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The Expression and Regulation of Prostaglandin H Synthase Type-2 in Preterm Labour Myometrium

Author: ZhangLiHong
Tutor: ChangQing
School: Third Military Medical University
Course: Obstetrics and Gynaecology
Keywords: PGHS-2 Premature birth Full-term PGE2 NFκB
CLC: R714.21
Type: Master's thesis
Year: 2010
Downloads: 35
Quote: 1
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Abstract


Preterm birth is a common complication of pregnancy, accounting for 5% to 15% of the entire pregnancy, accounting for 70% of neonatal deaths, 75% of neonatal morbidity. The incidence of preterm birth over the years has not reduced, no reliable prediction of preterm delivery in clinical diagnostic indicators, but also the lack of effective treatment measures. Most of the research on premature birth remains committed to clarify the mechanism of childbirth, especially biochemical changes in the onset of labor process, and hope in order to find a better forecasting and effective method for treatment of premature. Prostaglandins can change the fetal membrane structure, promote cervical ripening and lead to the contraction of the uterus, is the the childbirth occurs most important stimulus factors. PGHS-2 is the rate-limiting enzyme in prostaglandin synthesis, it arachidonic acid into prostaglandin precursor-PGG2 or PGH2 to form various forms of prostaglandins, prostacyclin and thromboxane. PGHS-2 is an inducible molecule, and its expression is subject to strict regulation of external stimuli, the role of factors such as IL-1β, LPS, cell-related signaling pathways such as MAPK, NFκB activation, thereby activating transcription factor, after into the nucleus, binding to promote transcription regulatory elements in the nucleus. The adjustment mechanism to increase the expression of PGHS-2 is more complex, and the different types of tissue and different types of stimuli, the expression regulation mechanism of the PGHS-2 was significantly different. Gestational PGHS-2 is mainly present in the uterine smooth muscle cells and endometrial cells, respectively, have a role to induce of childbirth and maintain pregnancy. With the approaching of childbirth and launch of inflammatory cytokines such as IL-1β activates MAPK and NFκB signal, causing the PGHS-2 expression was significantly upregulated prostaglandin synthesis, stimulate uterine muscle cell contraction, leading to the occurrence of childbirth. Preterm birth is premature delivery caused by a variety of factors to launch the uterus by a resting period of the activation of the premature conversion of PGHS-2 expression and regulation characteristics may be differences in preterm and full-term. In this study, the preliminary study of the PGHS-2 expression and regulation in preterm uterine muscle characteristics, provide a theoretical basis for further revealing the mechanisms of preterm birth. Order to clarify the PGHS-2 expression and regulation characteristics of preterm labor, the study selected preterm labor in labor (PTL) and not in labor (PTNL) as a study group, full-term labor (TL) of pregnant women not in labor (TNL) as a control group of pregnant women biopsy taken of the lower uterine segment myometrial tissue immunohistochemistry - immunofluorescence PGHS-2 expression in uterine muscle morphology was observed by real-time quantitative PCR (qRT-PCR) and Western blot assay myometrium PGHS-2, IL-8, OTR etc. childbirth-related factor mRNA and protein expression by enzyme-linked immunosorbent assay (ELISA) detecting myometrial prostaglandin PGE2 content, combined with the clinical data for statistical analysis of the test results . Meanwhile, the sub-preterm myometrium PGHS-2 signaling pathway, MAPK and NFκB protein molecules expression in uterine smooth muscle cells isolated and cultured, observation prostaglandin expression and activation of NF-kB by Western blot analysis. We randomly selected Southwest Hospital, July 2008 to 32 to 366 weeks of pregnancy preterm cesarean delivery in April 2010, and 37 to 416 weeks of full-term pregnant women, 80 cases during the same period. By real-time quantitative PCR (qRT-PCR) and Western blot assay myometrial PGHS-2, IL-8, OTR childbirth related factor mRNA and protein expression. The results showed that the PGHS-2 mRNA and protein levels in preterm labor and normal full-term presence of significant differences in the the premature pregnancy was significantly higher than that of full-term pregnancy, and premature birth labor, its expression in the myometrium and obvious lower than full-term labor. Our results suggest that preterm uterine smooth muscle in PGHS-2 expression and regulation characteristics differences may be related to the full-term births. Enzyme-linked immunosorbent assay (ELISA) to detect preterm labor is not in labor and term not in labor myometrium prostaglandin PGE2 content. The results show not labor in preterm and term not in labor PGE2 are expressed, and the former is significantly higher than the latter, suggesting that preterm birth is not in labor upregulation of PGHS-2 can promote the synthesis of PGE2. We take fresh preterm not in labor and term not in labor myometrium, the phosphorylation JNK1/2/3, p38, ERK1 / 2 levels of the kinase and NFκB p65 and IkBa protein expression levels were detected by Western blot. The results showed that preterm birth not in labor myometrium, JNK1/2/3 and p38 MAPK signaling pathway kinase phosphorylation levels significantly higher than the term not in labor, and ERK1 / 2 phosphorylation levels of contrast no significant differences. NFκB p65 and IkBa expression levels between prematurity and the full-term no significant differences, suggesting that the upregulation of PGHS-2 expression probably by MAPK signaling pathway JNK1/2/3 and p38 achieve. Isolation, culture and identification of the smooth muscle cells of the uterus, to take a fresh myometrium, Ophthalmology small scissors repeatedly chopped organization 2mm × 2mm × 2mm size, will organize inhalation centrifuge tube, added to the culture medium 5ml (containing 0.2% collagenase Ⅱ), 37 ℃ water bath oscillation digest about 12 ~~ 16h. The centrifugal supernatant plus DMEM medium containing 20% ??FBS (containing penicillin 100U/ml and streptomycin 0.1mg/ml) wind and percussion mix inoculated cell culture plates, 37 ℃ 5% CO2 incubator stand for 1 to 2d, cell spreading adherent fusiform growth of about 8 ~ 10d cells reached 90% confluence, can be passaged. Laser confocal microscope observation of α-actin expression in cultured cells. The results show that the uterine smooth muscle cells isolated and cultured adherent growth, fusiform, significant expression of α-actin protein, suggesting that the isolation and culture of success. In order to detect the impact of PGE2 on uterine muscle cells, NF-kB signaling pathway, first with PGE2 stimulates uterine smooth muscle cells 8h, 16h, 24h of NFκB p65 protein expression by Western blot to detect cell; then to stimulate IL-1β and LPS with the PGE2 stimulation or no stimulation of uterine smooth muscle cells, laser confocal observation NFκB p65 nuclear transfer. The results showed that PGE2 significantly inhibited NFκB p65 protein expression, and most significant in 16h. PGE2 significantly inhibited the NFkB p65 molecules induced by IL-1β and LPS stimulation in vitro transfer to the nucleus, suggesting that PGE2 significantly inhibited NFκB p65 expression in uterine smooth muscle cells. PGE2 can inhibit IL-1β, LPS-induced uterine smooth muscle cells in the NFκB p65 nuclear transfer, display during pregnancy, PGHS-2 may be provided by regulating the synthesis of prostaglandins to participate in regulation of the NFκB pathway activation. The above studies suggest that PGHS-2 expression and regulation in preterm and full-term births, there is a significant difference, and provides a theoretical support for further elucidate the mechanism of childbirth and premature.

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CLC: > Medicine, health > Obstetrics and Gynaecology > Obstetrics > Pathological pregnancy ( abnormal pregnancy ) > Miscarriage, premature labor and prolonged pregnancy
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