【作者】 陈舒华；

【导师】 田道法；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2008， 博士

【摘要】 目的探讨鼻咽癌高癌家系成员及其鼻咽癌患者的体质证候、遗传学特征,比较鼻咽癌高癌家系鼻咽癌患者不同体质证候之间的鼻咽组织差异表达蛋白质谱,以及与散发性鼻咽癌患者鼻咽原发灶组织、正常人鼻咽组织蛋白质谱的表达差异,以期发现鼻咽癌高癌家系鼻咽上皮细胞癌变的相关蛋白质及其与患者病理体质特征的相关性,试图从蛋白质组表达特性角度对鼻咽癌高癌家系鼻咽癌患者不同中医体质证候的病理本质进行解释。方法1.建立体质证候分类标准,采用“闭式问题调查卷”方法,分别调查鼻咽癌高癌家系核心成员、鼻咽癌高癌家系鼻咽癌患者的体质类型,并以散发性鼻咽癌患者及高癌家系对照成员进行中医病理体质的对照研究。2.获取鼻咽癌高癌家系鼻咽癌患者、散发性鼻咽癌患者及正常对照者的鼻咽活检组织标本,提取细胞总蛋白进行二维电泳,应用Image Master 2D Elite4.01软件对2-DE胶上的差异表达蛋白质进行分析,确定体质证型特异性差异表达蛋白质,分析其表达活性与各自体质特征的相关性。3.从不同组别2-DE胶上准确切取30个差异表达蛋白质斑点,分别进行MALDI-TOF质谱分析。再通过差异表达蛋白质肽指纹图数据的生物信息学分析,结合各自的体质证型特征,分析鼻咽癌高癌家系鼻咽癌患者不同中医体质证型的蛋白质组表达特性,探讨其病理意义。结果1.高癌家系核心成员体质类型与家系对照成员比较,除正常质外,高癌家系核心成员病理体质主要表现为气虚质和复合质,而家系对照成员则主要表现为正常质,病理体质则以失调热质为多见,组间比较具有统计学意义(P<0.05)。高癌家系鼻咽癌患者体质类型主要表现为气虚质、失调热质和复合质,与高癌家系核心成员组比较,P<0.05,统计学检验有显著性意义。散发性鼻咽癌患者组体质类型主要表现为气虚质、复合质和失调湿质,与高癌家系核心成员组比较,P<0.01,统计学意义非常显著。而将高癌家系鼻咽癌患者体质类型与散发性鼻咽癌患者比较,P>0.05,统计学意义不显著。2.五组研究对象共获得12张2-DE凝胶图谱,其中高癌家系鼻咽癌患者、高癌家系鼻咽癌患者气虚体质组和高癌家系鼻咽癌患者湿热体质组各2张图谱,散发性鼻咽癌患者组和正常人组各3张。各组凝胶电泳图谱的组内匹配率都达到了95%以上,而组间的匹配率亦在89%以上；每张凝胶图谱的蛋白质斑点平均数为871个,在IEF方向的点偏移率为1.87±0.21mm,在SDS-PAGE方向的偏移率为2.43±0.52 mm。3.从三个对比体系(高癌家系鼻咽癌患者组对散发性鼻咽癌患者组,高癌家系鼻咽癌患者组对正常人组,高癌家系鼻咽癌患者气虚体质组对高癌家系鼻咽癌患者湿热体质组)的2-DE凝胶图谱中,分别选取10个蛋白质表达水平差异在3倍以上的蛋白质斑点用作比较分析。结果显示,在高癌家系鼻咽癌患者湿热体质组对高癌家系鼻咽癌患者气虚体质组2-DE细节图谱差异表达蛋白质点的比较中,高癌家系鼻咽癌患者湿热体质组在1、2、4号蛋白质斑点位置无表达；在3、5、9、10号蛋白质斑点位置,高癌家系鼻咽癌患者湿热体质组表达活性下调；在6、7、8号蛋白质斑点位置,高癌家系鼻咽癌患者湿热体质组表达活性上调；结果说明,两组在蛋白质组表达特征上存在着明显差异。在高癌家系鼻咽癌患者组对正常人组2-DE细节图谱蛋白质差异斑点的比较中发现,正常人组在11、12、13、14号蛋白质斑点位置无表达；在15、16、19、20号蛋白质斑点位置,高癌家系鼻咽癌患者组表达活性下调：在17、18号蛋白质斑点位置,高癌家系鼻咽癌患者组表达活性上调；结果说明,两组在蛋白质组表达特征上存在着显著差异。在高癌家系鼻咽癌患者组与散发性鼻咽癌患者组2-DE细节图谱蛋白质差异斑点的比较中显示,高癌家系鼻咽癌患者组在21、22、27、28、29、30号蛋白质斑点位置的表达活性上调；而在23、24、25、26号蛋白质点位置,高癌家系鼻咽癌患者组的表达活性下调；结果说明,两组标本在蛋白质组特征上也存在着明显差异。4.分别从上述三个对比体系的2-DE凝胶图谱中,各选取10个蛋白质表达活性差异在3倍以上的蛋白质斑点,共计30个,上机进行MALDI-TOF-MS分析,鉴定出24个差异表达蛋白质。其中,对高癌家系鼻咽癌患者湿热体质组对高癌家系鼻咽癌患者气虚体质组差异表达蛋白质点的分析显示,其湿热体质组有两个蛋白质无表达,即F-肌动蛋白戴帽蛋白亚基和4号未知蛋白质；表达活性下调的蛋白质有Rho-GDP解离抑制蛋白、Alpha-烯醇化酶；表达活性上调的蛋白质有神经元特异性X11蛋白、热休克蛋白70 kDa。在高癌家系鼻咽癌患者组对正常人组差异表达蛋白质点的分析中显示,高癌家系鼻咽癌患者组表达活性下调的蛋白质有神经元特异性X11蛋白、热休克蛋白70 kDa、热休克蛋白90 ATP酶激活蛋白和Annexin A7；表达活性上调的蛋白质有角蛋白-1和硫氧还原蛋白过氧化物酶；正常人组有三个蛋白质无表达,即运铁蛋白受体、角蛋白-10和无名蛋白。在高癌家系鼻咽癌患者组对散发性鼻咽癌组差异表达蛋白质点的分析中显示,高癌家系鼻咽癌患者组表达活性下调的蛋白质有Intelectin-1和免疫球蛋白K型轻链C区；表达活性上调的蛋白质有葡萄糖-6-磷酸脱氢酶、Rho GDP解离抑制蛋白、腺苷酸环化酶结合蛋白、乳转铁蛋白。结论1.鼻咽癌高癌家系核心成员的体质类型中,除正常质外,病理体质主要表现为气虚质和复合质,而高癌家系鼻咽癌患者的体质类型主要表现为气虚质、失调热质和复合质。2.在三个不同组别对比体系(高癌家系鼻咽癌患者组对散发性鼻咽癌患者组,高癌家系鼻咽癌患者组对正常人组,高癌家系鼻咽癌患者气虚体质组对高癌家系鼻咽癌患者湿热体质组)中,分别存在不同的差异表达蛋白质特性。3.对质谱鉴定出的差异蛋白质按功能可分为：①、与鼻咽癌发生相关蛋白质,包括F-肌动蛋白戴帽蛋白亚基、神经特异性X11蛋白,角蛋白和Annex in A7;②、与遗传及转录有关的蛋白质,包括RhO-GDP解离抑制蛋白、Alpha-烯醇化酶、腺苷酸环化酶关联蛋白和免疫球蛋白K型轻链C区；③、与代谢有关的蛋白质,包括运铁蛋白受体蛋白、G6PD、Rho-GDP解离抑制蛋白、内凝集蛋白1、乳转铁蛋白；④、与ATP结合有关的伴侣蛋白质,包括热休克蛋白70 kDa,热休克90kDa蛋白ATP酶同源物-1。4.在质谱分析基础上,对差异表达蛋白质进行的功能分析显示,与散发性鼻咽癌组相比较,鼻咽高癌家系鼻咽癌患者原发灶肿瘤细胞的侵袭能力较强,而且气虚体质组患者鼻咽原发灶肿瘤细胞的侵袭能力又明显高于湿热体质组,结果提示,即使是同一类型的鼻咽癌病灶,在由禀赋因素所决定的不同病理体质类型个体,其肿瘤细胞生物学特性显然存在差异,因而表现不同的临床特征。深入探讨病理体质与鼻咽癌遗传易感性的相关性基础及其临床意义,应该有助于提高鼻咽癌的整体防治水平。更多还原

【Abstract】 ObjectivesIt is well known that there is a very close relationship between the development of nasopharyngeal carcinoma (NPC) and the related genetic characteristics and such a genetic event may cause very important implications in the population with very high risk to develop the lesion of NPC on the basis of constitutional patters. Therefore, it should be reasonable to explore the related basis of mechnanisms underlying such an event. In this regard, the study reported here was mainly aimed to investigate the constitutional patterns and the related genetic characteristics among the core familial members of familial NPC and the cases with NPC developed among these familial members at first, with the scattered distributed patients of NPC as case controls and healthy people as normal controls respectively. Then, proteomics analysis was carried out to their nasopharyngeal tissue samples in a comparative way to find out the