Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000, USA.
Curr Eye Res. 2010 Jan;35(1):80-90. doi: 10.3109/02713680903421194.
Homocysteine is implicated in ganglion cell death associated with glaucoma. To understand mechanisms of homocysteine-induced cell death, we analyzed the sensitivity of the RGC-5 cell line, differentiated using staurosporine, to physiologically-relevant levels of the excitotoxic amino acid homocysteine.
RGC-5 cells were differentiated 24 hr using 316 nM staurosporine and tested for expression of Thy 1.2 via immunodetection, RT-PCR, and immunoblotting. The sensitivity of staurosporine-differentiated RGC-5 cells to physiological levels of homocysteine (50, 100, 250 microM) and to high levels of homocysteine (1 mM), glutamate (1 mM), and oxidative stress (25 microM:10 mU/ml xanthine:xanthine oxidase) was assessed by TUNEL assay and by immunodetection of cleaved caspase-3. The sensitivity of undifferentiated RGC-5 cells to high (1, 5, and 10 mM) homocysteine was also examined.
Undifferentiated RGC-5 cells express Thy 1.2 mRNA and protein. Staurosporine-differentiated RGC-5 cells extend neurite processes and express Thy 1.2 after 24 hr differentiation; they express NF-L after 1 and 3 days differentiation. Treatment of staurosporine -differentiated RGC-5 cells with 50, 100, or 250 microM homocysteine did not alter neurite processes nor induce cell death (detected by TUNEL and active caspase-3) to a level greater than that observed in the control (non-homocysteine-treated, staurosporine-differentiated) cells. The 1 mM dosage of homocysteine in staurosporine-differentiated RGC-5 cells also did not induce cell death above control levels, although 18 hr treatment of non-differentiated RGC-5 cells with 5 mM homocysteine decreased survival by 50%.
RGC-5 cells differentiated for 24 hr with 316 nM staurosporine project robust neurite processes and are positive for ganglion cell markers consistent with a more neuronal phenotype than non-staurosporine-differentiated RGC-5 cells. However, concentrations of homocysteine known to induce ganglion cell death in vivo and in primary ganglion cells are not sufficient to induce death of RGC-5 cells, even when they are differentiated with staurosporine.
同型半胱氨酸与青光眼相关的神经节细胞死亡有关。为了了解同型半胱氨酸诱导细胞死亡的机制，我们分析了使用星形孢菌素分化的 RGC-5 细胞系对生理相关水平的兴奋性氨基酸同型半胱氨酸的敏感性。
使用 316 nM 星形孢菌素将 RGC-5 细胞分化 24 小时，并通过免疫检测、RT-PCR 和免疫印迹检测 Thy 1.2 的表达。使用 TUNEL 测定法和切割 caspase-3 的免疫检测法评估星形孢菌素分化的 RGC-5 细胞对生理水平的同型半胱氨酸（50、100、250 μM）和高水平的同型半胱氨酸（1 mM）、谷氨酸（1 mM）和氧化应激（25 μM：10 mU/ml 黄嘌呤：黄嘌呤氧化酶）的敏感性。还检查了未分化的 RGC-5 细胞对高浓度（1、5 和 10 mM）同型半胱氨酸的敏感性。
未分化的 RGC-5 细胞表达 Thy 1.2 mRNA 和蛋白。星形孢菌素分化的 RGC-5 细胞在 24 小时分化后伸出神经突过程并表达 Thy 1.2；它们在 1 天和 3 天后分化时表达 NF-L。用 50、100 或 250 μM 同型半胱氨酸处理星形孢菌素分化的 RGC-5 细胞不会改变神经突过程，也不会诱导细胞死亡（通过 TUNEL 和活性 caspase-3 检测），超过对照（未用同型半胱氨酸处理的星形孢菌素分化的）细胞观察到的水平。星形孢菌素分化的 RGC-5 细胞中的 1 mM 同型半胱氨酸剂量也不会诱导细胞死亡超过对照水平，尽管用 5 mM 同型半胱氨酸处理非分化的 RGC-5 细胞 18 小时会使存活率降低 50%。
用 316 nM 星形孢菌素分化 24 小时的 RGC-5 细胞伸出强壮的神经突过程，并对神经节细胞标志物呈阳性，与非星形孢菌素分化的 RGC-5 细胞相比具有更神经元表型。然而，体内和原代神经节细胞中已知诱导神经节细胞死亡的同型半胱氨酸浓度不足以诱导 RGC-5 细胞死亡，即使它们用星形孢菌素分化。
