A Study On The Molecular Mechanism Of Phlegm Stagnation And Its Role In The Process Of Laryngeal Carcinogenesis

ObjectiveLaryngeal cancer is one common malignant tumour of the Head and Neck, Precancerous lesion is the center link of carcinogenesis,it is very important to study the molecular mechanism of carcinogenesis for the preventing and treatment of cancer.The preliminary study showed that Phlegm stagnation play a very important role in the process of laryngeal carcinogenesis,but its molecular mechanism is still unknown.To investigate the role and molecular mechanism of Phlegm stagnation in the process of laryngeal carcinogenesis.The project intends to study on laryngeal precancerous lesions from three aspects:the literature research,clinical investigation and experimental research.As to get a preliminary pathogenesis study of laryngeal precancerous lesions with comprehensive analysis,to explore the basic characteristics of Traditional Chinese Medicine syndrome of laryngeal precancerous lesions,and the characteristics and evolution changes of Traditional Chinese Medicine designate in the process of laryngeal carcinogenesis,as to identify the higher malignant potential Traditional Chinese Medicine syndrome type from molecular level,to explore the role and molecular biology mechanism of Phlegm stagnation syndrome in the process of laryngeal carcinogenesis.And to provide direction and ideas for research and treatment of laryngeal precancerous lesions and laryngeal cancer with Traditional Chinese Medicine.The study will accumulate date on study the pathogenic factors and pathogenesis of laryngeal precancerous lesions,and it provides a scientific evidence for the preventing and treatment of laryngeal precancerous lesions with Traditional Chinese Medicine,it undoubtedly has an important scientific significance and practical value.Methods Clinical Research1.The literature cases research use the CNKI(the Web version) and China biomedicine literature database(CBMdisc),application software SPSS11.5 to analysis the laryngeal precancerous lesions in the literature of Traditional Chinese medicine,about the syndromes factors,the characteristics of four diagnostic methods and treatment prescription etc.2.The study of clinical investigation using the clinical popular survey method,combined with clinical information questionnaire of laryngeal precancerous lesions to collecte informations.To make a comprehensive analysis of the four diagnostic information of 88 cases of laryngeal precancerous lesions,simultaneously takes 100 vocal cord polyp cases and 60 laryngeal cancer cases as the progresses of diseases comparison research.A preliminary summary of laryngeal precancerous lesions of the characteristics of four diagnostic and the evolution characteristic of syndromes of Traditional Chinese medicine.3.The clinical observation research was to compare the therapeutic methods of Replenishing Qi and Activating blood circulation Resolving phlegm used in treating the laryngeal precancerous lesions.34 laryngeal precancerous lesions cases were divided into Replenishing Qi group and Activating blood circulation Resolving phlegm group,symptoms and signs such as a comprehensive quantitative score was observed,to compare therapeutic effect between Replenishing Qi group and Activating blood circulation Resolving phlegm group,as in improving symptoms and signs,and prevention recurrence of malignant aspects of laryngeal precancerous lesions.Experimental studyDefects in cell cycle regulation are common causes of the abnormal proliferation of cancer cells.The project use immunohistochemical method to detect expression of cyclin E,cyclin D1,P21,P16,and tumor suppressor gene PTEN,P53 in 50 cases of laryngeal precancerous lesions with Phlegm stagnation syndrome,simultaneously takes laryngeal precancerous lesions cases with Phlegm syndrome,the vocal cord polyp and the early laryngeal cancer specimens as control.ResultsClinical Research1.Judging from the literature,the current Traditional Chinese medicine study of laryngeal precancerous lesions is not deep enough,according to the clinical cases reported in the literature,the therapeutic methods is effective,accumulated some valuable clinical experience,and is worth for learning and reference.2.Phlegm stagnation syndrome is a main and important pathogenesis factor of laryngeal precancerous lesions,but not a single pathogenesis factor,there are Yin deficiency,Qi deficiency,heat or miscellaneous at the same time,as same as the previous literature study.Compared with vocal cord polyps and laryngeal cancer,Syndrome factors of laryngeal precancerous lesions is relatively complex(P＜0.05).3.Laryngeal precancerous lesions have some common pathological characteristics:the sex ratio is extremely unbalanced,vast majority of men and aged over 40 years old,over-pronunciation,smoking may be the risk factors.Its main symptoms are hoarseness,almost 95%of cases have hoarseness,followed by foreign body sensation or sputum viscosity sensation in throat,dry or sore throat flu,less cough expectoration,there will be little difficulty swallowing or breathing difficulties.Dark tongue with spots ecchymosis,greasy moss,pulse string or thin slip mainly.It also supports Phlegm and blood stasis syndrome is the main pathogenesis factor of Laryngeal precancerous lesions.From vocal cord polyps,laryngeal precancerous lesions to laryngeal cancer,the cases showed an upward trend in miscellaneous syndromes(P＜0.05).4.In terms of gender composition,age distribution in polyp of vocal cord, laryngeal precancerous lesions and laryngeal cancer was significant difference(P＜0.05).The lead cause of vocal cord polyp may closely related to the excessive use of voice,while the risk factors of laryngeal precancerous lesions and laryngeal cancer may be long-term history of alcohol and smoking.The positive rate of treponemapallidum syphilisimmune body in cases of vocal cord polyps,laryngeal precancerous lesions and laryngeal cancer is 0%,1.92%,5%by statistics,compared with laryngeal cancer,the positive rate of treponemapallidum syphilisimmune body in laryngeal precancerous lesions cases was no significant inter-group,while compared with vocal cord polyps cases,the positive rates of treponemapallidum