Caspase-8及Bcl-2表达与胎膜早破的相关性研究

目的:探讨Caspase-8及Bcl-2表达与胎膜早破的关系。方法:选择2003年6月~2004年5月我院足月妊娠自然分娩的孕妇48例,其中24例发生胎膜早破(胎膜早破组),24例未发生胎膜早破(对照组),病例均于阴道分娩后取胎膜破裂口处的胎膜组织5cm×5cm大,同时胎膜早破组在距胎膜破口处10cm以上的部位再取同样大的胎膜组织,胎膜组织均经石蜡包埋切片后采用免疫组化法测定Caspase-8及Bcl-2的表达。结果:(1)两组病例的胎膜组织均可见Caspase-8及Bcl-2的表达;(2)在胎膜早破组的胎膜组织中Caspase-8表达的阳性单位为6.89±0.19,对照组为2.33±0.06(P<0.01);而Bcl-2表达的阳性单位为9.55±0.24,对照组为21.37±0.32(P<0.01);(3)在胎膜早破组非破口部位胎膜组织中Caspase-8及Bcl-2表达的阳性单位分别为6.93±0.17和9·66±0.19,与破口部位胎膜组织中Caspase-8及Bcl-2表达的阳性单位比较无差异(P>0·05)。结论:胎膜早破的发生与Caspase-8的过度表达及Bcl-2表达的下调相关。     

胎膜早破绒毛膜羊膜bFGF与Bax、Bcl-2的表达

目的探讨碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)与凋亡调控基因Bax、Bcl-2在胎膜上的表达及与胎膜早破(premature rupture of membranes,PROM)发生的关系。方法采用免疫组化染色方法检测30例PROM及30例正常妊娠者的绒毛膜羊膜上bFGF与Bax、Bcl-2的表达。结果bFGF和Bax在滋养细胞、上皮细胞、间充质细胞和成纤维细胞胞浆内有表达,Bcl-2在绒毛膜合体滋养细胞上有表达;PROM组bFGF阳性平均积分光密度值(0.051±0.012)与正常组(0.098±0.027)比较明显降低(P<0.01),PROM组Bax阳性平均积分光密度值(0.086±0.021)与正常组(0.046±0.018)比较明显增高(P<0.01),PROM组Bcl-2阳性平均积分光密度值(0.058±0.020)与正常组(0.050±0.014)比较差异无统计学意义(P>0.05);PROM组bFGF与Bax呈负相关(r=-0.616,P<0.01),正常组bFGF与Bax间无相关性(r=-0.302,P>0.05)。结论绒毛膜羊膜上bFGF的低水平表达及过度细胞凋亡对PROM的发生有重要促进作用。     

Cx43、Bax和Bcl-2在胎膜早破患者胎膜组织中的表达及意义

目的检测间隙连接蛋白43（Cx43）在胎膜早破患者胎膜组织中的表达,分析其与凋亡相关蛋白Bax、Bcl-2表达的相关性,探讨三者与胎膜早破发生的关系。方法随机选择本院剖宫产分娩的孕32-42周胎膜早破孕妇30例（胎膜早破组）,其中未足月胎膜早破（pPROM）15例,足月胎膜早破（tPROM）15例;无胎膜早破孕妇30例（对照组）。采用免疫组化法（PV法）检测Cx43、Bax和Bcl-2的表达并进行图像分析。结果①各组胎膜组织中均可见Cx43、Bax、Bcl-2不同程度的表达。②tPROM组Cx43、Bcl-2的表达明显低于对照组（P〈0．01）,而Bax的表达高于对照组,差异均有显著性（P〈0．05）。tPROM组Cx43的表达高于pPROM组,差异有显著性（P〈0．01）;而Bcl-2、Bax在两组中比较,差异无显著性（P〉0．05）。③PROM组人工胎膜破口附近Cx43、Bcl-2的表达低于非人工胎膜破口附近,Bax的表达则相反,两组比较,差异有显著性（均P〈0．01）。④PROM组Cx43的表达水平与Bax呈负相关（r=-0．309,P〈0．05）。结论胎膜组织中Cx43的低表达及细胞的过度凋亡可能对胎膜早破的发生起一定的促进作用。     

