| ObjectiveLaryngeal cancer is one of the most common kinds of the neck and head cancer with increasing morbidity.In spite of the treatment of radiotherapy or chemotherapy,laryngeal cancer always results in serious death and recurrence rate.It has been proved that the Constitution Type in TCM is closely related to incidence and progress of carcinoma.Although there is few study on the relationship between the TCM constitution type and laryngeal cancer.If we find out the TCM constitution type which tend to get laryngeal cancer,it would be helpful to pevent laryngeal cancer by improving the TCM constitution type.In the past 20 years,professor Li and her team had found out the main TCM syndromes of laryngeal cancer patients are blood stasis syndrome,phlem syndrome,phlem and blood stasis syndrome and had proved their foundation.It’s very important to find out the microcosmic mechanism between phlem and blood stasis syndrome and laryngeal cancer for the therapy to laryngeal cancer combined TCM with western medicine.In the first place of this study is reviewing past researches on TCM constitution type and carcinomas.The second part is to find out the the constitution types in TCM of laryngeal cancer patients by clinical investigation.The last part is to seek out the influence of phlegm and blood stagnation on the expression of miRNAs of nude mice with laryngeal cancer cells.Methods1.Literature reviewBy literature review,the author would summarize the definition,types and interference of constitution in traditional Chinese medicine and then analyse the relationship between TCM constitution types and carcinomas.2.Clinical researchTCM constitution questionnaire was built by 9 types of TCM constitution from professor Wang.From 1/2014 to 2/2017,after TCM constitution questionnaire survey on 67 laryngeal cancer patients diagnosised that went to the Second Affiliated Hospital of Guangzhou University of TCM and 53 healthy people from Guangzhou Sanyuanli street citizen as control group,the TCM constitution type would be determined by questionnaire grades and then database would be built.So the distribution feature of the TCM constitution type of laryngeal patients became clear.3.Experimental researchThe Hep-2 laryngeal cancer cells were developed and copied.At the same time the nude mice with blood stasis syndrome、nude mice with phlegm syndrome and nude mice with phlegm and blood stagnation syndrome were built and proven.Then the Hep-2 laryngeal cancer cells were vaccinate under the skin of the right armpit of all nude mice in four groups.The growth of laryngeal cancer cells on nude mice were observed.After taking off the tumor from nude mice,vitality of cancer cells was tested by MTT,expression of miRNAs was tested by high-throughput sequencing and the target genes were proven by real time PCR.Results1.Clinical researchThe TCM constitution type of laryngeal cancer patients including 67 patients was compared with healthy people as control group including 53 people and it showed significantly differences.The main TCM constitution types of laryngeal cancer patients were blood stasis constitution and phlegm-dampness constitution and the population showed larger than the control group,P<0.05.In the group of laryngeal cancer patients,the account of peace constitution showed fewer than that of the control group,P<0.05.Besides peace constitution,the oter eight TCM constitutions are non-peace constitutions.In the group of laryngeal cancer patients there were 7 people with peace constitution,accounted 10.4%,60 people with non-peace constitutions,accounted 89.6%,while in the group of healthy people there were 27 people with peace constitution,accounted 50.9%,26 people with non-peace constitutions,accounted 49.1%.Eight non-peace constitutions appear lonely or combined with other non-peace constitutions which named combined constitution.There were 60 people with non-peace constitutions in the group of laryngeal cancer patients and it included 27 single non-peace constitution that accounted 40.30%and 33 combined non-peace constitutions that accounted 49.3%.There were 26 people with non-peace constitutions in the control group and it included 11 single non-peace constitution that accounted 20.8%and 15 combined non-peace constitutions that accounted 28.3%.In the group of laryngeal cancer patients there were 24 people with blood stasis constitution(included 6 people with single blood stasis constitution and 18 people with combined blood stasis constitution),while in the control group there were 3 people with blood stasis constitution(no people with single blood stasis constitution and 3 people with combined blood stasis constitution).In the group of laryngeal cancer patients there were 25 people with phlegm-dampness constitution(3 people with single phlegm-dampness constitution and 22 people with combined phlegm-dampness constitution),while in the control group there were 6 people with phlegm-dampness constitution(no people with single phlegm-dampness constitution and 6 people with combined phlegm-dampness constitution).The other six TCM constitutions(qi-deficiency constitution and yang-deficiency constitution and yin-deficiency constitution and damp-heat constitution and qi-depression constitution and special endowment constitution)between two groups showed little difference,P>0.05.Qi-deficiency