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。     

维甲酸对人子宫颈癌细胞系HeLa细胞间隙连接蛋白转导途径调控作用的研究

目的：探讨维甲酸对人子宫颈癌细胞系HeLa细胞间隙连接蛋白（Cx）43信号转导途径的调控作用。方法：应用特异性钙指示剂（Fluo-3 AM)在激光扫描共聚焦显微镜下动态观察维甲酸作用后的细胞质内信号转导分子游离钙的分布及强度变化。采用流式细胞仪、结合蛋白印迹技术分析，检测外源信号分子对Cx43的表达以及蛋白酪氨酸磷酸化状态的影响。结果：HeLa细胞内游离钙经维甲酸作用后明显超载，细胞内游离钙浓度（[Ca^2 ]i)由静息状态下的35.73μmol/L上升至58.16μmol/L。流式细胞仪分析，Cx43阳性细胞表达率由1.9%上升至26.3%。蛋白印迹技术分析，HeLa细胞出现Cx43酪氨酸磷酸化。结论：维甲酸对HeLa细胞Cx43信号转导途径的调控是在细胞质内游离钙的参与下，使Cx43阳性细胞表达率上升，并在酪氨酸位点出现明显的磷酸化作用下实现的。     

HeLa细胞中抑癌基因-间隙连接基因Cx43及其蛋白产物表达缺失

目的：探讨新一代肿瘤抑制基因-间隙连接基因Cx43及其蛋白产物在人子宫颈癌细胞系HeLa中的表达及意义。方法：应用核酸分子原位杂交和流式细胞仪分析技术,研究人子宫颈癌细胞系HeLa及阳性对照细胞人正常肝细胞系QZG中,间隙连接基因Cx43mRNA及其蛋白产物的表达规律。结果：原位杂交显示,Cx43mRNA在HeLa细胞中呈阴性,在QZG细胞中呈强阳性。流式细胞仪分析示Cx43蛋白在HeLa、QZG细胞中阳性细胞计数率分别为1．9％和99．0％,差异有高度显著性（P＜0．01）。结论：CX43基因及其蛋白产物在HeLa中表达缺失可能与子宫颈癌发生恶性表型相关。     

间隙连接蛋白43的磷酸化调节在卵巢上皮性癌化疗耐药中的作用

目的 通过检测卵巢上皮性癌(卵巢癌)间隙连接蛋白43(Cx43)、非磷酸化Cx43及蛋白激酶C(PKC)的表达,探讨Cx43的磷酸化调节在卵巢癌化疗耐药中的作用.方法 采用免疫组化法检测29例化疗敏感(化疗敏感组)和28例化疗耐药(化疗耐药组)卵巢癌患者癌组织中以及PKC抑制剂--星孢菌素处理后的卵巢癌顺铂耐药细胞株SKOV3/DDP细胞中Cx43、非磷酸化Cx43及PKC蛋白的表达,并采用三磷酸腺苷-生物荧光肿瘤药敏实验(ATP-TCA)检测SKOV3/DDP细胞对化疗药物的敏感性.结果 (1)免疫组化法检测显示,化疗耐药组Cx43和非磷酸化Cx43蛋白的阳性表达率(分别为54%和14%)明显低于化疗敏感组(分别为83%和59%,P<0.05);化疗耐药组PKC蛋白的阳性表达率明显高于化疗敏感组(分别为64%和31%,P<0.05).卵巢癌组织中,PKC蛋白的阳性表达率与Cx43、非磷酸化Cx43蛋白的阳性表达率呈明显负相关关系(r=-0.626和-0.714,P<0.05).(2)免疫组化法检测显示,星孢菌素处理后SKOV3/DDP细胞中PKC蛋白的表达强度减弱,Cx43及非磷酸化Cx43蛋白的表达强度增强,随着星孢菌素处理时间的延长,Cx43蛋白的表达强度进一步增强.(3)ATP-TCA检测显示,体外培养的SKOV3/DDP细胞对紫杉醇和顺铂均耐药;紫杉醇和顺铂分别联合星孢菌素处理后细胞敏感度增加,其中联合低浓度(1×10-8 mol/L)星孢菌素者为中度敏感.联合高浓度(1×10-7 moL/L)星孢菌素者为高度敏感.结论 PKC通过对Cx43的磷酸化作用导致Cx43蛋白的表达下降,降低卵巢癌对化疗药物的敏感性,这种效应可以被星孢菌素逆转.     

