| Traditional Chinese medicine?TCM?plays an important role in the diagnosis and treatment of diseases.However,syndrome diagnosis which is key process in the TCM,has always been a hot and difficult point in the clinical research.On the one hand,syndrome diagnosis relates to the improvement of clinical diagnosis and treatment level of TCM.On the other hand,it relates to the development and usage of Chinese patent medicine.It remains controversy about the standards of TCM syndromes in process of development of new Chinese patent medicine.Qi and blood are the basic material of human body to maintain the activity and function of human life.Qi and blood are closely related to each other.The spectrum of qi and blood diseases are wide,the research on TCM syndromes related to qi and blood in the process of development Chinese patent medicine would have broad prospects.Among them qi stagnation and blood stasis syndrome?QSBSS?is a common syndrome,involving a wide spectrum of diseases,could be seen in many diseases including gynecology,surgery and other diseases.At present,the clinical research on QSBSS is still difficult to evaluation of clinical efficacy by the unclear diagnosis.Therefore,this study carried out the following work from three aspects:theoretical research,clinical research and experimental research to study the diagnostic criteria of QSBSS.METHODSTheoretical ResearchReviewed classical literatures on the syndrome of QSBSS which related to pathogenesis,symptoms and treatment with fifth edition of Chinese medical classics.Used CNKI to search the literatures which were limited to 1974-2016,contained the title of QSBSS.Used NoteExpress2.0 software to manage and analyze literatures.Clinical ResearchA closed and open questionnaire was designed for senior physicians and researchers to acquaint with the form of diagnosis of QSBSS and establish the entry pool for designing the clinical research tool,which was done in the conference on October 24,2015.And then,a cross-sectional study was designed to collect 1076 patients from April to November in 2016 in Guang'anmen hospital.The qualification of researchers and the standard of research criteria should be explicit before the research.Patient's age,gender,place of birth,duration,living habits,symptoms,RT and DIC were collected.Clinical data were analyzed by frequency analysis,correlation analysis,cluster analysis,principal component analysis,factor analysis,discrimination,correlation coefficient,logistic stepwise regression,etc.ROC curve and Youden index was used to determine the diagnostic value of the QSBSS scale.Sensitivity,specificity and accuracy were used to evaluate the performance of the QSBSS scale.All these data statistics were used SPSS20.0 and R software.Experimental Research25 patients were collected from October to December in 2016 of which be divided into 5 groups including coronary heart disease with QSBSS,coronary heart disease with non-QSBSS,chronic gastritis with QSBSS,rheumatoid arthritis with QSBSS and healthy control group.Each group had 5 cases whose age,gender,height,weight,blood pressure and past medical history were collected after obtaining their informed consent.All cases were taken peripheral blood.And then the red blood cell lysate was used to separate white blood cells to extract the total RNA.Agarose gel electrophoresis and ultraviolet analysis were used to analyze and determine the purity,concentration and integrity of the total RNA.While the quality of total RNA was detected by Nano-Drop and Agilent 2100.The high-throughput sequencing technology was employed to sequence the circRNA of QSBSS.The quality of sequencing was evaluated by the base mass distribution,GC content distribution,sequencing saturation analysis,sequencing random assessment,reads enrichment analysis in different components,gene coverage analysis and reads distribution on the reference genome.Compared the sequencing results with the circBase data to obtain the known circRNA and newly measured circRNA with CIRI software.According to the position of the circRNA chromosome to annotate the sorce gene of circRNA.RPM was used to quantify the circRNA,and Venn were used to analyze the differential expression circRNA for QSBSS.KEGG enrichment analysis was used to analyze the differential expression circRNA.In the process of screening differential expression circRNA,the negative binomial distribution and edgeR were mainly used.RESULTSTheoretical ResearchRefering to 101 classical literatures,QSBSS could be