發(fā)布時間：2018-06-10 11:44

  本文選題：JWA + 老化　； 參考：《南京醫(yī)科大學》2011年博士論文

【摘要】：隨著人口年齡結構改變,老齡化趨勢日益凸顯。由于人口老化帶來生產力下降,并增加罹患年齡相關疾病如癌癥及神經退行性疾病的風險,人口老齡化問題已經引起全世界范圍的關注。在細胞及分子水平上,老化是損傷和修復間平衡的結果。損傷隨著年齡的增長不斷積累,而對損傷進行修復的效率卻不斷下降,并最終導致組織器官功能的改變或喪失。機體針對不同損傷類型存在許多種修復途徑,包括堿基切除修復、核苷酸切除修復、非同源末端連接及同源重組等。這些通路中基因功能的突變或缺失通常導致早衰。 JWA是一種廣譜的環(huán)境應答基因,在多種應激條件下JWA表達升高。前期的研究已證實JWA參與氧化應激誘導的DNA損傷修復；在氧化應激條件下,JWA從細胞漿易位到細胞核,而且和XRCC1和LIG3共定位；在氧化應激誘導的BER修復過程中,JWA正調控XRCC1, LIG3的表達。之前的結果提示我們JWA是一個潛在的DNA損傷修復分子,但JWA是否參與DNA雙鏈損傷修復,在整體水平上JWA具有什么樣的生理功能如是否參與調控機體老化我們依然不知道。 目的：探討JWA是否更廣泛的參與DNA損傷修復并從整體水平上影響機體的各項生命進程,從而為更加深入的了解JWA的功能提供新的科學依據。 方法：采用VP16和CPT處理細胞產生DNA雙鏈損傷,檢測JWA的表達及其細胞內定位情況；采用基于Cre-LoxP條件性基因剔除策略構建全身性JWA基因剔除小鼠；使用PCR、RT-PCR、基因測序及蛋白印跡從DNA、RNA及蛋白水平上鑒定JWA基因剔除小鼠基因型；采用肉眼觀察、生長隊列觀察、體重測量等觀察JWA基因剔除對小鼠基本指標如壽命、體重、外觀的影響；采用X線攝影、顯微攝影技術(Micro-CT)、HE染色等分析JWA基因缺失對小鼠不同組織器官病理形態(tài)的影響；采用FACS及ELISA分析JWA基因缺失對免疫功能細胞及生長因子水平的影響；采用開闊場實驗評估JWA基因缺失對小鼠活動能力的影響；采用SA-β-gal分析及流式分析術分析JWA基因缺失對細胞衰老的影響；采用基因芯片技術分析JWA基因缺失對基因表達譜的影響；采用寡核苷酸依賴的轉錄因子活性分析技術分析JWA基因缺失對轉錄因子活性的影響；采用生物信息學工具GSEA、DAVID及oPOSSUM分析JWA基因缺失基因表達譜中基因集富集情況、GO分布情況；我們進一步采用EMSA、Q-PCR、報告基因、免疫印跡及免疫沉淀等方法檢測JWA對NF-κB信號通路的影響。 結果：1.VP16及CPT誘導JWA表達分別升高2.4和2.3倍(P0.05),部分JWA蛋白在DNA損傷過程中從胞漿易位到胞核,進入細胞核的JWA與DNA損傷位點共定位,免疫熒光實驗發(fā)現細胞損傷越重,進入細胞核的JWA越多。 2.通過基因型鑒定表明采用Cre-LoxP基因剔除小鼠策略可以成功獲得JWA基因第二外顯子剔除的JWA基因缺失小鼠。 3.JWA基因剔除小鼠提早出現體重減輕(P0.05)、骨骼畸形(JWA+/+小鼠側彎Cobb's角為6.5±1.2,JWA-/-小鼠側彎Cobb's角為25±2.5,P0.001。而JWA+/+小鼠后凸Cobb's角為32.7±1.6,JWA-/-小鼠后凸Cobb's角為80.7±5.8,P0.001)、骨質疏松(JWA-/-小鼠骨密度為635.56±25.5mgHA/ccm,而JWA+/+小鼠骨密度為662.43±15.67mgHA/ccm,P0.05)、皮下脂肪層及免疫器官萎縮、活動能力下降(JWA-/-小鼠在10 min內跑動的距離為29.5±17.21m,JWA+/+小鼠為62.3±23.99m,P=0.012)、IGF-1水平降低及壽命縮短(JWA-/-小鼠一年生存率為22.7%,與JWA+/+小鼠相比,P=0.0003)等表型。 4.JWA基因剔除小鼠肝臟組織中β-Gal染色陽性細胞比例為10.6±2.6%,而JWA+/+肝臟衰老細胞比例為0.29±0.49%,P0.01;P6代JWA-/-MEF細胞中衰老細胞的比例為27.4±2.7%,而JWA+/+細胞中比例為17±2.4%,P0.01；JWA基因缺失MEF細胞G2期細胞比例為22.4±2.8%,比例顯著高于JWA+/+細胞中的比例12.8±0.7%,P0.05；JWA基因缺失MEF細胞中凋亡細胞所占的比例為2.3±0.67%,明顯低于JWA+/+MEF細胞中的比例6.27±1.12%,P0.01；p53、p16、p21等分子在JWA基因缺失的肝臟及MEF細胞中表達升高；高通量分析結果顯示部分衰老相關分泌因子在JWA基因缺失小鼠的脾臟、肝臟及MEF細胞中表達上升。 5.彗星實驗發(fā)現損傷細胞的比例在JWA基因缺失的原代脾臟細胞及MEF細胞中增多；JWA基因缺失小鼠中端粒酶活性水平下降但JWA基因缺失不影響體內活性氧族生成。 6. GSEA分析發(fā)現NF-κB及E2Fs轉錄因子下游基因在JWA基因缺失的脾臟中富集,p值均小于0.01,leading edge分析顯示大部分NF-κB的靶基因在JWA基因缺失后是上調的,而大部分E2Fs的靶基因則是下調的。 7. OATFA分析發(fā)現一系列轉錄因子的活性在JWA基因缺失的肝臟組織中發(fā)生改變,進一步運用oPOSSUM分析發(fā)現在這些轉錄因子中,NF-κB轉錄因子(Rela, c-Rel)可能起著重要作用。 8.DNA損傷狀態(tài)下,JWA基因缺失細胞中NF-κB的轉錄活性增強,表現為報告基因活性增強、靶基因Bcl-xL、Icam1、IL1a、Nfkb1、Nfkb2及Nfkbia表達上調,而恢復JWA表達可有效逆轉靶基因Icam1、IL1a及Bcl-xL的表達。JWA與IKKβ形成復合物,在DNA損傷的情況下兩者的相互作用明顯增強。干涉p65的表達可逆轉氧化應激所致JWA基因缺失MEF細胞的細胞衰老。而且,DNA損傷狀態(tài)下,JWA基因缺失增加細胞內的糖基化水平。抑制細胞糖基化,逆轉DNA損傷狀態(tài)下JWA基因缺失所致的NF-κB信號通路的激活。 結論：基于JWA廣泛地參與多種類型的DNA修復過程,本實驗通過Cre-loxp條件性基因剔除策略,成功構建了條件性JWA基因全身剔除小鼠,首次發(fā)現.JWA基因剔除小鼠出現早衰,這可能是JWA基因缺失導致DNA損傷積聚,端粒酶活性降低并激活NF-κB信號通路和引起細胞衰老導致的。我們的工作為全面認識JWA的生理功能提供了新的科學依據,也將為衰老的干預提供新的潛在靶點。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R363

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