Ⅲ型胶原及CTGF在胎膜早破发病机制中的作用

目的探讨Ⅲ型胶原、结缔组织生长因子（CTGF）在人胎膜组织中的表达及在胎膜早破发病机制中的作用。方法38例胎膜早破孕妇为胎膜早破组，其中未足月胎膜早破组（pPROM组）18例，足月胎膜早破组（tPROM组）20例；与胎膜早破组孕周相对应的非胎膜早破孕妇作为对照组，早产对照组18例，足月对照组20例。应用免疫组化及图像分析法检测胎膜Ⅲ型胶原、CTGF表达水平，以阳性区平均灰度值为检测依据。将各组孕妇胎膜Ⅲ型胶原、CTGF测定结果进行直线相关分析。结果①四组胎膜中均存在不同程度的Ⅲ型胶原、CTGF的表达；②pPROM组Ⅲ型胶原（88．81±5．25）、CTGF水平（85．45±6．91）均低于早产对照组（95．99±8．41，90．30±5．74），差异有统计学意义（P〈0．01，P〈0．05）；tPROM组Ⅲ型胶原（94．53±6．43）、CTGF（88．15±4．93）均低于足月对照组（100．80±9．77，93．20±5．33），（P〈0．05）；pPROM组Ⅲ型胶原（88．81±5．25）表达低于tPROM组（94．53±6．43），两者比较差异有统计学意义（P〈0．01）；pPROM组CT—GF灰度值（85．45±6．91）表达低于tPROM组（88．15±4．93）．但无统计学差异（P〉0．05）；③Ⅲ型胶原、CTGF水平在pPROM组中的表达呈正相关性，r=0．830（P〈0．01），而tPROM组中两者则无相关性。结论pPROM组与tPROM组中存在Ⅲ型胶原与CTGF的低表达。tPROM的发生可能与胎膜的退行性变有关，而pPROM的发生与胎膜本身结构病变密切相关。     

卵巢癌自杀基因治疗的旁观者效应及其与间隙连接蛋白43表达的关系

目的 探讨单纯疱疹病毒－胸苷激酶／更昔洛韦（HSV－TK／GCV）在卵巢癌治疗中的旁观者效应及其与间隙连接蛋白（Cx)43表达的关系,同时观察全反式维甲酸（RA）对两者关系的影响。方法 四甲基偶氮唑蓝（MTT）法比较经HSV－TK／GCV治疗及RA作用前后卵巢癌细胞株OVCAR3、CAOV3细胞旁观者效应的强弱;采用流式细胞仪,蛋白质免疫印迹法（Western Blot)、间接免疫荧光染色检测两卵巢癌细胞株RA作用前后Cx43的表达,Cx43的表达以平均荧光强度表示。结果 （1）HSV－TK／GCV对OVCAR3细胞产生较明显的旁观者效应,而对CAOV3细胞旁观者效应弱,两者比较,差异有显著性（P＜0．05）。（2）RA对OVCAR3细胞的旁观者效应有增强作用（P＜0．05）,但不影响CAOV3细胞的旁观者效应（P＞0．05）。（3）流式细胞仪检测提示,OVCAR3细胞中Cx43的表达为4．45,而CAOV3细胞中Cx43的表达为0．89,两者比较,差异有显著性（P＜0．05）;Western Blot、间接免疫荧光染色检测结果也表明,OVCAR3细胞有Cx43的表达,且定位于细胞膜,而CAOV3细胞中无Cx43的表达。（4）RA作用后,Western Blot、间接免疫荧光染色检测均显示,两卵巢癌细胞株的Cx43表达增加;流式细胞仪检测提示,细胞中Cx43表达均明显增加,OVCAR3细胞由4．45增至9．83,CAOV3细胞由0．89增至3．15（P均＜0．05）,Cx43仍定位于OVCAR3的细胞膜和CAOV3的细胞浆。结论 卵巢癌HSV－TK／GCV治疗的旁观者效应与其Cx43表达以及定位有关,增强细胞膜Cx43表达,将增强卵巢癌HSV－TK／GCV的旁观者效应,提高其疗效。     

