解剖学报

解剖学报 ›› 2019, Vol. 50 ›› Issue (2): 158-165.doi: 10.16098/j.issn.0529-1356.2019.02.004

• 神经生物学 • 上一篇    下一篇

半胱氨酸蛋白酶抑制剂C 对脑缺血再灌注大鼠脑皮质中热休克蛋白70、微管相关蛋白轻链3蛋白表达和神经元超微结构的影响

陈萌1*杜超2 田焕娜3 王爱乐4 夏晓晓5   

  1. 1.承德医学院基础医学院电子显微镜室; 2.承德医学院基础医学院生物化学教研室; 3.承德医学院基础医学院基础医学研究所 4.承德市中医院ICU室； 5.承德医学院2016级临床本科，河北 承德 067000
  • 收稿日期:2018-05-03 修回日期:2018-07-10 出版日期:2019-04-06 发布日期:2019-04-06
  • 通讯作者: 陈萌 E-mail:chenmeng_glia@163.com
  • 基金资助:
    河北省自然科学青年基金项目;河北省卫生计生委项目;承德医学院大学生科研项目

Effects of cystatin C pretreatment on expression of heat shock protein 70，microtubule-associated protein light chain-3 and ultrastructure in rats with cerebral cortex after ischemia and reperfusion

 CHEN Meng 1* DU Chao2 TIAN Huan-na3 WANG Ai-le4 XIA Xiao-xiao5   

  1. 1.Department of Electron Microscope; 2.Department of Biochemistry; 3.Institute of Basic Medical Sciences, Basic Medical College, Chengde Medical College;
     4.ICU Room of Chengde Traditional Chinese Medicine Hospital; 5.Grade 2016 Undergraduate Medical Student, Chengde Medical College, Hebei Chengde 067000, China
  • Received:2018-05-03 Revised:2018-07-10 Online:2019-04-06 Published:2019-04-06
  • Contact: CHEN Meng E-mail:chenmeng_glia@163.com

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摘要：

目的 应用不同浓度的半胱氨酸蛋白酶抑制剂C(Cys C)干预脑缺血再灌注大鼠，检测热休克蛋白70(HSP70)和微管相关蛋白轻链3(LC3) 蛋白的表达，观察其对神经元超微结构的影响。 方法 成年雄性SD大鼠60只，随机分成5组：假手术组(sham)、缺血再灌注组(IR)，Cys C低浓度组(Cys C1)、Cys C中浓度组(Cys C2)、Cys C高浓度组(Cys C3)，每组12只。采用线栓法制备脑缺血再灌注损伤模型，缺血2 h再灌注24 h后进行改良神经功能损伤严重程度评分(mNSS)。Western blotting半定量检测损伤中心脑皮质组织中HSP70、LC3-Ⅱ/LC3-Ⅰ蛋白表达；免疫组织化学法检测HSP70、LC3的阳性细胞个数；免疫荧光双标记法检测皮质神经元自噬相关蛋白LC3和神经元核抗原(NeuN)共染的阳性细胞平均吸光度值；透射电子显微镜下观察神经元超微结构的变化。 结果 与IR组相比，Cys C1、Cys C2组mNSS评分明显减少(P<0.05)，HSP70的表达较IR组明显升高(P<0.01)；而Cys C3组mNSS评分升高，与IR组差别无显著性 (P>0.05)；Cys C3组HSP70的表达明显降低(P<0.01)；Cys C各浓度组LC3-Ⅱ/LC3-Ⅰ的表达有不同程度的升高(P<0.01)；LC3和NeuN共染阳性细胞平均吸光度逐渐增强(P<0.05)；透射电子显微镜结果显示，Cys C1、Cys C2组神经细胞的超微结构有所改善，而Cys C3组脑皮质神经细胞损伤较重。 结论 Cys C可能在一定浓度范围内对缺血再灌注损伤神经细胞有保护作用。浓度过高则导致神经细胞自噬性死亡。

关键词: 脑缺血再灌注, 自噬, 热休克蛋白70, 微管相关蛋白轻链3, 免疫印迹法, 免疫荧光, 大鼠

Abstract:

Objective To study the effect of different concentrations of cystatin C (Cys C) pretreatment on apoptosis related proteins, the expression of heat shock protein 70(HSP70) and autophagy related proteins, the expression of microtubule-associated protein light chain-3(LC3) in rats following cerebral ischemia reperfusion injury. Methods The healthy sixty male SD rats were randomly divided into five groups: the sham operation group(sham), the cerebral ischemia reperfusion injury group(IR) and low concentration of Cys C group(Cys C1), middle concentration of Cys C group(Cys C2) and high concentration of Cys C group(Cys C3), 12 rats in each group. The model of cerebral ischemia reperfusion injury was established by a suture method. After ischemia for 2 hours and reperfusion 24 hours, the modified neurological severity scores were detected. Using Western blotting method to detect the expression of HSP70, LC3-Ⅱ/LC3-Ⅰprotein. The number of HSP70 and LC3 positive cells were detected by immunohistochemical SP method . The average absorbance of autophagy related protein LC3/neuronal nuclei (NeuN) in corticalneurons was detected by immunofluorescence double labeling assay. The structural changes of the neurons were observed under transmission electron microscope. Results Compared with the IR group, the mNSS score significantly reduced(P<0.05), and upregulated the expression of HSP70 in Cys C1, Cys C2 group(P<0.01). While the mNSS score obviously increased in Cys C3 group, there was no significantly different(P>0.05) between Cys C3 group and IR group; the expression of HSP70 decreased in the Cys C3 group(P<0.01); the expression of LC3-Ⅱ/LC3-Ⅰprotein in each concentration cystatin C group increased gradually(P<0.01). The average absorbance of LC3/NeuN in the each concentration cystatin C group increased gradually (P<0.01). The results of transmission electron microscope showed that the ultrastructure pathological changes of cerebral cortex neurons could alleviated in Cys C1 and Cys C2 group, there were many obvious autophagic lysosomes. While the ultrastructural damage of cerebral cortex neurons in Cys C3 group was very serious. Conclusion Within a certain concentration range, Cyst C has a protective effects on ischemia reperfusion injury neurons. Otherwise, excessive autophagy maybe leads to autophagic death.

Key words: Cerebral ischemia reperfusion, Autophagy, Heat shock protein 70, Microtubule-associated protein light chain-3, Western blotting, Immunofluorescence, Rat