Centre of Ophthalmology, University Eye Hospital Tübingen, Tübingen, Germany.
Graefes Arch Clin Exp Ophthalmol. 2012 Aug;250(8):1221-9. doi: 10.1007/s00417-011-1906-3. Epub 2012 Jan 6.
Supplementation of staurosporine is the method of choice for differentiating the solely existing retinal ganglion cell (RGC)-5 cell line. This differentiation was initially claimed to be in the absence of apoptosis, but some publications supposed the induction of apoptosis during staurosporine induced RGC-5 differentiation. In respect to these inconsistencies in the literature, we investigated in detail whether RGC-5 cell differentiation by staurosporine induces apoptosis or not.
Amounts of 50 nM, 200 nM, 300 nM, and 600 nM of staurosporine were supplemented on RGC-5 cells for 24 h. Cell morphology and cell death, via propidium iodide staining, were evaluated with phase contrast and fluorescence microscopy, respectively. Cell amount, cell proliferation, and cell viability were analyzed by crystal violet staining, CFSE flow cytometry, and MTS assay, respectively. Apoptosis was determined by analyzing caspase 3/7 activity, Annexin-V+/ 7AAD- cells and the quotient of Bax to Bcl-2 mRNA expression via caspase 3/7 activity assay, flow cytometry, and real-time PCR, respectively.
RGC-5 cells started to change their morphology and their expression of neuronal markers at 50 nM of staurosporine. This was associated with apoptosis and cell death, as indicated by a 2.1-fold (p < 0.0005) increase in caspase 3/7 activity, a 1.2-fold (p < 0.05) induction of Annexin-V+/ 7AAD- cells, and a 12-fold (p < 0.0005) increase in propidium iodide positive cells, respectively. Furthermore, staurosporine led to a dose-dependent increase in apoptosis and reduction in cell viability, cell density, and cell proliferation.
The lowest staurosporine concentration inducing RGC-5 cell differentiation is accompanied by apoptosis and cell death.
添加司他夫定是分化唯一存在的视网膜神经节细胞（RGC-5）细胞系的首选方法。最初声称这种分化不存在细胞凋亡，但一些出版物认为在司他夫定诱导的 RGC-5 分化过程中会诱导细胞凋亡。鉴于文献中的这些不一致之处，我们详细研究了司他夫定诱导的 RGC-5 细胞分化是否会诱导细胞凋亡。
将 50 nM、200 nM、300 nM 和 600 nM 的司他夫定添加到 RGC-5 细胞中 24 小时。通过相差显微镜和荧光显微镜分别评估细胞形态和通过碘化丙啶染色评估细胞死亡。通过结晶紫染色分别分析细胞数量、细胞增殖和细胞活力，通过 CFSE 流式细胞术和 MTS 测定分别分析细胞增殖和细胞活力。通过分析 caspase 3/7 活性、Annexin-V+/7AAD-细胞和 Bax/Bcl-2 mRNA 表达的比率，分别通过 caspase 3/7 活性测定、流式细胞术和实时 PCR 测定来确定细胞凋亡。
RGC-5 细胞在 50 nM 司他夫定时开始改变其形态和神经元标志物的表达。这与细胞凋亡和细胞死亡有关，如 caspase 3/7 活性增加 2.1 倍（p < 0.0005）、Annexin-V+/7AAD-细胞增加 1.2 倍（p < 0.05）和碘化丙啶阳性细胞增加 12 倍（p < 0.0005）分别表示。此外，司他夫定导致细胞凋亡和细胞活力、细胞密度和细胞增殖呈剂量依赖性降低。
诱导 RGC-5 细胞分化的最低司他夫定浓度伴随着细胞凋亡和细胞死亡。
