Research On VEGF-MMPs Pathway In Estrogen Induced Dysfunctional Uterine Bleeding And It’s Serum Biomarker

Dysfunctional uterine bleeding(Dysfunctional Uterine Bleeding, DUB) called dysfunctional uterine bleeding is due to the hypothalamus- pituitary-ovarian axis dysfunction caused by abnormal uterine bleeding, including anovulatory uterine bleeding(anovulatory dysfunctional uterine bleeding, ADUB) and ovulation DUB(ovulatory dysfunctional uterine bleeding, ODUB). Which accounted for 80%,, ADUB serious harm to women’s health. Studies have shown that found that estrogen and matrix metalloproteinase(MMP-2/9) increased levels ADUB in most patients, but in its course of estrogen is not always at a high level state, sometimes very low, it is not certain estrogen Causation and MMP-2/9’s. To investigate whether estrogen can directly stimulate the expression of MMP-2/9 endometrial epithelial cells, suggesting that the new bleeding ADUB mechanism, under the auspices of the Hainan Provincial Natural Science Foundation of China(30633 \ 812148), the preliminary findings in patients with endometrial MMP ADUB-2/9 increased expression of estrogen can induce primary endometrial epithelial cells MMP-2/9 increased expression, and by upregulation of vascular endothelial growth factor(vascular endothelial growth factor, VEGF) expression of activated signaling pathways to regulate MMP ErK-2/9 expression levels, VEGF play a central regulatory role; on this basis, MMP-2/9 and the tight junctions were investigated estrogen stimulation from cellular and animal level after protein Occludin, Claudin-5, the expression of Zo-1 while after endometrial endothelial monolayers and endometrial vascular permeability, increased estrogen stimulation determine MMP-2 expression / 9, and increased expression of MMP-2/9’s lead to endometrial vascular pass increased permeability, and MMP-2/9 inhibitor SB-3CT can reduce endometrial vascular permeability, blocking intravascular material leakage, provide a scientific basis for the treatment of anovulatory DUB drug development; again were screened using proteomic-SELDI identified ADUB and ODUB common serum markers serum amyloid A(SAA), found high concentrations of SAA reduce ESC cell viability, in a certain concentration range, SAA dose- and time-dependent down-regulated expression of VEGF and the clinical significance in revealing SAA DUB diagnosis, treatment and prognosis.Part Ⅰ: VEGF Plays a Central Regulatory Role in Estrogen-induced MMP-2/9 ExpressionObjective: To study the estrogen-induced molecular mechanisms of blood work, because VEGF is associated with estrogen-induced recognized DUB happen, but how VEGF causes uterine bleeding occurs, is not clear. The DUB occur ultimately by MMPs break down collagen and ultimately by dysfunctional endometrial bleeding, so the focus of the study DUB mechanism should be placed on the expression of VEGF regulates MMPs, so this part of the proposed study in anovulatory function DUB Research(ADUB) DUB patients on endometrial epithelial cell signaling pathways regulated VEGF expression of MMPs, analyze and identify the molecular mechanism.Methods: In situ detection of ADUB Zymography patient with endometrial matrix metalloproteinase 2 and 9(MMP2 / 9) activity and expression level of primary cultured human uterine epithelial cells, cells were stimulated by estrogen, real-time RT-PCR and western blot were used to detect mRNA and protein levels, gelatin zymography to detect the activity of MMP-2/9’s, P44 / 42 MAPK pathway silence cell changes observed after VEGF and MMP-2/9 expression levels, and P44 / 42 MAPK specific inhibitor U0126 blocked the phosphorylation of Erk effect on MMP-2/9 and VEGF expression.Results: In situ Zymography technical discovery activity and expression levels in patients with endometrial ADUB matrix metalloproteinase 2 and 9(MMP2 / 9) are increased in primary cultured uterine epithelial cells found that estrogen can induce MMP-2 / 9 and VEGF expression increased, meanwhile, found that estrogen can activate P44 / 42 MAPK(Erk1 / 2) signaling pathway, after RNA interference silencing VEGF expression of cell P44 / 42 MAPK pathway is blocked, and MMP-2/9 expression down, U0126 blocked the phosphorylation of Erk can down-regulate the expression of MMP-2/9, but does not decrease the expression of VEGF.Conclusions: VEGF first activated Erk1 / 2 signaling pathway, then expressed through phosphorylation stimulate Erk1 / 2 of MMP-2/9, VEGF expression in estrogen-induced MMP-2/9 in the central regulation. Part Ⅱ: Effect of Estrogen on Endometrial Endothelial Cell Permeability andIts Molecular MechanismObjective: To study the effect of estrogen on cultured human endometrial endothelial cells and rat endometrial vascular permeability, and permeability changes and MMP2 / 9 expression levels of tight junction protein, VEGF expression levels relationship.Methods:Transwell device for measuring endothelial cell monolayer permeability, qRT-PCR and western-blotting were used to detect MMP2 / 9 mRNA and protein expression of tight junction proteins(Occludin, Claudin-5, ZO-1) levels and, Determination of MMP2 / 9 active gelatin zymography, MTT cell viability analysis, PI staining frozen sections measuring endometrial vascular permeability in rat tissues, immunofluorescence assay endothelial cells monolayer tight junction protein ZO-1 distribution, using specific inhibitors interfere MMP2 / 9 and observed changes in the index case.Results: estrogen dose-dependent and time-dependent increase in endometrial vascular endothelial cell permeability, can be inhibited by SB-3CT MMP-2/9 inhibitor; with E2-induced MMP-2/9 expression levels rise and its activity increased, connexin Occludin between endothelial cells, Claudin-5, ZO-1 expression levels decreased, thereby enabling the space between cells increases, increased