【作者】 陈文琦；

【导师】 毕志刚；

【作者基本信息】 南京医科大学 ， 皮肤病与性病学， 2008， 博士

【摘要】 中波紫外线（ultraviolet B,UVB）可诱导皮肤肿瘤发生,又可诱导人皮肤成纤维细胞（humn skin fibroblast,HSF）发生应激诱导的提前衰老（stressinducedpremature senescence,SIPS）。目前认为细胞衰老是抑制肿瘤发生的机制之一,抑癌基因p53是其中关键的调控因子,但是在UVB诱导的细胞衰老中未见报道,因此,研究p53在UVB诱导的提前衰老中的调控作用和肿瘤抑制作用具有重要的意义。本研究分四个部分探讨了UVB照射诱导HSF发生SIPS中p53的调控作用及肿瘤抑制作用。研究目的:第一部分探讨中波紫外线（UVB）诱导人皮肤成纤维细胞（HSF）衰老与UVB辐射剂量的关系,建立UVB诱导的成纤维细胞早衰模型,为深入研究UVB诱导的细胞衰老与肿瘤发生的关系奠定基础。第二部分探讨UVB诱导SIPS后细胞周期调控的变化。探讨p53相关的生长停滞、细胞凋亡和肿瘤发生相关基因表达在UVB诱导的SIPS中的变化情况。初步探讨UVB诱导SIPS在肿瘤发生机制中的作用。第三部分,转染针对p53的短发夹RNA（short hairpin RNA,shRNA）质粒建立抑制p53表达的HSF细胞系。探讨抑制p53表达对UVB诱导HSF发生SIPS及其肿瘤抑制作用的影响。第四部分建立稳定过表达野生型p53基因（wtp53）的人皮肤成纤维细胞系。探讨过表达p53对UVB诱导SIPS及其肿瘤抑制作用的影响。研究方法:1.以亚毒性剂量UVB反复照射HSF以诱导其衰老。2.衰老相关的β-半乳糖苷酶（senescence-associatedβ-galactosidase,SAβ-gal）染色法检测照射后细胞的SAβ-gal活性。3.四甲基偶氮唑盐微量酶反应比色法（methyl thiazolyl tetrazolium,MTT法）检测HSF经UVB辐射后的细胞增殖情况。4.逆转录聚合酶链反应（reverse transcription-polymerase chainraction,RT-PCR）检测三种衰老相关基因即纤维结合素（fibronectin,FN）,骨结合素（osteonectin,ON）和平滑肌22（smooth muscle 22,SM22）基因的表达。5.流式细胞仪检测细胞周期。6.蛋白印迹法（western blot,WB）检测细胞周期蛋白p53、p21、p16的表达。7.实时荧光定量PCR芯片的方法检测与p53相关的生长停滞,凋亡及肿瘤发生相关的基因的mRNA表达水平。包括p53、p21、p16、p19、bax、bcl-2、缺氧诱导因子1α（hypoxia-induciblefactor 1 alpha subunit,HIF-1α）、血管内皮生长因子（vascular endothelialgrowth factor,VEGF）、人双微球蛋白2基因（human double minute 2,hdm2）等。8.在脂质体lipofectamine^TM2000介导下,分别将p53的shRNA的真核表达质粒pGCsi-p53和含有野生型p53的真核表达质粒pCMV-p53导入HSF中,经G418筛选抗性克隆,扩大培养,建立稳定转染克隆细胞系。用RT-PCR、WB和实时定量PCR分析目的基因及其蛋白表达。9.建立的抑制p53表达和p53过表达的HSF模型分别在UVB照射后进行SAβ-gal染色、MTT法、实时荧光定量PCR芯片测定。研究结果:第一部分:筛选出适宜的诱导衰老的剂量和次数,以10 mJ/cm²的亚毒性UVB剂量连续5次照射HSF后,衰老的生物学特征得以明显表现,包括细胞生长停滞,SAβ-gal活性,衰老相关基因的表达等。在建立的衰老模型中,第一,MTT法检测显示了细胞增生能力的减弱;第二,具有SAβ-gal活性的阳性细胞明显增加;第三,三种衰老相关基因,FN,ON和SM22的表达亦明显增强。第二部分:流式细胞仪对UVB诱导的SIPS模型进行细胞周期检测发现,大部分细胞发生了G1期阻滞;衰老通路中p53,p21和p16的表达都明显增加;实时荧光定量PCR芯片检测发现,与生长停滞相关的抑癌基因p53、p21、p16、p19的表达均上调;p53下游的凋亡相关基因中的抑凋亡基因bcl-2表达稍上调,而凋亡基因bax表达下调;在肿瘤发生相关基因中,癌基因HIF-1α、VEGF、hdm2的表达均下调。第三部分:成功建立RNA干扰抑制p53表达的HSF细胞系,转染细胞中p53基因及蛋白水平明显下调。抑制p53表达的细胞株经UVB照射后SAβ-gal活性降低,具有抑制UVB诱导的细胞生长停滞的作用。肿瘤发生相关基因检测结果表明抑制p53表达可抑制衰老基因的表达,但p16通路并不受之影响,在UVB作用下仍可发挥作用。抑制p53的表达可使抑凋亡基因bcl-2上调及促凋亡基因bax下调,癌基因HIF-1α、VEGF、hdm2表达明显增加。这些p53依赖的细胞凋亡和抑制肿瘤作用被抑制。UVB照射与否对这两种作用无明显影响。第四部分:成功建立过表达p53的HSF细胞系,转染细胞中存在p53基因及蛋白的稳定过表达。过表达p53的细胞株经UVB照射后SAβ-gal活性未见增高,对UVB诱导的细胞生长停滞的影响并不显著。肿瘤发生相关基因检测结果表明过表达p53未能提高衰老基因的表达;过表达p53可使抑凋亡基因bcl-2下调及促凋亡基因bax上调;癌基因HIF-1α、VEGF、hdm2表达明显降低。这些p53依赖的细胞凋亡和抑制肿瘤作用因p53的过表达而更为显著。UVB照射可加强这两种作用。结论:第一部分:反复亚毒性剂量UVB照射HSF可诱导其SIPS发生,表现出与复制衰老（RS）的成纤维细胞许多共同的特征。实验成功建立了一种UVB诱导的HSF的SIPS模型,可用于进一步的相关研究。第二部分:G1期阻滞和衰老信号通路蛋白表达增加进一步印证了UVB诱导HSF发生的SIPS。p53信号通路相关基因的表达变化提示UVB诱导的SIPS可能具有与p53相关的抗凋亡和肿瘤抑制的作用,但其中p53信号通路所起的作用还需更进一步的实验探索。第三部分:抑制p53表达能够延迟或部分抑制HSF的复制衰老和UVB诱导的SIPS。UVB诱导的SIPS中的部分肿瘤抑制机制确由p53所调控,抑制p53表达可使部分p53依赖的细胞凋亡和抑制肿瘤作用明显缺失,从而有助于肿瘤的发生。第四部分:过表达p53不能促进HSF的复制衰老和UVB诱导的SIPS。过表达p53可使p53依赖的细胞凋亡和抑制肿瘤作用明显增强,UVB还可加强这种作用,从而有助于抑制肿瘤发生。在UVB作用下,过表达p53可能更倾向于诱导凋亡而非衰老。更多还原

