摘要
目的探讨Src信号通路在室管膜下区（subventricular zone，SVZ）表达胆碱乙酰基转移酶（choline acetyltransferase，ChAT）的胆碱能神经元促进脑缺血后神经再生中的作用。方法成年雄性昆明小鼠84只，按随机数字表法分为假手术±溶剂组（18只）、脑缺血±溶剂组（22只）、脑缺血±多奈哌齐（胆碱酯酶抑制剂）组（21只）和脑缺血±多奈哌齐±KX2—391（Src阻断剂）组（23只）。用改良神经功能评分（modified neurological severity scores，mNSS）评价神经功能改善情况。采用Ki67标记增殖的细胞，双肾上腺皮质激素（doublecortin，DCX）标记神经母细胞，免疫荧光观察各组小鼠SVZ内Ki67’／DCX’细胞的表达。Westernblot分析各组小鼠SVZ中Ki67、磷酸化表皮生长因子受体（phos—pho—epidermal growth factor receptor，p-EGFR）、p-Raf、Src和P—Akt蛋白表达量的变化。结果脑缺血±多奈哌齐±KX2—391组小鼠在各评估时间点mNSS分值较脑缺血±多奈哌齐组明显降低，差异有统计学意义（P〈0．05）。术后第10天，脑缺血±多奈哌齐组小鼠SVZ中Ki67’／DCX’细胞数为（125．33±13．71）个／视野，脑缺血±多奈哌齐±KX2．391组细胞数为（71．67±18．35）个／视野，差异有统计学意义（P〈0．05）。此外，脑缺血±多奈哌齐组小鼠Ki67、p-EGFR、p-Raf、Src和p-Akt蛋白表达量显著升高，其相应光密度为1．39±0．23，1．42±0．19，0．88±0．13，1．14±0．19，1．04±0．18，脑缺血±多奈哌齐±KX2—391组小鼠Ki67、P-EGFR、P—Raf、Src和P—Akt蛋白表达量显著减少，其相应光密度为0．84±0．26，0．94±0．26，0．73±0．15，0．71±0．18，0．81±0．19（P〈0．05）。结论脑缺血后SVZ内ChAT±神经元可能通过Src信使间接上调表皮生长因子受体信号通路，进而促进SVZ神经干细胞增殖。
Objective To investigate the role of Src signaling pathway in neurogenesis promoted by choline acetyhransferase （CHAT） ＋ neurons in the subventricular zone （ SVZ ） after ischemic stroke. Methods The eighty-four mice were randomly assigned into four groups：sham-operated mice treated with vehicle （Sham＋vehicle, n= 18）, middle cerebral artery occlusion （MCAO）-operated mice treated with vehicle （ MCAO＋vehicle, n= 22） , MCAO mice treated with donepezil （ MCAO＋donepezil, n= 21 ）, MCAO mice treated with donepezil and Src inhibitor KX2-391 （MCAO＋donepezil＋KX2-391 ,n= 23 ）.Mice were subjected to the temporary MCAO model of ischemic stroke.Modified neurological severity score （mNSS） was used to assess neurologic function of the mice. Proliferative cells were labeled with Ki67, and neuroblasts with douhlecortin （DCX）.The expression of Ki67＋/DCX＋ in the SVZ was detected by immunofluorescence.The ex- pression of Ki67, phospho-epidermal growth factor receptor （p-EGFR） , p-Raf, Src and p-Akt in the SVZ were quantified by Western blot. Results MCAO±donepezil＋KX2-391 group showed worse performance in the mNSS test than MCAO＋donepezil group （P〈0.05）.Ten days after MCAO,the number of Ki67＋/DCX＋ cells in the SVZ of MCAO±donepezil group was 125.33± 13.71/area,which was 71.67± 18.35/area in MCAO＋donepezil＋KX2-391 group （P〈0.05）.What＇ s more, the expression of proteins Ki67, p-EGFR, p-Raf, Src and p-Akt in mice of M CAO＋donepezil group was markedly increased, which was （ 1.39± 0.23 ） , （ 1.42± 0.19 ） , （0.88±0.13） ,（1.14±0.19）, （1.04±0.18） and it was decreased in MCAO＋donepezil＋KX2-391 group, which was 0.84±0.26,0.94±0.26,0.73±0.15,0.71±0.18,0.81±0.19（P〈0.05）.Conclusion CHAT＋ neurons in SVZ may promote neurogenesis after stroke via Srcepidermal growth factor receptor （EGFR） signaling pathway.
出处
《中华行为医学与脑科学杂志》
CAS
CSCD
北大核心
2017年第10期922-926,共5页
Chinese Journal of Behavioral Medicine and Brain Science
基金
国家自然科学基金项目（81571137）
河南省科技创新人才计划项目（154200510014）
河南省医学科技攻关计划项目（201501005）
