DNA修复蛋白JWA在小鼠衰老中的作用及机制研究

发布时间：2018-06-10 11:44

本文选题：JWA + 老化　；参考：《南京医科大学》2011年博士论文

【摘要】：随着人口年龄结构改变,老龄化趋势日益凸显。由于人口老化带来生产力下降,并增加罹患年龄相关疾病如癌症及神经退行性疾病的风险,人口老龄化问题已经引起全世界范围的关注。在细胞及分子水平上,老化是损伤和修复间平衡的结果。损伤随着年龄的增长不断积累,而对损伤进行修复的效率却不断下降,并最终导致组织器官功能的改变或丧失。机体针对不同损伤类型存在许多种修复途径,包括碱基切除修复、核苷酸切除修复、非同源末端连接及同源重组等。这些通路中基因功能的突变或缺失通常导致早衰。 JWA是一种广谱的环境应答基因,在多种应激条件下JWA表达升高。前期的研究已证实JWA参与氧化应激诱导的DNA损伤修复；在氧化应激条件下,JWA从细胞浆易位到细胞核,而且和XRCC1和LIG3共定位；在氧化应激诱导的BER修复过程中,JWA正调控XRCC1, LIG3的表达。之前的结果提示我们JWA是一个潜在的DNA损伤修复分子,但JWA是否参与DNA双链损伤修复,在整体水平上JWA具有什么样的生理功能如是否参与调控机体老化我们依然不知道。目的：探讨JWA是否更广泛的参与DNA损伤修复并从整体水平上影响机体的各项生命进程,从而为更加深入的了解JWA的功能提供新的科学依据。方法：采用VP16和CPT处理细胞产生DNA双链损伤,检测JWA的表达及其细胞内定位情况；采用基于Cre-LoxP条件性基因剔除策略构建全身性JWA基因剔除小鼠；使用PCR、RT-PCR、基因测序及蛋白印迹从DNA、RNA及蛋白水平上鉴定JWA基因剔除小鼠基因型；采用肉眼观察、生长队列观察、体重测量等观察JWA基因剔除对小鼠基本指标如寿命、体重、外观的影响；采用X线摄影、显微摄影技术(Micro-CT)、HE染色等分析JWA基因缺失对小鼠不同组织器官病理形态的影响；采用FACS及ELISA分析JWA基因缺失对免疫功能细胞及生长因子水平的影响；采用开阔场实验评估JWA基因缺失对小鼠活动能力的影响；采用SA-β-gal分析及流式分析术分析JWA基因缺失对细胞衰老的影响；采用基因芯片技术分析JWA基因缺失对基因表达谱的影响；采用寡核苷酸依赖的转录因子活性分析技术分析JWA基因缺失对转录因子活性的影响；采用生物信息学工具GSEA、DAVID及oPOSSUM分析JWA基因缺失基因表达谱中基因集富集情况、GO分布情况；我们进一步采用EMSA、Q-PCR、报告基因、免疫印迹及免疫沉淀等方法检测JWA对NF-κB信号通路的影响。结果：1.VP16及CPT诱导JWA表达分别升高2.4和2.3倍(P0.05),部分JWA蛋白在DNA损伤过程中从胞浆易位到胞核,进入细胞核的JWA与DNA损伤位点共定位,免疫荧光实验发现细胞损伤越重,进入细胞核的JWA越多。 2.通过基因型鉴定表明采用Cre-LoxP基因剔除小鼠策略可以成功获得JWA基因第二外显子剔除的JWA基因缺失小鼠。 3.JWA基因剔除小鼠提早出现体重减轻(P0.05)、骨骼畸形(JWA+/+小鼠侧弯Cobb's角为6.5±1.2,JWA-/-小鼠侧弯Cobb's角为25±2.5,P0.001。而JWA+/+小鼠后凸Cobb's角为32.7±1.6,JWA-/-小鼠后凸Cobb's角为80.7±5.8,P0.001)、骨质疏松(JWA-/-小鼠骨密度为635.56±25.5mgHA/ccm,而JWA+/+小鼠骨密度为662.43±15.67mgHA/ccm,P0.05)、皮下脂肪层及免疫器官萎缩、活动能力下降(JWA-/-小鼠在10 min内跑动的距离为29.5±17.21m,JWA+/+小鼠为62.3±23.99m,P=0.012)、IGF-1水平降低及寿命缩短(JWA-/-小鼠一年生存率为22.7%,与JWA+/+小鼠相比,P=0.0003)等表型。 4.JWA基因剔除小鼠肝脏组织中β-Gal染色阳性细胞比例为10.6±2.6%,而JWA+/+肝脏衰老细胞比例为0.29±0.49%,P0.01;P6代JWA-/-MEF细胞中衰老细胞的比例为27.4±2.7%,而JWA+/+细胞中比例为17±2.4%,P0.01；JWA基因缺失MEF细胞G2期细胞比例为22.4±2.8%,比例显著高于JWA+/+细胞中的比例12.8±0.7%,P0.05；JWA基因缺失MEF细胞中凋亡细胞所占的比例为2.3±0.67%,明显低于JWA+/+MEF细胞中的比例6.27±1.12%,P0.01；p53、p16、p21等分子在JWA基因缺失的肝脏及MEF细胞中表达升高；高通量分析结果显示部分衰老相关分泌因子在JWA基因缺失小鼠的脾脏、肝脏及MEF细胞中表达上升。 5.彗星实验发现损伤细胞的比例在JWA基因缺失的原代脾脏细胞及MEF细胞中增多；JWA基因缺失小鼠中端粒酶活性水平下降但JWA基因缺失不影响体内活性氧族生成。 6. GSEA分析发现NF-κB及E2Fs转录因子下游基因在JWA基因缺失的脾脏中富集,p值均小于0.01,leading edge分析显示大部分NF-κB的靶基因在JWA基因缺失后是上调的,而大部分E2Fs的靶基因则是下调的。 7. OATFA分析发现一系列转录因子的活性在JWA基因缺失的肝脏组织中发生改变,进一步运用oPOSSUM分析发现在这些转录因子中,NF-κB转录因子(Rela, c-Rel)可能起着重要作用。 8.DNA损伤状态下,JWA基因缺失细胞中NF-κB的转录活性增强,表现为报告基因活性增强、靶基因Bcl-xL、Icam1、IL1a、Nfkb1、Nfkb2及Nfkbia表达上调,而恢复JWA表达可有效逆转靶基因Icam1、IL1a及Bcl-xL的表达。JWA与IKKβ形成复合物,在DNA损伤的情况下两者的相互作用明显增强。干涉p65的表达可逆转氧化应激所致JWA基因缺失MEF细胞的细胞衰老。而且,DNA损伤状态下,JWA基因缺失增加细胞内的糖基化水平。抑制细胞糖基化,逆转DNA损伤状态下JWA基因缺失所致的NF-κB信号通路的激活。结论：基于JWA广泛地参与多种类型的DNA修复过程,本实验通过Cre-loxp条件性基因剔除策略,成功构建了条件性JWA基因全身剔除小鼠,首次发现.JWA基因剔除小鼠出现早衰,这可能是JWA基因缺失导致DNA损伤积聚,端粒酶活性降低并激活NF-κB信号通路和引起细胞衰老导致的。我们的工作为全面认识JWA的生理功能提供了新的科学依据,也将为衰老的干预提供新的潜在靶点。
