Studies On The Program Of Standardized Quantitative Pattern Differentiation Based On Pattern Elements For Nasopharyngeal Precancerous Lesion And Its Proteomic Basis

ObjectiveTo establish a standardized program for associated clinical information collection during the diagnostic course of cases with nasopharyngeal precancerous lesion (NPL) by means of Four Diagnostic Methods in order to set up the related standards for quantitative pattern differentiation among NPL cases based on the principles of pattern elements and to explore the molecular basis underlying these patterns of NPL on the basis of proteomics carried out with peripheral blood leukocytes.Materials and methods1. Establishment of survey forms for pattern elements On the basis of systematic literature review and discussion conclusions of specialist group, associated symptoms, pattern elements and patterns for nasopharyngeal precancerous lesion were screened out from these results. And then, survey forms were designed for doing such investigation on symptoms, pattern elements and patterns for NPL with reference of internationally popular designing strategy for such a survey, with their reliability and validity being tested for these forms.2. Data bank establishment A data bank was established on the basis of clinical epidemic survey on symptoms, pattern elements and patterns of NPL, with restricted statistical analyzing procedures used to calculate the contribution levels of these elements for pattern differentiation. Then, diagnostic standards (estimating thresholds) were set up for pattern elements to use in practice. Followed was the use of pattern elements in practical survey among the cases with NPL in a quantitative way, with these results comparatively analyzed with those made by a group of specialist to verify the concordance between them, to explore the distributing characteristics of patterns in this kind of lesion. 3. Analysis on the sub-cellular proteomic features of patterns among NPL cases Some groups of NPL cases with different pattern were randomly screened out from surveyed population to do the proteomic studies including 2-DE procedures and MALDI-TOF mass spectrography to explore the molecular basis underlying the effects of pattern elements during the pattern differentiation in clinical practice.Results1. The survey forms were established with very higher quality in their reliability and validity practical survey, especially with very higher qualities in accordance with the basic principles of Traditional Chinese Medicine.2. The database of pattern differentiation for nasopharyngeal precancerous lesion was established, with their contribution levels and diagnostic standards (verifying thresholds) for pattern elements determined on the basis of comparative study between quantitative pattern differentiation with pattern elements and that made out of specialists showing very higher concordance.3. It was shown that both of Qi deficient pattern and dampness heat pattern were the commonly seen patterns in clinical practice among cases with NPL as revealed by both of pattern differentiating course made by either specialists or via pattern elements.4. Ten 2-DE images were obtained from the samples among these 3 groups, with the mean protein spots being 447, the deflection rates being 1.67±0.19 in IEF direction and 2.37±0.53 in SDS-PAGE direction, and with their intra-group variations very less showing the matching rates of intra-group being more than 89% and that of inter-group more than 79%.5. Six spots were selected from each group of 2-DE gels with their variation of protein expressing levels more than 3 times, when compared among different groups respectively, to do the expressing level analysis based on their optical density comparison. It was shown that spots 1,2,3,4 and 5 were the predominantly highly expressed proteins in Qi-deficient group but no expression for spots 1,2 and 4 and lower expression for spots 3 and 5 in the controlling one when compared it with that of healthy one. However, spots 7,8,9 and 10 were expressed in dampness-heat group but not in healthy one, with spot 11 down-regulated and spot 12 up-regulated in their expressing levels, when compared with that of healthy group. On the other hand, spots 13 and 14 were up-regulated and spots 15,16,17 and 18 were down-regulated in their expressing levels, when compared between the group of Qi-deficiency and that of dampness-heat.6. At the last, six spots were cut down from each group of 2-DE gels with their variation of protein expressing levels more than 3 times and 18 spots collected in total, when compared among different groups respectively, to do MALDI-TOF mass spectrography. The proteins were identified finally through SWISS-PROT database and ExPASy Proteomics Server search based on the results of 2-DE analysis. Ten kinds of protein were identified from this step of study. When compared between Qi-deficient group and healthy one, there was one kind of protein identified as an kind of unknown protein, one out of the up-regulated proteins was one nuclear ribonuclear protein expressed in a way of heterogeneity, and two out of the down-regulated ones were as nucleolins B23 and hPOT1. When compared among the group of dampness-heat and that of health, the down-regulated protein was Ser/Thr protein kinase, the up-regulated protein included adenyl cyclase combining protein-1, and the un-expressed proteins were sulphuroxygeon hydrogenised protein peroxydase and counter-proliferin in the samples of case group, whiles the expression tendency of these proteins were in the right reversed direction with that former group. Furthermore, when compared among the lesion groups of Qi-deficient group and dampness-heat group, the proteins up-regulated in their expressing levels were receptor-related protein/RNA polymerase-associated protein, and those with their expression down-regulated were Rho GDP dissociation-depression protein and Ras, while it was in the right reversed tendency with that the former group.Conclusions1. It has been confirmed that the reliability and validity of these survey forms for symptoms, pattern elements and clinical patterns are very high for a practical use to research at the purposes of standardized operating program of information collection during the caring out course of four diagnostic procedures for diagnosis of cases with nasopharyngeal precancerous lesion.2. The outcomes of clinical pattern differentiation can be improved to a higher level with the use of standardized operating program of information collection during the caring out course of four diagnostic procedures for such cases in a quantitative way as well as for a better guiding effect on the standardized study for the associated molecular basis of clinical patterns of the lesion. Furthermore, some testing indexes can be integrated into the clinical practice operation to improve its validity better with some of closely related micro-assaying indicators at higher specificity on the basis of this kind of associated molecular basis for clinical patterns, with very important implications in its realistic usage.3. It can be summed up from these studies that Qi-deficiency pattern should be the predominant clinical pattern for cases with nasopharyngeal precancerous lesion, suggesting that Q-deficiency should be the key target in the pathogenesis ring of intervening therapeutic course with such a lesion.4. There are different expressing properties for specifically expressed proteins at the sub-cellular proteomic level among these surveyed patterns as Qi-deficiency, dampness-heat and healthy controls respectively.5. To explore their implications of differentiated expressed proteins on the basis of mass spectrography and bio-database searching for their biological functions, it can be deducted that the down-regulated expressing level of transcribing activity of rRNA associated protein in Qi-deficient group may be responsible for the suppression of function among lymphocytes so as to induce the reduced status of immune function in the whole body. Therefore, this group of cases with a Qi-deficient pattern may be put into a relatively status of pathogenesis with much higher risk of cancer development from their precancerous lesion in the nasopharyngeal epithelia.6. To deduct more reliable conclusions on these proteins in the terms of pattern associated molecular basis, more studies are required to be carried out in this kind of population in future.