constitution and yang-deficiency constitution and yin-deficiency constitution are deficiency constitutions.Population with deficiency constitutions were combined in two groups and TCM constitutions were ranked from more to less:in the group of laryngeal cancer patients,deficiency constitutions>phlegm-dampness constitution>blood stasis constitution>damp-heat constitution>qi-depression constitution>peace constitution>special endowment constitution,while in the control group,peace constitution>deficiency constitutions>damp-heat constitution>qi-depression constitution>phlegm-dampness constitution>blood stasis constitution>special endowment constitution.Deficiency constitutions occupied a large portion in both groups,46.32%in the group of laryngeal cancer patients and 32.1%in the control group.2.Experimental research(1)Nude mice with blood stasis syndrome、nude mice with phlegm syndrome、nude mice with phlegm and blood stagnation syndrome were built and proven.The nude mice of blood stasis syndrome showed petechiae and ecchymosis somewhere,dark red color around the lip,dark purple of tongue color and they put up weight slower and reduced water intake and food intake.Bleeding time of nude mice tail and blood coagulation time by glass tube of the model group were obviously shorter than bleeding time of control group.All those were typical syndromes of blood stasis syndrome.The nude mice of phlegm syndrome exhibited typical syndromes of phlegm syndrome.They put up weight faster with more sleepiness,lowered activities,more love for collective,loose stool,reduced water intake and reduced food intake.Blood lipids:TC,LDL-C and HDL-C of model group nude mice appeared to be higher than that of control group nude mice.The nude mice of phlegm and blood stagnation syndrome exhibited typical syndromes of phlegm-blood stagnation syndrome.They showed petechiae and ecchymosis somewhere,dark red color around the lip,dark purple of tongue color and they put up weight faster with more sleepiness,lowered activities,more love for collective,loose stool and reduced water intake and food intake.Bleeding time of nude mice tail and blood coagulation time by glass tube of the model group were obviously shorter than that of the control group.Blood lipids:TC,LDL-C and HDL-C of model group nude mice appeared to be higher than control group.(2)The Hep-2 laryngeal cancer cells were vaccinate under the skin of the right armpit of model nude mice successfully.The growth of laryngeal cancer cells on nude mice of blood stasis syndrome showed faster than other three groups.The time point was the 20th day(nearly third week)after vaccinating.Volume and weight of tumors from the four groups showed no difference,P>0.05.(3)By MTT,vitality of cancer cells of nude mice with blood stasis syndrome、nude mice with phlegm syndrome、nude mice with phlegm and blood stagnation syndrome showed stronger than that of the control group.(4)The high-throughput sequencing results showed that there were many different miRNAs expressed in the three model groups than in the control group,especially in the nude mice group with blood stasis syndrome and nude mice group with phlegm-blood stagnation syndrome.After reviewing literature and deleting the repeated miRNAs,10 miRNAs were thought to be the the target genes.So the 10 miRNAs were tested by real time PCR and 5 miRNAs were proven to be different from the control group:miR-27a-5p、miR-708-5p、miR-30c-5p、miR-22-3p、miR-19a-3p.In the nude mice group with blood stasis syndrome and nude mice group with phlegm and blood stagnation syndrome,miR-27a-5p was up and miR-708-5p、miR-30c-5p、miR-22-3p、miR-19a-3p showed down expressed.In the group of phlegm syndrome,miR-27a-5p showed up and miR-708-5p showed down expressed.Conclusion1.Clinical researchThe main TCM constitution types of laryngeal cancer patients were blood stasis constitution and phlegm-dampness constitution and there were many deficiency constitutions as well.So laryngeal cancer may be closely related to blood stasis constitution and phlegm-dampness constitution and zhengqi-deficiency constitutions.Zhengqi-deficiency constitutions may act as basic and blood stasis constitution and phlegm-dampness constitution act as external.2.Experimental researchBlood stasis syndrome、phlegm syndrome、phlegm-blood stagnation syndrome might enchance vitality of laryngeal cancer cells through MTT results.The gene mechanism may be blood stasis syndrome、phlegm syndrome、phlegm-blood stagnation syndrome affecting miRNAs expression in laryngeal cancer cells:miR-27a-5p expressed up in nude mice with blood stasis syndrome、nude mice with phlegm syndrome and nude mice with phlegm-blood stagnation syndrome,so the gene miR-27a-5p may be the promotion gene to laryngeal cancer cells;while the miRNAs as miR-708-5p、miR-30c-5p、miR-22-3p、miR-19a-3p may be the suppressive miRNAs to laryngeal cancer cells.3.ProspectsIt may be helpful to regulate blood stasis constitution and phlegm-dampness constitution and deficiency constitutions for the prevention from carcinoma of larynx.In the therapy of laryngeal cancer patients it may be useful to use the thrapy of " huatan and quyu " to transform the gene expression especially to patients with blood stasis syndrome、phlegm syndrome or phlegm-blood stasis syndrome. |
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