SSeCKS在施万细胞炎症反应中的作用

发布时间：2018-02-08 14:47

  本文关键词： 施万细胞 炎症 SSeCKS 分泌 脱髓鞘 大鼠　出处：《南通大学》2010年硕士论文　论文类型：学位论文

【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)在炎症应激下的施万细胞中,肿瘤坏死因子-α(tumor necrosis factor-alpha,TNF-α)自分泌、增殖抑制及髓鞘形成异常中的作用及其机制。 方法 1、为了研究SSeCKS在施万细胞TNF-α自分泌中的作用,本研究通过体外培养及纯化大鼠施万细胞,运用逆转录-多聚酶链反应(reverse transcription polymerase chain reaction,RT-PCR)及酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测施万细胞TNF-α自分泌的能力,运用RT-PCR及蛋白免疫印迹检测TNF-α诱导SSeCKS的表达及p38与Jun氨基末端激酶(Jun N-terminal kinase,JNK)信号转导通路的激活情况。在此基础上,构建SSeCKS干扰及过表达载体,转染施万细胞后,检测干扰及过表达SSeCKS后施万细胞TNF-α自分泌及p38与JNK信号转导通路激活的变化。 2、为了研究SSeCKS在TNF-α诱导施万细胞的增殖抑制中的作用,本研究利用重组大鼠TNF-α处理施万细胞,运用5’-溴脱氧尿核苷(5’-bromodeoxyuridine,BrdU)掺入法,分析施万细胞增殖的变化,利用RT-PCR及蛋白免疫印迹,检测TNF-α诱导SSeCKS的表达及磷酸化水平的变化。在此基础上,通过干扰大鼠施万细胞SSeCKS的表达,分析施万细胞增殖的改变。接着通过分析细胞外信号调节激酶(extracellular signal-regulated kinase,ERK1/2)的活性、cyclin D1的表达及其报告基因活性的改变明确SSeCKS对cyclin D1表达水平的调节及与ERK1/2激活的相关性;同时通过分析SSeCKS与cyclin D1结合能力及cyclin D1细胞亚定位的改变,明确SSeCKS对cyclin D1的定位的调节及其与两者结合能力的相关性。 3、为了研究SSeCKS在施万细胞分化及髓鞘形成中的作用,本研究通过分离培养大鼠施万细胞及背根神经节(dorsal root ganglion,DRG)神经元,并在体外建立环单磷酸腺苷(cyclic adenosine monophosphate,cAMP)诱导施万细胞分化模型及施万细胞与DRG神经元体外共培养成髓鞘模型,分析施万细胞分化过程中SSeCKS的表达变化。在此基础上,干预SSeCKS的表达,分析其对施万细胞分化的形态学改变、分化相关标记物的表达及髓鞘形成能力的影响,并通过分析蛋白激酶B(protein kinase B,PKB/Akt)的磷酸化水平,明确SSeCKS发挥上述作用的细胞信号机制。 结果 1、重组大鼠TNF-α刺激施万细胞24 h后,RT-PCR及ELISA分析发现施万细胞内TNF-α的mRNA水平及培养基中自分泌的TNF-α含量较未处理组显著增高,RT-PCR及蛋白免疫印迹也显示TNF-α处理后的施万细胞内SSeCKS的α亚型的mRNA及蛋白水平显著增加。干扰施万细胞中SSeCKS的表达发现,TNF-α诱导的自分泌量较未干扰组显著降低,过表达SSeCKS的α亚型后,施万细胞TNF-α诱导的自分泌显著提高。蛋白质免疫印迹显示,TNF-α可以激活p38和JNK信号通路,抑制p38及JNK可以有效地抑制TNF-α的自分泌。进一步的研究发现,干扰SSeCKS的α亚型的表达可以抑制TNF-α诱导的p38和JNK的激活,过表达施万细胞中的SSeCKS的α亚型后,TNF-α诱导的自分泌可以被p38和JNK的抑制剂SB202190和SP600125显著抑制。 2、BrdU掺入法测定细胞增殖率发现用不同浓度的TNF-α处理施万细胞12 h后,细胞增殖率较正常组相比显著降低,在1 ng/ml被抑制50%,在10 ng/ml被抑制75%。流式细胞术显示,在予TNF-α处理后,72.2%的细胞被阻滞在G0/G1期,而在未处理的细胞中,仅有52.9%的细胞处于G0/G1期。RT-PCR及蛋白质免疫印迹显示在予TNF-α处理后,施万细胞内SSeCKS的α亚型的mRNA水平、蛋白水平及磷酸化水平较正常细胞以剂量依赖的方式增加。在予TNF-α处理SSeCKS的α亚型干扰的施万细胞,细胞增殖分析及流式细胞分析术发现降低SSeCKS的α亚型的表达可以逆转TNF-α诱导的施万细胞的增殖抑制及G0/G1期的阻滞。进一步分析其原因,发现cyclin D1的表达及ERK1/2的激活水平随着TNF-α的浓度增加而降低,干扰SSeCKS的α亚型表达可以逆转TNF-α诱导的cyclin D1的表达水平及ERK1/2的激活水平的降低。同时,TNF-α可以诱导施万细胞中cyclin D1的细胞核定位减少及cyclin D1与SSeCKS结合能力的增加,干扰SSeCKS的α亚型的表达可以逆转cyclin D1细胞核定位的减少。 