【作者】 刘军；

【导师】 韩英浩；

【作者基本信息】 黑龙江八一农垦大学 ， 细胞生物学， 2016， 硕士

【摘要】 Prdx2(Peroxiredoxin Ⅱ)是一种广泛存在于机体组织细胞中的过氧化物还原酶,具有及时清除体内较低浓度活性氧(ROS)的作用。经过长时间的研究发现,Prdx2基因敲除小鼠的胚胎成纤维细胞(MEF)表现出严重的细胞衰老特征,而且Prdx2基因敲除鼠出现了明显的皮肤衰老症状,表现为全层皮肤厚度减少、表皮角质层增厚、真皮层明显变薄,这些现象显示出了Prdx2在皮肤衰老过程中可能的重要作用。虽然Prdx2的功能得到了较为广泛的研究,但是缺失Prdx2引起皮肤衰老的原因和Prdx2在真皮间充质干细胞修复衰老皮肤组织中的作用机理仍然未知。为了进一步探究Prdx2在延缓皮肤衰老和保护真皮间充质干细胞(DMSCs)中的分子机制,本实验利用Prdx2基因敲除模式鼠,研究Prdx2在真皮间充质干细胞延缓皮肤衰老过程中的作用,阐明Prdx2保护真皮间充质干细胞防止皮肤提前衰老的分子机制。首先,在体内部分,本实验利用3月龄(Young)、8月龄(Middle Age)、11月龄(Aged)Prdx2基因敲除型小鼠和野生型小鼠,采用H&E染色和Masson胶原染色的方法观察其皮肤组织变化;通过背部全层皮肤损伤修复情况评估皮肤功能;通过蛋白免疫印迹(Western Blot)的方法检测皮肤组织中与衰老和增殖相关的蛋白表达水平。其次,在细胞部分,本实验利用流式细胞术(FACS)对真皮间充质干细胞进行鉴定,并检测不同代次(Passages)真皮间充质干细胞的细胞周期(Cell Cycle)、细胞凋亡(Apoptosis)水平;利用SA-β-gal染色方法检测真皮间充质干细胞衰老情况;利用MTT绘制生长曲线;利用Western Blot检测相关蛋白表达水平。第三,在分子部分,通过细胞免疫荧光染色,通过核蛋白抽提(Nucleoprotein extraction)和蛋白免疫印迹观察β-catenin入核水平和其他关键信号蛋白表达水平,阐明Prdx2调控经典Wnt/β-catenin信号的分子机制。在体内水平H&E染色及Masson胶原染色结果显示随着小鼠月龄的增加真皮层厚度逐渐减少,且相同月龄的Prdx2基因敲除型小鼠的真皮厚度小于野生型小鼠的真皮层厚度;皮肤损伤愈合实验显示基因敲除型小鼠背部全层皮肤伤口愈合速度慢于野生型小鼠;Western Blot结果显示随着小鼠月龄的增加皮肤组织中p21、p16的表达量增加且相同月龄敲除型高于野生型,stat3的磷酸化水平和PCNA表达量降低且敲除型低于野生型。在细胞水平,随着小鼠真皮间充质干细胞的代次增加,细胞周期发生了G0/G1期阻滞且相同代次敲除型阻滞比例更高;SA-β-gal染色结果显示随着代次增加细胞衰老比例增加,相同代次敲除型小鼠真皮间充质干细胞细胞衰老比例明显高于野生型;增殖曲线显示,3P时两基因型小鼠真皮间充质干细胞增殖速度无差异,随代次增多敲除型增殖速度明显低于野生型;Western Blot结果显示随代次增加p21、p16的表达量增加且相同代次敲除型高于野生型stat3的磷酸化水平和PCNA表达量降低且敲除型低于野生型。在分子水平,细胞免疫荧光染色和Western Blot结果显示,Prdx2基因敲除型小鼠真皮间充质干细胞的β-catenin入核增加,GSK3β(Ser9)和Akt磷酸化水平增加。以上结果表明,Prdx2可以调控经典Wnt/β-catenin信号通路保护小鼠真皮间充质干细胞使其维持正常的数量和功能,Prdx2的缺失会导致Wnt/β-catenin信号通路的异常激活,从而造成真皮间充质干细胞的提前衰老,进而导致了Prdx2敲除型小鼠皮肤的提前衰老和伤口愈合能力的下降。更多还原

