微丝聚合剂重构大鼠脊髓星形胶质细胞骨架对水通道蛋白4与K+通道蛋白4.1基因表达的影响

Effects of gene expression of aquaporins-4 and K+ ion channel protein-4.1 after cytoskeleton reconstruction by jasplakinolide in rat spinal cord astrocytes

中文关键词:  星形胶质细胞  微丝  增殖

英文关键词:Astrocytes  Microfilament  Proliferation

中文摘要:

  【摘要】 目的：探讨不同浓度微丝聚合剂（Jasplakinolide，JSK）对大鼠脊髓星形胶质细胞水通道蛋白4（aquapor?鄄ins-4，AQP4）和K+通道蛋白4.1（potassium ion channel protein-4.1，Kir4.1）基因表达的影响。方法：分离培养原代大鼠脊髓星形胶质细胞并鉴定，采用浓度为0.05μg/ml、0.10μg/ml、0.20μg/ml、0.40μg/ml的JSK干预细胞2h。噻唑蓝比色法（3-4，5-Dimethylthiazol-2-yl-25-diphenyltetrazolium brom，MTT）检测细胞的增殖情况，激光共聚焦显微镜观察细胞骨架的变化和Real-time PCR法检测AQP4和Kir4.1 mRNA表达的变化。结果：0.05μg/ml、0.10μg/ml、0.20μg/ml和0.40μg/ml浓度的JSK在干预细胞2h时与空白对照组相比对细胞的增殖无明显差异（P＞0.05），各浓度间相比无统计学意义。激光共聚焦显微镜观察0.05μg/ml、0.10μg/ml和0.20μg/ml 浓度的JSK可以使得微丝空间结构发生重构，而0.40μg/ml浓度则使细胞微丝发生破坏。Real-time PCR检测显示，0.05μg/ml、0.10μg/ml、0.20μg/ml和0.40μg/ml浓度的JSK均能显著降低AQP4和Kir4.1基因的表达，并与对照组相比有统计学意义（P＜0.05），且0.05μg/ml、0.10μg/ml、0.20μg/ml和0.40μg/ml浓度间相比也有统计学意义。结论：0.05μg/ml、0.10μg/ml和0.20μg/ml浓度的JSK可使大鼠脊髓星形胶质细胞微丝发生重构且可以下调AQP4和Kir4.1基因的表达。

英文摘要:

  【Abstract】 Objectives: To investigate the mRNA expression of aquaporins-4(AQP4) and potassium ion channel protein-4.1(Kir4.1) by different concentration of jasplakinolide(JSK), a microfilament polymerization agent, in spinal cord astrocytes of rats. Methods: The primary spinal cord astrocytes from new born rats were extracted and cultured. 3-4, 5-Dimethylthiazol-2-yl-25-diphenyltetrazolium brom(MTT) was used to measure the cytotoxicity of JSK by different concentrations(0.05μg/ml, 0.10μg/ml, 0.20μg/ml and 0.40μg/ml). The changes of cytoskeleton were monitored under the Laser Scanning Confocal Microscopy(LSCM). The mRNA levels of AQP4 and Kir4.1 were measured after JSK with different concentrations by real-time PCR. Results: MTT assay showed no significant difference in 0.05μg/ml, 0.10μg/ml, 0.20μg/ml and 0.40μg/ml groups compared with control group at 2h. LSCM showed that JSK of 0.05μg/m, 0.10μg/ml and 0.20μg/ml could remodel the spinal cord astrocyte cytoskeleton, nevertheless, 0.40μg/ml could damage cytoskeleton. Real-time PCR analysis showed that JSK of 0.05μg/ml, 0.10μg/ml, 0.20μg/ml and 0.40μg/ml concentrations could significantly decrease the gene expression of AQP4 and Kir4.1, which was statistically significant compared with control group(P<0.05). Furthermore, there were significant differences among 0.05μg/ml, 0.10μg/ml, 0.20μg/ml and 0.40μg/ml groups. Conclusion: JSK of 0.05μg/ml, 0.10μg/ml and 0.20μg/ml can remodel the cytoskeleton and decrease the mRNA expression of AQP4 and Kir4.1.