differentially expressed proteins on 2-DE gels, and then, followed was mass spectrographic analysis on these differentially expressed proteins to explore their functional and pathogenesis implications in the development of NPC on the basis of various patterns of constitution so as to investigate the correlation of genetic factors with constitutional patterns and their pathogenesis affection on the development of NPC in terms of Traditional Chinese Medicine for developing more reliable preventive and therapeutic procedures for NPC in practice usage in future.Methods1. A clinical epidemic survey was carried out to investigate the distributing pattern of constitutional patterns among the members of familial NPC on the basis of clinical features of the studied subjects based on the standards for constitutional pattern classifying, put forward by previous literatures with a little modified survey system through a closed questionnaire program among the members of core pedigree, in which two or more cases with NPC had been diagnosed pathologically before. A group of scatteredly distributed NPC cases and a group of healthy people were taken as the case controlling and normal controlling respectively.2. To detect the differentially expressed proteins in different constitutional patterns, tissue samples were taken from the nasopharynx of all subjects included in this study and total cellular proteins were extracted from these samples respectively to carry out two dimensional electrophoresis (2-DE) to find out the differentially expressed special proteins for different groups on the 2-DE gel map following an image comparative analysis with the software of Image Master 2D Elite 4.01. Then, a correlative exploration was made to see into the constitutional pattern specific proteins differentially expressed in nasopharyngeal tissue and their correlation with their own constitutional patterns in the consideration of pathogenesis implications underlying the development of NPC among high risk population with this lesion in familial nasopharyngeal carcinoma.3. Thirty differentially expressed protein spots specific for different groups were exactly cut down from each of these 2-DE gels to carry out mass spectrographic analysis for identification of specific proteins by MALDI-TOF and their function confirmation through bioinformatics analysis on the peptide finger-print maps on line. Then, correlative exploration was made to discuss the special features of differentially expressed proteins and their implications in special constitutional patterns based on their functional aspects and different expressive activities in various patterns of constitution correlated with different levels of risk suffering from the developing process of NPC in terms of TCM pathogenesis.Results1. The results of this study showed that the constitutional patterns of core members in the pedigrees of familial NPC were mainly composed of Qi deficient pattern and complex pattern other than the normal pattern of constitution, while those people as controls in the surveyed pedigrees showed their constitutional patterns mainly being normal one with fewer cases showing disharmony heat pattern as the un-normal status of constitution, with very significant difference in the distributing pattern in this aspect (P<0.05). However, those cases with NPC among the core members in these surveyed pedigrees were shown mainly being such constitutional patterns as Qi deficiency, imbalanced heat and complex one, also with a much remarked difference in the distribution of patterns when compared with that of core members in these pedigrees (P<0.05). On the other hand, those scatteredly distributed NPC patients among common population were mainly composed of such constitutional patterns as Qi deficiency, complex and imbalanced dampness, with a very significant difference here (P<0.01) when