syphilisimmune body between group is significant,whether the occurrence of laryngeal cancer has certain relationship with treponemapallidum infection,it is worth discussing.The statistics shows the rate of carcinogenesis of laryngeal precancerous lesions is 2.63%,and the time for carcinogenesis of laryngeal precancerous lesions appeares to be 2-5 years.5.The therapeutic methods of Activate blood circulation and Resolving phlegm applied after laryngomicro-surgery on laryngeal precancerous lesions shows a certain therapeutic effect,such as to alleviate symptoms,improve the signs,slow down or reduce the recurrence(P＜0.05)etc. Experimental study1.P21^Waf1,cyclinD1,cyclin E,P16 are mainly expressed in cell nucleus, in vocal cord polyps cases,P21^Waf1,Pl6 protein expression dyeing coloration is deep,and the masculine cell is crowded.In laryngeal precancerous lesions P21^Waf1,P16 protein expression dyeing shoal,and the depth varies,the masculine cell reduces,positive cells decreased.From vocal cord polyps,laryngeal laryngeal precancerous lesions to laryngeal cancer,the positive intensity was gradually decreasing(P＜0.05).Just on the contrary,the positive strength expression of cyclin E,cyclinD1 protein in vocal cord polyps,laryngeal precancerous lesions and laryngeal cancer is gradually increased(P＜0.05).2.PTEN mainly expressed in cytoplasm,P53,PCNA expression in the nucleus mainly.PTEN protein expression in vocal cord polyps is deep dye staining and positive cell density.PTEN protein expression in laryngeal precancerous lesions is dyeing shoal and masculine cell reduces.PTEN protein expression in laryngeal cancer staining shallow,less-positive cells.From vocal cord polyps,laryngeal precancerous lesions to laryngeal cancer,positive intensity was gradually decreasing(P＜0.01).The positive intensity expression of PCNA and P53 protein in vocal cord polyp,laryngeal precancerous lesions and laryngeal cancer showed a upward trend(P＜0.05).3.In laryngeal precancerous lesions of Phlegm stagnation group and phlegm group,the positive rate of P21^Waf1protein expression was of no statistically significant(P=1.0000),weak and strong masculine gende(+,++)is of the same(P＞0.05).Expression of the strong masculine gender(+++)was significant(P=0.0228).Positive rate of cyclinD1 protein expression levels in Laryngeal precancerous lesions with Phlegm stagnation or phlegm was of no statistically significant difference(P=1.0000,P=0.6618,P=0.6132).4.In laryngeal precancerous lesions specimens with Phlegm stagnation or phlegm syndrome,all levels of masculine genders rate of cyclin E,P16 protein expression was of non-statistics significance(P＞0.05).Conclusions1.Phlegm stagnation play an important role in the process of the occurrence and development of laryngeal precancerous lesions,it runs through all the process of carcinogenesis.Phlegm stagnation is one of the basic etiology and pathogenesis of laryngeal precancerous lesions.With the malignant developing of laryngeal precancerous lesions,the miscellaneous syndromes become more common and complex.The variety of syndromes and miscellaneous complex situation of laryngeal precancerous lesions may prompt its malignant transformation.Clinical use of Activate blood circulation Resolving phlegm should be a right direction.Tobacco and alcohol may be the risk factors of malignant transformation of laryngeal precancerous lesions.2.From benign lesions(vocal polyps)to laryngeal precancerous lesions to laryngeal carcinoma in situ,there is a dynamic changes of the cell cycle-related factor,apoptosis factor.P21^Waf1,Pl6 expression in vocal cord polyp was of the deepest dye staining and positive cell density,In laryngeal precancerous lesions,P21^Waf1,P16 protein expression staining shallow and positive cells decrease.In laryngeal cancer the proteins expression was superficial staining and positive cells less.From vocal cord polyps,laryngeal precancerous lesions to laryngeal cancer,positive rate was of gradually decreasing intensity.And the expression of cyclin E,cyclinD1 protein in vocal cord polyps,laryngeal precancerous lesions and laryngeal cancer is just on the contrary,the positive gradient strength is gradually increased (P＜0.05).The relevant gene protein may be the prognosis markers of laryngeal precancerous lesions,can be used to as a more reliable indicator of carcinogenesis.3.As to strong positive(+++)expression of P21 in laryngeal precancerous lesions,there were significant differences between group with Phlegm stagnation and phlegm syndrome.It shows that laryngeal precancerous lesions with Phlegm stagnation syndrome was of the higher malignant potential,while group with Phlegm syndrome was of less malignant potential.Suggesting that need to strengthening monitoring and intervention of laryngeal precancerous lesions with Phlegm stagnation syndrome.4.Tongue can reflect healthy condition of the body,The tongue from normal pale red to dark red indicates the process of pathogenic fire and phlegm coagulation with stagnation.Tongue fur from whitish to thin greasy yellow to yellow,indicating the progress of disease.When color of patients' tongue with laryngeal precancerous lesions deepened,thickens thinly,presents deep red tongue,purple tongue,crack tongue,or yellow greasy moss,attention should be given to the tendency of malignant transformation,and it is necessary to follow-up closely and take preventing interventions.5.The study on laryngeal precancerous lesions exploring the relationship between the occurrence of laryngeal cancer and Phlegm stagnation from perspective of cell cycle regulation,identification which Syndrome type of laryngeal precancerous lesions is of higher malignant potential.To get a initial summary of the basic pathogenesis of laryngeal precancerous lesions,exploring the molecular mechanism of carcinogenesis from the level of molecular biology.At present,there was no systematic research domestic and foreign about these,the project will build a good foundation for cancer prevention and treatment with Chinese medicine,and have some reference value for future study on laryngeal precancerous lesions.Due to few research time and cases of laryngeal precancerous lesions,there are many unavoidable deficiencies,it needs for further research work.