permeability, these effects can be MMP-2/9 inhibition The inhibitor SB-3CT; the results of in vivo studies have found that E2-induced upregulation of VEGF, although the SB-3CT can inhibit the elevated expression level of MMP-2/9 and activity increased, but not inhibit E2-induced upregulation of VEGF; this study found that blocking the MMP-2/9 can efficiently blocking estrogen E2 causes an increase in endometrial vascular endothelial cell permeability, in order to MMP-2/9 DUB treatment provides a new potential drug targets for efficient intervention.Conclusions: Estrogen can induce VEGF production, promote MMP-2/9 expression, decomposition cell tight junction proteins, lead to endometrial endothelial cell permeability, which can be inhibited by inhibitors of MMP-2/9’s.Part Ⅲ: Anovulatory DUB Serum Marker Screening, Identification and Expression of MenstruationObjective: The diagnosis of dysfunctional uterine bleeding mainly based on history, menstrual history, BBT, cervical mucus ultrasound and endometrial biopsy, but from the onset to diagnosis may experience a long time delay treatment therapy, cause considerable pain to the patient. So, how early screening and diagnosis of DUB DUB is still an important research topic. This study used proteomics technology-SELDI to ADUB patients and normal women serum marker protein for screening.Methods: SELDI for ADUB patients and normal women serum marker proteins were screened at the same time, the use of Tricine- SDS-PAGE gel separation, mass spectrometry and immunoprecipitation to detect the differences in the protein peak protein identification, using ELISA, RT-PCR and Western-blotting further verification was identified proteins in the blood of patients and normal work menstruating women expression.Results: Application of SELDI technology at home and abroad for the first time Biomarker ADUB screening and screening two different proteins, further using Tricine-SDS-PAGE gel separation, mass spectrometry and immunoprecipitation they identified as SAA and VEGF, By ELISA, RT-PCR, Western-blotting detection, confirmation SAA upregulated in patients with menstrual ADUB, whereas upregulation of VEGF in normal menstruation in women, consistent with the results and SELDI protein identification technology screening results.Conclusion: SAA and VEGF are important serum markers of ADUB and DUB patients Part Ⅳ:A Comparative Study of Blood Serum Protein Markers of ADUB and ODUBObjective: To explore the pathogenesis of the disease protein level, more directly illuminate the changes in the mechanism of life under physiological or pathological conditions of the two kinds of serum markers in the blood work were more likely to find their common serum markers.Methods: SELDI for ADUB patients and normal women serum marker proteins were screened at the same time, the use of Tricine- SDS-PAGE gel separation, mass spectrometry and immunoprecipitation to detect the differences in the protein peak protein identification, using ELISA, RT-PCR and Western-blotting further verification was identified proteins in the blood of patients and normal work menstruating women expression.Results: This study is the first application of SELDI-TOF-MS Screening dysfunctional uterine bleeding serum protein markers, and screened to three different proteins, and further identified as SAA, VEGF and VKOR, by ELISA, RT-PCR, Western-blotting testing, confirmed that SAA ADUB and ODUB upregulated in patients with menstrual, VEGF expression down-regulated in patients with menstrual ADUB rather VKOR downregulated in ODUB menstrual patients, suggesting that SAA, VEGF and VKOR may be associated with uterine bleeding.Conclusion: SAA are common serum markers of DUB.Part Ⅴ: Impact of SAA on VEGF Expression of Human Endometrial Stromal CellsObjective: To explore the power of blood serum markers SAA on VEGF expression in human endometrial stromal cells, preliminary analysis of SAA pathophysiology of blood in power.Methods: MTT cell viability analysis, and the use of qRT-PCR, Western-blotting and ELISA were used to detect mRNA VEGF, the protein expression levels and secretion, cell immunohistochemistry assay vimenatin identified ESC cell purity.Results: The SAA after treatment, loosely connected cells, adherent cells is poor, some cells showed shrinkage, increased shedding of cells. But it has no effect on survival of low concentration SAA ESC, when the SAA concentration of 60 mg / L, ESC cell viability decreased significantly(P <0.05, compared with the control group), and with the SAA concentration increased, ESC cell viability further decline, when SAA concentration of 200 mg / L, the impact of ESC cell survival difference maximum, SAA concentration of 400 mg / L and 200 mg / L was no significant. Low concentration(10mg / L) could increase the mRNA and protein levels and secretion of VEGF, but with the SAA concentration increased, compared with the control group, mRNA levels of VEGF protein levels were significantly decreased secretion to 40 mg / The most significant effect of L. Results can be seen from the above, SAA within a certain range of concentrations concentration-dependent down-regulate the expression and secretion of low concentration SAA VEGF gene but have raised the gene expression of VEGF, which is noteworthy. Because of 20 mg / L of SAA significantly downregulated VEGF, so take time to study the effectiveness of the concentration of VEGF expression SAA regulation, showing a significant increase in VEGF expression levels in a short time, but over time, the expression levels gradually decreased, 36 h and 72 h of the expression level was significantly lower than the control group.Conclusions: The high concentration of SAA to make ESC cell viability decreased in a certain concentration range, SAA dose- and time-dependent down-regulate the expression of VEGF.