【Abstract】 Prdx2(Peroxiredoxin Ⅱ) is a kind of peroxidase which widly exists in tissues and cells of the body. Scavenging active oxygen species(ROS) at a relatively lower concentration at any time is one of the functions of Prdx2. After a long period observation, we found that Prdx2 knockout mouse embryonic fibroblasts(MEF) have an obvious cellular senescence feature and what’s more Prdx2 knockout mouse also appeared some skin aging symptoms, such as decreased thickness of skin, increased thickness of stratum corneum epidermidis, and decreased dermal thickness. These phenotypes indicate that Prdx2 may play an important role in skin aging.Although extensive researches have done to reveal the function of Prdx2, the reason of why absence of Prdx2 can induce premature skin aging and the molecular mechanism of Prdx2 promotes skin stem cells repair the aging tissues is unknown. In order to further explore the molecular mechanism of the role of Prdx2 in the process of resisting skin aging and protecting dermal mesenchymal stem cells(DMSCs) stemness, the Prdx2 knockout mouse and wild mouse are used to analysis the role of Prdx2 in the process of delay skin aging and clarify the mechanism of Prdx2 protect DMSCs to prevent premature skin aging.Firstly, in the bodily portion, 3, 8, 11 month age Prdx2 knockout mouse and wild mouse were used to observe skin structure changes by HE staining and Masson college staining;analysis skin function by skin wound healing assay; measure the proteins express level associate with aging, proliferation and apoptosis in skin tissue by western blotting. Secondly, in the cellular portion, Prdx2 knockout and wild DMSCs were used to identify by FACS; different passages of the two genotype cells were used to analysis cell cycle, apoptosis by FACS, obvious cellular senescence proportion by SA-β-gal staining, drawed growth curve by MTT assay, and measure relative proteins express levels by western blotting. Thirdly, in molecular portion, in order to reveal the mechanism of Prdx2 mediate canonical Wnt/β-catenin pathway, Prdx2 knockout and wild DMSCs were used to measure the β-catenin nuclear import situation and other relative protein express levels by cyto-immunofluorescence staining, Nucleoprotein extraction and western blotting.In the bodily portion, H&E staining and Masson collagen staining results show that dermal thickness decreased with mouse month age addition and in the same age Prdx2 knockout mouseeven thinner than wild mouse; skin wound healing results show that Prdx2 ablation mouse healing slower than wild mouse; Western blotting results show that p21, p16 expression increased with month age addition and in the same age Prdx2 ablation mouse are higher,phospho-stat3 and PCNA express decreased with month age addition and in same age Prdx2 ablation mouse are lower. In the cellular portion, with the month age addition, cell cycle assay shows that G0/G1 arrest is increasing in DMSCs, and if in the same month age Prdx2 knockout DMSCs have a higher arrest proportion. Apoptosis assay shows that the apoptosis level in these DMSCs are same; SA-β-gal staining results show that cellular senescence proportion is increasing with passage addition and if in the same passage Prdx2 knockout DMSCs have a higher proportion; growth curves show that there is no difference between Prdx2 knockout DMSCs and wild DMSCs at 3P, but with the passage addition the speed of Prdx2 knockout DMSCs proliferation is slower than wild DMSCs; Western blotting results show that with the addition of passage, p21, p16 express is increased and in the same passage the expressions are higher in Prdx2 knockout DMSCs, the expression of Phospho-stata3 and PCNA is decreased with passage addition and if in the same passage the expressions in prdx2 knockout DMSCs are lower. In the molecular proportion, cyto-immunofluorescence staining and western blotting results show that β-catenin nuclear import is increased in Prdx2 knockout mouse, the expression of Phosopho-GSK3β(Ser9) and Phospho-Akt is increased.These results indicate that Prdx2 mediates canonical Wnt/β-catenin pathway to protect mouse dermal mesenchymal stem cells. If Prdx2 is deficient, Wnt/β-catenin pathway will inappropriate activating that induces DMSCs premature senescence and then induces mouse skin premature aging and function insufficient.更多还原