Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198-6025, USA.
J Ocul Pharmacol Ther. 2011 Feb;27(1):7-15. doi: 10.1089/jop.2010.0051. Epub 2010 Nov 20.
The purpose of this study was to investigate the effects of hyperglycemia, its fluctuations, and glucose starvation on the expression of glucose-regulated protein 78/binding immunoglobulin protein (GRP78/BiP), one of the most commonly used markers of endoplasmic reticulum stress, in rat capillary pericytes and endothelial cells cultured separately and together.
Conditionally immortalized rat retinal pericyte and endothelial cell lines were cultured in dishes coated with collagen type I in Dulbecco's modified Eagle's medium containing 5.5 mM glucose. For cocultures, pericytes and endothelial cells were seeded together on rat tail collagen type I-coated cell culture plates. After 24 h of initial culture, the medium was replaced with serum-free medium containing 0-100 mM glucose for periods of up to 72 h. GRP78/BiP, caspase-3, and nuclear factor-κB expression were investigated using western blots.
No significant increase in GRP78/BiP expression was observed when pericytes, endothelial cells, or cocultures were exposed to either 25, 50, or 100 mM glucose for 48 h compared with the control level of 5.5 mM glucose. Similarly, no change in expression of GRP78/BiP was observed when media glucose levels were reduced from either 5.5 or 25 to 1 mM. GRP78/BiP expression significantly increased when cells were cultured for 24 h in glucose-deprived medium. This was accompanied by a time-dependent increase in the expression of caspase-3 and nuclear factor-κB.
In diabetic retinopathy, hyperglycemia has been reported to induce apoptosis in retinal capillary vascular cells, but these studies suggest that the apoptosis is not linked to the expression of GRP78/BiP, one of the most commonly used markers of endoplasmic reticulum stress. However, GRP78/BiP-linked apoptosis may play a role in vascular changes associated with retinal ischemia/reperfusion.
本研究旨在探讨高血糖及其波动和葡萄糖饥饿对分别培养和共培养的大鼠毛细血管周细胞和内皮细胞中葡萄糖调节蛋白 78/结合免疫球蛋白蛋白（GRP78/BiP）表达的影响，GRP78/BiP 是内质网应激最常用的标志物之一。
条件永生化大鼠视网膜周细胞和内皮细胞系在含有 5.5mmol/L 葡萄糖的 Dulbecco 改良 Eagle 培养基中涂有 I 型胶原的培养皿中培养。进行共培养时，将周细胞和内皮细胞一起接种在涂有大鼠尾巴 I 型胶原的细胞培养板上。初始培养 24 小时后，将培养基更换为含有 0-100mmol/L 葡萄糖的无血清培养基，培养时间长达 72 小时。使用 Western blot 检测 GRP78/BiP、caspase-3 和核因子-κB 的表达。
与 5.5mmol/L 葡萄糖的对照水平相比，周细胞、内皮细胞或共培养物暴露于 25、50 或 100mmol/L 葡萄糖 48 小时后，GRP78/BiP 的表达均无明显增加。同样，当培养基葡萄糖水平从 5.5 或 25 降至 1mmol/L 时，GRP78/BiP 的表达也没有变化。当细胞在无葡萄糖培养基中培养 24 小时时，GRP78/BiP 的表达显著增加。这伴随着 caspase-3 和核因子-κB 的表达呈时间依赖性增加。
在糖尿病性视网膜病变中，高血糖已被报道可诱导视网膜毛细血管血管细胞凋亡，但这些研究表明，凋亡与内质网应激最常用的标志物之一 GRP78/BiP 的表达无关。然而，与 GRP78/BiP 相关的凋亡可能在与视网膜缺血/再灌注相关的血管变化中发挥作用。
