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目的观察大鼠海马注射溶血磷脂酸(LPA)后磷酸化Rb蛋白的表达及神经元凋亡的变化。方法将60只Wistar大鼠分为实验组(n=20)注射LPA、对照组(n=20)注射PBS和拮抗剂组(n=20)注射LPA+苏拉明,利用脑立体定位技术在大鼠双侧海马微量注射溶血磷脂酸溶剂,于注射后12、24、48和72h各不同时间点采用免疫荧光组织化学方法测定该区域神经细胞中ser795位点磷酸化Rb蛋白(p-Rb,ser795)的表达水平;TUNEL技术检测神经细胞凋亡情况。结果实验组注射LPA后12h海马CA3区神经细胞中p-Rb表达增加,注射LPA24h后表达到达高峰,且均高于拮抗剂组和对照组(P<0.05)。LPA注射后12h可检测到凋亡细胞,24h TUNEL阳性细胞数达高峰,且阳性细胞数均多于拮抗剂组和对照组(P<0.05)。LPA注射后的12h和24h可观察到p-Rb和TUNEL分别以镶嵌和重叠的方式共定位。结论 LPA在动物水平上诱导了Rb蛋白的磷酸化并导致了神经细胞早期的凋亡,可能间接或者不参与其晚期凋亡过程。
Objective To investigate the expression of phosphorylated Rb protein and neuronal apoptosis in hippocampus of rats after injection of lysophosphatidic acid (LPA). Methods Sixty Wistar rats were divided into experimental group (n = 20), LPA injection group (n = 20), PBS and antagonist group (n = 20) The bilateral hippocampal microinjection of lysophosphatidic acid solvent was used to detect the phosphorylation of Rb protein at the ser795 site (p-Rb, ser795) by immunofluorescence histochemistry at different time points of 12, 24, 48 and 72 h after injection ). The TUNEL technique was used to detect the neuronal apoptosis. Results The expression of p-Rb in neurons of hippocampal CA3 region increased 12 h after injection of LPA in experimental group, and peaked at 24 h after injection of LPA, both of which were higher than those of antagonist group and control group (P <0.05). Apoptotic cells were detected at 12h after injection of LPA. The number of TUNEL positive cells reached the peak at 24h, and the number of positive cells was more than that of antagonist group and control group (P <0.05). At 12 h and 24 h after LPA injection, p-Rb and TUNEL were observed to co-localize in a mosaic and overlapping manner, respectively. Conclusion LPA induces the phosphorylation of Rb protein at the animal level and leads to the early apoptosis of nerve cells, which may or may not be involved in the late apoptosis process.