caused by traumatism,emotional frustration,cold-evil,excessively comfortable,improper sexual life,ingestion of drugs,malpractice and postpartum disorders.Among them,traumatism,emotional frustration and cold-evil are common factors.Pain is a common symptom of QSBSS,including chest pain,stomachache,abdominal pain,joint pain,etc.Meanwhile,it could be seen tumor and other symptoms.Among 1121 literatures with QSBSS,841 were related to diseases,involving 76 kinds of diseases,and gynecology diseases,surgery/orthopedics diseases,cardiovascular diseases and digestion diseases were mainly.It is meaningful to do the research of QSBSS diagnosis standard for the lack of diagnostic criteria of QSBSS.Clinical Research1 Expert questionnaireThere were 29 males and 16 females in expert group with the average age of 48.67 years old.49%of them got the doctor degree,while 29%of them got master degree,and 22%of them got bachelor degree.Among them 29 experts had ever participated in the research on the diagnosis of TCM syndrome,accounting for 64.44%;According to the results,42 pools were established for the research tool.2 Clinical cross-sectional study2.1 General situation1076 patients included 667 QSBSS patients and 409 Non-QSBSS patients with 467 males?43.40%?and 609 females?56.60%?.Among them,the oldest was 89 years old,the youngest was 18 years old,and the average age was 57.44 years,the place of birth involved 21 provinces and municipalities,while mainly in North China included Beijing?751?,Hebei province?94?,the Inner Mongolia Autonomous Region?32?.And the shortest duration was 0.1 year,the longest duration was 57 years,the average duration was 6.6 years.There were 120 kinds of diseases involved with the diagnosis in the western medicine while 59 kinds of diseases involved with TCM.There were 147 symptoms,and the top frequency of them were pain?871?,irritability?671?,dizziness?419?,fatigue?393?,chest tightness?389?,dry mouth?356?,etc.There were 20 kinds of tongue,top three were the dark red?476?,thin white moss?399?and thin greasy moss?210?.There were 12 kinds of pulse,top four were thready pulse?414?,string pulse?290?,sink pulse?187?and uneven pulse?149?.1076 patients,illness associated with mood within a month?732?,participated in daily physical activity?weekly>2 times,each time>30 minutes??451?,daily sedentary?except the normal sleep habits,weekly>3 times,every time>1 hours??744?,smoking?364?,drinking alcohol?281?,traumatism?175?.871 patients had pain symptoms,and sore fixation?648 cases?,distending pain?532 cases?,stabbing pain?264 cases?were common.2.2 Correlation analysis Emotional factors?P=0.000?,drinking alcohol?P=0.46?,distending pain?P=0.001?,scurrying pain?P=0.027?,cutting pains?P=0.035?,PLT?P=0.042?,PT?P=0.042?,INR?P=0.024?were positively related to QSBSS.While dull pain?P=0.028?,RBC?P=0.014?,HBG?P=0.008?,HCT?P=0.008?were negative related to QSBSS.2.3 Classification and dimensionality reduction With cluster analysis symptoms could be divided into four categories,which pain and irritability were one of the categories.With factor and principal component analysis,symptoms could be divided into nine factors,and according to the characteristics of the variable,pain,irritability,dysmenorrhea,dark menstrual,abdominal pain,chest tightness,chest pain,stomach pain and dysuria could be considered as a potential factor of QSBSS.With cluster analysis pulse could be divided into three categories,deep pulse and uneven pulse were one of the categories.With factor and principal component analysis pulse could be divided into three factors,deep pulse and uneven pulse could be considered as a potential factor of QSBSS.With cluster analysis tongue could be divided into three categories,dark purple tongue,petechia or ecchymosis tongue and mauve pale tongue were one of the categories;With factor and principal component analysis for 667 cases of QSBSS patients,purple tongue could be considered as a potential factor of QSBSS.2.4 Screening Pools were screened and determined to build the diagnosis model of QSBSS by using the methods which were combined with expert consultation,correlation coefficient,discrimination,classical literature and guidelines for the clinical research of Chinese medicine new drugs.While pools met over two aspects could enter the model building.There were 44 pools met the condition.While considered the purpose of this study was to develop a universality QSBSS diagnosis scale,dark menstrual,menstrual with blood clot were excluded,back pain,stomach