compared with that of core members in these surveyed pedigrees but with no such significant difference (P>0.05) when compared with that of cases of NPC among the core members in these surveyed pedigrees.2. From the studied 5 groups of subjects,12 pieces of 2-DE gel maps were successfully obtained, in which 2 ones got from each group of the three groups of NPC cases and 3 ones from the group of scattered distributed NPC cases in common population and 3 ones from the group of healthy controls respectively. There was an intra-group matching rate above 95% for each group of maps and the inter-group matching rates were all larger than 89% following the protein spots matching analysis. The averaged protein spots were 871 for each 2-DE gel map, with a deviation rate of 1.87±0.21 mm in the direction of IEF and a deviation rate of 2.43±0.52 mm in the direction of SDS-PAGE.3. Ten differentially expressed protein spots with the expressive activity differences larger than 3 times among the compared groups were selected for a more detailed comparative analysis from the 2-DE gel maps of 3 comparing systems composed of familial NPC cases group in the surveyed pedigrees vs. the group of scattered distributed NPC cases in common population, familial NPC cases group in the surveyed pedigrees vs. the group of healthy people, and the group of NPC cases with a constitutional pattern of Qi deficiency vs. that of such cases with a constitutional one of dampness-heat in the surveyed pedigrees of family NPC. As showed from this kind of comparative analysis on the detailed 2-DE gel maps between these two kinds of constitutional patterns among familial NPC cases of pedigrees, those cases with a dampness-heat pattern showed no expressive activities on the protein spots 1,2 and 4, down-regulated expressive activities on the protein spots 3,5,9 and 10, and up-regulated expressive activities on the protein spots 6,7 and 8, suggesting that there being a very significant difference in the features of expression at the level of proteomics between these two kinds of constitutional pattern even though with the same lesion. In the comparative analysis system of detailed 2-DE gel maps made among familial NPC cases group in the surveyed pedigrees and the group of healthy people, there were no expressive activities observed on the protein spots 11,12,13 and 14 in healthy people group, while down-regulated expressive activities seen on the protein spots 15,16,19 and 20, and up-regulated expressive activities shown on the protein spots 17 and 18 in the paired comparing familial NPC cases group in the surveyed pedigrees, suggesting that there being a very significant difference in the characteristics of expression at the level of proteomics between these two groups as well. In the comparative analysis on the detailed 2-DE gel maps of comparing system of familial NPC cases group in the surveyed pedigrees vs. the group of scattered distributed NPC cases, family NPC cases in the surveyed pedigrees showed up-regulated expressive activities shown on the protein spots 21,22,27,28,29 and 30, and down-regulated expressive activities seen on the protein spots 23,24, 25 and 26, suggesting that there being also a very significant difference in the expressive file modes of proteins between these two groups of samples.4. Ten differentially expressed protein spots with the expressive activity differences larger than 3 times among the compared groups were exactly cut down from the 2-DE gel maps of each comparing system for mass spectrographic analysis with the procedures of MALDI-TOF-MS, 30 protein spots in total for these three comparing systems, with 24 kinds of differentially expressive protein identified following this analyzing process. Among these differentially expressive proteins identified here, two were shown with no expressive activities, i.e. F-actin-capping