甘油二酯激酶&gamma;参与大鼠胚胎脑神经上皮细胞的发育

牛宝贝 崔慧林* 曹锡梅 师亮 马晋峰 乔从进

doi:10.16098/j.issn.0529-1356.2016.04.004

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解剖学报 ›› 2016, Vol. 47 ›› Issue (4) : 456-461. DOI: 10.16098/j.issn.0529-1356.2016.04.004

神经生物学

甘油二酯激酶γ参与大鼠胚胎脑神经上皮细胞的发育

牛宝贝¹ 崔慧林^1* 曹锡梅¹师亮¹马晋峰² 乔从进¹

作者信息 +

Diacylglycerol kinase γ is involved in the neuroepithelium development of rat embryonic brain

NIU Bao-bei¹ CUI Hui-lin ^1* CAO Xi-mei¹SHI Liang¹ MA Jin-feng² QIAO Cong-jin¹

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文章历史 +

摘要

目的探讨甘油二酯激酶γ(DGKγ)在大鼠胚胎脑神经上皮发育中的作用及可能机制。方法 18只胎龄（E）11.5、E12.5、E14.5、E16.5、E18.5大鼠胚胎用于Western blotting方法检测DGKγ在大鼠胚胎脑组织的表达，E9.5～E18.5大鼠胚胎各5只用于免疫组织化学和免疫荧光染色方法检测DGKγ在大鼠胚胎脑的分布以及DGKγ与Ki67、乙酰胆碱转移酶（ChAT）及酪氨酸羟化酶（TH）的细胞内共定位。结果 DGKγ蛋白含量在E11.5～E14.5表达逐渐增多，E16.5表达较E14.5显著减少，E18.5表达显著增多且高于E14.5水平。E10.5～E12.5，DGKγ在脑泡壁出现表达，并随着脑泡的发育表达在端脑、除大脑脚外的中脑、后脑和末脑；E13.5～E14.5，DGKγ的强阳性表达从海马及周围的新皮质、苍白球、嗅脑和大脑脚阳性延伸到除新皮质外的脑的各区域；E15.5至出生前，DGKγ在新皮质阳性表达增强，但在大脑脚和脑桥表达减弱。免疫荧光染色显示，E14.5，与海马和苍白球相比，嗅脑的DGKγ阳性细胞中Ki67阳性率较高（87%），ChAT 阳性率较低（9%）（均P＜0.05），中脑顶盖的DGKγ阳性细胞中62%为TH阳性；E16.5，新皮质的DGKγ阳性细胞中Ki67阳性率为26%，较E14.5时（49%）显著降低（P＜0.05），ChAT阳性率为70%，较E14.5时（45%）显著增高（P＜0.05）；DGKγ多位于神经上皮细胞的胞质，也可见于胞膜上，或者同时位于胞质和胞核。结论 DGKγ在胚胎脑的表达模式和亚细胞分布，提示其参与了胚胎脑神经上皮的增殖、迁移和分化后神经元的发育。

Abstract

Objective To investigate the effects and possible mechanisms of diacylglyceral kinase γ (DGKγ) in the neuroepithelium development of the rat embryonic brain.Methods Western blotting was performed to examine the expression of DGKγ in 18 rat embryonic brains at embryonic day(E)11.5, E12.5, E14.5, E16.5 and E18.5. Five rat embryos each day from E9.5 to E18.5 were stained by immunohistochemistry and immunofluorescence to detect the distribution of DGKγ in rat embryonic brains and the colocalization of DGKγ with Ki67/choline acetyltramsferase(ChAT)/tyrosine hydroxylase(TH) in neuroepithelial cells. Results The expression of DGKγ protein increased gradually from E11.5 to E14.5, decreased significantly at E16.5 than that at E14.5, and the expression of DGKγ at E18.5 increased obviously to the level and higher than that at E14.5. At E10.5-E12.5, DGKγ was expressed from one brain vesicle to telencephalon, mesencephalon, rhombencephalon and myelencephalon except the tegmentum. At E13.5-E14.5, DGKγ was strongly expressed in hippocampus and its adjacent neocortex, pallidum, rhinencephalon and tegmentum, and then extending to all regions of brain except the neocortex. From E15.5 to before birth, the expression of DGKγ increased in neocortex and weakened in tegmentum and pons. Immunofluorescence staining showed that in DGKγ positive cells of rhinencephalon, Ki67 positive rate was higher (87%) and ChAT positive rate was lower (9%) than that of hippocampus and pallidum (all P<0.05). In tectum of E14.5 rat, 62% DGKγ positive cells showed TH positive reaction. In DGKγ positive cells of neocortex, Ki67 positive rate significantly decreased from 49% (E14.5) to 26% (E16.5), while ChAT positive rate was significantly increased from 45% (E14.5) to 70% (E16.5) (all P<0.05). The expression of DGKγ was observed in cytoplasm of neuroepithelial cells, or in membrane, or in both nucleus and cytoplasm. Conclusion The expression pattern of DGKγ in the rat embryonic brain and the subcellular distribution of DGKγ suggest that DGKγ is involved in the proliferation, migration and development of neurons.

