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为检测以LRP6受体为激活开关的Wnt-β-catenin信号通路与PrP106-126毒性多肽引起的神经元损伤之间的相互关系,并研究该信号通路与神经保护性蛋白REST的相关性,从而进一步阐明Wnt-LRP6-REST对毒性多肽引起的神经元损伤的影响。利用Wnt信号通路的激活剂Licl、抑制剂DKK1或PrP106-126毒性多肽刺激原代神经元,通过免疫印迹检测神经保护性蛋白REST及Wnt信号通路下游相关蛋白的变化情况,通过激光共聚焦观察REST与Wnt信号通路正相关蛋白β-catenin的共定位情况。分别构建LRP6的过表达质粒pCMV-C-HALRP和干扰质粒pGPH1/GFP/Neo-LRP6-Rat shRNA-1和shRNA-2,将已经构建好的质粒转染进入原代神经元。通过免疫印迹检测PrP106-126毒性多肽对Wnt信号通路标志性蛋白(β-catenin及GSK3β)的影响,检测LRP6的过表达或干扰对REST或Wnt信号通路下游蛋白的影响。通过免疫荧光观察LRP6对由毒性多肽诱导的神经纤维损伤的影响,用流式细胞仪检测相应的细胞活力。由LRP6受体激活的Wnt-β-catenin信号通路在一定程度上正向调控神经保护性蛋白REST的表达,LRP6在信号调节的过程中起到了关键作用,并且LRP6对由PrP106-126毒性多肽引起的神经元损伤有缓解作用,LRP6的过表达增加了神经元细胞的活力,起到了神经保护作用。
To examine the relationship between the Wnt-|Â-catenin signaling pathway activated by the LRP6 receptor and the neuronal damage induced by the PrP106-126 toxic polypeptide and to investigate the correlation of this signaling pathway with the neuroprotective protein REST Further elucidate the effect of Wnt-LRP6-REST on neuronal damage induced by toxic peptides. The primary neurons were stimulated by activator Wnt signaling pathway inhibitor Licl, inhibitor DKK1 or PrP106-126. Western blotting was used to detect the changes of downstream proteins related to REST and Wnt signaling pathways. Western blotting was used to detect REST Co-localization with β-catenin, a positive correlate of Wnt signaling pathway. The recombinant plasmids pCMV-C-HALRP and interference plasmids pGPH1 / GFP / Neo-LRP6-Rat shRNA-1 and shRNA-2 were constructed respectively and transfected into primary neurons. The effects of PrP106-126 cytotoxicity on the Wnt signaling proteins (β-catenin and GSK3β) were detected by Western blotting. The effect of LRP6 overexpression or interference on downstream proteins of REST or Wnt signaling pathway was detected. The effect of LRP6 on nerve fiber injury induced by toxic polypeptide was observed by immunofluorescence, and the corresponding cell viability was detected by flow cytometry. The Wnt-|Â-catenin signaling pathway activated by the LRP6 receptor positively regulates the expression of the protective protein REST to a certain extent. LRP6 plays a key role in signal regulation, and LRP6 is caused by the PrP106-126 toxic polypeptide Of neuronal damage have a mitigating effect, LRP6 overexpression increased neuronal cell viability, played a neuroprotective effect.