【作者】 李美香；

【导师】 陈主初；

【作者基本信息】 中南大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 间质在肿瘤发生发展中的作用越来越受到重视,现已成为当今肿瘤研究的一个热门话题。鼻咽癌（nasopharyngeal carcinoma,NPC）是我国南方常见的一种上皮性恶性肿瘤,其发病率和死亡率均居世界首位,严重危害国人的生命和健康。为了从蛋白质组水平探讨间质细胞在肿瘤发生发展过程中的作用及其机制,本研究采用激光捕获显微切割（laser capture microdissection,LCM）技术分别从活检的NPC组织和正常鼻咽粘膜（normalnasopharyngal mucosa,NNM）组织中切割纯化NPC间质和NNM间质,应用荧光差异双向凝胶电泳（Fluorescent two-dimensional difference gelelectrophoresis,2D-DIGE）结合质谱技术分离、鉴定间质相关蛋白。建立了LCM纯化的NPC间质和NNM间质蛋白的荧光差异蛋白表达图谱,高通量筛选与肿瘤发生发展相关的间质蛋白,共得到34个有统计学意义的蛋白质点,质谱鉴定得到20个差异蛋白。其中在NPC间质中高表达的蛋白有Periostin,S100A9,CapG,PYCARD等,低表达的蛋白有L-plastin,Rho-GDI-β,B23,hnRNP K等。采用Western blot验证了其中部分差异蛋白（Periostin,S100A9,CapG,L-plastin）在NPC和NNM组织中的表达水平,证实了2D-DIGE结果的可靠性。为探讨NPC间质差异表达蛋白质的功能和临床病理学意义,采用免疫组织化学染色检测部分差异蛋白质在存档石蜡包埋组织（30例正常鼻咽粘膜上皮组织、66例原发NPC及20例颈淋巴结转移NPC）中的表达,统计学分析差异蛋白质表达水平与临床病理特征的关系。结果显示:Periostin,S100A9和CapG在NPC组织中的表达较NNM组织中明显上调（p<0.01）,L-plastin在NPC组织中的表达较NNM组织中明显下调（p<0.01）;Periostin和S100A9在颈淋巴结转移NPC中的表达较原发NPC明显上调（p<0.05）,CapG和L-plastin在颈淋巴结转移鼻咽癌中的表达与原发鼻咽癌比较无显著差异（p>0.05）。统计学分析显示:Periostin,S100A9,CapG上调和L-plastin下调与NPC组织学分化及临床分期相关（p<0.01）,Periostin和S100A9上调还与NPC淋巴结转移密切相关（p<0.01）。为进一步探讨NPC间质差异表达蛋白质的功能和临床病理学意义,本研究选取差异蛋白质Periostin为研究对象,首先采用Western blot检测其在不同分化程度或转移潜能的NPC细胞（CNE1、CNE2、5-8F和6-10B）及NIH 3T3成纤维细胞中的表达水平,结果发现Periostin在CNE1、CNE2、6-10B和3T3细胞中不表达,在5-8F细胞中弱表达。随后采用脂质体转染方法将真核表达载体pCMV-neo（+）-Periostin及其相应空白载体pCMV-neo（+）转染6-10B细胞（低转移潜能细胞）,建立稳定表达Periostin的细胞系,同时将pCMV-neo（+）-Periostin及其相应空白载体pCMV-neo（+）瞬时转染NIH3T3成纤维细胞。然后以稳定转染6-10B细胞及瞬时转然的NIH 3T3细胞为样本,观察6-10B细胞中Periostin高表达后的细胞生物学特性的改变及对基质金属蛋白酶（matrix metalloproteinases,MMPs）活性的影响。研究结果如下:成功建立了稳定表达Periostin的6-10B细胞系(6-10B^periostin)和空白载体转染的6-10B细胞系(6-10B^vector)。生物学特性分析结果显示:（1）与对照组细胞比较,6-10B^periostin细胞周期进程加快,G1期减少,S期、G2期增加;（2）高表达Periostin的6-10B细胞较对照组细胞生长速度明显加快;（3）6-10B^periostin细胞的平板集落形成数明显增多;（4）6-10B^periostin细胞的软琼脂集落形成率显著提高;（5）6-10B^periostin细胞的侵袭、迁移能力明显增强;（6）明胶酶谱实验显示,与对照组细胞比较,Periostin高表达的细胞分泌的MMP-2、MMP-9活性增加。接着用免疫组织化学的方法检测了α_vβ₅整合素（integrinα_vβ₅）在NPC和NNM组织、6-10B^periostin和6-10B^vector、6-10B细胞中的表达水平,发现integrinα_vβ₅在NPC组织和6-10B^periostin细胞中表达均较对照组增强,Spearman相关性分析显示integrinα_vβ₅和Periostin在NPC组织中的表达强度呈正相关（r=0.682,p=0.000）。结果提示:Periostin可能是通过与NPC细胞膜上integrinα_vβ₅受体结合来促进肿瘤细胞的生长,提高MMPs的活性,促进肿瘤细胞的侵袭和转移。综上所述,本研究采用LCM结合定量蛋白质组学技术从NPC间质和NNM间质中筛选到20个差异蛋白,并通过临床病理标本到细胞模型对差异蛋白Periostin的功能及作用机理进行了探讨。实验结果显示periostin在NPC病程演进中发挥正向调控作用:Periostin与NPC的分化程度、转移相关,Periostin高表达可促进NPC的侵袭和转移,Periostin可能通过与NPC细胞膜上的integrin-α_vβ₅受体结合发挥其功能。Periostin促进NPC转移与其影响MMPs的活性有关。研究结果提示:这些差异表达的蛋白将有助于阐明鼻咽癌细胞和周围间质的关系,对间质蛋白功能的进一步研究,将有助于解析间质在肿瘤发生发展中的作用并为从间质途径寻找肿瘤治疗靶标提供新思路。更多还原