3、用cAMP诱导施万细胞体外分化中,SSeCKS的表达随着诱导的时间的延长而降低。用EGFP标记的SSeCKS siRNA的慢病毒载体干扰施万细胞中SSeCKS的表达可以加快体外诱导的细胞分化的形态改变,同时在SSeCKS表达干扰的施万细胞中,分化的标记髓鞘蛋白0(myelin protein zero,P0)及髓磷脂相关糖蛋白(myelin associated glycoprotein,MAG)的表达较未干扰组增加。将干扰SSeCKS表达的施万细胞与神经元共培养诱导髓鞘的形成,发现干扰了施万细胞中SSeCKS的表达可以促进体外形成髓鞘的数量。进一步分析其机制,发现干扰SSeCKS的表达可以促进Akt 473号位丝氨酸的磷酸化。 结论 1、施万细胞中存在着TNF-α的自分泌循环,其分泌的TNF-α可以作用于自身,诱导自身的JNK和p38信号转导通路的激活,促进TNF-α的合成和分泌;TNF-α作用于施万细胞后,表达增多的SSeCKS可以通过促进JNK和p38的激活,在TNF-α的自分泌循环中发挥着正向调控作用。 2、TNF-α以剂量依赖的方式抑制施万细胞的增殖,诱导施万细胞中SSeCKS的mRNA水平、蛋白水平及磷酸化水平的上调,同时可以抑制cyclin D1的表达及细胞核定位。SSeCKS在TNF-α诱导的施万细胞增殖抑制中发挥着负性调控作用。这主要是通过抑制ERK1/2激酶的活性,下调cyclin D1的表达及增加与cycling D1结合,抑制cyclin D1的细胞核转位发挥作用。 3、SSeCKS在施万细胞的分化过程中表达下调,其可以抑制施万细胞的体外分化及髓鞘的形成;SSeCKS在施万细胞体外分化及髓鞘形成中的负性调控作用主要是通过抑制Akt的活化发挥作用。
[Abstract]:Objective To study the role and mechanism of tumor necrosis factor - alpha ( TNF - 伪 ) 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 - 伪 in Schwann cells , the expression of TNF - 伪 induced by TNF - 伪 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 - 伪 , 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 - 伪 induced by TNF - 伪 increased significantly . The expression of TNF - 伪 induced by TNF - 伪 increased significantly . The expression of TNF - 伪 induced the activation of TNF - 伪 and inhibited the activation of TNF - 伪 . After overexpression of SSeCKS , the self - secretion of TNF - 伪 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 - 伪 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 - 伪 plays a positive role in the self - secretion cycle of TNF - 伪 . TNF - 伪 acts on itself , induces its activation and promotes the synthesis and secretion of TNF - 伪 . After the action of TNF - 伪 on Schwann cells , the expression of SSeCKS can promote the activation of p38 and exert positive regulation in the autosecretion cycle of TNF - 伪 . 2 . TNF - 伪 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 - 伪 . 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 .

【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

【共引文献】

相关博士学位论文 前2条

1 李建萍;EAN大鼠神经轴膜与淋巴细胞离子通道及雷公藤多甙干预的研究[D];复旦大学;2004年

2 赵春梅;过量表达LeHSP21.5减缓番茄的UPR应答并增强其耐逆性[D];山东师范大学;2006年

相关硕士学位论文 前1条

1 刘海鸥;内皮细胞表达SSeCKS功能的初步研究[D];南通大学;2006年

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