本文選題：坐骨神經(jīng)損傷　切入點(diǎn)：施萬細(xì)胞　出處：《蘇州大學(xué)》2015年博士論文

【摘要】：目的研究KHSRP在坐骨神經(jīng)損傷后的表達(dá)情況,探究KHSRP與施萬細(xì)胞分化和神經(jīng)元軸突再生的關(guān)系,分析KHSRP與β-catenin的相互調(diào)節(jié)關(guān)系及兩者在施萬細(xì)胞分化和神經(jīng)元軸突再生過程中的作用,進(jìn)一步揭示KHSRP在周圍神經(jīng)損傷后的再生機(jī)制。方法1.為了研究KHSRP在損傷后坐骨神經(jīng)中的表達(dá),本研究通過構(gòu)建大鼠坐骨神經(jīng)夾傷模型,利用western blot,免疫組織化學(xué)技術(shù)檢測(cè)了KHSRP在正常組織及損傷組織中的表達(dá)；采用免疫熒光方法檢測(cè)了KHSRP的細(xì)胞定位情況,分析其與施萬細(xì)胞分化和神經(jīng)元軸突再生的關(guān)系2.為了研究KHSRP在施萬細(xì)胞分化過程中的作用,本研究通過構(gòu)建原代施萬細(xì)胞體外分化模型,western blot及RT-PCR技術(shù)檢測(cè)KHSRP、β-catenin及相關(guān)蛋白在此過程中的表達(dá)情況；通過核漿分離檢測(cè)KHSRP的胞漿轉(zhuǎn)移情況；通過構(gòu)建KHSRP小干擾RNA及β-catenin過表達(dá)質(zhì)粒并結(jié)合免疫細(xì)胞熒光技術(shù)檢測(cè)KHSRP是否通過調(diào)節(jié)β-cateni n的穩(wěn)定性從而介導(dǎo)施萬細(xì)胞的分化。3.為了研究KHSRP在神經(jīng)元突起生長(zhǎng)過程中的作用,采用100ng/ml NGF刺激未分化的PC12細(xì)胞,模擬神經(jīng)元體外分化過程,以及構(gòu)建原代DRG神經(jīng)元體外發(fā)育模型,檢測(cè)KHSRP在神經(jīng)元延伸過程中的表達(dá)、胞漿轉(zhuǎn)運(yùn)及對(duì)神經(jīng)元突起延伸的影響。4.原代施萬細(xì)胞和神經(jīng)元的共培養(yǎng),模擬坐骨神經(jīng)損傷后施萬細(xì)胞的再成髓鞘化過程,檢測(cè)KHSRP、β-catenin的表達(dá)及KHSRP的胞漿運(yùn)輸對(duì)β-catenin穩(wěn)定性的影響。結(jié)果1. western blot免疫組化分析顯示,KHSRP在坐骨神經(jīng)損傷后表達(dá)升高,在第5天達(dá)到最高,隨后逐漸回復(fù)。免疫熒光結(jié)果顯示KHSRP NF200、S100及Oct-6陽性的細(xì)胞中均有表達(dá),提示KHSRP可能在施萬細(xì)胞分化和神經(jīng)元軸突再生過程中發(fā)揮作用。2.在cAMP誘導(dǎo)的施萬細(xì)胞分化過程中, KHSRP的表達(dá)升高,而β-catenin的表達(dá)降低。核漿分離結(jié)果顯示KHSRP在分化過程中胞漿蛋白水平增加,相應(yīng)此時(shí)β-catenin的胞漿蛋白水平降低,提示KHSRP可能通過調(diào)節(jié)β-catenin的穩(wěn)定性來介導(dǎo)施萬細(xì)胞的分化。KHSRP的小干擾RNA轉(zhuǎn)染施萬細(xì)胞后抑制了施萬細(xì)胞分化的形態(tài)發(fā)生,該效應(yīng)與過表達(dá)β-catenin的施萬細(xì)胞形態(tài)相似。3.NGF刺激PC12細(xì)胞分化和體外培養(yǎng)的原代DRG神經(jīng)元發(fā)育的過程中,KHSRP的蛋白及mRNA水平的表達(dá)均升高,而β-catenin成相反趨勢(shì)。此外也相應(yīng)觀察到KHSRP的胞漿聚集及β-catenin的胞漿穩(wěn)定性降低現(xiàn)象。KHSRP的小干擾RNA轉(zhuǎn)染的PC12或DRG神經(jīng)元突起延伸也受到影響。4.施萬細(xì)胞和DRG神經(jīng)元共培養(yǎng)過程中,KHSRP在1d表達(dá)最低,隨后隨著施萬細(xì)胞髓鞘化,表達(dá)逐漸增高,β-catenin的表達(dá)呈相反趨勢(shì)。核漿分離也顯示出KHSRP的胞漿轉(zhuǎn)移及β-catenin的穩(wěn)定性衰退現(xiàn)象。結(jié)論1.KHSRP在坐骨神經(jīng)損傷后參與施萬細(xì)胞分化和神經(jīng)元軸突再生過程。2.KHSRP通過調(diào)節(jié)自身的核漿分布來影響β-catenin的穩(wěn)定性,從而介導(dǎo)周圍神經(jīng)的再生過程。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R688

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本文編號(hào)：1711826