ΔFosB通过MMP-9调控山羊乳腺上皮细胞凋亡的分子机制

发布时间：2018-03-05 20:25

  本文选题：奶山羊　切入点：ΔFosB　出处：《西北农林科技大学》2015年硕士论文　论文类型：学位论文

【摘要】：ΔFosB是FosB(Finkel-Biskis-Jinkins murine osteosarcoma B)基因的自然截短型,是AP-1(activator protein 1)家族的重要成员,在细胞的增殖、凋亡及分化过程起关键作用。MMP-9(Matrix metalloproteinase-9,明胶酶B)是MMPs家族最大成员之一,参与调节细胞外基质的降解,并与细胞的增殖和凋亡、细胞迁移、炎症的发生及乳腺组织重塑等关系密切。过表达AP-1家族转录因子c-Fos和c-Jun,可增加MMP-9的mRNA转录水平,并使其活性增加。STATs家族对细胞增殖和生存相关基因的表达以及乳腺功能的调控具有重要作用。在恶性乳腺肿瘤细胞中,STAT5可激活下游EGF信号通路,而EGFR激活后可以促进MMP-9的表达。研究表明STAT5可诱导山羊乳腺上皮细胞发生凋亡,在患乳腺炎的山羊乳腺上皮细胞中MMP-9的表达及活性均增加,并与乳汁中的体细胞数呈正相关。通过数据库比对从奶山羊基因组中找到MMP-9基因的5’端,对其前约3500bp序列进行生物信息学分析发现其启动子序列中存在多个AP-1(Fos家族)结合位点。虽然ΔFosB、STATs和MMP-9均与细胞增殖和凋亡密切相关,但有关其在山羊乳腺上皮细胞凋亡过程中的调控机理研究还未见报道。因此,本研究旨在证明在山羊乳腺上皮细胞中,ΔFosB对MMP-9是否具有调控作用及两者对山羊乳腺上皮细胞增殖和凋亡的影响。试验采用qRT-PCR、Western blot、流式细胞仪分析等技术,分别用ΔFosB重组腺病毒、MMP-9抑制剂(SB-3CT)、STAT5激动剂和抑制剂(Pimozide和PRL)和CaCl2处理山羊乳腺上皮细胞,然后检测细胞凋亡相关基因的表达变化及细胞的增殖活性和凋亡情况,以揭示FosB和MMP-9调控山羊乳腺上皮细胞凋亡的分子机制,为阐明山羊乳腺上皮细胞在泌乳期和退化期的凋亡分子机理积累资料。获得的主要结果如下:1.MMP-9抑制剂SB-3CT处理山羊乳腺上皮细胞24h后,mRNA水平上,MMP-9表达下降,Bcl-2、Bcl-xl和CyclinD1表达下降、Bax、Bad、Caspase-3、Caspase-9和P53表达升高,细胞的增殖活性下降,凋亡加剧;2.超表达ΔFosB重组腺病毒处理山羊乳腺上皮细胞48h后,mRNA水平上,ΔFosB、MMP-9、Bcl-2、Bcl-xl和CyclinD1表达量上升,Bax、Bad、Caspase-3、Caspase-9和P53表达量减少,细胞的增殖活性上升,凋亡减少;而经干扰FosB重组腺病毒处理后,结果与之相反;3.与单独使用SB-3CT处理相比,若预先经超表达ΔFosB重组腺病毒处理,MMP-9、Bcl-2、Bcl-xl和CyclinD1表达升高、Bax、Bad、Caspase-3、Caspase-9和P53表达下降,细胞增殖活性上升,凋亡下降;而经干扰ΔFosB重组腺病毒处理,结果与之相反;4.使用CaCl2处理山羊乳腺上皮细胞后,随Ca2+浓度的增加,Bcl-2、Bcl-xl和CyclinD1表达下降,Bax、Bad、Caspase-3、Caspase-9和P53的表达增加,Bcl-2/Bax和Bcl-xl/Bax减小,细胞增殖活性增大,促进细胞凋亡;若预先经超表达ΔFosB重组腺病毒处理,Bcl-2、Bcl-xl和CyclinD1表达有所上升,Bax、Bad、Caspase-3、Caspase-9和P53的表达下降,Bcl-2/Bax和Bcl-xl/Bax的有所上升,细胞增殖活性上升,凋亡减少;5.PRL诱导STAT5激活后,MMP-9表达下降,Bcl-2、Bcl-xl和CyclinD1表达上升,Bax、Bad和P53的表达下降,细胞增殖活性升高,凋亡减少;使用Pimozide抑制STAT5的表达后,MMP-9和EGFR的表达增加,Bcl-2、Bcl-xl和CyclinD1表达下降,Bad和Bax表达上升,细胞增殖活性下降,凋亡增多。综上所述,在奶山羊乳腺上皮细胞中,ΔFosB促进MMP-9的表达;ΔFosB分别抑制由SB-3CT和Ca2+诱导的细胞凋亡;STAT5参与由MMP-9诱导的细胞增殖和凋亡,该调控作用是通过EGFR/STAT信号途径进行的。
[Abstract]:FosB is FosB (Finkel-Biskis-Jinkins murine osteosarcoma B) natural truncated gene, AP-1 (activator protein 1) is an important member of the family, in the process of cell proliferation, apoptosis and differentiation plays a key role in.MMP-9 (Matrix metalloproteinase-9, gelatinase B) MMPs family is the largest one of the members involved in the regulation of degradation of the extracellular matrix, and with the proliferation and apoptosis of cells, cell migration, inflammation and breast tissue remodeling. There is close relationship between overexpression