本文選題：奶山羊　切入點：ΔFosB　出處：《西北農(nóng)林科技大學》2015年碩士論文　論文類型：學位論文

【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)家族的重要成員,在細胞的增殖、凋亡及分化過程起關(guān)鍵作用。MMP-9(Matrix metalloproteinase-9,明膠酶B)是MMPs家族最大成員之一,參與調(diào)節(jié)細胞外基質(zhì)的降解,并與細胞的增殖和凋亡、細胞遷移、炎癥的發(fā)生及乳腺組織重塑等關(guān)系密切。過表達AP-1家族轉(zhuǎn)錄因子c-Fos和c-Jun,可增加MMP-9的mRNA轉(zhuǎn)錄水平,并使其活性增加。STATs家族對細胞增殖和生存相關(guān)基因的表達以及乳腺功能的調(diào)控具有重要作用。在惡性乳腺腫瘤細胞中,STAT5可激活下游EGF信號通路,而EGFR激活后可以促進MMP-9的表達。研究表明STAT5可誘導山羊乳腺上皮細胞發(fā)生凋亡,在患乳腺炎的山羊乳腺上皮細胞中MMP-9的表達及活性均增加,并與乳汁中的體細胞數(shù)呈正相關(guān)。通過數(shù)據(jù)庫比對從奶山羊基因組中找到MMP-9基因的5’端,對其前約3500bp序列進行生物信息學分析發(fā)現(xiàn)其啟動子序列中存在多個AP-1(Fos家族)結(jié)合位點。雖然ΔFosB、STATs和MMP-9均與細胞增殖和凋亡密切相關(guān),但有關(guān)其在山羊乳腺上皮細胞凋亡過程中的調(diào)控機理研究還未見報道。因此,本研究旨在證明在山羊乳腺上皮細胞中,ΔFosB對MMP-9是否具有調(diào)控作用及兩者對山羊乳腺上皮細胞增殖和凋亡的影響。試驗采用qRT-PCR、Western blot、流式細胞儀分析等技術(shù),分別用ΔFosB重組腺病毒、MMP-9抑制劑(SB-3CT)、STAT5激動劑和抑制劑(Pimozide和PRL)和CaCl2處理山羊乳腺上皮細胞,然后檢測細胞凋亡相關(guān)基因的表達變化及細胞的增殖活性和凋亡情況,以揭示FosB和MMP-9調(diào)控山羊乳腺上皮細胞凋亡的分子機制,為闡明山羊乳腺上皮細胞在泌乳期和退化期的凋亡分子機理積累資料。獲得的主要結(jié)果如下:1.MMP-9抑制劑SB-3CT處理山羊乳腺上皮細胞24h后,mRNA水平上,MMP-9表達下降,Bcl-2、Bcl-xl和CyclinD1表達下降、Bax、Bad、Caspase-3、Caspase-9和P53表達升高,細胞的增殖活性下降,凋亡加劇;2.超表達ΔFosB重組腺病毒處理山羊乳腺上皮細胞48h后,mRNA水平上,ΔFosB、MMP-9、Bcl-2、Bcl-xl和CyclinD1表達量上升,Bax、Bad、Caspase-3、Caspase-9和P53表達量減少,細胞的增殖活性上升,凋亡減少;而經(jīng)干擾FosB重組腺病毒處理后,結(jié)果與之相反;3.與單獨使用SB-3CT處理相比,若預先經(jīng)超表達ΔFosB重組腺病毒處理,MMP-9、Bcl-2、Bcl-xl和CyclinD1表達升高、Bax、Bad、Caspase-3、Caspase-9和P53表達下降,細胞增殖活性上升,凋亡下降;而經(jīng)干擾ΔFosB重組腺病毒處理,結(jié)果與之相反;4.使用CaCl2處理山羊乳腺上皮細胞后,隨Ca2+濃度的增加,Bcl-2、Bcl-xl和CyclinD1表達下降,Bax、Bad、Caspase-3、Caspase-9和P53的表達增加,Bcl-2/Bax和Bcl-xl/Bax減小,細胞增殖活性增大,促進細胞凋亡;若預先經(jīng)超表達ΔFosB重組腺病毒處理,Bcl-2、Bcl-xl和CyclinD1表達有所上升,Bax、Bad、Caspase-3、Caspase-9和P53的表達下降,Bcl-2/Bax和Bcl-xl/Bax的有所上升,細胞增殖活性上升,凋亡減少;5.PRL誘導STAT5激活后,MMP-9表達下降,Bcl-2、Bcl-xl和CyclinD1表達上升,Bax、Bad和P53的表達下降,細胞增殖活性升高,凋亡減少;使用Pimozide抑制STAT5的表達后,MMP-9和EGFR的表達增加,Bcl-2、Bcl-xl和CyclinD1表達下降,Bad和Bax表達上升,細胞增殖活性下降,凋亡增多。綜上所述,在奶山羊乳腺上皮細胞中,ΔFosB促進MMP-9的表達;ΔFosB分別抑制由SB-3CT和Ca2+誘導的細胞凋亡;STAT5參與由MMP-9誘導的細胞增殖和凋亡,該調(diào)控作用是通過EGFR/STAT信號途徑進行的。
[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揪閲忓噺灝，

本文編號：1571746