本文選題：坐骨神經損傷　切入點：施萬細胞　出處：《蘇州大學》2015年博士論文

【摘要】：目的研究KHSRP在坐骨神經損傷后的表達情況,探究KHSRP與施萬細胞分化和神經元軸突再生的關系,分析KHSRP與β-catenin的相互調節(jié)關系及兩者在施萬細胞分化和神經元軸突再生過程中的作用,進一步揭示KHSRP在周圍神經損傷后的再生機制。方法1.為了研究KHSRP在損傷后坐骨神經中的表達,本研究通過構建大鼠坐骨神經夾傷模型,利用western blot,免疫組織化學技術檢測了KHSRP在正常組織及損傷組織中的表達；采用免疫熒光方法檢測了KHSRP的細胞定位情況,分析其與施萬細胞分化和神經元軸突再生的關系2.為了研究KHSRP在施萬細胞分化過程中的作用,本研究通過構建原代施萬細胞體外分化模型,western blot及RT-PCR技術檢測KHSRP、β-catenin及相關蛋白在此過程中的表達情況；通過核漿分離檢測KHSRP的胞漿轉移情況；通過構建KHSRP小干擾RNA及β-catenin過表達質粒并結合免疫細胞熒光技術檢測KHSRP是否通過調節(jié)β-cateni n的穩(wěn)定性從而介導施萬細胞的分化。3.為了研究KHSRP在神經元突起生長過程中的作用,采用100ng/ml NGF刺激未分化的PC12細胞,模擬神經元體外分化過程,以及構建原代DRG神經元體外發(fā)育模型,檢測KHSRP在神經元延伸過程中的表達、胞漿轉運及對神經元突起延伸的影響。4.原代施萬細胞和神經元的共培養(yǎng),模擬坐骨神經損傷后施萬細胞的再成髓鞘化過程,檢測KHSRP、β-catenin的表達及KHSRP的胞漿運輸對β-catenin穩(wěn)定性的影響。結果1. western blot免疫組化分析顯示,KHSRP在坐骨神經損傷后表達升高,在第5天達到最高,隨后逐漸回復。免疫熒光結果顯示KHSRP NF200、S100及Oct-6陽性的細胞中均有表達,提示KHSRP可能在施萬細胞分化和神經元軸突再生過程中發(fā)揮作用。2.在cAMP誘導的施萬細胞分化過程中, KHSRP的表達升高,而β-catenin的表達降低。核漿分離結果顯示KHSRP在分化過程中胞漿蛋白水平增加,相應此時β-catenin的胞漿蛋白水平降低,提示KHSRP可能通過調節(jié)β-catenin的穩(wěn)定性來介導施萬細胞的分化。KHSRP的小干擾RNA轉染施萬細胞后抑制了施萬細胞分化的形態(tài)發(fā)生,該效應與過表達β-catenin的施萬細胞形態(tài)相似。3.NGF刺激PC12細胞分化和體外培養(yǎng)的原代DRG神經元發(fā)育的過程中,KHSRP的蛋白及mRNA水平的表達均升高,而β-catenin成相反趨勢。此外也相應觀察到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轉染的PC12或DRG神經元突起延伸也受到影響。4.施萬細胞和DRG神經元共培養(yǎng)過程中,KHSRP在1d表達最低,隨后隨著施萬細胞髓鞘化,表達逐漸增高,β-catenin的表達呈相反趨勢。核漿分離也顯示出KHSRP的胞漿轉移及β-catenin的穩(wěn)定性衰退現(xiàn)象。結論1.KHSRP在坐骨神經損傷后參與施萬細胞分化和神經元軸突再生過程。2.KHSRP通過調節(jié)自身的核漿分布來影響β-catenin的穩(wěn)定性,從而介導周圍神經的再生過程。
[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.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R688

【共引文獻】

相關期刊論文 前10條

1 陳紅江;駱文龍;;周圍神經損傷再生的分子機制[J];重慶醫(yī)學;2007年07期

2 何家全,蔡文琴,張可成;少突膠質細胞與神經元相互作用時神經元細胞內鈣離子濃度的變化[J];第三軍醫(yī)大學學報;2001年07期

3 劉鋮,吳祖澤;膠質瘢痕與脊髓損傷修復[J];國外醫(yī)學.神經病學神經外科學分冊;2004年04期

4 鐘建;陽明明;蔣電明;;含川芎嗪UW液保存不同時間對異體神經再生的影響[J];第三軍醫(yī)大學學報;2013年19期

5 許永志;陳彬;劉惠娜;陳燕紅;林淳崢;;血清Cys C、PEDF、VEGF檢測在糖尿病腎病診斷及治療中的意義[J];國際檢驗醫(yī)學雜志;2014年02期

6 唐芳;馬武開;姚血明;;Wnt/β-catenin信號通路與骨關節(jié)炎[J];風濕病與關節(jié)炎;2014年02期

7 張振輝;江子欣;楊其霖;陳偉燕;熊旭明;;miR-142-3p對THP-1細胞釋放炎癥因子的調控作用[J];廣東醫(yī)學;2014年02期

8 王敏;李春輝;李宏杰;;胃癌組織SOX2、TGF-β/Smad信號傳導通路研究進展[J];承德醫(yī)學院學報;2014年01期

9 胡惠雯;侯欣瑜;蔡兼;鈕俊;項爭;安基永;林善枝;;低溫脅迫響應的甜楊miR475靶基因的預測及表達[J];基因組學與應用生物學;2014年03期

10 王琳;許能貴;;針刺對腦缺血后大腦可塑性促進作用的研究進展[J];中華中醫(yī)藥雜志;2014年08期

相關會議論文 前2條

1 蔡敏;羅成剛;方神權;;Dickkopf-3在早期非小細胞肺癌組織中的表達與臨床預后關系的探討[A];醫(yī)藥導報(2013年8月第32卷增刊)[C];2013年

2 嚴春花;張文風;;糖尿病大鼠坐骨神經NGF、MNCV的相關性及益氣活血通絡方的干預[A];中華中醫(yī)藥學會第十四次中醫(yī)方劑學學術年會論文集[C];2014年

相關博士學位論文 前10條

1 馬小川;精神分裂癥相關蛋白dysbindin-1在神經發(fā)育中的作用[D];中國科學技術大學;2011年

2 張水華;金屬蛋白酶在大鼠視神經擠壓傷的表達及意義[D];中國人民解放軍軍醫(yī)進修學院;2003年

3 于卉影;PI3K/PKB介導的bFGF對細胞生長的影響[D];中國醫(yī)科大學;2003年

4 楊興龍;PDK分子PH結構域與蛋白激酶B在胃癌細胞抗脫落凋亡中的相互作用[D];中國人民解放軍第一軍醫(yī)大學;2003年

5 劉鋮;腺病毒載體介導肝細胞生長因子對脊髓損傷的基因治療及相關機理探討[D];中國人民解放軍軍事醫(yī)學科學院;2004年

6 朱國臣;化學萃取神經支架修復面神經缺損的實驗研究及腦橋小腦角區(qū)內鏡解剖學研究[D];鄭州大學;2004年

7 董紅讓;外消旋聚乳酸/神經生長因子復合導管修復周圍神經缺損實驗研究[D];第一軍醫(yī)大學;2006年

8 楊鵬;AKT2在胰腺癌發(fā)生發(fā)展中的作用及其機制研究[D];華中科技大學;2006年

9 殷中罡;軟骨素酶ABC促進自體神經移植后軸突再生[D];天津醫(yī)科大學;2007年

10 湯婛;PI-3K/Akt信號轉導在樹，

本文編號：1711826