protein subunit and unknown protein spot 4, two were down-regulated in their expressive activities, i.e. ARHGDIA protein and alpha-enolase, and two were up-regulated in their expressive activities, i.e. neuron-specific X11 protein and heat shock 70 kDa protein, among the cases with a constitutional pattern of dampness-heat when compared them with that of Qi deficiency among the cases of familial NPC in the surveyed pedigrees in the regard of constitution specific proteomic expression features. In the comparative analysis on the cases of familial NPC in the surveyed pedigrees vs. healthy controls, it was shown that there were three proteins down-regulated in their expressive activities, i.e. neuron-specific X11 protein,70 kDa heat shock protein and activator of 90 kDa heat shock protein ATPase homolog, and one protein up-regulated in its expressive activity, i.e. peroxiredoxin TSA1, among the cases of NPC in the surveyed pedigrees, while two proteins were confirmed with no expressive activities, i.e. transferrin receptor protein 1 and putative one, among the healthy controls. Moreover, the comparing analysis, made on familial NPC cases of surveyed pedigrees vs scatteredly distributed NPC patients in common population, showed that there was one protein, Intelectin-1, being down-regulated in its expressive activity, and there were four proteins, i.e. glucose-6-phosphate 1-dehydrogenase, Rho GDP dissociation inhibitor (GDI) beta, CAP, adenylate cyclase-associated protein 1 and lactotransferrin, being up-regulated in their expressive activities among the former group of cases.Conclusions1. The main pathological constitution patterns, other than the normal one for most members, of the core members in the surveyed pedigrees of familial NPC are composed of Qi deficient one and complex one among fewer people of these pedigrees, while those NPC cases out of these surveyed pedigrees are mainly observed with Qi deficient pattern, imbalanced heat pattern and complex one.2. There are different modes in the differentially expressed proteins at proteomic level shown respectively for the studied three comparing systems, i.e. the group of familial NPC cases of surveyed pedigrees vs the one of scatteredly distributed NPC patients in common population, the group of familial NPC cases surveyed pedigrees vs the one of healthy people and the sub-group of cases with a constitutional pattern of Qi deficiency vs. the one of cases with a constitutional pattern of dampness-heat among the NPC patients out of the surveyed familial nasopharyngeal carcinoma pedigrees.3. On the basis of functional exploration made on differentially expressive proteins according to mass spectrographic analysis and with a comparison with that of scatteredly distributed NPC patients in common population, it can be concluded that the tumor cells in the primary focus of nasopharynx hold much stronger invasive potentiality among the familial NPC cases in the studied pedigrees than usual, especially among those with a constitutional pattern of Qi deficiency, much higher than that among those with a constitutional one of dampness-heat. This suggests that, even though with a same type of nasopharyngeal lesion in the same quality, there may be different biological behaviors as well as different clinical features present in the same type of tumor cells among various individuals due to different kinds pathological constitutional patterns in terms of TCM mainly dependent upon the related genetic factors. Therefore, it should be helpful to the promotion of preventive and therapeutic levels of nasopharyngeal carcinoma and the other associated conditions as a whole through further systematic investigation on the correlation of pathological constitutional patterns with genetic susceptibility for the development of NPC in both fields of basic research and clinical investigation.更多还原