pain,abdominal pain,limb pain,headache,back pain,joint pain and dysmenorrhea,breast pain were merged into pain,irritability and depression were merged into emotional frustration.Finally,there were 33 pools were put into the logistic stepwise regression model including pain,emotional frustration,sore fixation,distending pain,stabbing pain,scurrying pain,cutting pain,local activity limitation,lump or mass,belching,sour regurgitation,heartburn,fatigue,polydipsia,abdominal distension,dry mouth,chest tightness,chest fullness,frequent micturition,squamous and dry skin,darkish complexion,cyanosis nail,skin ecchymosis,lumbago,breast tenderness,swelling,dark purple lips,petechia or ecchymosis tongue,dark purple tongue,dark red tongue,uneven pulse,string pulse,deep pulse,thready pulse,tight pulse.2.5 Weighted and value determinate.Logistic stepwise regression was used to analyze 33 pools to build the diagnosis model of QSBSS.There were 15 steps,and third step was the best than others for the forecast total percentage was 81.5%,H-L P=0.105>0.05.Among them,pain?P=0.000?,emotional frustration?P=0.000?,lump or mass?P=0.001?,dark purple tongue?P=0.039?,fatigue?P=0.023?,local activity limitation?P=0.035?,dark purple lips?P=0.024?,petechia or ecchymosis tongue?P=0.002?,deep pulse?P=0.011?had statistical significance.However,the regression coefficient of fatigue,local activity limitation and dark purple lips were negative which were unsuited to enter the model.Excluded these three pools,put distending pain,scurrying pain,chest tightness and uneven pulse into the model.Finally used logistic regression to build the model.The ROC curve was drawn by the diagnosis of model and doctors.And the area under the ROC curve was 0.851.The Youden index of the model was 0.63,the sensitivity was 79.9%,the specificity was 83.1%.Based on the boundary value and logistic regression model,the diagnosis of syndrome QSBSS could be explained as 1.55 X pain+2.673 × emotional frustration+0.330 X distending pain+1.081 × scurrying pain+0.700 × petechia or ecchymosis tongue+0.207 X dark purple tongue+0.603 × uneven pulse+0.318 × deep pulse>3.356.2.6 Scale construction and performance evaluation The highest diagnosis score of QSBSS is 51.5 points,when it would be up to 20 points could be diagnosed as QSBSS.The sensitivity of the scale is 80.35%,the specificity is 81.91%,the accuracy rate is 80.94%,which means the results were achieved the expected goal.Experimental ResearchBaseline comparison among 25 subjects,gender?P=0.42?,diabetes mellitus?P=0.114?,cancer?P=0.384?,weight?P=0.324?and diastolic blood pressure?P=0.469?,P>0.05.While high blood pressure?P=0.017?,age?P=0.008?and systolic blood pressure?P=0.14?,P<0.05.RIN in 8.6?10.0 and 28S/18S between 1.1?1.8 which was showed the total RNA extraction had met the experimental requirements.And the quality of sequencing results were good enough to the identification,annotation and quantitative of the circRNA.There were 28059 circRNAs measured by 25 samples of which the total length were 66844321 bp,the average length was 2382.2bp.Among them,10955 were in the circBase,while the other 17104 were predict circRNA.The number of sense-overlapping circRNA were 24632,accounting for 87.79%,and exonic circRNA were 1217,accounting for 4.34%.The similarity of samples in each group was low,and there existed clustering between different groups Finally,there were 4 differential expression of circRNAs in QSBSS which included circRNA09849,circRNA11523,circRNA18046,circRNA24450,and circRNA11523 could be related to hsacirc0005860 in circBase while other three circRNAs were predict circRNAs.KEGG was used to analyze the differential expression of circRNAs in QSBSS.Five enrichment paths were found which included path:hsa05144,path:hsa04914,path:hsa04650,path:hsa04110 and path:hsa05203,and they mainly involved in the natural killer cell mediated cytotoxicity pathway and the cell cycle pathway.CONCLUSIONSQSBSS is a common TCM syndrome,involves a wide spectrum of diseases which is the primary cause are traumatism,emotional frustration and cold-evil.And pain is a common symptom of QSBSS,clinical researches on the diagnosis QSBSS are necessary and meaningful.The diagnosis scale of QSBSS is universality,and its sensitivity,specificity and accuracy rate could reach the expected goal,which could be provided a basis for the diagnosis of QSBSS.And QSBSS exists differential expression in circRNA to be provided for later identification of qPCR. |
PDF Full Text Request