【Abstract】 Solar ultraviolet B irradiation can induce skin photocarcinogenesis and it also can induce stress-induced premature senescence in human skin fibroblasts with the key regulation factor-p53.In this study(including four parts),we explored the regulation of p53 in Ultraviolet B-induced premature senescent human skin fibroblasts with function of tumor suppression.Objective:In partⅠ,we developed a model of UVB-induced premature senescence in human skin fibroblast(HSF)to facilitate further study about relationship among stress-induced premature senescence and tumorigenesis induced by UVB.In partⅡ,to explore the effects of UVB-induced SIPS on cell cycle regulation in HSF.To explore the effects of on mRNA expression of p53-related genes involved in growth arrest,apoptosis and tumorigenesis.To further elucidate the role of UVB-induced SIPS in molecular mechanisms of tumorigenesis.In partⅢ,to transfect p53 short hairpin RNA(shRNA)plasmid into human skin fibroblasts for establishing a cell line with repressed expression of p53.To explore the effect of repressesd expression of p53 on UVB-induced SIPS and tumor suppression activity in HSF.In partⅣ,to establish a cell line that exogenous wild-type p53(wtp53)gene transfected human skin fibroblasts.To explore the effect of over expression of p53 on UVB-induced SIPS and tumor suppression activity in HSF.Methods:1.We chosed a subcytotoxic dose from different dosages of UVB and radiated HSF five times at this level to induce senescence.2.SAβ-gal activity was investigated.3.Cell viability was determined by MTT assay.4. Three senescence- associated genes,fibronectin(FN),osteonectin(ON)and smooth muscle 22(SM22)mRNA expression was determined by RT-PCR.5.In UVB-stressed premature HSF,cell cycle assay was analyzed with fluorescence-activated cell sorting(FACS)by flow cytometer.6.Protein expression of p53,p21 and p16 was determined by western blot(WB).7.A real time PCR array was performed to investigate mRNA level of a number of genes involved in growth arrest,apoptosis and tumorigenesis induced by UVB. 8.Eukaryotic expressing plasmids pGCsi-p53 containing short hairpin RNA of p53 gene and pCMV-p53 containing wild type p53 gene were introduced by lipofectamine-mediated gene transfection into human skin fibroblasts.After transfection,the cells were selected by G418.Then resistant clones were chosen and expanded in DMEM culture medium.RT-PCR, real-time quantitative PCR,WB were used to determine the expression of p53 gene and p53 protein.9.SAβ-gal activity,MTT assay and PCR array were investingated in UVB-stressed HSF lines which repressesd expression of p53 and overexpresssion of p53.Results: PartⅠ:We found suitable subcytotoxic dosage and radiation times for inducing premature senescence.After repetitive five subcytotoxic exposures to UVB at the dose of 10mJ/cm~2,biomarkers of senescence were markedly established:cell growth arrest,senescence-associatedβ-galactosidase(SAβ-gal)activity,,and overexpression of senescence-associated genes.First,there was a loss of replicative potential as assessed by MTT assay.Second,there was an increase in the proportion of cells positive for SAβ-gal activity.Third,the levels of the mRNA of three senescence-associated genes,FN,ON and SM22, were also upregulated.PartⅡ:FACS analysis showed that UVB-stressed HSF are blocked mostly in