【作者】 王燏婵；

【导师】 程纯；

【作者基本信息】 南通大学 ， 免疫学， 2008， 硕士

【摘要】 目的1.分别研究p27^kip1 2个主要磷酸化位点—187位苏氨酸（Thr）和10位丝氨酸（Ser）,在非霍奇金淋巴瘤（non-Hodgkin’s lymphoma,NHL）病例中的异常磷酸化情况,结合增殖指标Ki-67、p27^kip1相关蛋白Skp2、Jab1及临床相关资料,初步探讨p27^kip1异常磷酸化与NHL发生、发展的关系。2.在细胞水平,分别探讨p27^kip1Thr187、Ser10磷酸化异常与淋巴瘤细胞生长周期的关系。方法1.首先在组织水平,选取116例NHL,其中惰性NHL18例,包括11例滤泡性淋巴瘤（Follicular center cell lymphoma）和7例胃肠黏膜相关淋巴组织淋巴瘤（Marginal zone B-cell lymphoma of MALT type）;侵袭性NHL98例,包括37例弥漫性大B细胞淋巴瘤（Diffuse large B-cell lymphoma）,49例鼻NK/T细胞淋巴瘤（NK/T lymphoma）,12例外周非特殊型T细胞淋巴瘤（Unspecified peripheral T-cell lymphoma）。另取同时期保存的15例良性增生淋巴结作为对照。采用免疫组化方法,检测Thr187磷酸化p27^kip1、Ser10磷酸化p27^kip1及p27^kip1相关分子Jab1表达情况,SPSS13.0统计软件分析数据。2.采用RT-PCR技术扩增人类Jab1全长序列,通过DNA重组技术分别将其重组于pcDNA3.1-Myc及pcDNA3.1载体,构建Jab1正义表达质粒,酶切电泳分析及DNA测序的方法对重组表达质粒进行鉴定。3.采用血清饥饿合并释放同步化处理淋巴瘤细胞,流式细胞仪检测细胞周期,Western blot、免疫荧光、免疫沉淀及核浆分离技术检测Thr187磷酸化p27^kip1、Ser10磷酸化p27^kip1及p27^kip1相关分子Skp2、Jab1等在细胞内的表达、亚细胞定位。应用pcDNA3.1-Myc-Jab1表达质粒采用脂质体瞬时转染淋巴瘤细胞,流式细胞仪分析增加Jab1表达对淋巴瘤细胞周期的影响,Western blot、免疫荧光、核浆分离及免疫沉淀检测Jab1过表达对p27^kip1的表达、亚细胞定位及功能的影响。结果1.免疫组化结果显示:1) NHL中Thr187磷酸化p27^kip1阳性率明显高于良性增生性淋巴结中（生发中心以外）的表达水平。结合临床资料分析显示:Thr187磷酸化p27^kip1与NHL病人的年龄、临床分期有相关性。病人年龄越大,临床分期越晚,其阳性率越高;与NHL病人性别、淋巴结转移情况无明显相关性。相关分析显示:Thr187磷酸化p27^kip1与Ki-67表达呈正相关（rs = 0.639,P<0.001）;Thr187磷酸化p27^kip1与Skp2表达呈正相关（rs= 0.594,P<0.001）;Thr187磷酸化p27^kip1与p27^kip1的表达呈正相关（rs = 0.175,P<0.001）。生存分析显示:Thr187p27^kip1磷酸化阳性（>10%）的病人,其五年生存率明显低于阴性（≤10%）的病人（P < 0.001）。2) Ser10磷酸化p27^kip1、Jab1蛋白阳性率均明显高于良性增生性淋巴结中（生发中心以外）的表达水平。结合临床资料分析显示:Ser10磷酸化p27^kip1、Jab1蛋白表达与NHL临床分期有相关性。与NHL病人年龄、性别、淋巴结转移情况无明显相关性。相关分析显示:Ser10磷酸化p27^kip1与Ki-67表达无明显相关性;Ser10磷酸化p27^kip1与Jab1表达呈正相关（rs= 0.696,P<0.001）;Ser10磷酸化p27^kip1与p27^kip1的表达呈负相关（rs = -0.457,P<0.001）;Jab1蛋白与增殖指标Ki-67表达呈正相关（rs = 0.870,P<0.001）;Jab1蛋白与p27^kip1蛋白表达呈明显负相关（rs = -0.380,P<0.001）。生存分析显示:Ser10磷酸化p27^kip1阳性（>10%）、Jab1阳性（>10%）的病人,其五年生存率均明显低于阴性（≤10%）的病人（P < 0.001）。2.构建pcDNA3.1-Myc-Jab1正义表达质粒,通过酶切电泳分析及DNA测序的方法对重组表达质粒进行鉴定,证实构建成功。3.血清饥饿合并释放能够刺激淋巴瘤细胞同步化生长,随着细胞周期进程:1)Thr187磷酸化p27^kip1表达增加,并且主要位于胞核内;p27^kip1对cyclinE/CDK2复合物抑制作用减弱,CDK2激酶活性增强。2)Ser10磷酸化p27^kip1与Jab1表达明显增加;Jab1过表达导致p27^kip1磷酸化形式改变,胞浆分布增多,与CDK分子的亲和能力改变,最终促进细胞进入S期。结论1. p27^kip1Thr187、Ser10两个位点的磷酸化异常与NHL病例恶性程度密切相关。提示:p27^kip1Thr187、Ser10两个磷酸化位点可能分别通过不同途径使p27^kip1表达及功能发生异常,导致细胞周期失控并促进细胞异常增殖,从而参与了NHL的发生和发展。2.成功构建了人类Jab1正义表达质粒,为进一步探讨Jab1基因在NHL发生发展中的作用奠定了实验基础。3.在细胞水平,Thr187磷酸化p27^kip1可能通过泛素-蛋白酶体途径影响p27^kip1的降解,而Ser10磷酸化p27^kip1与Jab1从介导p27^kip1出核这一途径影响p27^kip1的核内外分布,两个磷酸化位点通过各自的功能特点调节p27^kip1的表达及功能,从而参与对淋巴瘤细胞生长的调控。更多还原

