鉴定、克隆及筛选与慢性高眼压视网膜损害相关的基因表达片段。方法　于兔眼前房注射1%甲基纤维素每周1次,连续5周,制作慢性高眼压模型。提取实验组和对照组兔眼视网膜组织总RNA及纯化mRNA。采用原位杂交技术,进行视网膜组织差异表达基因抑制性消减文库的构建及初步的基因筛选。对一个上调的cDNA片段用地高辛标记,采用原位杂交的方法在实验组和对照组的视网膜中进行相关基因片段的细胞定位。结果　成功构建慢性高眼压条件下兔眼视网膜损害相关基因的抑制性消减cDNA文库,经基因片段测序及同源性检索,得到有效基因序列16个,经NCBI BLAST信息途径分析,发现高度同源序列(同源性>98% )2个。其中一个编号F9基因片段与Ras家族基因相似,原位杂交结果确定其大量表达于实验组视网膜神经节细胞及内核层细胞中,而对照组视网膜组织未表达。结论　慢性高眼压条件下视网膜组织基因表达失调,F9片段在慢性高眼压视网膜损害过程中表达明显增高。建立慢性高眼压条件下视网膜损害相关基因的抑制性消减cDNA文库,可为今后青光眼视神经损害机制及视神经保护的研究奠定基础。

Cloning and screening of retina damage related genes in experimental rabbit chronic ocular hypertension

Objective To construct the subtractive differentially expressed genes cDNA library of retina in rabbits with chronic ocular hypertension then to clone and screen the genes that related with retina damage. Methods Rabbit chronic ocular hypertension model was Established by injecting 1% methylcellulose into anterior chamber once a week for 5 weeks. Total RNA was extracted from retina of experimental and control rabbit eyes. Differentially expressed cDNAs libraries between chronic ocular hypertension eyes and control eyes were constructed using suppression subtraction hybridization(SSH) and then the preliminary data was screened for gene expresion. One of upregulation expressed sequence tags(ESTs) was labeled with biotin as a probe. Cellular location of the interested genes in retina was identified by in situ hybridization. Results The constructed subtractive cDNA library that related to retina damage of chronic ocular hypertension was established and sixteen effective sequences were obtained. Two known expressed sequence tags (similarity over 98%) were found by BLAST Analyze in NCBI. The EST(number F9)was 99% similar to Ras genes family. Through ISH cellular location it was found the gene highly expressed in retina ganglion cells and inner nuclear layer of experimental retina but not in controls. Conclusions There was abnormal gene expression in rabbit retina in response to chronic ocular hypertension. Our result suggests EST F9 may play an important role in retina damage under chronic ocular hypertension. The subtractive differentially expressed genes cDNA library could provide valuable information in the research of optical neuro damage and protection in glaucoma.