G1 phase of the cell cycle.Protein expressions of p53,p21 and p16 in senescence pathways were increased significantly.PCR array indicated some genes are differentially expressed in UVB-induced SIPS.First,anti-oncogenes involved in growth arrest p53,p21,p16,p19 were all overexpressed.Second, among genes involved in p53-dependent apoptosis,bax was downregulated while bcl-2 was up-regulated slightly.Third,among genes involved in tumorgensis induced by UVB,HIF-1α,VEGF and hdm2 were down-regulated.PartⅢ:A repressed expression of p53 HSF cell clone was established successfully.In the transfected HSF cell p53 mRNA and p53 protein expression were repressesd sigmficantly.SAβ-gal activity was reduced and growth arrest was inhibited in UVB-treated HSF-pGCsi-p53 cells.Repressed expression of p53 downregulated senescence associated genes without influencing p16 pathway under UVB.mRNA expression of genes involved in apoptosis and tumorigenesis in HSF-pGCsi-p53 cells showed up-regulation of bcl-2,downregulation of bax,up-regulation of HIF-1α,VEGF and hdm2. Repressed expression of p53 inhibitored p53-related apoptosis and tumor repression activity without influence of UVB.PartⅣ:An anti-G418 cell clone was established and expanded in culture. The transfected wtp53 gene HSF-p53 cells showed overexpression of p53 mRNA and p53 protein.Overexpression of p53 in UVB-treated HSF-wtp53 cells couldn’t induce SAβ-gal activity and growth arrest.Expression of genes involved in tumorigenesis showed overexpression of p53 couldn’t up-regulated senescence associated genes and function of p16 pathway under UVB was. negligible.mRNA expression of genes involved in apoptosis and tumorigenesis in HSF-pCMV-p53 cells showed downregulation of bcl-2, up-regulation of bax,downregulation of HIF-1α,VEGF and hdm2. Overexpression of p53 enhanced p53-related apoptosis and tumor repression activity and UVB radiation could even promoted them.Conclusions:PartⅠ:SIPS could be induced by repeated subcytotoxic UVB in HSF,which shared many features with replicative senescence(RS).We could use this UVB-induced SIPS model in further related studies.PartⅡ:G1 stage arrest and expression of proteins involved in senescence pathways further confirm UVB-induced SIPS in HSF.Expression of p53-related genes suggest that UVB-induced SIPS may play an important role in p53-related apoptosis resistance and tumor suppression activity.The role of p53 pathwys in related mechanisms should be further explored.PartⅢ:RS and UVB-stessed SIPS in HSF could be reserved or partly inhibitored by repressed expression of p53.Repressed expression of p53 inhibitor p53-related apoptosis and tumor repression activity with a promoting tumorigenesis function,which confirmed tumorigenesis regulation of p53 in UVB-induced SIPS.PartⅣ:RS and UVB-stessed SIPS in HSF couldn’t be induced by overexpression of p53.Overexpression of p53 can enhance p53-related apoptosis and tumor repression activity with a tumorigenesis repression function,and UVB can even promote it.Overexpression of p53 may prefer to induce apotosis but not senescence under UVB radiation更多还原