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目的探讨Wnt5a/Frizzled-2信号在创伤性颅脑损伤(TBI)后神经细胞钙超载过程中的作用。方法体内实验:成年Sprague-Dawley大鼠(96只),随机分为正常组A(32只),单纯损伤组B(32只)和RNAi抑制组C(32只)。B组和C组Fenny式法制作中型颅脑损伤模型。其中C组模型建立前48 h采用立体定向海马区注射RNAi以抑制Frizzled-2的表达。伤后24 h处死大鼠,取伤侧海马组织,用western blotting方法检测其中Frizzled-2和Wnt5a含量,用免疫荧光染色和激光共聚焦显微镜检测其中钙离子浓度。单因素方差分析比较各组间的差别。结果 A组大鼠海马组织细胞中稳定表达着Wnt5a/Frizzled-2信号。TBI后,与A组相比,B组海马组织中的Wnt5a和Frizzled-2的表达分别升高了2倍和5倍(P<0.01),海马细胞中钙离子的含量显著增高(P<0.01)。与B组相比,随着Frizzled-2的抑制,C组海马细胞中的Wnt5a和Frizzled-2的表达分别降低1倍、3.5倍(P<0.01),同时细胞中的钙离子含量显著降低1.5倍,接近A组(P<0.01)。结论在生理和病理条件下,Wnt5a/Frizzled-2信号在调节神经细胞中的钙离子浓度变化中起到重要的作用,同时我们的研究提示该信号的相关重要组成因子Wnt5a,Fzd2和P-CamKII以及针对Frizzled-2设计的特异性RNAi可以作为日后TBI研究和治疗的相关靶点。
Objective To investigate the role of Wnt5a / Frizzled-2 signaling in the process of calcium overload in neural cells after traumatic brain injury (TBI). Methods In vivo, 96 adult Sprague-Dawley rats were randomly divided into normal control group (32 rats), simple injury group (32 rats) and RNAi inhibition group (32 rats). B group and C group Fenny method of making medium-sized brain injury model. In group C, RNAi was injected into the hippocampus 48 h before establishment to inhibit Frizzled-2 expression. The rats were sacrificed at 24 hours after injury and the injured hippocampus tissues were harvested. The contents of Frizzled-2 and Wnt5a were detected by western blotting. The concentration of calcium was detected by immunofluorescence staining and confocal laser scanning microscopy. One-way ANOVA was used to compare the differences among the groups. Results The Wnt5a / Frizzled-2 signal was stably expressed in hippocampal cells of group A. Compared with group A, the expression of Wnt5a and Frizzled-2 in group B were increased by 2-fold and 5-fold respectively (P <0.01), and the content of calcium in hippocampus was significantly increased after TBI (P <0.01) ). Compared with group B, the expression of Wnt5a and Frizzled-2 in C group was decreased by 1 time and 3.5 times (P <0.01) with the inhibition of Frizzled-2, and the content of calcium in cells was significantly reduced by 1.5 Times, close to the A group (P <0.01). Conclusions Wnt5a / Frizzled-2 signaling plays an important role in regulating the changes of calcium concentration in nerve cells under physiological and pathological conditions. Meanwhile, our study suggests that Wnt5a, Fzd2 and P-CamKII As well as specific RNAi designed for Frizzled-2 can serve as a relevant target for future TBI research and therapy.