【Abstract】 The role which stroma plays in tumor carcinogenesis is more and more important and now is becoming a hotspot.Nasopharyngeal carcinoma（NPC） is one of the most common malignant tumors in southern China,its incidence and mortality occupies the first place of the world,and is a great threat to people’s health and lives.To delineate the stromal proteins involved in NPC carcinogenesis,we assessed differences in protein expression of the stroma from NPC and normal nasopharyngeal mucosa（NNM） using a quantitative proteomic approach combined with laser capture microdissection（LCM）.LCM was performed to purify stromal tissue from the NPC and NNM,respectively.The protein expression profiles of the stroma tissue from NPC and NNM were compared by fluorescent two-dimensional difference gel electrophoresis（2D-DIGE）,and 34 differential protein spots between tumor stroma（TS） and normal stroma （NS） were chosen to be identified by mass spectrometry（MS）.2D-DIGE patterns of the purified stroma of NPC and NNM were established.A total of 20 differential proteins were identified,and part of the differential proteins （periostin,S100A9,CapG and L-plastin） were selectively further analyzed by Western blotting to validate the results of 2D-DIGE.Of all the identified proteins,periostin,S100A9,CapG,PYCARD et al.were up-regulated in NPC stroma,whereas L-plastin,Rho-GDI-β,B23,hnRNP K et al.were down-regulated in NPC stroma.To explore the function and clincopathological significances of the differential expression proteins, immunohistochemical（IHC） analysis was performed to detect the expression levels of the differential proteins in paraffin-embedded archival tissue specimens,including 66 cases of primary NPC,30 cases of NNM,and 20 cases of cervical metastatic lymph node from NPC（LMNPC）,and the correlation of their expression levels with clinicopathologic features were evaluated.The expression levels of periostin,S100A and CapG in primary NPC were significantly higher than those in NNM（P＜0.01）,and L-plastin was significantly down-regulated in NPC versus NNET（P＜0.01）;the expression levels of periostin and S100A9 in LMNPC were significantly higher than those in primary NPC（P＜0.05）,but no significant difference in the expression level of CapG and L-plastin were observed in the primary NPC and LMNPC.The statistics analysis showed:periostin,S100A9,CapG up-regulated and L-plastin down-regulated were correlated with poor histologic type/grade,advanced clinical stage,periostin and S100A9 up-regulated were correlated with lymph node metastases（P＜0.01）.On the other hand,periostin was selected to explore the function in NPC cells.First,Western blotting was employed to detect the expression levels of periostin in four NPC cell lines（CNE1,CNE2,5-8F and 6-10B） with different differentiated degrees and/or metastatic potentials and NIH 3T3 fibroblasts, and found that periostin did not expressed in CNE1,CNE2,6-10B and NIH 3T3 cells,and 5-8F cells show weak expression.Sequently,the recombinant plasmids[pCMV-neo（+）-Periostin]and control plasmids[pCMV-neo（+）] were transfected into 6-10B cells（low metastatic potentials） using lipofectamine 2000^TM reagent to build stable transfection clone cells,at the same time,the recombinant plasmids[pCMV-neo（+）-Periostin]and control plasmids[pCMV-neo（+）]were also transfected into NIH 3T3 fibroblasts to obtain transient transfection cells.With stable transfection 6-10B cells (6-10B^periostin) and transient transfection NIH 3T3 cells,their biological characteristic analysis was performed as follows:（1） Flow cytometry（FCM） analysis of 6-10B^periostin cell revealed a significant decrease of G1 phase with a corresponding increase of S phase and G2 phase populations when compared with the control cell lines;（2） The MTT assay showed that up-regulated periostin in 6-10B cells was associated with a marked increase of cell growth; （3） The monolayer growth experiment showed that the clonoogenicity of 6-10B^periostin was significantly higher than those of the controls;（4） Soft agar growth experiments revealed that the colony formation abilities of 6-10B^periostin was higher than those of the controls;（5） Transwell chamber invasion assay showed that up-regulation of periostin expression in 6-10B cells was associated with increased in vitro cell invasion and migration;（6） A gelatin zymogram for MMPs activation demonstrated an increase in MMP-2 and MMP-9 activity in cultivated supernatant of 6-10B（periostin） and mixed cultured 6-10B and 3T3（+） cells.Furthermore,the expression of integrin-α_vβ₅ was also detected by IHC in NPC and NNM,6-10B（periostin） cells,6-10B（vector） cells and 6-10B cells,the expression levels of integrin-α_vβ₅ in primary NPC and 6-10B（periostin） cells were significantly higher than those in NNM and 6-10B（vector）, 6-10B cells.The expression in NPC of integrin-α_vβ₅ showed positively correlated with the expression of periostin（r=0.682,p=0.000）.Our results from above experiments demonstrated that periostin plays an important role in regulation of cell proliferation,the activity of MMPs,cell migration and invasion probably by combining with integrin-α_vβ₅。Taken together,we identified 20 differential proteins between the stroma from NPC and NNM by quantitative proteomic approach coupled with LCM. Sequently,the differential expression protein periostin was further analyzed by IHC,molecular biology technology and cell biology technology.The results indicate that Periostin plays a positive role in regulation of the evolution of NPC:periostin is related to the differentiation,metastasis and prognosis of NPC and exert its functions probably by combining with integrin-α_vβ₅. Periostin promotes the invasion of NPC through regulating the activity of MMPs.Our results will be helpful to study the role of stroma in the NPC carcinogenesis,as well as discover the interaction between NPC cells and their surrounding microenvironment,and provide a new idea for finding stromal targets of tumor therapy.更多还原