人β防御素3在胎膜早破妊娠妇女中的表达及其意义

目的:探讨人β防御素3(h BD3)在胎膜早破妊娠妇女中的表达及其临床意义。方法:随机选择2013年1—12月于湖北省孝感市中心医院妇产科住院分娩的初产胎膜早破患者45例作为胎膜早破组,取同期正常妊娠妇女45例作为对照组。应用免疫组织化学SP法检测胎膜组织中h BD3的表达,酶联免疫吸附法检测血清肿瘤坏死因子α(TNF-α)和h BD3的含量。结果:胎膜早破组h BD3阳性33例;对照组h BD3阳性13例,2组阳性率比较差异有统计学意义(P0.05);与对照组比较,胎膜早破组血清TNF-α、h BD3在入院及分娩时均升高(P0.05),且胎膜早破组分娩时血清TNF-α、h BD3较入院时均升高(P0.05)。结论:胎膜早破时h BD3可能是在TNF-α的介导下参与了抗微生物的天然免疫反应。     

脂多糖经线粒体途径诱导细胞滋养细胞凋亡

目的:探讨脂多糖(Lipopolysaccharide,LPS)诱导细胞滋养细胞凋亡的机制。方法:从正常早孕绒毛分离细胞滋养细胞,用无血清培养基培养。在培养基中加入不同浓度的LPS,使其终浓度分别为0、25、50、100、200ng/ml。用光镜和透射电镜观察细胞滋养细胞的形态学变化;用Western blot检测Bax、Bcl-2和Caspase-8的表达。结果:LPS作用后24h,光镜和电镜下见细胞滋养细胞出现凋亡的形态学变化;Western blot检测结果表明,LPS抑制Bcl-2表达,促进Bax和Caspase-8表达。结论:LPS能够经线粒体途径诱导细胞滋养细胞凋亡。     

雌激素对去势小鼠脾细胞凋亡的作用机制探讨

目的:探讨雌激素对去势小鼠脾细胞凋亡、凋亡基因Fas、FasL及ER亚型表达的影响。方法:流式细胞仪检测不同浓度E2时去势小鼠脾细胞的凋亡率;RT-PCR方法检测细胞内Fas、FasL及ERα和ERβmRNA的表达。结果:除最低浓度(10-5μmol/L)外,去势+E2组凋亡率及Fas、FasL基因的表达都高于青年组(P<0.05);与去势对照组相比,E2在10-3μmol/L、10-2μmol/L浓度时凋亡率及Fas、FasL基因的表达增高(均P<0.01)。E2在10-4μmol/L时ERα、ERβ基因的表达升高(ERα:P<0.05;ERβ:P<0.01),以后随浓度增加ERα和ERβmRNA水平有下降去势。结论:高浓度E2致脾细胞凋亡,与上调凋亡促进基因Fas、FasL基因表达有关,低浓度E2可能逆转去势小鼠脾细胞凋亡。不同浓度E2对去势小鼠脾细胞ERα和ERβ基因表达的影响支持雌激素对ER表达的调节作用:在生理范围内为正向调节,超生理范围为负向调节。     

基质金属蛋白酶-9和肿瘤坏死因子-α对胎膜早破患者绒毛膜羊膜炎预测的评价

目的 探讨检测孕妇血清中基质金属蛋白酶-9（MMP-9）和血清及羊水中肿瘤坏死因子-α（TNF-α）在诊断胎膜旱破孕妇绒毛膜羊膜炎中的作用。方法 选择2004-06—2004-12在汕头市潮南区人民医院和汕头大学医学院第一附属医院产科住院分娩的胎膜早破患者42例为研究组，并选择同期正常40例孕妇为对照组，采用酶联免疫吸附法和高敏感的放射免疫法检测血清的MMP-9和血清及羊水中的TNF-α水平，同时行产后胎膜病理检查。结果 胎膜早破孕妇羊水中TNF-α和血清中MMP-9水平明显高于对照组，差异显著（P〈0．05）；但血清中的TNF-α与对照组比较差异无显著性（P〉0．05）。血清中MMP-9水平在重度和轻中度绒毛膜羊膜炎组差异显著（P〈0.05）；而羊水中TNF-α差异无显著性（P〉0．05）。结论 测定母体血清的MMP-9含量和羊水中TNF-α水平，可作为胎膜早破患者是否患有绒毛羊膜炎的辅助诊断指标，尤其是血清的MMP-9含量的高低，还可以判定绒毛膜羊膜炎的严重程度。     

