| 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.
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