[Abstract]:As the age structure of the population changes, the trend of aging has become increasingly prominent. Population aging has attracted worldwide attention because of the declining productivity of population and increasing the risk of age related diseases such as cancer and neurodegenerative diseases. At the cellular and sub level, aging is the balance between damage and repair. As a result, damage accumulates as the age increases, and the efficiency of repair of damage continues to decline, and ultimately leads to changes or loss of tissue and organ function. There are many repair pathways for different types of injury, including base resection and repair, nucleotide excision repair, non homologous end connection and homologous recombination. Mutation or deletion of gene function in pathway usually leads to premature senility.
JWA is a broad-spectrum environmental response gene that increases the expression of JWA under a variety of stressful conditions. Previous studies have confirmed that JWA is involved in oxidative stress induced DNA damage repair. Under oxidative stress, JWA translocation from cytoplasm to nucleus and co localizes XRCC1 and LIG3; JWA is regulated in the process of oxidative stress induced BER repair. XRCC1, LIG3 expression. Previous results suggest that JWA is a potential DNA damage repair molecule, but whether JWA is involved in the repair of DNA double stranded damage, and what physiological functions of JWA at the overall level, such as whether or not to participate in the regulation of the aging of the body, we still do not know.
Objective: To explore whether JWA is more widely involved in the repair of DNA damage and affects the life process of the body from the overall level, so as to provide a new scientific basis for understanding the function of JWA in depth.
Methods: DNA double strand damage was produced by VP16 and CPT cells, and the expression of JWA was detected and its intracellular location was detected. The generalized JWA gene culling mice were constructed by Cre-LoxP conditional gene culling strategy, and PCR, RT-PCR, gene sequencing and Western blotting were used to identify the JWA gene knockout mice gene. The effects of JWA gene deletion on the basic indexes such as life, weight and appearance were observed by naked eye observation, growth queue observation and weight measurement, and the effects of JWA gene deletion on the pathological morphology of different organ organs in mice were analyzed by X-ray photography, microphotography (Micro-CT) and HE staining, and JWA gene was analyzed by FACS and ELISA. The effect of deletion on the level of immune functional cells and growth factors was studied. The effect of JWA gene deletion on the activity of mice was evaluated by open field experiment. The effect of JWA gene deletion on cell senescence was analyzed by SA- beta -gal analysis and flow analysis, and the effect of the deletion of JWA gene on the gene expression profile was analyzed by gene chip technique. The effect of JWA gene deletion on the activity of transcription factors was analyzed using the oligonucleotide dependent transcription factor activity analysis technique. The concentration of gene collection and GO distribution in the gene expression profiles of JWA gene deletion were analyzed by bioinformatics tool GSEA, DAVID and oPOSSUM; we further adopted EMSA, Q-PCR, reporter gene, Western blot and immunoblotting. The effects of JWA on the NF- kappa B signaling pathway were detected by pestilence sedimentation.