蛋白酶体抑制剂MG132诱导卵巢癌SKOV3细胞凋亡和自噬的作用机制

目的:观察MG132对卵巢癌SKOV3细胞株生长的影响,以及自噬、凋亡相关因子的表达,初步探讨MG132抑制卵巢癌细胞生长的作用机制。方法:以0.5,1.5,2.5,3.5μg/mL浓度MG132作用于SKOV3细胞,采用噻唑蓝(MTT)比色法检测细胞生长情况;流式细胞术检测细胞凋亡率;免疫组织化学(IHC)、蛋白质印迹(Western blotting)、逆转录聚合酶链反应(RT-PCR)检测自噬Beclin1因子、凋亡Caspase-3因子的表达。结果:MG132作用SKOV3细胞后细胞生长受到抑制;MG132的作用随细胞浓度和时间逐渐增加,细胞凋亡率逐渐增加;IHC检测发现Beclin1、Caspase-3阳性表达;Western blotting检测发现Beclin1、Caspase-3、Bim、Bax蛋白表达增加,与MG132浓度的增加呈正相关,Bcl-2蛋白表达减少,与MG132浓度的增加呈负相关;MG132组Caspase-3和Beclin1的mRNA表达量均高于对照组(P0.05)。结论:蛋白酶体抑制剂MG132对卵巢癌SKOV3细胞生长有抑制作用,呈浓度和时间依赖性,其抑制作用与凋亡和自噬有关。     

大鼠睾丸局部热作用对生精细胞Bcl-2及Bax蛋白表达的影响

目的:探讨睾丸局部热作用对大鼠生精细胞凋亡调节蛋白Bcl-2及Bax的影响。方法:雄性Sprague-Dawley大鼠40只,随机分热处理组和对照组,热处理组按热处理后0.5d、1d、3d和6d随机分成4个亚组(n=6),每个亚组相应设立对照组(n=4)。应用免疫组织化学方法检测Bcl-2和Bax在生精细胞的表达。结果:与对照组相比较,热作用后各组Bcl-2阳性细胞表达率及染色阳性细胞Bcl-2含量均显著下降(P<0.01),热作用后各组染色阳性细胞Bax含量均显著增高(P<0.01),除6d组Bax阳性细胞表达率显著下降外(P<0.01),其它各组Bax阳性细胞表达率无差别(P>0.05)。热作用后Bax阳性信号重新分布,从对照组分布在胞浆变成热作用后分布在核周及核。结论:热作用诱导睾丸生精细胞Bcl-2表达下降,Bax表达增高且重新分布。Bcl-2及Bax基因与热诱导生精细胞凋亡发生密切相关。     

miR-26a在妊娠期高血压疾病患者中的表达及其对滋养层细胞侵袭和凋亡的影响

目的:探讨miR-26a在妊娠期高血压疾病患者中的表达及其对滋养层细胞侵袭和凋亡的影响及其机制。方法:qRT-PCR法检测miR-26a表达,miRNA转染JAR细胞系构建control、miR-26a NC、miR-26、miR-26a inhibitor NC和miR-26a inhibitor组细胞。CCK-8法检测细胞活力,Brdu检测细胞增殖,Matrigel侵袭实验检测细胞侵袭,TUNEL检测细胞凋亡,Western blot法检测Bcl-2、Bax、Cleaved-caspase 9、E-cadherin、Vimentin表达。结果:妊娠期高血压疾病孕妇血浆中miR-26a表达量明显高于正常孕妇,差异有统计学意义(P0.05)。过表达miR-26a抑制JAR细胞活力、增殖与侵袭能力(P0.05);抑制miR-26a表达则增加JAR细胞活力、增殖与侵袭能力(P0.05)。过表达miRNA-26后,抗凋亡蛋白Bcl-2表达量下降(P0.05),凋亡蛋白Cleaved Caspase-9和Bax表达量升高(P0.05);抑制miRNA-26表达后,抗凋亡蛋白Bcl-2表达量增加(P0.05),凋亡蛋白Cleaved Caspase-9和Bax表达减少(P0.05)。结论:miR-26a在妊娠期高血压疾病患者中高表达,过表达miR-26a能抑制滋养层细胞细胞增殖和侵袭,促进凋亡,其可能机制是通过抑制EMT,调节Bcl-2/Caspase-9介导的。     

