發(fā)布時間：2018-06-03 21:20

  本文選題：PTHrP + ΔfosB�。� 參考：《西北農(nóng)林科技大學》2015年碩士論文

【摘要】：甲狀旁腺相關蛋白(PTHrP)在不同類型細胞的不同情況下,可以促進細胞凋亡也可以抑制細胞凋亡。激活蛋白-1(AP-1)參與多種細胞的增殖和分化過程,Δ-小鼠骨肉瘤基因B(ΔfosB)作為AP-1家族的一員,可能參與奶山羊乳腺上皮細胞的增殖和凋亡過程。研究發(fā)現(xiàn)PTH(1-34)誘導fosB/ΔfosB基因表達,PTHrP與PTH擁有相似的結構和共同的細胞表面受體,因此推測PTHrP可能調控ΔfosB的表達。但是目前兩者之間的關系以及它們在奶山羊乳腺上皮細胞增殖、凋亡和細胞周期過程中的作用尚不清楚。本研究用實驗室制備并保存的PTHrP和Δfos B重組腺病毒處理原代奶山羊乳腺上皮細胞,通過實時熒光定量PCR技術檢測目的基因的mRNA表達量變化,用CCK-8和酶標儀分析技術檢測奶山羊乳腺上皮細胞的增殖情況,用PI單染方法和流式細胞儀分析技術檢測奶山羊乳腺上皮細胞的凋亡和周期情況。該研究為進一步揭示泌乳期和退化期山羊乳腺上皮細胞凋亡的分子調控機制積累資料,為提高奶山羊產(chǎn)奶量和乳品質提供理論基礎和試驗依據(jù)。本研究獲得的主要結果如下:(1)在奶山羊乳腺上皮細胞中分別感染超表達PTHrP和干擾表達PTHrP重組腺病毒:Ad-PTHrP和Ad-PTHrP-572后,發(fā)現(xiàn)PTHrP抑制ΔfosB的mRNA表達。(2)在奶山羊乳腺上皮細胞中分別感染超表達PTHrP、干擾表達PTHrP、超表達ΔfosB和干擾表達ΔfosB重組腺病毒:Ad-PTHrP、Ad-PTHrP-322、Ad-ΔfosB和Ad-ΔfosB-572后,發(fā)現(xiàn)PTHrP和ΔfosB均增加抗凋亡基因Bcl-2、Bcl-xL、增值細胞核抗原基因PCNA、細胞周期基因Cyclin D1的mRNA表達,減少促凋亡基因Bax、p53、caspase-3的mRNA表達,增加Bcl-2/Bax、Bcl-xL/Bax比率,促進奶山羊乳腺上皮細胞增殖、增加S期與G2期的細胞比率,減少其細胞凋亡。(3)在奶山羊乳腺上皮細胞中分別感染Ad-ΔfosB+Ad-PTHrP、Ad-ΔfosB+Ad-PTHr P-322、Ad-ΔfosB-572+Ad-PTHrP和Ad-ΔfosB-572+Ad-PTHrP-322后,發(fā)現(xiàn)ΔfosB可以加強PTHrP對奶山羊乳腺上皮細胞的促增殖和抗凋亡作用。綜上所述,在奶山羊乳腺上皮細胞中,PTHrP抑制ΔfosB的mRNA表達,PTHrP和ΔfosB都有促增殖和抗凋亡的作用,PTHrP可能通過ΔfosB調控奶山羊乳腺上皮細胞的增殖、凋亡和細胞周期。
[Abstract]:PTHrP (parathyroid associated protein) can promote cell apoptosis and inhibit cell apoptosis in different cell types. Activator protein-1 (AP-1) is involved in the proliferation and differentiation of many kinds of cells. 螖 -mouse osteosarcoma gene B (螖 FOS B), as a member of the AP-1 family, may be involved in the proliferation and apoptosis of mammary epithelial cells in dairy goats. It was found that PTHrP could induce the expression of fosB/ 螖 fosB gene and PTHrP had similar structure and common cell surface receptor to PTH. Therefore, we speculated that PTHrP might regulate the expression of 螖 fosB. However, the relationship between them and their roles in the proliferation, apoptosis and cell cycle of mammary epithelial cells in dairy goats are unclear. In this study, PTHrP and 螖 fos B recombinant adenovirus prepared and preserved in laboratory were used to treat primary dairy goat mammary epithelial cells. The mRNA expression of the target gene was detected by real-time fluorescence quantitative PCR. The proliferation of mammary epithelial cells in dairy goats was detected by CCK-8 and enzymatic analysis. The apoptosis and cycle of mammary epithelial cells in dairy goats were detected by Pi single staining and flow cytometry. This study provided theoretical basis and experimental basis for improving milk yield and milk quality of dairy goats in order to further reveal the molecular regulatory mechanism of breast epithelial cell apoptosis in lactating and degenerated goats. The main results obtained in this study are as follows: (1) after infection with PTHrP and interfering PTHrP recombinant adenovirus, the recombinant adenovirus: Ad-PTHrP and Ad-PTHrP-572 were infected in milk goat mammary epithelial cells, respectively. It was found that PTHrP inhibited the mRNA expression of 螖 fosB in dairy goat mammary epithelial cells infected with PTHrP, interfered with PTHrP, overexpressed 螖 fosB and interfered with the recombinant adenovirus of 螖 fosB Ad-PTHrPn322Ad- 螖 fosB and Ad- 螖 fosB-572, respectively. It was found that both PTHrP and 螖 fosB increased the expression of anti-apoptotic gene Bcl-2Bcl-xL, proliferating cell nuclear antigen gene PCNA, mRNA expression of cell cycle gene Cyclin D1, decreased the mRNA expression of apoptosis-promoting gene Bax-p53 caspase-3, increased the ratio of Bcl-2 / Baxax-Bcl-xL / Bax, and promoted the proliferation of mammary epithelial cells in dairy goats. The ratio of S phase to G2 phase was increased, and apoptosis was reduced.) after Ad- 螖 fosB Ad-PTHrPnP Ad- 螖 fosB Ad-PTHr P-322Ad- 螖 fosB-572 Ad-PTHrP and Ad- 螖 fosB-572 Ad-PTHrP-322 were infected in dairy goat mammary epithelial cells, it was found that 螖 fosB could enhance the effect of PTHrP on the proliferation and anti-apoptosis of dairy goat mammary epithelial cells. In conclusion, in dairy goat mammary epithelial cells, both PTHrP and 螖 fosB inhibit the expression of 螖 fosB mRNA. PTHrP may regulate the proliferation, apoptosis and cell cycle of dairy goat mammary epithelial cells through 螖 fosB.
【學位授予單位】：西北農(nóng)林科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S827

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