of AP-1 family transcription factors c-Fos and c-Jun, MMP-9 can increase the level of mRNA transcription and its activity play an important role in regulating the expression of.STATs family to increase cell proliferation and survival related genes and mammary gland function. In malignant breast tumor cells, STAT5 can activate the EGF signaling pathway and activation of EGFR can promote the expression of MMP-9. The results show that STAT5 can induce the goat mammary gland Epithelial cell apoptosis, the expression and activity of MMP-9 in mastitis goat mammary epithelial cells were increased, and correlated with somatic cell count in milk was found in the MMP-9 gene. The 5 'end from dairy goat genome through the database, the former about 3500bp sequence bioinformatics analysis revealed the presence of multiple the AP-1 promoter sequence (Fos family) binding sites. Although FosB, STATs and MMP-9 were closely correlated with cell proliferation and apoptosis, but the apoptosis process in goat mammary epithelial cells in the regulation mechanism of research has not been reported. Therefore, this study aims to prove that in goat mammary epithelial cells, FosB of whether MMP-9 has effect of regulation and both on the proliferation and apoptosis of goat mammary epithelial cells. In this experiment, qRT-PCR, Western blot, flow cytometry analysis technique were used to FosB recombinant adenovirus, MM P-9 inhibitor (SB-3CT), STAT5 agonists and antagonists (Pimozide and PRL) and CaCl2 in goat mammary epithelial cell proliferation and apoptosis, then detect the expression and changes of apoptosis related genes, to reveal the molecular mechanism of FosB and MMP-9 regulation of goat mammary epithelial cell apoptosis, in order to elucidate the goat mammary epithelial cells in lactation and involution of the molecular mechanism of the apoptosis of accumulated data. The main results are as follows: 1.MMP-9 inhibitor SB-3CT in goat mammary epithelial cells after 24h, the level of mRNA, decreasing the expression of MMP-9, Bcl-2, Bcl-xl and CyclinD1 decreased and the expression of Bax, Bad, Caspase-3, Caspase-9 and P53 expression increased, decreased cell proliferation activity over expression of apoptosis increased; 2. FosB recombinant adenovirus of goat mammary epithelial cells after 48h treatment, the level of mRNA, FosB, MMP-9, Bcl-2, Bcl-xl and CyclinD1 expression increased, Bax, Bad, Caspase -3,Caspase-9鍜孭53琛ㄨ揪閲忓噺灏，

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