免疫性卵巢早衰小鼠卵巢组织中Cx43、Bcl-2表达的研究

目的:检测免疫性卵巢早衰(POF)小鼠卵巢组织中缝隙链接蛋白43(Cx43)和B细胞淋巴瘤-2(Bcl-2)的m RNA及蛋白水平,以探讨其在免疫性POF发病中的作用。方法:建立免疫性POF小鼠模型并选择同期佐剂对照组及正常对照组,分别于2周、4周、6周观察各组小鼠双侧卵巢组织学变化。ELISA法测定各组小鼠血清FSH水平。免疫组化法检测各组小鼠卵巢组织中Cx43和Bcl-2蛋白相对表达量,PCR法检测Cx43和Bcl-2 m RNA水平。结果:POF组4周及6周Cx43蛋白、Bcl-2蛋白相对表达量显著低于正常对照组和佐剂对照组(P0.05);POF组4周及6周Cx43蛋白、Bcl-2蛋白相对表达量较POF组2周降低(P0.05)。POF组4周及6周Cx43 m RNA、Bcl-2 m RNA表达水平显著低于正常对照组佐剂对照组(P0.05);POF组4周及6周Cx43 m RNA、Bcl-2 m RNA表达水平较POF组2周降低(P0.05)。结论:在免疫性POF小鼠卵巢组织中Cx43和Bcl-2蛋白及m RNA表达均下调,可能与在免疫性POF的发生发展有关。     

细胞外基质与生殖

细胞外基质(ECM)主要包括纤维粘连蛋白(FN),层粘连蛋白(LN),胶原纤维(CL).三者在生殖过程中均起着重要作用.胶原又分为Ⅰ～Ⅴ型胶原,其中Ⅳ型胶原与生殖关系最密切.LN主要分布于基质与基底膜,FN主要于基质及间质细胞,而Ⅳ型胶原主要于基底膜.ECM主要功能为促进细胞间粘附与增殖,从而促进卵泡发育,孕卵种植及维持妊娠.ECM异常可导致流产、早产、胎膜早破、妊高征等病理妊娠.另外,ECM尚与妇科肿瘤关系密切.本文主要阐述了ECM与生殖的关系.     

子宫平滑肌激活蛋白-1及间隙连接蛋白43在妊娠期的表达与分娩发动和早产的关系

目的　探讨激活蛋白-1(Activator protein-1,AP-1)、间隙连接蛋白43(connexin43,Cx43)在足月产和早产子宫平滑肌中的表达情况及其相关性. 方法　应用免疫组化法检测15例足月未临产、15例足月临产、10例早产临产产妇的子宫平滑肌中AP-1的两个亚单位c-Jun、c-Fos蛋白及Cx43的表达情况. 结果 (1)Cx43免疫组化积分在早产临产组(4.204±0.42)及足月临产组(4.33±0.51)表达水平显著高于足月未临产组(3.15±0.41)(P<0.01).(2)c-Jun蛋白在早产临产组、足月临产组、足月未临产组标记指数分别为(52.34±4.18)%、(45.25±5.24)%、(34.14±4.26)%,三组两两比较差异均有统计学意义(P<0.01).(3)c-Fos蛋白在早产临产组、足月临产组、足月未临产组标记指数分别为(53.48±4.36)%、(43.32±6.21)%、(31.29±3.34)%,三组两两比较差异均有统计学意义(P<0.01).(4)妊娠子宫平滑肌中Cx43表达与c-Jun、c-Fos蛋白表达呈正相关关系,(r=0.65、0.63,P<0.01). 结论 Cx43在分娩发动中发挥着重要的作用.妊娠子宫平滑肌中Cx43的表达水平与AP-1表达有一定的相关性.     

间隙连接在妇产科领域的研究进展

细胞间隙连接（ｇａｐｊｕｎｃｔｉｏｎ,ＧＪ）是相邻细胞间进行物质和信息交换的跨膜蛋白通道结构,对细胞增殖分化调控和内环境稳定起重要作用。间隙连接蛋白的表达直接影响子宫平滑肌瘤发生,分娩及卵泡发育等过程。间隙连接蛋白转录抑制,功能缺陷,与括宫颈癌在内的多种肿瘤的发生,发展及转移密切相关,其蛋白表达及功能的恢复答铁恶性表型逆转。ｃｘ基因可能是新近发现的一类非突变型抑癌基因。     

Preterm premature rupture of the membranes and antioxidants: the free radical connection

AIM: To discuss the role of oxidant stress in preterm, premature rupture of the membranes (PPROM). RESULTS: There is evidence to suggest that preterm, premature rupture of the membranes occurs secondary to focal collagen damage in the fetal membranes. CONCLUSION: Oxidant stress caused by increased ROS formation and/or antioxidant depletion may disrupt collagen and cause premature membrane rupture. We propose that supplementation with vitamins C and E may synergistically protect the fetal membranes, and decrease the risks of PPROM.     