Interleukin-6 and interleukin-1 receptor antagonist in amniotic fluid and cord blood in patients with pre-term, premature rupture of the membranes.

OBJECTIVE: We investigated the role of interleukin (IL)-6 and IL-1 receptor antagonist (IL-1ra) in pre-term premature rupture of the membranes (PROM). METHOD: Amniotic fluid samples were collected from 10 patients with pre-term PROM (group 1a), 13 patients undergoing genetic amniocentesis (group 2), seven patients with normal vaginal delivery (group 3a), and 11 patients with elective cesarean section (group 4a). Umbilical venous blood was collected from nine cases of pre-term PROM (group 1b), 19 cases of normal delivery (group 3b) and nine cases of elective cesarean section (group 4b). RESULTS: The concentration of IL-6 in the amniotic fluid in group 1a was significantly higher than in group 2 (P<0.001), and that in group 3a was significantly higher than in group 4a (P<0.001). The concentration of IL-1ra in amniotic fluid in group 1a was significantly higher than in group 2 (P<0.001). The concentrations of IL-6 in umbilical venous blood were not significant among the groups. The concentration of IL-1ra in umbilical venous blood in group 1b was significantly higher than in group 3b (P<0.05) and group 4b (P<0.05). CONCLUSIONS: High concentrations of amniotic fluid IL-6 present in pre-term PROM, are indicative of intrauterine inflammation, probably due to sub-clinical infection. The high concentrations of IL-1ra in amniotic fluid and umbilical venous blood suggest the possibility that the presence of this anti-inflammatory cytokine serves to prevent the development of overt inflammation.     

基质金属蛋白酶-9及其组织抑制物-1诊断胎膜早破并绒毛膜羊膜炎的临床价值

目的:探讨母血、羊水、脐血中基质金属蛋白酶-9(MMP-9)及其组织抑制物-1(TIMP-1)的水平变化与胎膜早破发生的相关性,分析它作为一种新型的生物学标志物对绒毛膜羊膜炎和新生儿预后判断的临床价值。方法:采用双抗体夹心酶联免疫吸附法检测58例胎膜早破及38例正常孕妇(对照组)母血、羊水、脐血中MMP-9/TIMP-1水平的动态变化,同时进行胎膜的病理检查。结果:胎膜早破组母血、羊水、脐血中MMP-9的含量均高于对照组,尤以羊水中MMP-9水平升高明显,与对照组差异有显著性(P<0.05,P<0.01);而TIMP-1水平变化则明显低于对照组,差异有显著性(P<0.05,P<0.01)。母血、羊水、脐血中MMP-9的含量随破膜时间的延长增高,破膜时间超过24h增高尤为明显。而TIMP-1则随破膜时间的延长而下降,破膜时间超过24h降低尤为明显。绒毛膜羊膜炎患者母血、羊水、脐血中MMP-9水平明显高于非绒毛膜羊膜炎患者,差异有显著性(P<0.05,P<0.01),而TIMP-1水平则明显低于非绒毛膜羊膜炎患者,差异具有显著性(P<0.05,P<0.01)。胎膜早破组产妇所生新生儿Apgar评分≤7分者的母血、羊水、脐血中MMP-9的含量显著高于Apgar≥8分的新生儿(P<0.05,P<0.01),胎膜早破组新生儿Apgar评分≤7分者的母血、羊水、脐血中TIMP-1含量则显著低于Apgar≥8分的新生儿(P<0.05,P<0.01)。结论:MMP-9的异常升高及其抑制物TIMP-1显著下降是胎膜早破发生的重要发病机制。MMP-9/TIMP-1含量变化可以成为一种新的生物学标志用于早期诊断胎膜早破并绒毛膜羊膜炎特别是尚处于亚临床感染状态的孕妇,并有助于评估新生儿预后。     

Amniotic fluid endothelin levels and the incidence of premature rupture of membranes.