【Abstract】 Objectives1. To investigate the expressions of 2 major phosphorylation formats of p27^kip1 respectively and its relative protein S-phase kinase-associated protein 2（Skp2） and Jun activation domain binding protein 1 （Jab1） in human non-Hodgkin’s Lymphomas and their clinicopathologic significance.2. To evaluate the specific effects of pThr187p27^kip1 and pSer10p27^kip1 during lymphoma cell growth.Methods1. Immunohistochemical technique was used to detect the expression levels of pThr187p27^kip1, pSer10p27^kip1p27^kip1, Skp2, Jab1 and Ki-67 in 116 cases of NHLs and 15 cases of reactive lymphoid tissue. The NHL cases characterized in this study were universal in oriental included: 11 follicular lymphomas （FL）, 7 mucosa-associated lymphoid tissue B cell lymphomas （MALT）, 37 diffuse large B-cell lymphomas （DLBCL）, 49 Nasal natural killer （NK）/T-cell lymphomas and 12 unspecified peripheral T-cell lymphomas （PT-un）.2. Human Jab1 complete cDNA was amplified by RT-PCR method, and was reconstructed into the eukaryotic expressive vector pcDNA3.1-Myc to form the Jab1 sense expressive plasmid. The reconstructed DNA was identified by enzyme digestion and DNA sequencing.3. Serum starvation and release used to synchronize lymphoma cells. Flow cytometer was used to analyze cell-cycles. The expression and subcellular localization of pThr187p27^kip1, pSer10p27^kip1, Skp2 and Jab1 were detected by western blot, immunoflurescence and subcellular fractionation. Transfection was performed using lipofectamine 2000 transfection reagent according to the manufacture’s protocol. The cells were harvested 48 h after transfection and cell-cycles were detected by Flow cytometry. The expression and subcellular localization of Jab1, p27^kip1 and p-Ser10p27^kip1 were investigated by western blot, immunoflurescence, immunoprecipitation and subcellular fractionation.Results1. Immunohistochemistry analysis showed: 1) The expression level of pThr187p27^kip1 in NHLs was higher than that in reactive lymphoid tissues （except germinal center）. Using a 10% cutoff for pThr187p27^kip1 expression, pThr187p27^kip1 positive expression was significantly associated with patients’age and clinical stage, but it was not associated with patients’sex and lymph node metastasis. In all cases of NHLs analyzed, pThr187p27^kip1 expression did correlate with expression of Skp2 （rs =0.594, P<0.001） and p27^kip1 （rs =0.175, P=0.062） as well as Ki-67 （rs =0.639, P<0.001）. By using the Kaplan-Meier analysis, patients with pThr187p27^kip1-positive tumor were significantly associated with short overall survival （P < 0.001）. 2) The expression levels of pSer10p27^kip1 and Jab1 in NHLs were higher than that in reactive lymphoid tissues （except germinal center）. Using a 10% for cutoff, pSer10p27^kip1 and Jab1 overexpression were both significantly associated with advanced stage, but they were not associated with patients’age, sex and lymph node metastasis. As continuous variables, pSer10p27^kip1 expression did positively correlated with expression of Jab1（rs= 0.696, P<0.001） and negatively correlated with expression of p27^kip1 （rs = -0.457,P<0.001）; Jab1 expression was negatively associated with p27^kip1 expression （rs = -0.380, P <0.001） and was positively associated with Ki-67 expression （rs = 0.870, P < 0.001） in all cases of NHLs analyzed. Survival analysis indicated that patients with pSer10p27^kip1 or Jab1-positive tumor were significantly associated with short overall survival （P < 0.001）.2. The cloned DNA was confirmed to be human Jab1 cDNA.3. Advances in the cell cycle study have revealed: 1) pThr187p27^kip1 increased as the cells were treated with serum addition as well as the same kinetics were observed at cyclin E and CDK2. Subcellular fractionation showed that pThr187p27^kip1 was mainly present in the nucleus of cells released from G1, leading to the dramatically reduced p27^kip1 accumulation. Immunoprecipitation studies suggested that the enhancement of phosphorylation at Thr187 of p27^kip1 mainly occurred in the CDK2/CyclinE complex. 2) pSer10p27^kip1 and Jab1 were also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. We’ve found that overexpression of Jab1 could increasing the Ser10 and Thr157 phosphorylation of p27^kip1 while the Thr187 phosphorylation was significantly decreased. Our preliminary data demonstrated a significant decrease of p27^kip1 expression level and a significant increase of S phase of the cell cycle in NHL cell lines that overexpressed Jab1. Immunoprecipitation studies suggested that Jab1 could change in p27^kip1 binding preferences to CDKs and relatively major p27^kip1preferentially bound to CDK4.Conclusions1. Increased expression levels of pThr187p27^kip1 and pSer10-p27^kip1 were closely correlated with the aggressiveness of NHLs. Both 2 major phosphrylation formats may influence the expression and function of p27^kip1 through different pathways, thus may play key roles in the occurrence and development of NHLs.2. We successfully cloned human Jab1 gene and constructed its eukaryotic expressive plasmid, which was an ideal model for further study.3. Since Thr187 phosphorylation is required for p27^kip1 ubiquitination and Ser10 phosphorylation is necessary for the nuclear export of the protein mediated by the exportin Jab1, both phosphorylation formats may be involved in the control of the G1-S transition and have oncogenic potential.更多还原