甘油二酯激酶ζ、蛋白激酶Cδ和Tis21在发育中大鼠脑的表达和定位

Expression and localization of diacylglycerol kinase ζ,protein kinase C δ and Tis21 in the developing rat brain

目的 探讨甘油二酯激酶ζ（DGKζ）、蛋白激酶Cδ（PKCδ）和Tis21在大鼠胚胎脑的时空表达模式以及与脑发育的关系。方法 胚龄（E）9.5-E18.5大鼠胚胎各5只,连续切片用于免疫组织化学方法检测DGKζ、p-PKCδ和Tis21在大鼠胚胎脑的表达;22只胚龄E11.5、E12.5、E14.5、E16.5和E18.5大鼠胚胎用于Western blotting,检测DGKζ、磷酸化PKCδ（p-PKCδ）、PKCδ和Tis21在大鼠胚胎脑组织的表达。结果 免疫组织化学显示,于E9.5 p-PKCδ和Tis21在神经沟神经上皮呈阳性表达,DGKζ为阴性;E10.5-E11.5,DGKζ、p-PKCδ和Tis21在脑泡壁均呈阳性表达;在E12.5三者均表达于端脑、间脑、中脑腹侧和脑桥处神经上皮,而中脑背侧、峡部和小脑原基仅DGKζ呈阳性表达,p-PKCδ和Tis21呈阴性;于E13.5三者沿海马、隔、苍白球及嗅脑呈阳性分布,在新皮质呈阴性,p-PKCδ和Tis21在峡部和小脑原基出现阳性表达;于E14.5 DGKζ表达延伸到新皮质,在脑的各个区域呈现出全阳性。而p-PKCδ和Tis21表达仅延伸到中脑顶盖,在新皮质仍为阴性;于E15.5 p-PKCδ和Tis21在新皮质出现阳性表达,在脑的各个区域均呈阳性;E16.5-E18.5,三者在小脑和脑桥表达范围缩小;DGKζ和p-PKCδ可表达在神经上皮细胞的胞质,或胞质和细胞核。Western blotting显示,DGKζ和Tis21在E11.5-E14.5表达逐渐增多,E12.5较E11.5显著增多,在E16.5和E18.5逐渐下降;p-PKCδ/PKCδ显示PKCδ活性变化趋势与DGKζ表达趋势一致。结论 DGKζ、p-PKCδ和Tis21时空表达与脑发育模式一致,DGKζ和p-PKCδ在神经上皮细胞的亚细胞分布提示它们主要通过DGKζ/Tis21和PKCδ/Tis21途径参与脑的发育。

Objective To investigate the spatio-temporal expression patterns and the relationship of diacylglyceral kinase ζ （ DGKζ ） , protein kinase CS（PKC 8） and Tis21 in the developing rat brain. Methods Serial sections of five rat embryos each day from E9. 5 to El8.5 were stained immunohistochemically to detect the expression of DGKζ, phosphated PKC 8（p-PKC 8） and Tis21 in rat embryonic brains. Western blotting was performed to examine the expression of DGKζ, p-PKCS, PKC8 and Tis21 in 22 rat embryonic brains at Ell. 5, E12.5, E14.5, E16. fi and E18.5. Results Immunohistochemistry staining showed that p-PKC8 and Tis21 were positively expressed in the neuroepithelium of neural groove while DGKζ was negatively expressed at E9.5. DGKζ, p-PKC8 and Tis21 were positively expressed in the neuroepithelium of brain vesicles at El0.5 and Ell. 5. At El2.5, DGKζ, p-PKC8 and Tis21 were positively expressed in the neuroepithelum of telencephalon, diencephalon, ventral mesencephalon and pons. DGKζ was positively expressed in dorsal mesencephal, isthmus and cerebellar primordium, while the expression of p-PKC8 and Tis21 was not observed until El3.5. At El3.5, the expression of all of these three proteins was positive in neuroepithelium and differentiating field of hippocampus, pallidum, septal and rhinencephalic and negative in neocortex. At El4. 5, with the positive expression extending to neocortex, DGKζ was expressed in the nervous tissue of whole brain. The expression of p-PKC8 and Tis21 onlyextended to rectum. DGKζ expression pattern happened to p-PKCζ and Tis21 at El5.5. At El6. 5-E18.5, their expression became weaker and was restricted to the cerebellum germinal layer and pontine nucleus. DGKζ and p-PKCζ were expressed in cytoplasm of neuroepithelial cells, or in both cytoplasm and nucleus. Western blotting showed that the expression of DGKζ and Tis21 protein increased gradually at Ell. 5, El2. 5 and El4. 5, and decreased gradually at El6.5 and El8.5. The expression was significantly increased at E12.5 compared with Ell. 5. The ratio of p-PKC8 to PKC8 showed that the change of PKC8 activity had the same tendency with DGKζ expression. Conclusion The spatio-temporal expression of DGKζ, p-PKC8 and Tis21 conforms the development pattern of rat embryonic brain. The subcellular distribution of DGK and p-PKC8 in neuroepithelial cells suggests that they may regulate the development of the embryonic brain via DGKζ Tis21 and PKCS/Tis21 signaling pathway.