本文關鍵詞： 施萬細胞 炎癥 SSeCKS 分泌 脫髓鞘 大鼠　出處：《南通大學》2010年碩士論文　論文類型：學位論文

【摘要】：目的研究Src抑制的蛋白激酶C的底物(Src-suppressed protein kinase C substrate,SSeCKS)在炎癥應激下的施萬細胞中,腫瘤壞死因子-α(tumor necrosis factor-alpha,TNF-α)自分泌、增殖抑制及髓鞘形成異常中的作用及其機制。 方法 1、為了研究SSeCKS在施萬細胞TNF-α自分泌中的作用,本研究通過體外培養(yǎng)及純化大鼠施萬細胞,運用逆轉錄-多聚酶鏈反應(reverse transcription polymerase chain reaction,RT-PCR)及酶聯(lián)免疫吸附試驗(enzyme linked immunosorbent assay,ELISA)檢測施萬細胞TNF-α自分泌的能力,運用RT-PCR及蛋白免疫印跡檢測TNF-α誘導SSeCKS的表達及p38與Jun氨基末端激酶(Jun N-terminal kinase,JNK)信號轉導通路的激活情況。在此基礎上,構建SSeCKS干擾及過表達載體,轉染施萬細胞后,檢測干擾及過表達SSeCKS后施萬細胞TNF-α自分泌及p38與JNK信號轉導通路激活的變化。 2、為了研究SSeCKS在TNF-α誘導施萬細胞的增殖抑制中的作用,本研究利用重組大鼠TNF-α處理施萬細胞,運用5’-溴脫氧尿核苷(5’-bromodeoxyuridine,BrdU)摻入法,分析施萬細胞增殖的變化,利用RT-PCR及蛋白免疫印跡,檢測TNF-α誘導SSeCKS的表達及磷酸化水平的變化。在此基礎上,通過干擾大鼠施萬細胞SSeCKS的表達,分析施萬細胞增殖的改變。接著通過分析細胞外信號調(diào)節(jié)激酶(extracellular signal-regulated kinase,ERK1/2)的活性、cyclin D1的表達及其報告基因活性的改變明確SSeCKS對cyclin D1表達水平的調(diào)節(jié)及與ERK1/2激活的相關性;同時通過分析SSeCKS與cyclin D1結合能力及cyclin D1細胞亞定位的改變,明確SSeCKS對cyclin D1的定位的調(diào)節(jié)及其與兩者結合能力的相關性。 3、為了研究SSeCKS在施萬細胞分化及髓鞘形成中的作用,本研究通過分離培養(yǎng)大鼠施萬細胞及背根神經(jīng)節(jié)(dorsal root ganglion,DRG)神經(jīng)元,并在體外建立環(huán)單磷酸腺苷(cyclic adenosine monophosphate,cAMP)誘導施萬細胞分化模型及施萬細胞與DRG神經(jīng)元體外共培養(yǎng)成髓鞘模型,分析施萬細胞分化過程中SSeCKS的表達變化。在此基礎上,干預SSeCKS的表達,分析其對施萬細胞分化的形態(tài)學改變、分化相關標記物的表達及髓鞘形成能力的影響,并通過分析蛋白激酶B(protein kinase B,PKB/Akt)的磷酸化水平,明確SSeCKS發(fā)揮上述作用的細胞信號機制。 結果 1、重組大鼠TNF-α刺激施萬細胞24 h后,RT-PCR及ELISA分析發(fā)現(xiàn)施萬細胞內(nèi)TNF-α的mRNA水平及培養(yǎng)基中自分泌的TNF-α含量較未處理組顯著增高,RT-PCR及蛋白免疫印跡也顯示TNF-α處理后的施萬細胞內(nèi)SSeCKS的α亞型的mRNA及蛋白水平顯著增加。干擾施萬細胞中SSeCKS的表達發(fā)現(xiàn),TNF-α誘導的自分泌量較未干擾組顯著降低,過表達SSeCKS的α亞型后,施萬細胞TNF-α誘導的自分泌顯著提高。蛋白質免疫印跡顯示,TNF-α可以激活p38和JNK信號通路,抑制p38及JNK可以有效地抑制TNF-α的自分泌。進一步的研究發(fā)現(xiàn),干擾SSeCKS的α亞型的表達可以抑制TNF-α誘導的p38和JNK的激活,過表達施萬細胞中的SSeCKS的α亞型后,TNF-α誘導的自分泌可以被p38和JNK的抑制劑SB202190和SP600125顯著抑制。 2、BrdU摻入法測定細胞增殖率發(fā)現(xiàn)用不同濃度的TNF-α處理施萬細胞12 h后,細胞增殖率較正常組相比顯著降低,在1 ng/ml被抑制50%,在10 ng/ml被抑制75%。流式細胞術顯示,在予TNF-α處理后,72.2%的細胞被阻滯在G0/G1期,而在未處理的細胞中,僅有52.9%的細胞處于G0/G1期。RT-PCR及蛋白質免疫印跡顯示在予TNF-α處理后,施萬細胞內(nèi)SSeCKS的α亞型的mRNA水平、蛋白水平及磷酸化水平較正常細胞以劑量依賴的方式增加。在予TNF-α處理SSeCKS的α亞型干擾的施萬細胞,細胞增殖分析及流式細胞分析術發(fā)現(xiàn)降低SSeCKS的α亞型的表達可以逆轉TNF-α誘導的施萬細胞的增殖抑制及G0/G1期的阻滯。進一步分析其原因,發(fā)現(xiàn)cyclin D1的表達及ERK1/2的激活水平隨著TNF-α的濃度增加而降低,干擾SSeCKS的α亞型表達可以逆轉TNF-α誘導的cyclin D1的表達水平及ERK1/2的激活水平的降低。