G蛋白偶联受体56基因敲除抑制少突胶质前体细胞成熟

Knockout of GPR56 gene inhibits maturation of oligodendrocyte progenitor cells

目的:探讨G蛋白偶联受体56(GPR56)基因敲除对小鼠脑胼胝体内轴突髓鞘化和少突胶质前体细胞(OPCs)成熟的影响。方法:筛选出GPR56基因杂合型(GPR56+/-)和敲除型(GPR56-/-)小鼠36只,分为GPR56+/-和GPR56-/-组,每组18只。每组根据小鼠出生后时间分为出生后7 d(P7)、14 d(P14)、21 d(P21)和28d(P28)4个亚组。应用FluoroMyelin染色观察P14、P21和P28 GPR56+/-和GPR56-/-小鼠脑胼胝体内髓鞘形成。用电镜观察P28 GPR56+/-和GPR56-/-小鼠胼胝体内轴突髓鞘化,比较髓鞘的厚度。用荧光免疫组化染色观察P7GPR56+/-和GPR56-/-小鼠胼胝体内血小板源性生长因子α受体阳性(PDGF-αR+)细胞(即OPCs)的数量。用原位杂交监测P28 GPR56+/-和GPR56-/-小鼠胼胝体内髓鞘蛋白脂质蛋白阳性(PLP+)细胞数。用出生后1 d的GPR56+/-和GPR56-/-小鼠脑皮质做体外OPCs培养并诱导其分化成熟,观察pro-oligodendroblast、immature oligodendrocyte和mature oligodendrocyte阶段O4+细胞百分比。结果:与GPR56+/-小鼠比较,在P14、P21和P28GPR56-/-小鼠脑胼胝体中髓鞘的形成明显减少。电镜见P28 GPR56-/-小鼠脑胼胝体内髓鞘化轴突的数量明显减少,髓鞘g-ratio值变大,髓鞘厚度变薄。荧光免疫组化和原位杂交结果显示P7 GPR56+/-和GPR56-/-小鼠胼胝体内PDGF-aR+细胞数量无差异,但P28 GPR56+/-小鼠胼胝体内PLP+细胞数明显多于P28 GPR56-/-小鼠。体外细胞培养结果显示在pro-oligodendroblast阶段GPR56-/-O4+细胞百分比明显多于GPR56+/-O4+细胞,在immature oligodendrocyte和mature oligodendrocyte阶段GPR56-/-O4+细胞百分比明显少于GPR56+/-O4+细胞。结论:GPR56蛋白可能参与了脑白质轴突髓鞘化和OPCs的成熟。

AIM： To explore the effect of G-protein-coupled protein 56 （GPR56） gene knockout on axonal my elination and the maturation of oligodendrocyte progenitor cells （OPCs） in the corpus callosum of mouse brain. METH ODS： Thirty-six GPR56 ^＋/- and GPR56^＋/- mice were selected and divided into GPR56 ^＋/- group and GPR56^＋/- group （18 mice in each group）. According to the postnatal days, the mice in each group were further divided into P7, P14, P21 and P28 subgroups. Myelin formation in the corpus callosum of P14, P21 and P28 GPR56 ^＋/- and GPR56 ^＋/- mice was ob- served by FluoroMyelin staining. The number of myelinated axons and thickness of myelin sheaths were measured by elec tron microscopy. The numbers of platelet-derived growth factor alpha receptor-positive （ PDGF-aR ＋ ） and proteolipid pro tein-positive （ PLP~ ） ceils in the corpus callosum of GPR56^＋/-and GPR56^＋/- mice were compared by the methods of im- munofluorescence and in situ hybridization. GPR56^＋/- and GPR56^＋/- OPCs were cultured using P1 GPR56^＋/- and GPR56^＋/- mouse brain cortex and induced differentiation and maturation in vitro. The percentage of GPR56^＋/- and GPR56-/- 04 ＋ cells in pro-oligodendroblast, immature oligodendrocyte and mature oligodendrocyte stages was compared by04 RESULTS： The myelin formation was obviously reduced in the corpus callosum of P14, P21 andP28 GPR.56^＋/- mice as compared with GPR56~z- mice. The number of myelinated axons was obviously reduced and the g ratio value was increased significantly in the corpus callosum of P28 GPR56-/- mice. No significant difference of the PDGF-aR ＋ cell number in the corpus callosum between P7 d GPR56＋/- and GPR56-/- mouse brains was observed. The number of PLP~ cells was significantly decreased in the corpus callosum of P28 GPR.56-/- mice as compared with GPR56 ＋/- mice. The percentage of GPR56 -/- 04 ＋ cells in pro-oligodendroblast stages was obviously higher than that of GPR56^＋/- 04＋ cells. On the contrary, the percentages of GPR.56^＋/- 04＋ ceils in immature oligodendrocyte and mature oligodendrocyte stages were significantly reduced. CONCLUSION： GPR.56 may be involved in axonal myelination and OPCs maturation in the corpus callosum of mouse brain.