Objective. The purpose of this prospective study was to record Endothelin 1 (ET1) concentrations in the second trimester amniotic fluid and in women who develop premature rupture of membranes (PROM), preterm premature rupture of the membranes (PPROM) and in women with uneventful pregnancies. Method. Amniotic fluid was retrieved by amniocentesis from 125 women in the second trimester of pregnancy. The levels of Endothelin were measured by a sensitive and specific radioimmunoassay. Results. From the 125 women included in the study 20 developed PROM and preterm PROM (13 PPROM and 7 PROM). The ET1 concentration was significantly higher (P<0.001) in PROM and PPROM than in normal pregnancy (96.4 vs. 43 pg/ml). The sub-analysis of the two rupture of membranes groups found that the concentration of ET1 was higher in the PPROM than in PROM (118 vs. 72 pg/ml). Conclusion. The amniotic fluid concentration of ET1 is elevated by the second trimester in women who later develop preterm PROM or term PROM.     

孕期接触褪黑素对细菌脂多糖引起的小鼠炎性细胞因子释放的影响

目的:研究孕期接触褪黑素(MT)对细菌脂多糖(LPS)引起小鼠炎性细胞因子释放的影响。方法:孕17d鼠被随机分为对照组、LPS组和MT+LPS组,每组12只。LPS组、MT+LPS组孕鼠均给予LPS(500μg/kg,i.p.);MT+LPS组在LPS处理前0.5h给予MT(5mg/kg,i.p.);对照组孕鼠给予等容积生理盐水。于LPS或生理盐水处理1.5h后摘眼球取血,处死孕鼠,并留取羊水、胎肝、胎脑。用ELISA法分别测定母血、羊水、胎肝和胎脑中TNF-α、IL-1β、IL-6及IL-10含量。结果:LPS能显著升高母血、羊水和胎肝中TNF-α、IL-1β、IL-6及IL-10含量,MT+LPS组母血和胎肝中IL-10含量明显高于LPS组,MT预处理显著抑制LPS引起的母血TNF-α释放,但MT对LPS引起的母血和羊水中IL-1β、IL-6含量的变化无明显影响。此外,LPS显著升高胎脑中TNF-α和IL-10含量,而MT预处理却明显降低LPS引起的胎脑TNF-α释放。结论:孕期接触MT可多向调节LPS诱发的小鼠母血、羊水、胎肝和胎脑中促炎细胞因子与抗炎细胞因子释放。     

孕产妇血清中MMP-3、TNF-α、IL-10的表达与早产和早产胎膜早破的关系

目的探讨基质金属蛋白酶-3（MMP-3）、肿瘤坏死因子-α（TNF-α）、白细胞介素-10（lL-10）在孕产妇血清中的表达与早产、胎膜早破的关系。方法选择单胎头位初产妇80例作为研究对象,按孕周、胎膜是否破裂和产妇是否临产分为早产临产组（sPTD）、早产胎膜早破组（PPROM）、先兆早产组（TPL）和妊娠28～36＋6周无产兆组（对照组）,每组各20例。用ELISA法检测孕妇血清中MMP-3及TNF-α、lL-10的水平。结果①早产临产组、早产胎膜早破组、先兆早产组和对照组血清中MMP-3的浓度分别为（242.25±72.40）ng/ml、（225.95±85.43）ng/ml、（197.85±57.08）ng/ml、（186.80±54.33）ng/ml;TNF-α的浓度分别为（1332.35±346.65）pg/ml、（1365.00±211.80）pg/ml、（1188.15±269.43）pg/ml、（1061.85±210.02）pg/ml;IL-10的浓度分别为（563.65±116.50）pg/ml、（566.80±123.03）pg/ml、（521.00±105.14）pg/ml、（483.50±119.17）pg/ml;②早产组血清中MMP-3,TNF-α浓度高于对照组,以TNF-α升高更明显（P〈0.01）;而IL-10在前两组中有增高趋势,但与后两组相比差异无统计学意义（P〉0.05）;③血清中MMP-3、TNF-α、IL-10浓度呈两两正相关。结论①孕产妇血清中MMP-3及TNF-α浓度与早产、胎膜早破密切相关;②孕产妇血清中MMP-3、TNF-α及IL-10在临产、胎膜早破中可能起协同作用。     

Amniotic fluid concentrations of adrenomedullin in preterm labor.

