| Background and Objective Endometriosis(EM),referes to a normal endometrium gland and interstitium appearing outside the uterine cavity,the pathogenesis and pathophysiological process of endometriosis are not clear,nor specific laboratory tests to confirm the diagnosis.Therefore,in the first part of this study,we compared the differences of serum carbohydrate Antigen 125(CA125)and neutrophil-to-lymphocyte ratio(NLR)between EM patients and healthy subjects,to explore the correlation between CA125,NLR and endometriosis.In the second part,we analyzed the expression difference of NOD1-2 in ectopic and eutopic endometrial mesenchymal stem cells,the abnormal expression of NOD1-2 is involved in the occurrence and development of ectopic lesions,thereby supplementing immune and inflammatory mechanism of endometriosis.The ectopic stem cells origin is a new pathogenesis of EM.Stem cells have strong self-renewal ability and participate in endometrial regeneration after menstruation.Ectopic lesions contain dysfunctional stem cells,but how stem cells participate in the occurrence of endometriosis is not clear.It?s still controversial whether exist differences in biological characteristics between eutopic and ectopic endometrial mesenchymal stem cells.Endometriosis is a chronic inflammatory disease,nucleotide-binding oligomerization domain like protein 1-2(NOD1-2)are new pattern recognition receptors which can recognize various pathogen-related molecular patterns,initiate innate immune responses,stimulate macrophage activation and inflammatory signal transduction,and participate in the occurrence and development of various diseases.Materials and Methods 1.Select 100 cases of EM patients who underwent selective operation in the gynecology department of Henan Provincial People's Hospital from November 2017 to December 2018 as the endometriosis group,and select 100 healthy people as the control group during the same period.Analysis the correlation between CA125,NLR and endometriosis.2.Collection tissues(endometriosis,n=43;normal,n=16;eutopic-EM,n=12;ectopic-EM,n=12).Real-time quantitative PCR(qPCR)and immunohistochemistry(IHC)detected the expression differences of NOD1 and NOD2 in eutopic and ectopic endometrial tissues.Collagenase? digested tissues to obtain eutopic and ectopic endometrial stromal cells.Flow cytometry analyzed the expression of stem cell markers CD44,CD73,CD140 b,CD146,SUSD2,CD271 for identifing the purity of mesenchymal stem cells;Three-line differentiation experiments identified the adipogenic-genic,osteogenic-genic and chondrogenic-genic of eutopic and ectopic endometrial stromal cells.Cloning formation experiments identified colony form unit ability of eutopic and ectopic endometrial stromal cells;qPCR detected the transcription level of NOD1 in ectopic and eutopic endometrial stromal cells;CCK8,Transwell,apoptosis experiment and Clone formation experiments detected the proliferation,invasion,migration and apoptosis and colony form unit of eutopic-mesenchymal stem cells(eut-MSC)and ectopic-mesenchymal stem cells(ect-MSC),because these functional differences may cause the formation of ectopic lesions.Up-regulate or inhibit NOD1 expression,observed the proliferation,colony form unit,invasion,migration and apoptosis of eut-MSCs and ect-MSCs.qPCR detected the transcription levels of eut-MSCs and ect-MSCs invasion-related genes MMP2,MMP3,angiogenesis-related factors VEGFA,IL-8 and anti-inflammatory cytokine TGF-?1,to explore the microenvironment difference of eutopic and ectopic endometrial.Results: 1.The levels of serum CA125 and NLR in endometriosis were significantly higher than those in control;as the r-AFS stage increasing,serum CA125 and NLR levels increasing as well.2.qPCR showed NOD1 m RNA expression level in EM was significantly higher than that in EN,ect-EM was significantly higher than eut-EM(P <0.05).The NOD2 m RNA expression level in EM was