导出引用

牛宝贝崔慧林曹锡梅师亮马晋峰乔从进. 甘油二酯激酶γ参与大鼠胚胎脑神经上皮细胞的发育[J]. 解剖学报. 2016, 47(4): 456-461 https://doi.org/10.16098/j.issn.0529-1356.2016.04.004

NIU Bao-bei CUI Hui-lin CAO Xi-mei SHI Liang MA Jin-feng QIAO Cong-jin. Diacylglycerol kinase γ is involved in the neuroepithelium development of rat embryonic brain[J]. Acta Anatomica Sinica. 2016, 47(4): 456-461 https://doi.org/10.16098/j.issn.0529-1356.2016.04.004

参考文献

［1］Ishisaka M, Hara H. The roles of diacylglycerol kinases in the central nervous system: review of genetic studies in mice ［J］. J Pharmacol Sci, 2014, 124(3):336-343.

［2］Goto K, Hozumi Y, Nakano T, et al. Cell biology and pathophysiology of the diacylglycerol kinase family: morphological aspects in tissues and organs［J］. Int Rev Cytol, 2007, 264:25-63.

［3］Adachi N, Oyasu M, Taniguchi T, et al. Immunocytochemical localization of a neuron-specific diacylglycerol kinase beta and gamma in the developing rat brain［J］. Brain Res Mol Brain Res, 2005, 139(2):288-299.

［4］Matsubara T, Shirai Y, Myiasaka K, et al. Nuclear transportation of diacylglycerol kinase γ and its possible function in the nucleus［J］. J Biol Chem, 2006, 281(10):6152-6164.

［5］Yamada K, Sakane F, Imai Si, et al. Regulatory role of diacylglycerol kinase gamma in macrophage differentiation of leukemia cells［J］. Biochem Biophys Res Commun, 2003, 305(1):101-107.

［6］Tanino F, Maeda Y, Sakai H, et al. Induction of filopodia-like protrusions in N1E-115 neuroblastoma cells by diacylglycerol kinase gamma independent of its enzymatic activity: potential novel function of the C-terminal region containing the catalytic domain of diacylglycerol kinase gamma［J］. Mol Cell Biochem, 2013, 373(1-2):85-93.

［7］Altman J, Bayer SA. Altas of Prenatal Rat Brain Development［M］. Boca Raton: CRC Press Inc, 1994:1-453.

［8］Wolpert L, Jessell T, Lawrene P, et al. Principles of Development ［M］. Oxford: Oxford University Press, 2006:387-396.

［9］Kee N, Sivalingam S, Boonstra R, et al. The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis［J］. J Neurosci Methods, 2002, 115(1):97-105.

［10］Groves NJ, Bradford D, Sullivan RK, et al. Behavioral effects of adult vitamin D deficiency in BALB/c mice are not associated with proliferation or survival of neurons in the adult hippocampus［J］. PLoS One, 2016, 11(4):e0152328.

［11］Goto K, Funayama M, Kondo H. Cloning and expression of a cytoskeleton-associated diacylglycerol kinase that is dominantly expressed in cerebellum［J］. Proc Natl Acad Sci, 1994, 91(26):13042-13046.

［12］Shirai Y, Segawa S, Kuriyama M, et al. Subtype-specific translocation of diacylglycerol kinase alpha and gamma and its correlation with protein kinase C［J］. J Biol Chem, 2000, 275(32):24760-24766.

［13］Batista EL Jr, Warbington M, Badwey JA, et al. Differentiation of HL-60 cells to granulocytes involves regulation of select diacylglycerol kinases (DGKs)［J］. J Cell Biochem, 2005, 94(4):774-793.

［14］Kamiya K, Ryer E, Sakakibara K, et al. Protein kinase C delta activated adhesion regulates vascular smooth muscle cell migration［J］.J Surg Res, 2007, 141(1):91-96.

［15］Li J, Niu BB, Liu HX, et al. Expression and localization of diacylglycerol kinase ζ, protein kinase C δ and Tis21 in the developing rat brain［J］. Acta Anatomica Sinica, 2015, 46(5):609-615.(in Chinese)

李佳, 牛宝贝, 刘慧霞, 等. 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［J］. 解剖学报, 2015, 46(5):609-615.

［16］Takeshita E, Kume A, Maeda Y, et al. Diacylglvcerol kinase γ is a novel anionic phospholipid binding protein with a selective binding preference［J］. Biochem Biophys Res Commun, 2014, 444(4):617-621.

［17］Schambra UB, Sulik KK, Petrusz P, et al. Ontogeny of cholinergic neurons in the mouse forebrain［J］. J Comp Neurol, 1989, 288(1): 101-122.

［18］Messinqham KA, Aust S, Helfenberqer J, et al. Autoantibodies to collagen XVII are present in Parkinson’s disease and localize to tyrosine-hydroxylase positive neurons［J］. J Invest Dermatol, 2016, 136(3):721-723.