同時,TNF-α可以誘導施萬細胞中cyclin D1的細胞核定位減少及cyclin D1與SSeCKS結合能力的增加,干擾SSeCKS的α亞型的表達可以逆轉cyclin D1細胞核定位的減少。 3、用cAMP誘導施萬細胞體外分化中,SSeCKS的表達隨著誘導的時間的延長而降低。用EGFP標記的SSeCKS siRNA的慢病毒載體干擾施萬細胞中SSeCKS的表達可以加快體外誘導的細胞分化的形態(tài)改變,同時在SSeCKS表達干擾的施萬細胞中,分化的標記髓鞘蛋白0(myelin protein zero,P0)及髓磷脂相關糖蛋白(myelin associated glycoprotein,MAG)的表達較未干擾組增加。將干擾SSeCKS表達的施萬細胞與神經(jīng)元共培養(yǎng)誘導髓鞘的形成,發(fā)現(xiàn)干擾了施萬細胞中SSeCKS的表達可以促進體外形成髓鞘的數(shù)量。進一步分析其機制,發(fā)現(xiàn)干擾SSeCKS的表達可以促進Akt 473號位絲氨酸的磷酸化。 結論 1、施萬細胞中存在著TNF-α的自分泌循環(huán),其分泌的TNF-α可以作用于自身,誘導自身的JNK和p38信號轉導通路的激活,促進TNF-α的合成和分泌;TNF-α作用于施萬細胞后,表達增多的SSeCKS可以通過促進JNK和p38的激活,在TNF-α的自分泌循環(huán)中發(fā)揮著正向調(diào)控作用。 2、TNF-α以劑量依賴的方式抑制施萬細胞的增殖,誘導施萬細胞中SSeCKS的mRNA水平、蛋白水平及磷酸化水平的上調(diào),同時可以抑制cyclin D1的表達及細胞核定位。SSeCKS在TNF-α誘導的施萬細胞增殖抑制中發(fā)揮著負性調(diào)控作用。這主要是通過抑制ERK1/2激酶的活性,下調(diào)cyclin D1的表達及增加與cycling D1結合,抑制cyclin D1的細胞核轉位發(fā)揮作用。 3、SSeCKS在施萬細胞的分化過程中表達下調(diào),其可以抑制施萬細胞的體外分化及髓鞘的形成;SSeCKS在施萬細胞體外分化及髓鞘形成中的負性調(diào)控作用主要是通過抑制Akt的活化發(fā)揮作用。
[Abstract]:Objective To study the role and mechanism of tumor necrosis factor - alpha ( TNF - 偽 ) self - secretion , proliferation inhibition and myelination in Schwann cells in inflammatory stress . method 1 . In order to study the role of SSeCKS in the self - secretion of TNF - 偽 in Schwann cells , the expression of TNF - 偽 induced by TNF - 偽 was detected by reverse transcription polymerase chain reaction ( RT - PCR ) and enzyme linked immunosorbent assay ( ELISA ) . 2 . In order to study the role of SSeCKS in the inhibition of proliferation of Schwann cells induced by TNF - 偽 , the changes of the proliferation of Schwann cells were analyzed by using 5 ' - bromodeoxyuridine ( 5 ' - bromodeoxyuridine ) , and the changes of expression and phosphorylation of SSeCKS were detected by RT - PCR and Western blot . 3 . In order to study the role of SSeCKS in Schwann cell differentiation and myelination , we isolated cultured rat Schwann cells and dorsal root ganglia ( DRG ) neurons , and established cyclic adenosine monophosphate ( cAMP ) induced Schwann cell differentiation model and Schwann cells and DRG neurons in vitro . Results It was found that the expression of TNF - 偽 induced by TNF - 偽 increased significantly . The expression of TNF - 偽 