【作者】 毛玲；

【导师】 梅元武； 胡波；

【作者基本信息】 华中科技大学 ， 神经病学， 2010， 博士

【摘要】 第一部分维甲酸诱导神经母细胞瘤细胞分化的分子机制目的研究维甲酸诱导神经母细胞瘤细胞分化的分子机制。方法用维甲酸体外诱导神经母细胞瘤细胞BE(2)-C分化后,在普通光学显微镜下观察细胞形态变化,采用流式细胞仪检测细胞周期变化,分别采用实时定量PCR(Real-time PCR)和免疫印迹法(Western blotting)观察各种细胞周期素(cyclinA、cyclinB1、cyclinD1、cyclinE1和cyclinE2)和细胞周期素依赖性激酶(CDK1、CDK2、CDK4和CDK6)的mRNA水平和蛋白水平变化。用放线菌酮(Cycloheximide, CHX)抑制细胞蛋白质的合成后,使用免疫印迹法及Image J软件分析细胞周期素(cyclinA、cyclinB1和cyclinE2)半衰期的变化。结果维甲酸体外诱导神经母细胞瘤细胞12天后细胞长出长的突起,出现神经元样分化,细胞周期检测结果显示维甲酸诱导组G0／G1期细胞占总细胞的78.2%,较对照组的50.9%增加了27.3%,提示细胞周期阻滞,增殖减少。实时定量PCR和免疫印迹结果显示,细胞周期素cyclinA、cyclinB1和cyclinE2及细胞周期素依赖性激酶CDK1的mRNA水平和蛋白水平均随维甲酸诱导时间的延长而逐渐降低,而蛋白半衰期检测发现这些蛋白的半衰期并没有明显变化,这说明维甲酸处理后,cyclinA、cyclinB1、cyclinE2和CDK1蛋白水平的降低发生在转录水平。cyclinD1的mRNA水平和蛋白水平有增高的趋势,而cyclinE1和其他CDKs (CDK2、CDK4和CDK6)的mRNA水平和蛋白水平均没有明显变化。结论维甲酸能够诱导神经母细胞瘤细胞分化,并且在转录水平上抑制cyclinA、cyclinB1、cyclinE2和CDK1的表达,从而导致细胞周期阻滞。第二部分Hoxc9在维甲酸诱导神经母细胞瘤细胞分化中的作用第一节维甲酸诱导神经母细胞瘤细胞Hoxc9表达增高目的观察维甲酸诱导神经母细胞瘤细胞分化后基因表达谱的变化,寻找维甲酸诱导细胞分化的关键分子。方法采用基因芯片技术观察维甲酸诱导神经母细胞瘤细胞分化后基因表达谱的变化。通过实时定量PCR和免疫印迹法进一步验证基因芯片检查结果。结果基因芯片检查发现维甲酸诱导神经母细胞瘤细胞分化后,一些Hox基因水平上调。通过实时定量PCR和免疫印迹法进一步验证,发现Hoxc9的mRNA水平和蛋白水平随着维甲酸诱导时间的延长明显增高。结论Hoxc9可能与维甲酸诱导的神经母细胞瘤细胞分化有关。第二节Hoxc9过表达抑制神经母细胞瘤细胞生长目的观察Hoxc9过表达对神经母细胞瘤细胞生长的影响。方法采用逆转录病毒转染法及四环素诱导表达系统使神经母细胞瘤细胞系(BE(2)-C、SK-N-AS、SK-N-DZ和SK-N-F1)过表达Hoxc9,用MTT法、Ki67染色、流式细胞术和细胞衰老实验检测Hoxc9对神经母细胞瘤细胞生长的影响,采用软琼脂实验及裸鼠成瘤实验观察Hoxc9对神经母细胞瘤细胞致瘤性的影响。采用实时定量PCR和免疫印迹法检测神经母细胞瘤细胞过表达Hoxc9后,细胞周期素cyclinA、cyclinB1、cyclinD1、cyclinE1、cyclinE2和细胞周期素依赖性激酶CDK1、CDK2、CDK4和CDK6表达水平的变化。结果过表达Hoxc9的神经母细胞瘤细胞生长明显减慢,细胞增殖标志物Ki67阳性细胞数减少,流式细胞术发现细胞发生G1期阻滞,细胞衰老实验提示细胞衰老加快。软琼脂实验中过表达Hoxc9的细胞较对照组细胞集落形成能力下降,裸鼠成瘤实验发现过表达Hoxc9的细胞致瘤性降低。细胞周期调控因子检测发现Hoxc9能够抑制cyclinA、cyclinB1、cyclinE2和CDK1 mRNA水平和蛋白水平的表达。结论Hoxc9过表达抑制神经母细胞瘤细胞的生长。第三节Hoxc9是维甲酸诱导神经母细胞瘤细胞分化的关键因子目的观察Hoxc9在维甲酸诱导神经母细胞瘤细胞分化中的作用。方法采用慢病毒感染法下调神经母细胞瘤细胞中Hoxc9后,用维甲酸诱导对照组和Hoxc9下调组细胞,在普通光学显微镜下观察细胞形态变化,用台盼蓝染色法和Ki67染色法观察细胞的增殖情况,用免疫印迹法观察细胞周期素cyclinA、cyclinB1、cyclinD1和cyclinE2表达水平的变化。结果抑制Hoxc9的表达能够阻止维甲酸诱导的神经母细胞瘤细胞分化,抑制维甲酸诱导的细胞周期素cyclinA、cyclinB1和cyclinE2表达水平的下调,而对细胞周期素cyclinD1表达水平无明显影响。结论Hoxc9是维甲酸诱导神经母细胞瘤细胞分化的关键因子。第三部分Hoxc9在神经母细胞瘤细胞周期调控中的作用目的观察Hoxc9在神经母细胞瘤细胞周期调控中的作用。方法采用慢病毒感染法下调神经母细胞瘤细胞中Hoxc9和／或cyclinB1的表达,在普通光学显微镜下观察细胞形态的变化,采用流式细胞术及免疫荧光法检测细胞周期的变化,采用免疫印迹法观察pHH3、Hoxc9、cyclinA、cyclinB1蛋白水平的变化。结果Hoxc9下调至基础水平的10%时,贴壁的神经母细胞瘤细胞逐渐变圆、脱离培养皿底,最后死亡。流式细胞仪及免疫荧光法检测细胞周期的变化发现随着Hoxc9表达水平的下调,细胞出现M期阻滞,细胞分裂障碍,cyclinB1表达水平增高,而下调cyclinB1的表达能够逆转细胞M期阻滞。结论Hoxc9通过调节cyclin B1的水平调控神经母细胞瘤细胞周期。更多还原

