摘要
目的 评价抗凋亡基因bcl XL 对视网膜光感受器细胞的抗凋亡作用。方法 首先建立谷氨酸损伤的SD大鼠视网膜光感受器细胞凋亡模型。将体外培养的光感受器细胞分为A组 (正常对照组 )、B组 (谷氨酸组 )及C组 (rAd gfp bcl XL 转染 +谷氨酸组 ) ;其中C组在加入谷氨酸前 4 8h用滴度为 6 5×l0 12 pfu/mL的重组腺病毒rAd gfp bcl XL 转染光感受器细胞 ;荧光显微镜下观察光感受器细胞中的绿色荧光蛋白表达情况 ;用免疫组化法分析rAd gfp bcl XL 转染细胞和未转染细胞Bcl XL蛋白水平 ;DNA琼脂糖凝胶电泳以评判 3个组神经元凋亡发生与否及凋亡程度 ;采用Hoechst332 5 8染色做正常核和凋亡核的形态学检测。结果 免疫组化检测结果表明转染细胞与未转染细胞的Bcl XL 蛋白表达水平有差异 ,DNA电泳分析发现B组呈典型的DNA“梯度”条带 ,而A组和C组几乎无DNA“梯度”条带 ,核形态学检测结果亦证实转染组较未转染组的核有明显差异。结论重组腺病毒介导转染的bcl XL 对体外培养的视网膜光感受器细胞有抗凋亡作用 ,提高视网膜光感受器细胞bcl XL 的表达水平可能为视网膜变性疾病提供潜在有效的治疗方法。
Objective To assess the effect of bcl-X L, an anti-apoptotic gene, on glutamate-induced apoptosis in cultured retinal photoreceptors. Methods Glutamate-induced apoptosis in cultured retinal photoreceptors was established. The experiment was divided into three groups: control, glutamate treatment and rAd-gfp-bcl-X L+glutamate transfection group, the protection of bcl-X L on retinal photoreceptors from apoptosis was evaluated. 6.5×10 12 pfu/L rAd-gfp-bcl-X L were transfected into retinal photoreceptors in the rAd-gfp-bcl-X L+glutamate group 48 h before Glutamate-induced apoptosis was established. The positive photoreceptors with green fluorescence were identified under fluorescence microscopy. The expression of Bcl-X 2 protein in rAd-gfp-bcl-X L transfected and non-transfected neurons were assessed by immunohistochemistry assay. DNA fragment was detected by agarose gel electrophoresis. Nuclei were revealed by hoechst33258 staining. Results rAd-mediated gene delivery can transfect retinal photoreceptors effectively. Increased expression of Bcl-X L protein was demonstrated in the rAd-transfecting neurons. Transfection of bcl-X L significantly decreased the number of apoptotic retinal photoreceptors in vitro. Conclusion Transfection of bcl-X L protects cultured retinal photoreceptors from apoptosis induced by glutamate. Transfection the gene of bcl-X L may be a potential gene therapy for retinal degenerative diseases.
出处
《中华眼科杂志》
CAS
CSCD
北大核心
2003年第8期490-494,共5页
Chinese Journal of Ophthalmology
基金
国家自然科学基金资助项目 (3 9770 790 )
"2 11工程"重点学科基金资助项目 (980 0 6)
