PTHrP通过ΔfosB调控奶山羊乳腺上皮细胞的增殖、凋亡和细胞周期的分子机制

发布时间：2018-06-03 21:20

本文选题：PTHrP + ΔfosB　；参考：《西北农林科技大学》2015年硕士论文

【摘要】：甲状旁腺相关蛋白(PTHrP)在不同类型细胞的不同情况下,可以促进细胞凋亡也可以抑制细胞凋亡。激活蛋白-1(AP-1)参与多种细胞的增殖和分化过程,Δ-小鼠骨肉瘤基因B(ΔfosB)作为AP-1家族的一员,可能参与奶山羊乳腺上皮细胞的增殖和凋亡过程。研究发现PTH(1-34)诱导fosB/ΔfosB基因表达,PTHrP与PTH拥有相似的结构和共同的细胞表面受体,因此推测PTHrP可能调控ΔfosB的表达。但是目前两者之间的关系以及它们在奶山羊乳腺上皮细胞增殖、凋亡和细胞周期过程中的作用尚不清楚。本研究用实验室制备并保存的PTHrP和Δfos B重组腺病毒处理原代奶山羊乳腺上皮细胞,通过实时荧光定量PCR技术检测目的基因的mRNA表达量变化,用CCK-8和酶标仪分析技术检测奶山羊乳腺上皮细胞的增殖情况,用PI单染方法和流式细胞仪分析技术检测奶山羊乳腺上皮细胞的凋亡和周期情况。该研究为进一步揭示泌乳期和退化期山羊乳腺上皮细胞凋亡的分子调控机制积累资料,为提高奶山羊产奶量和乳品质提供理论基础和试验依据。本研究获得的主要结果如下:(1)在奶山羊乳腺上皮细胞中分别感染超表达PTHrP和干扰表达PTHrP重组腺病毒:Ad-PTHrP和Ad-PTHrP-572后,发现PTHrP抑制ΔfosB的mRNA表达。(2)在奶山羊乳腺上皮细胞中分别感染超表达PTHrP、干扰表达PTHrP、超表达ΔfosB和干扰表达ΔfosB重组腺病毒:Ad-PTHrP、Ad-PTHrP-322、Ad-ΔfosB和Ad-ΔfosB-572后,发现PTHrP和ΔfosB均增加抗凋亡基因Bcl-2、Bcl-xL、增值细胞核抗原基因PCNA、细胞周期基因Cyclin D1的mRNA表达,减少促凋亡基因Bax、p53、caspase-3的mRNA表达,增加Bcl-2/Bax、Bcl-xL/Bax比率,促进奶山羊乳腺上皮细胞增殖、增加S期与G2期的细胞比率,减少其细胞凋亡。(3)在奶山羊乳腺上皮细胞中分别感染Ad-ΔfosB+Ad-PTHrP、Ad-ΔfosB+Ad-PTHr P-322、Ad-ΔfosB-572+Ad-PTHrP和Ad-ΔfosB-572+Ad-PTHrP-322后,发现ΔfosB可以加强PTHrP对奶山羊乳腺上皮细胞的促增殖和抗凋亡作用。综上所述,在奶山羊乳腺上皮细胞中,PTHrP抑制ΔfosB的mRNA表达,PTHrP和ΔfosB都有促增殖和抗凋亡的作用,PTHrP可能通过ΔfosB调控奶山羊乳腺上皮细胞的增殖、凋亡和细胞周期。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827

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