【Abstract】 PART 1 The molecular mechanisms of retinoic acid-induced differentiation of neuroblastoma cellsObjective To investigate the molecular mechanisms of retinoic acid-induced differentiation of neuroblastoma cellsMethods Retinoic acid was used to induce the differentiation of neuroblastoma cells. The cell cycle of cells was measured by flow cytometry after PI staining. Real time-PCR and western blotting were used to detect the expression of cyclins (cyclinA, cyclinB1, cyclinD1, cyclinE1, and cyclinE2) and CDKs (CDK1, CDK2, CDK4, and CDK6). The half-life of cyclins was assessed by western blotting and Image J software after the biosynthesis of cyclins was inhibited by protein synthesis inhibitor cycloheximide.Results RA induced differentiation and G1 arrest of neuroblastoma BE (2)-C cells. The levels of cyclinA, cyclinB1, cyclinE2 and CDK1 declined substantially with time following RA treatment. The half-life of cyclins (cyclinA, cyclinB1, and cyclinE2) was not apparently affected by RA treatment.Conclusions Retinoic acid induces growth arrest and differentiation of neuroblastoma BE (2)-C cells by downregulating the expression of cyclins (cyclinA, cyclinB1, and cyclinE2) and CDK1.PART 2 The role of Hoxc9 in retinoic acid-induced differentiation of neuroblastoma cellsSection 1 Upregulation of Hoxc9 expression in RA-induced differentiation of neuroblastoma cellsObjective To investigate the effect of RA on genes expression of neuroblastoma cells. Methods Microarray was used to detect the changes of cDNA level in BE (2)-C cells treated with RA. Real time PCR and Western blotting were used to confirm the result of microarray.Results Microarray showed that the expression of certain Hox genes were upregulated in BE (2)-C cells after exposure to RA. Real time PCR and Western blotting showed the mRNA and protein level of Hoxc9 increased substantially with time.Conclusions Hoxc9 may have a role in RA-induced differentiation of neuroblastoma cells.Section 2 Ectopic expression of Hoxc9 inhibites growth of neuroblastoma cellsObjective To investigate the effect of Hoxc9 overexpression in neuroblastoma cells.Methods After generating BE (2)-C-derived cells with inducible expression of Hoxc9 in the absence of doxycycline with the Retro-X Tet-Off advanced inducible gene expression system, MTT assay, Ki67 staining, flow cytometry, senescence assay, soft agar and xenograft assay were used to investigate the functional consequence of Hoxc9 upregulation in neuroblastoma cells. Real time PCR and western blotting were used to detect the molecular basis of Hoxc9 upregulation in neuroblastoma.Results Ectopic expression of Hoxc9 markedly inhibited the growth of neuroblastoma cells in culture, soft agar and immunodeficient mice. Hoxc9 overexpression also resulted in a marked reduction in levels of cyclinA, B1, E2 and CDK1 in BE (2)-C cells.Conclusions Ectopic expression of Hoxc9 inhibited the growth of neuroblastoma cells. Section 3 Hoxc9 is the key mediator of RA-induced differentiation of neuroblastoma cellsObjective To investigate the effect of Hoxc9 in RA-induced differentiation of neuroblastoma cells.Methods After downregulation of Hoxc9 in neuroblastoma cells with lentiviruses, we detected the changes of neuroblastoma cells treated with RA by trypan blue exclusion assay and Ki67 staining. The expression of cyclins (cyclinA, cyclinB1, cyclinD1, and cyclinE2) were assessed by Western blotting.Results Abrogation of Hoxc9 attenuated the RA-induced differentiation of neuroblastoma cells. RA-induced downregulation of cyclinA, cyclinB1, and cyclinE2 was also attenuated after inhibiting the downregulation of Hoxc9. Conclusions Hoxc9 is required for RA-induced G1 arrest and differentiation of NB cells.PART 3 The role of Hoxc9 in neuroblastoma cell cycle controlObjective To investigate the role of Hoxc9 in neuroblastoma cell cycle control.Methods After elimination of Hoxc9 and/or cyclin B1 expression by RNA interference, we investigated the morphologic changes of NB cell lines with microscope. Flow cytometry and immunofluorescence were used to detect cell cycle. The changes of the protein levels were detected by western blotting.Results Downregulation of Hoxc9 resulted in a dramatic increase in the number of cells that appeared round and refractile. These cells apparently arrested in M phase as indicated by FACS analysis and by their expression of pHH3, a marker for mitotic cells. The expression of cyclinB increased in Hoxc9 knockdown cells. RNAi-mediated downregulation of cyclin B1 was able to rescue the phenotype of Hoxc9 knockdown in NB cells.Conclusions Hoxc9 control the divition of NB cells by regulating cyclinB1 transcription.更多还原