slightly higher than EN,and the ect-EM was slightly higher than eut-EM,however no statistical difference between the two groups(P> 0.05).Immunohistochemical technique showed that NOD1 mainly expressed in ectopic lesion tissues,NOD1 mainly expressed in the cytoplasm of ectopic endometrial stromal cells.3.Flow analysis showed that the average positive percentage of CD44,CD73,CD146,CD140 b in ect-MSCs and eut-MSCs was more than 90%;the expression gap of SUSD2 among individuals of the same cell type was very large,and the average positive percentage was about 50%;both ect-MSC and eut-MSCs lowly expressed CD271,and the positive percentage was less than 10%.4.Three-line differentiation experiment showed eutopic and ectopic endometrial stromal cells could differentiate into adipocytes,osteoblasts and chondrocytes after 21 days of differentiation induction culture.5.CCK8 showed eut-MSCs possess more significantly proliferative capacity than ect-MSCs.6.Tri-DAP promoted the proliferation of eut-MSCs in a time-and concentration-dependent manner.1ng/m L,10ng/m L,100ng/m L,1?g/m L,10?g/m L concentration gradients Tri-DAP stimulated eut-MSCs 24 h,48h,72 h respectively.eut-MSCs began to significantly proliferation after 24 h at a concentration of 1?g/m L(P <0.05),10 ?g/m L no longer promoted proliferation;after 48 h,Tri-DAP significantly promoted eut-MSCs proliferation(P <0.05),1?g/m L Tri-DAP most noticeably promoted proliferation,10?g/m L can still promote proliferation but the effect was reduced;after 72 h,100ng/m L Tri-DAP had the most obvious effect of promoting proliferation,as the concentration increased,the cell proliferation decreased.ML130 inhibited the proliferation of ect-MSCs in a time-and concentration-dependent manner.0,0.28 ?m/m L,0.56 ?m/m L ML130 stimulated ect-MSCs 24 h,48h,72 h respectively,no significant inhibitory effect after 24h;0.56 ?m/m L ML130 significantly inhibited ect-MSCs after 48h(P <0.05);the inhibitory effect disappeared after 72 h.7.Clone formation experiments showed Tri-DAP stimulated eut-MSCs form colony-forming units,however no statistical difference compared with control;0.56?m/m L ML130 stimulated ect-MSCs,colony-forming units significantly decreased(P <0.05).8.Transwell showed invasion and migration capacity of eut-MSCs significantly higher than ect-MSCs(P <0.05).9.ML130 inhibited invasion and migration of ect-MSCs in a concentration-dependent manner.0.56?m/m L ML130 significantly inhibited invasion and migration of ect-MSCs(P <0.05).10.qPCR showed the m RNA expression of NOD1,IL-8 and VEGFA in ect-MSCs were higher than those in eut-MSCs,while MMP2,MMP3 and TGF-?1 m RNA were lower than those in eut-MSCs.11.Apoptosis experiment showed ect-MSCs and eut-MSCs no difference in apoptosis rate.0.28?m/m L and 0.56?m/m L ML130 stimulated ect-MSCs 24 h,48h respectively,no obvious apoptotic cells.Inhibition of NOD1 had no significantly effect on the apoptosis of ect-MSCs(P> 0.05).Conclusion: Serum CA125 and NLR levels were positively correlated with endometriosis and r-AFS stage.Joint detection of CA125 and NLR,the positive rate of EM detection is higher.NOD1 mainly expressed in ectopic endometrial tissue,the expression of NOD2 between eutopic and ectopic endometrial tissue have no statistical differences.The proliferation,invasion and migration capacity of eut-MSCs higher than ect-MSCs,however the apoptosis rate no difference between eut-MSCs and ect-MSCs;1?g/m L Tri-DAP stimulation for 48 h significantly promoted proliferation of eut-MSCs;0.56?m/m L ML130 inhibited ect-MSCs proliferation,invasion,migration and colony-forming units significantly,however no obvious inhibitory effect on the apoptosis rate.The expression of NOD1,IL-8,VEGFA in ect-MSCs increased,while MMP2,MMP3 and TGF-?1 decreased.NOD1 plays an important role in participating in the innate immune response of endometriosis. |
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