OBJECTIVE: To determine whether adrenomedullin levels in amniotic fluid were associated with preterm labor. METHODS: We measured immunoreactive adrenomedullin in amniotic fluid collected by amniocentesis from 36 women with clinical diagnosis of preterm labor or preterm premature rupture of membranes (PROM) and from 18 normal pregnant women. RESULTS: Amniotic fluid from cases of PROM and failure to respond to tocolysis were associated significantly with higher amniotic fluid adrenomedullin concentrations (177.0 +/- 22.5 pg/mL and 182.7 +/- 22.0 pg/mL, respectively, P < .01) than that from uncomplicated pregnancies (101.2 +/- 28.1 pg/mL) or preterm labor responsive to tocolysis (102.3 +/- 26.8 pg/mL). CONCLUSION: Amniotic fluid adrenomedullin is higher than normal in cases of PROM and preterm labor unresponsive to tocolysis, perhaps indicating enhanced synthesis from placenta or fetal membranes being stimulated by bacterial products.     

Fetal plasma MMP-9 concentrations are elevated in preterm premature rupture of the membranes

OBJECTIVE: The objective of this study was to determine whether the concentrations of matrix metalloproteinase-9 (MMP-9) in the fetal (fetal plasma and amniotic fluid) and maternal compartments (plasma) are different in patients presenting with preterm premature rupture of membranes (PROM) than in those with preterm labor and intact membranes. STUDY DESIGN: Fetal plasma MMP-9, interleukin-1beta (IL-1beta), IL-6, soluble tumor necrosis factor receptors 1 (sTNF-R1) and 2 (sTNF-R2) were measured in fetuses with preterm labor and intact membranes (n = 96) and preterm PROM (n = 43). The concentrations of analytes were determined with sensitive and specific immunoassays. A P value <.05 was considered significant. RESULTS: (1) The median fetal plasma MMP-9 concentration was significantly higher in fetuses with preterm PROM than in those with preterm labor (P =.035). (2) In contrast, fetal plasma IL-1beta, sTNF-R1, and sTNF-R2 were significantly higher in patients with preterm labor than in those with preterm PROM (IL-1beta, P =.01; sTNF-R1, P =.003; and sTNF-R2, P =.02). (3) The median amniotic fluid concentration of MMP-9 was higher in patients with preterm PROM than in those with preterm labor (P <.001). CONCLUSION: Fetuses with preterm PROM have increased concentrations of an enzyme (MMP-9) implicated in the mechanism of membrane rupture but lower concentrations of IL-1beta, sTNF-R1, and sTNF-R2 than fetuses with preterm labor and intact membranes. A role for the fetus in the genesis of preterm PROM deserves consideration.     

Amniotic fluid concentrations of fibronectin and intra-amniotic infection

A cohort study with four groups of pregnant women was designed to evaluate if a decrease in the concentration of fibronectin (FN) in amniotic fluid plays a role in the genesis of premature rupture of the membranes (PROM) and/or intra-amniotic infection: group 1, 15 patients with PROM and intra-amniotic infection; group 2, 15 patients with PROM but without intra-amniotic infection; group 3, 15 patients with preterm labor without PROM or intra-amniotic infection; and group 4, 15 patients who underwent elective amniocentesis for fetal lung maturity studies. No significant differences in amniotic fluid concentrations of FN among any of the four groups was observed (F = 0.146; P = 0.931). These results suggest that a deficiency of FN in amniotic fluid is not a critical factor in determining which patients may be affected by PROM or which of them will develop an intra-amniotic infection.