KHSRP在坐骨神经损伤后的表达及功能研究

发布时间：2018-04-04 21:49

本文选题：坐骨神经损伤　切入点：施万细胞　出处：《苏州大学》2015年博士论文

【摘要】：目的研究KHSRP在坐骨神经损伤后的表达情况,探究KHSRP与施万细胞分化和神经元轴突再生的关系,分析KHSRP与β-catenin的相互调节关系及两者在施万细胞分化和神经元轴突再生过程中的作用,进一步揭示KHSRP在周围神经损伤后的再生机制。方法1.为了研究KHSRP在损伤后坐骨神经中的表达,本研究通过构建大鼠坐骨神经夹伤模型,利用western blot,免疫组织化学技术检测了KHSRP在正常组织及损伤组织中的表达；采用免疫荧光方法检测了KHSRP的细胞定位情况,分析其与施万细胞分化和神经元轴突再生的关系2.为了研究KHSRP在施万细胞分化过程中的作用,本研究通过构建原代施万细胞体外分化模型,western blot及RT-PCR技术检测KHSRP、β-catenin及相关蛋白在此过程中的表达情况；通过核浆分离检测KHSRP的胞浆转移情况；通过构建KHSRP小干扰RNA及β-catenin过表达质粒并结合免疫细胞荧光技术检测KHSRP是否通过调节β-cateni n的稳定性从而介导施万细胞的分化。3.为了研究KHSRP在神经元突起生长过程中的作用,采用100ng/ml NGF刺激未分化的PC12细胞,模拟神经元体外分化过程,以及构建原代DRG神经元体外发育模型,检测KHSRP在神经元延伸过程中的表达、胞浆转运及对神经元突起延伸的影响。4.原代施万细胞和神经元的共培养,模拟坐骨神经损伤后施万细胞的再成髓鞘化过程,检测KHSRP、β-catenin的表达及KHSRP的胞浆运输对β-catenin稳定性的影响。结果1. western blot免疫组化分析显示,KHSRP在坐骨神经损伤后表达升高,在第5天达到最高,随后逐渐回复。免疫荧光结果显示KHSRP NF200、S100及Oct-6阳性的细胞中均有表达,提示KHSRP可能在施万细胞分化和神经元轴突再生过程中发挥作用。2.在cAMP诱导的施万细胞分化过程中, KHSRP的表达升高,而β-catenin的表达降低。核浆分离结果显示KHSRP在分化过程中胞浆蛋白水平增加,相应此时β-catenin的胞浆蛋白水平降低,提示KHSRP可能通过调节β-catenin的稳定性来介导施万细胞的分化。KHSRP的小干扰RNA转染施万细胞后抑制了施万细胞分化的形态发生,该效应与过表达β-catenin的施万细胞形态相似。3.NGF刺激PC12细胞分化和体外培养的原代DRG神经元发育的过程中,KHSRP的蛋白及mRNA水平的表达均升高,而β-catenin成相反趋势。此外也相应观察到KHSRP的胞浆聚集及β-catenin的胞浆稳定性降低现象。KHSRP的小干扰RNA转染的PC12或DRG神经元突起延伸也受到影响。4.施万细胞和DRG神经元共培养过程中,KHSRP在1d表达最低,随后随着施万细胞髓鞘化,表达逐渐增高,β-catenin的表达呈相反趋势。核浆分离也显示出KHSRP的胞浆转移及β-catenin的稳定性衰退现象。结论1.KHSRP在坐骨神经损伤后参与施万细胞分化和神经元轴突再生过程。2.KHSRP通过调节自身的核浆分布来影响β-catenin的稳定性,从而介导周围神经的再生过程。
[Abstract]:Objective to study the expression of KHSRP after sciatic nerve injury and to explore the relationship between KHSRP and Schwann cell differentiation and axonal regeneration.The relationship between KHSRP and 尾 -catenin and their roles in Schwann cell differentiation and axonal regeneration were analyzed, and the mechanism of KHSRP regeneration after peripheral nerve injury was revealed.Method 1.In order to study the expression of KHSRP in sciatic nerve after injury, the expression of KHSRP in normal and injured tissues was detected by western blot and immunohistochemistry.The cellular localization of KHSRP was detected by immunofluorescence method, and its relationship with Schwann cell differentiation and neuronal axon regeneration was analyzed.In order to study the role of KHSRP in the differentiation of Schwann cells, the expression of KHSRP, 尾 -catenin and related proteins was detected by western blot and RT-PCR techniques.The cytoplasmic metastasis of KHSRP was detected by nuclear and cytoplasmic separation, the differentiation of Schwann cells was mediated by the regulation of stability of 尾 -cateneni n by KHSRP by constructing KHSRP small interfering RNA and 尾 -catenin overexpression plasmids and immunofluorescence technique.In order to study the role of KHSRP in neuronal process, undifferentiated PC12 cells were stimulated by 100ng/ml NGF. The process of neuronal differentiation in vitro was simulated, and the model of primary DRG neuron development in vitro was constructed.To detect the expression of KHSRP during neuronal extension, cytoplasmic transport and its effect on neuronal neurite extension. 4.Primary Schwann cells and neurons were co-cultured to simulate the process of remyelination of Schwann cells after sciatic nerve injury. The effects of the expression of KHSRP, 尾 -catenin and the cytoplasmic transport of KHSRP on the stability of 尾 -catenin were detected.Results 1. Immunohistochemical analysis of western blot showed that the expression of KHSRP increased after sciatic nerve injury, reached its highest level on the 5th day, and then recovered gradually.The results of immunofluorescence showed that both KHSRP NF200mS100 and Oct-6 positive cells were expressed, suggesting that KHSRP may play an important role in Schwann cell differentiation and axonal regeneration.During the differentiation of Schwann cells induced by cAMP, the expression of KHSRP increased, while the expression of 尾 -catenin decreased.The results of nuclear and cytoplasmic separation showed that the level of cytoplasmic protein increased during the differentiation of KHSRP, and the cytoplasmic protein level of 尾 -catenin decreased correspondingly.These results suggest that KHSRP may mediate the differentiation of Schwann cells by regulating the stability of 尾 -catenin. KHSRP's small interfering RNA transfection inhibits the morphogenesis of Schwann cells.This effect was similar to that of Schwann cells overexpression of 尾 -catenin. 3. NGF stimulated the differentiation of PC12 cells and the expression of KHSRP protein and mRNA during the development of primary DRG neurons in vitro, while 尾 -catenin showed a reverse trend.It was also observed that the cytoplasmic aggregation of KHSRP and the decrease of cytoplasmic stability of 尾 -catenin. The neurite extension of PC12 or DRG neurons transfected with small interfering RNA of KHSRP was also affected by .4.During co-culture of Schwann cells and DRG neurons, the expression of KHSRP was the lowest in 1 day, then increased with the myelination of Schwann cells, and the expression of 尾 -catenin showed a reverse trend.Nuclear and cytoplasmic segregation also showed cytoplasmic transfer of KHSRP and stability decline of 尾-catenin.Conclusion 1.KHSRP is involved in Schwann cell differentiation and axonal regeneration after sciatic nerve injury. 2. KHSRP mediates the regeneration process of peripheral nerve by regulating its nuclear and cytoplasmic distribution and influencing the stability of 尾 -catenin.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R688

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