背景:内皮细胞对于维持血脑屏障的稳态至关重要。肌动蛋白丝(Girdin)和磷光体(p)-Girdin的大梁对于将人脑微血管内皮细胞(HBMECs)吞噬成血小板(PLT)至关重要。但潜在机制尚不清楚,需要进一步研究.
方法:在PLT和细胞松弛素D治疗后,Hoechst33,342检测到细胞凋亡。使用蛋白质印迹测定靶向Girdin(sh-Girdin)或过表达Girdin(OE-Girdin)的短发夹RNA的转染效率。Sh-Girdin,OE-Girdin,突变的Girdin(m-Girdin),和微丝结合区缺失的Girdin(Del-Girdin)在PLT条件下转染到HBMECs中。随后,流式细胞术和透射电镜检测PBMECs对PLT的吞噬。通过蛋白质印迹定量Girdin和磷酸化(P)-Girdin水平。免疫组织化学(IHC)检测Girdin的阳性表达。PLT的本地化,Girdin,通过共聚焦显微镜分析了p-Girdin和HBMECs在PLT中的吞噬。
结果:细胞松弛素D逆转了PLT对细胞凋亡的抑制作用。OE-Girdin增强了PLT标记的荧光强度和PLT对HBMECs的吞噬,而sh-Girdin,m-Girdin,Del-Girdin反向地跑了.OE-Girdin提高了Girdin和p-Girdin水平,而sh-Girdin和Del-Girdin正好相反,但m-Girdin并不影响p-Girdin和Girdin水平。
结论:Girdin和p-Girdin在HBMECs中与PLT共同定位。Girdin的过表达被鉴定为与PTL的吞噬增加有关。Girdin可能是缓解内皮细胞凋亡的有效靶点。
BACKGROUND: Endothelial cells are crucial in maintaining the homeostasis of the blood-brain barrier. Girders of actin filament (Girdin) and phosphor (p)-Girdin are essential for the engulfment of human brain microvascular endothelial cells (HBMECs) into platelets (PLTs), but the potential mechanism remains unclear and requires further study.
METHODS: Following PLT and cytochalasin D treatment, Hoechst 33,342 detected apoptosis. The transfection efficiency of the short hairpin RNA targeting Girdin (sh-Girdin) or overexpressing Girdin (OE-Girdin) was determined using western blotting. Sh-Girdin, OE-Girdin, mutated Girdin (m-Girdin), and microfilament binding region deleted Girdin (Del-Girdin) were transfected into HBMECs under PLT conditions. Subsequently, the engulfment of HBMECs by PLTs was detected by flow cytometry and transmission electron microscopy. Girdin and phosphorylated (p)-Girdin levels were quantified by western blot. The positive expression of Girdin was measured by immunohistochemistry (IHC). The localization of PLT, Girdin, and p-Girdin and the engulfment of HBMECs in PLTs were analyzed by confocal microscopy.
RESULTS: Cytochalasin D overturned the inhibitory effect of PLT on cell apoptosis. OE-Girdin enhanced the fluorescent intensity of PLT-labelling and the engulfment of HBMECs by PLTs, while sh-Girdin, m-Girdin, and Del-Girdin ran reversely. OE-Girdin elevated the Girdin and p-Girdin levels, while sh-Girdin and Del-Girdin were the opposite, but m-Girdin did not affect the p-Girdin and Girdin levels.
CONCLUSIONS: Girdin and p-Girdin were co-located with PLTs in HBMECs. The over-expression of Girdin was identified as being associated with the increasing engulfment of PTLs. Girdin may be an effective target to alleviate endothelial cell apoptosis.