| Part Ⅰ Proteomic analysis of uterine smooth muscle tissue in patients with adenomyosis based on iTRAQ TechnologyObjectiveWe screened proteins expressed differentially in patients with adenomyosis(AM)based on isobaric tags for relative and absolute quantitation(iTRAQ),and analyzed their involved life activity pathways in patients with adenomyosis(AM).Screening differentially expressed proteins and pathways related to contraction to provide target proteins and research pathways for further study of the mechanism of abnormal uterine contraction in AM patients.MethodUterine smooth muscle tissue samples were extracted from AM patients and cervical intraepithelial neoplasia patients,whose basic information were consistent.Using homogenized SDT lysis method to prepare homogenized proteins.FASP proteolysis digested the sample into peptides.ITRAQ reagent was used to label the protein of each uterine smooth muscle strip sample.We screened differential proteins based on high PH RP fractionation,mass spectrometry identification and protein search.Blast2 GO was used for GO analysis of differential proteins.The KAAS database was compared with the KEGG GENE database to KO classify the target protein and obtain the pathways involved in the target protein.GO term and KEGG enrichment analysis were performed by Fisher exact test.Gluster3.0 software classified samples and protein expression.Finally,a hierarchical clustering Heatmap was generated using Java Trewview.Result(1)Compared with the non-adenomyosis group,there were 93 differential proteins in the adenomyosis group,including 70 up-regulated proteins and 23 down-regulated proteins.Thirteen contraction-related differential proteins were screened,including phospholamban,Serine/threonine-protein kinase 26,Serine/threonine-protein phosphatase 4 regulatory subunit 2,Actin-like protein(Fragment)involved in oxytocin signaling regulation(accession numbers:Q562T7,Q562M5),Putative beta-actin-like protein 3,Cytochrome).Coxidase subunit 2(Fragment)and the proteins SWI/SNF related,matrix associated,actin dependent regulator of chromatin,subfamily C that proved to be associated with the development of related myocardial smooth muscle diseases,Member 1,Epididymis luminal protein 176,Ribosome biogenesis regulatory protein homolog,Mevalonate kinase(Fragment),Uncharacterized protein,Alkyl-dihydroxyacetone phosphate synthase,peroxisomal.(2)Combining GO functional annotation and enrichment analysis with KEGG pathway.105 enrichment pathways were found.And 8 possible contraction-related pathways were preliminarily screened,including Calcium signaling pathway,Oxytocin signaling pathway,Regulation of actin cytoskeleton,Cardiac muscle contraction,Hypertrophic cardiomyopathy(HCM),Viral myocarditis,Arrhythmogenic.Right ventricular cardiomyopathy(ARVC),Dilated cardiomyopathy.ConclusionThere were 93 differential proteins in patients with adenomyosis,among which 13 were contraction-related differential proteins.And 8 pathways were contraction-related,including calcium signal regulation pathway,oxytocin signal regulation pathway and related pathways have been confirmed to be related to cardiac smooth muscle contraction.Part Ⅱ Contractile function test of uterine smooth muscle in patients with adenomyosisObjectiveMulti-channel biological signal analysis system was used to record shrinkage curve of uterine muscle strip collected from adenomyosis(AM)and non-adenomyosis patients in vitro.The contraction of muscle strips after using oxytocin and oxytocin receptor antagonist(atosiban)was compared to provide direct evidence for the relationship between abnormal uterine contraction and oxytocin signaling pathway in patients with adenomyosis,and to provide theoretical basis for further study of the pathogenesis and clinical treatment of adenomyosis.MethodA total of 11 myometrial tissues were collected from patients underwent hysterectomy due to AM.10 myometrial tissues were collected from patients underwent hysterectomy due to cervical intraepithelial neoplasia as controls and matched with basic information.10 myometrial tissues were collected from patients underwent hysterectomy due to cervical intraepithelial neoplasia as controls whose basic information matched with the AM group.Sampling method:Immediately after hysterectomy,three muscle strips were cut along the transverse axis of the uterus and divided into basic group(without administration),OTgroup(the concentration of oxytocin was 10-7mol/L),OTR-anta+OTgroup(the concentration of the Atosiban was 10-5 mol/L,and then added oxytocin which concentration was 10-7 mol/L immediately).The muscle strip contractile function test was performed on the uterine smooth muscle tissue by an in vitro experimental device.And the contraction curve of uterine muscle strip was recorded by MPA 2000 ex vivo experimental system software.The contraction frequency,amplitude and area under contraction curve(AUC)of uterine muscle strips in AM and control groups under different conditions were analyzed and compared.Result(1)In the basic state,the amplitude of uterine contraction in the AM group(2.41±1.34g)was not different from the control group(2.69±0.97g,P=0.103).But the contraction frequency was higher in the AM group(5.88±1.74 times/10 min)than in the control group(3.53±1.25 times/10 min,P=0.001).The AUC of AM group(1334.43±242.90 kg·s/10 min)was larger than that of control group(884.91±18.26kg·s/10min,P=0.032).(2)Under the influence of the same concentration of oxytocin,the amplitude of contraction in the AM group was(3.34±0.87g),which was larger than that in the control group(2.69±0.97g,P=0.026).And the contraction frequency in the AM group(7.60±2.82/10min)was higher than that in the control group(4.03±1.58/10min,P=0.008).Moreover,the AUC of the AM group(1882.76±326.54 kg·s/10 min)was larger than that of control group(1287.24±235.24 kg·s/10 min,P=0.002).(3)The contraction amplitude of the AM group(2.68±0.76g)was not different from that of the control group(2.44±0.43g,P=0.126),While the oxytocin was added immediately after Atosiban.And the contraction frequency of the AM group(4.56±1.53 times/10 min)was higher than that of the control group(2.76±1.87 times/10 min,P=0.038).Besides,the AUC of the AM group(1153.83±137.72 kg·s/10 min)was larger than that of control group 925.52±93.52 kg·s/10min,P=0.042).(4)We Compared the contraction of the AM group in different states.After using oxytocin,the contraction amplitude of the AM group(3.34±0.87g)was larger than that of the basic state(2.41±1.34g,P=0.007).And the contraction frequency of the AM group(7.60±2.82 times/10min)was higher than that of the basic state(5.88±1.74 times/10min,P=0.008).AUC of the AM group(1882.76±326.54 kg·s/10min)was larger than that of the basic state(1334.43±242.90 kg·s/10min,P=0.042).After blockaded with oxytocin receptor antagonists,there was no significant difference between the AM group(2.68±0.76g)and the basic state(2.41±1.34g,P=0.686).And there was no significant difference in contraction frequency between the AM group(4.56±1.53/10min)and the basic state(5.88±1.74/10min,P=0.029).What’s more,the AUC of the AM group(1153.83±137.72 kg·s/10min)was smaller than that in the basic state(1334.43±242.90 kg s/10min,P=0.017).After blockaded with oxytocin receptor antagonists,the contraction curve of the AM group was close to that of the basic state.ConclusionIn the basal state,there was abnormal contraction of uterine smooth muscle existed in patients with adenomyosis,which was mainly manifested by higher contraction frequency and AUC than that in patients without adenomyosis.The uterine smooth muscle of the patients with adenomyosis was more sensitive to oxytocin than that of patients without adenomyosis.Under the stimulation of oxytocin,the contraction frequency and amplitude were higher in patients with adenomyosis. |
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