缺氧缺血损伤新生大鼠星形胶质细胞β_1整合素的表达及FDP干预的实验研究

The study on the expression of β_1 integrin in cultured neonatal rat astrocytes during hypoxia-ischemia and fructose-1,6-disphosphate intervention

目的探讨星形胶质细胞(AC)β1整合素在缺氧缺血损伤中的作用和外源性1,6-二磷酸果糖(FDP)对AC缺氧缺血损伤β1整合素表达的影响,为缺氧缺血性脑损伤的机制和治疗提供实验及理论依据。方法原代培养成熟纯化的新生大鼠AC随机分成3组,①缺氧缺血组:AC在1%氧,无糖无血清的培养基中分别培养12 h和24 h;②FDP干预组:AC在1%氧,无糖无血清、加入FDP 20μl/ml的培养基中分别培养12 h和24 h;③对照组:常氧培养组。每一个样品采用光镜和扫描电镜分别观察AC胞体、足突和相互连接的改变;实时定量RT-PCR检测β1整合素mRNA表达水平;免疫荧光检测β1整合素蛋白表达水平。结果随着缺氧缺血时间延长,β1整合素mRNA和蛋白在AC的表达明显降低(P<0.05);FDP干预组较相应时段缺氧缺血组β1整合素mRNA和蛋白表达均明显增加(P<0.05)。随着缺氧缺血时间延长,AC形态变化明显,主要表现为细胞间连接减少,胞体缩小,足突减少;FDP干预12 h组,AC形态变化明显减小。结论 AC缺氧缺血损伤时,β1整合素表达下降,AC间的相互连接破坏,其可能影响AC正常形态、功能的维持。FDP可能通过增加β1整合素的表达,维持AC间相互连接和AC的正常形态,减轻缺氧缺血对AC的损伤。

Objectives To detect the expression of β1 integrin in cultured neonatal rat astrocytes（AC）during experimental hypoxia-ischemia（HI）and fructose-1,6-disphosphate（FDP）intervention,to explore the effect of β1 integrin in AC with HI injury and the effect of FDP intervention on the expression of β1 integrin in AC with HI injury,to provide experimental and theoretical data for understanding mechanisms and management of hypoxia-ischemia brain damage.Methods The primary culture of purified mature AC were randomly divided into five groups,① HI group,the AC was exposed to 1% O2,and cultured in medium without FBS and glucose for 12 h and 24 h respectively.② FDP intervention group,the AC was exposed to 1% O2,cultured in medium without FBS and glucose but with FDP 20 μl/ml for 12 h and 24 h respectively.③ Control group,the AC was exposed to normal oxygen.In each sample,the changes of AC cell body,foot processes,and interconnected were observed by light microscope and scanning electron microscope.The expression of β1 integrin mRNA were determined by real-time PCR and the expression of β1 integrin protein were determined by immunofluorescence.Results As the time of HI was prolonged,the expression level of β1 integrin mRNA and protein in AC was significantly decreased（P〈 0.05）.The expression levels of β1 integrin mRNA and protein in FDP intervention group were significantly higher than that in HI group at the same period（P 〈0.05）.As the time of HI was prolonged,obvious morphological changes was observed in AC,mainly including the decreasing of the connections between cells,the constracting of cell body,and reducing of cell foot processes.However,in FDP intervention group with 12 h intervention,only slight morphological changes was observed in AC.Conclusions During HI injury,the expression of β1 integrin in AC was decreased and the connections between ACs were broken,whichmay affect the normal morphology and function of AC.FDP could maintain connections between AC and normal morphology of AC to reduce the HI damage by increasing the expression of β1 integrin.