induced by TNF - 偽 increased significantly . The expression of TNF - 偽 induced the activation of TNF - 偽 and inhibited the activation of TNF - 偽 . After overexpression of SSeCKS , the self - secretion of TNF - 偽 could be inhibited by p38 MAPK and SP600125 . Cell proliferation assay and flow cytometry showed that 72.2 % of cells were blocked in G0 / G1 phase after TNF - 偽 treatment . 3 . In vitro differentiation of Schwann cells induced by cAMP , the expression of SSeCKS decreased with the increase of induction time . The expression of SSeCKS in SSeCKS siRNA induced by EGFP could accelerate the morphological change of cell differentiation induced in vitro . At the same time , the expression of SSeCKS in SSeCKS expression could accelerate the formation of myelin . The mechanism was further analyzed . It was found that the expression of SSeCKS could promote the phosphorylation of serine at position 473 . Conclusion TNF - 偽 plays a positive role in the self - secretion cycle of TNF - 偽 . TNF - 偽 acts on itself , induces its activation and promotes the synthesis and secretion of TNF - 偽 . After the action of TNF - 偽 on Schwann cells , the expression of SSeCKS can promote the activation of p38 and exert positive regulation in the autosecretion cycle of TNF - 偽 . 2 . TNF - 偽 inhibited the proliferation of Schwann cells in a dose - dependent manner , induced up - regulation of SSeCKS mRNA level , protein level and phosphorylation level in Schwann cells , while inhibiting the expression of cyclin D1 and nuclear localization . SSeCKS plays a negative role in inhibiting the proliferation of Schwann cells induced by TNF - 偽 . 3 . SSeCKS was down - regulated during differentiation of Schwann cells , which could inhibit the in vitro differentiation of Schwann cells and the formation of myelin . The negative regulation of SSeCKS in the differentiation of Schwann cells and the formation of myelin sheaths was mainly by inhibition of activation .

【學位授予單位】：南通大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R363

【共引文獻】

相關博士學位論文 前2條

1 李建萍;EAN大鼠神經(jīng)軸膜與淋巴細胞離子通道及雷公藤多甙干預的研究[D];復旦大學;2004年

2 趙春梅;過量表達LeHSP21.5減緩番茄的UPR應答并增強其耐逆性[D];山東師范大學;2006年

相關碩士學位論文 前1條

1 劉海鷗;內(nèi)皮細胞表達SSeCKS功能的初步研究[D];南通大學;2006年

，

本文編號：1495693