Results: 1.VP16 and CPT induced JWA expression increased by 2.4 and 2.3 times respectively (P0.05). Part of JWA protein was translocated from cytoplasm to nucleus during DNA damage. The JWA and DNA damage site entered the nucleus, and the immunofluorescence test found that the more serious the cell injury was, the more JWA entered the nucleus.
2. genotype identification showed that Cre-LoxP gene knockout mice could successfully obtain JWA gene deletion mice deleted from the second exon of JWA gene.
3.JWA gene knockout mice appeared early weight loss (P0.05), bone malformation (JWA+/+ mouse side bend Cobb's angle was 6.5 + 1.2, JWA-/- mouse side bend Cobb's angle was 25 + 2.5, P0.001. while JWA+/+ mice were 32.7 + 1.6, JWA-/- mice 80.7 + 5.8, P0.001), osteoporosis (635.56 + bone density of mice) M, JWA+/+ mice bone density was 662.43 + 15.67mgHA/ccm, P0.05), subcutaneous fat layer and immune organ atrophy, activity decreased (the distance of JWA-/- mice running in 10 min was 29.5 + 17.21m, JWA+/+ mice were 62.3 + 23.99m, P=0.012), IGF-1 level decreased and life shortened (22.7% of one year survival rate of JWA-/- mice, compared with mice. 0.0003) phenotypes.
The proportion of beta -Gal staining positive cells in the liver tissues of 4.JWA gene knockout mice was 10.6 + 2.6%, while the proportion of JWA+/+ liver senescent cells was 0.29 + 0.49%, P0.01, and the proportion of senescent cells in P6 generation JWA-/-MEF cells was 27.4 + 2.7%, while the proportion of JWA+/+ cells was 17 + 2.4%, P0.01, and JWA based missing MEF cells were 22.4 + 2.8%, and the proportion was 22.4 + 2.8%. The proportion of JWA+/+ cells was 12.8 + 0.7%, P0.05, and the proportion of apoptotic cells in MEF cells with JWA deletion was 2.3 + 0.67%, which was significantly lower than that in JWA+/+MEF cells, 6.27 + 1.12%, P0.01; p53, p16, p21 and other molecules increased in the liver and MEF cells of JWA gene deletion; high throughput analysis showed partial senescence The expression of related secretory factors increased in spleen, liver and MEF cells of JWA deficient mice.
5. comet experiment found that the proportion of damaged cells increased in the primary spleen cells and MEF cells with JWA gene deletion, and the telomerase activity in JWA gene deletion mice decreased, but the deletion of JWA gene did not affect the formation of ROS in the body.
6. GSEA analysis found that the downstream genes of NF- kappa B and E2Fs transcription factors were enriched in the spleen of the JWA gene, and P values were less than 0.01. Leading edge analysis showed that most of the target genes of NF- kappa B were up-regulated after the JWA gene deletion, while most of the E2Fs target genes were down-regulated.
7. OATFA analysis found that the activity of a series of transcriptional factors changes in the liver tissues of the JWA gene deletion. Further oPOSSUM analysis found that the NF- kappa B transcription factor (Rela, c-Rel) may play an important role in these transcription factors.
Under the condition of 8.DNA damage, the transcriptional activity of NF- kappa B in the JWA gene deletion cells enhanced, the expression of the reporter gene activity was enhanced, the expression of target gene Bcl-xL, Icam1, IL1a, Nfkb1, Nfkb2 and Nfkbia was up regulated, while the resumed JWA expression could effectively reverse the target gene Icam1. The expression of the interferometric p65 can reverse the cell senescence of JWA gene missing MEF cells induced by oxidative stress. Moreover, the deletion of JWA gene increases the level of glycosylation in the cell under the condition of DNA damage. It inhibits the glycosylation of cells and reverses the activation of the NF- kappa B signaling pathway caused by the deletion of the JWA gene in the DNA damage state.
Conclusion: Based on the extensive participation of JWA in a variety of DNA repair processes, this experiment successfully constructed a conditioned JWA gene knockout mouse through the Cre-loxp conditional gene culling strategy. It was the first time to find premature failure in the.JWA gene knockout mice. This may be the accumulation of DNA damage in the JWA gene deletion, the decrease of telomerase activity and the activation of NF- kappa B. Our work provides a new scientific basis for a comprehensive understanding of the physiological functions of JWA, and will also provide new potential targets for the intervention of aging.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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