Cui Huilin, Du Jiazheng, Xie Jianshan, Zhang Jixia, Tao Yun, Huang Yige, Li Lei, Cao Ximei, Zhang Yu
Department of Histology and Embryology, Shanxi Medical University, Taiyuan 030001, China.
Key Laboratory of Cellular Physiology, Ministry of Education, and the Department of Physiology, Shanxi Medical University, Taiyuan 030001, China.
Acta Biochim Biophys Sin (Shanghai). 2024 Oct 23;57(2):250-260. doi: 10.3724/abbs.2024156.
In this study, we aim to investigate diacylglycerol kinase (DGK) γ expression in developing neural tubes (NTs) and its effects on neural stem cell (NSC) proliferation and migration. Whole-mount hybridization (WMISH) and immunohistochemistry are performed to explore DGKγ localization in developing NTs . NSCs are treated with sh-DGKγ, R59949, or PMA . Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and neurosphere formation assay are utilized to evaluate NSC proliferation. Neurosphere migration assay and a transwell chamber assay are used to assess NSC migration. The diacylglycerol (DAG) content is detected via enzyme-linked immunosorbent assay (ELISA). The mRNA expression of DGKγ is detected via quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression levels of DGKγ, protein kinase C (PKC) and phosphorylated PKC (p-PKC) are detected via western blot analysis. The results show that DGKγ mRNA is expressed predominantly in developing NTs. The neuroepithelium in developing NTs is positive for NSC markers, including Nestin, glial fibrillary acidic protein (GFAP), and DGKγ. DGKγ is expressed in the cytoplasm and nucleus of the neuroepithelium and is coexpressed with p-PKCγ and p-PKCδ. The proliferation of NSCs, the number of EdU-positive NSCs, and the number of neurospheres are decreased by sh-DGKγ and R59949 but increased by PMA. There is a shorter migration distance of NSCs and fewer migrated NSCs in the sh-DGKγ, R59949 and PMA groups. DAG content and the p-PKCδ/PKCδ ratio are increased by sh-DGKγ, R59949 and PMA, whereas the p-PKCγ/PKCγ ratio is decreased by PMA. Taken together, our findings indicate that DGKγ facilitates NSC proliferation and migration, which is responsible for the participation of DGK in NT development. DGKγ facilitates NSC migration via the DAG/PKCδ pathway.
在本研究中，我们旨在研究二酰基甘油激酶（DGK）γ在发育中的神经管（NT）中的表达及其对神经干细胞（NSC）增殖和迁移的影响。进行全胚胎原位杂交（WMISH）和免疫组织化学以探索DGKγ在发育中的NT中的定位。用sh-DGKγ、R59949或佛波酯（PMA）处理NSC。利用细胞计数试剂盒-8（CCK-8）检测、5-乙炔基-2'-脱氧尿苷（EdU）检测和神经球形成检测来评估NSC增殖。使用神经球迁移检测和Transwell小室检测来评估NSC迁移。通过酶联免疫吸附测定（ELISA）检测二酰基甘油（DAG）含量。通过定量实时聚合酶链反应（qRT-PCR）检测DGKγ的mRNA表达。通过蛋白质印迹分析检测DGKγ、蛋白激酶C（PKC）和磷酸化PKC（p-PKC）的蛋白表达水平。结果显示，DGKγ mRNA主要在发育中的NT中表达。发育中的NT中的神经上皮对NSC标志物呈阳性，包括巢蛋白、胶质纤维酸性蛋白（GFAP）和DGKγ。DGKγ在神经上皮的细胞质和细胞核中表达，并与p-PKCγ和p-PKCδ共表达。sh-DGKγ和R59949降低了NSC的增殖、EdU阳性NSC的数量和神经球的数量，但PMA使其增加。在sh-DGKγ、R59949和PMA组中，NSC的迁移距离较短，迁移的NSC较少。sh-DGKγ、R59949和PMA增加了DAG含量和p-PKCδ/PKCδ比值，而PMA降低了p-PKCγ/PKCγ比值。综上所述，我们的研究结果表明，DGKγ促进NSC增殖和迁移，这是DGK参与NT发育的原因。DGKγ通过DAG/PKCδ途径促进NSC迁移。
