PCBP2在大鼠坐骨神经损伤后的时空表达变化对雪旺细胞增殖的影响

发布时间：2019-03-24 14:01

【摘要】：目的研究PCBP2在大鼠坐骨神经损伤过程中的表达变化、作用,分析PCBP2与雪旺细胞增殖之间的关系,探讨PCBP2在周围神经损伤和修复过程中的作用机制,也为周围神经损伤的治疗提供依据。方法1.选用健康成年雄性SD大鼠(220-275 g左右)54只,构建大鼠坐骨神经夹伤模型,根据损伤时间的不同,将大鼠随机分为9组:正常组(假手术组)、6 h、12 h、1 d、3 d、5 d、1 w、2 w和4w。每组6只,其中3只用于组织切片,3只用于提取神经组织蛋白,运用Western Blot方法测定不同损伤时间点PCBP2的表达变化,初步确定PCBP2的表达在周围神经损伤后的变化趋势。2.通过免疫组化的方法观察PCBP2在周围神经损伤后相应组织中的表达情况,通过免疫荧光双标方法分析PCBP2在坐骨神经中的分布和细胞定位。3.筛选PCBP2干扰的雪旺细胞稳转株,在稳转株中验证干扰PCBP2后是否会影响FHL3、p21信号的表达。运用流式细胞技术及CCK-8分析干扰PCBP2后对雪旺细胞增殖周期的影响。结果1.Western bolt结果显示PCBP2蛋白水平在神经损伤后3d开始上调,在7d达到高峰,在损伤后4w恢复到正常水平。2.免疫组化结果表明坐骨神经夹伤后,神经组织中的PCBP2阳性率增强;免疫荧光双标分析表明在坐骨神经夹伤后,PCBP2主要表达于S100标记的雪旺细胞中,与NF200标记的轴突没有共定位。此外,PCBP2与雪旺细胞增殖标记物PCNA也有共定位。3.TNF-α诱导的雪旺细胞增殖模型中,PCBP2的表达逐渐增加,与细胞增殖标记物PCNA的表达趋势一致,但FHL3及p21的表达逐渐降低。流式细胞分析技术及CCK-8结果表明:干预PCBP2的表达能抑制雪旺细胞的增殖。4.雪旺细胞中干预PCBP2的表达后,FHL3及p21的表达增加,同时干预FHL3的表达后,p21的表达减少,并且免疫荧光双标实验表明PCBP2与FHL3及p21有共同的定位。结论1.PCBP2的表达在急性损伤后3-5d明显升高,4w恢复到正常水平。其表达主要定位在雪旺细胞中,在轴突中表达量较少,且与雪旺细胞的增殖相关。2.PCBP2调节雪旺细胞的增殖过程可能通过FHL3-p21信号通路完成。
[Abstract]:Objective to study the expression and effect of PCBP2 during sciatic nerve injury in rats, to analyze the relationship between PCBP2 and Schwann cell proliferation, and to explore the mechanism of PCBP2 in peripheral nerve injury and repair. It also provides the basis for the treatment of peripheral nerve injury. Method 1. 54 healthy adult male SD rats (220? 275g) were used to construct sciatic nerve injury model of rats. According to the different time of injury, the rats were randomly divided into 9 groups: normal group (sham-operated group), 6 h, 12 h, 1 d, 3 d, 6 h, 12 h, 1 d, 3 d, respectively. 5 d, 1 w, 2 w and 4 w. There were 6 rats in each group, of which 3 were used for tissue sections and 3 for extracting nerve tissue proteins. The expression of PCBP2 at different time points after injury was measured by Western Blot method, and the change trend of PCBP2 expression after peripheral nerve injury was preliminarily determined. 2. The expression of PCBP2 in the corresponding tissues after peripheral nerve injury was observed by immunohistochemical method, and the distribution and cellular localization of PCBP2 in sciatic nerve were analyzed by immunofluorescence double labeling method. 3. The stable transformation of Schwann cells with PCBP2 interference was screened and the effect of PCBP2 interference on the expression of FHL3,p21 signal was verified in the stable transformation strain. Flow cytometry and CCK-8 were used to analyze the effect of PCBP2 interference on Schwann cell cycle. Results the results of 1.Western bolt showed that the level of PCBP2 protein was up-regulated at 3 days after nerve injury, reached its peak at 7 days, and returned to normal level at 4 weeks after injury. The results of immunohistochemistry showed that the positive rate of PCBP2 in nerve tissue increased after sciatic nerve injury, and immunofluorescence double labeling analysis showed that PCBP2 was mainly expressed in S100-labeled Schwann cells after sciatic nerve injury and did not co-locate with NF200-labeled axons. 3. In Schwann cell proliferation model induced by TNF-伪, the expression of PCBP2 increased gradually, which was consistent with the expression trend of cell proliferation marker PCNA, but the expression of FHL3 and p21 decreased gradually. In addition, PCBP2 co-located with Schwann cell proliferation marker PCNA. Flow cytometry and CCK-8 showed that intervention of PCBP2 expression could inhibit Schwann cell proliferation. 4. The expression of FHL3 and p21 increased after intervention of PCBP2 in Schwann cells, and the expression of p21 decreased after intervention of FHL3. The double immunofluorescence staining showed that PCBP2 was co-located with FHL3 and p21. Conclusion the expression of 1.PCBP2 increased significantly 3 days after acute injury and returned to normal level at 4 weeks. The expression of PCBP2 in Schwann cells was mainly localized in Schwann cells, and was less expressed in axons, which was related to the proliferation of Schwann cells. 2.PCBP2 regulated the proliferation of Schwann cells through FHL3-p21 signaling pathway.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R745.4

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