Is the collagen content reduced when the fetal membranes rupture? A clinical study of term and prematurely ruptured membranes

In 15 patients experiencing premature rupture of the fetal membranes (PROM) and in 15 control subjects having delivered spontaneously at term, the collagen content of the membranes was determined by the hydroxyproline method. From each patient two membrane specimens were obtained, one from the rupture margin and another from the membranes in close relation to the placental margin. No significant difference in the collagen content was demonstrated between the two groups of patients. Moreover, no significant difference was observed comparing the collagen content within the paired membrane specimens of each patient in each group. Neither was there any obvious change in the membrane collagen content in relation to clinical signs of chorioamnionitis or microbiological findings. It is concluded that changes in the collagen content of the fetal membranes bear no significance as to the etiology of PROM, neither is such a change involved in the mechanism of membrane rupture at term.     

Collagen types in normal and prematurely ruptured amniotic membranes

Collagen content in preterm amnions with premature rupture of the membranes was significantly lower than that of preterm amnions without premature rupture of the membranes. Collagen types were studied through sodium dodecyl sulfate-polyacrylamide gel electrophoresis in human amnions from pregnant women with or without premature rupture of the membranes. Collagen types I, III, and V were recognized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in all samples. In samples taken from preterm patients with premature rupture of the membranes, the ratios of III/I, III/V, and III/total collagen were significantly lower than those from ones without premature rupture of the membranes. The ratios of I/V, I/total collagen, and V/total collagen showed no change in gestations with and without premature rupture of the membranes, respectively. In term samples there was no significant difference in the ratios of all collagen types between those with and those without premature rupture of the membranes. Elastins were not demonstrable in amnion with and without premature rupture of the membranes. These studies suggest that the reduction of type III collagen content in amnion is related to the cause of premature rupture of the membranes, particularly in preterm samples.     

Does an intracervical infection influence the fibrinolytic activity and the collagen content of the fetal membranes? A study of ascending infections in pregnant ewes

Apart from solely mechanical explanations, premature rupture of the membranes (PROM) has been suggested to be caused by an ascending infection. In order to investigate the role of infection in the mechanism of PROM, pregnant ewes were experimentally inoculated endocervically with either Bacteroides fragilis, Streptococcus intermedius or group B streptococci. These microorganisms were previously reported to be implicated in PROM in humans. The present investigation concerns the possible effect of an experimentally induced ascending infection on the collagen content and fibrinolytic activity (FA) of the fetal membranes. No relationship was observed between an ascending infection during pregnancy and the collagen content content of the fetal membrane specimens. It was concluded that changes in the collagen content bear no etiological significance in the mechanism of premature membrane rupture irrespective of an ascending infection's being present or not. Concerning FA in only one case, experiencing a Strept. intermedius amnionitis, was an elevated FA value observed. This finding indicates that the involvement of FA in the process of membrane rupture following ascending infection during pregnancy cannot be ruled out.     

Vitamins C and E: missing links in preventing preterm premature rupture of membranes?

We propose that generation of reactive oxygen species may be a potentially reversible pathophysiologic pathway leading to preterm premature rupture of the membranes. Reactive oxygen species generated by the body's response to diverse insults such as infection, cigarette smoking, bleeding, or cocaine use can activate collagenolytic enzymes and impair fetal membrane integrity. Vitamin E, a lipid-soluble antioxidant, inhibits membrane-damaging effects of reactive oxygen species-induced lipid peroxidation. Vitamin C, a water-soluble antioxidant in plasma, stimulates and protects collagen synthesis while recycling vitamin E. Prior evidence shows that (1) damage by reactive oxygen species can impair fetal membrane integrity, (2) reduced midgestation levels of vitamin C are associated with preterm premature rupture of membranes, and (3) these vitamins can be safely and effectively absorbed and delivered to gestational tissues. Current prenatal vitamin preparations contain vitamins C and E in concentrations that are less than 1/3 and 1/10, respectively; these levels have been suggested for effective antioxidant protection. We hypothesize that increased dietary consumption or supplementation of vitamins C and E during pregnancy may reduce physiologically the risks of that portion of preterm premature rupture of membranes that is mediated by excessive or undamped peroxidation of fetal membranes. This hypothesis, if confirmed, should stimulate initiation of therapeutic trials to test the efficacy of enhanced supplementation with vitamins C and E during pregnancy to prevent preterm premature rupture of membranes.     

Second harmonic generation microscopy of fetal membranes under deformation: Normal and altered morphology

Introduction

Insight into the microstructure of fetal membrane and its response to deformation is important for understanding causes of preterm premature rupture of the membrane. However, the microstructure of fetal membranes under deformation has not been visualized yet. Second harmonic generation microscopy, combined with an in-situ stretching device, can provide this valuable information.

Methods

Eight fetal membranes were marked over the cervix with methylene blue during elective caesarean section. One sample per membrane of reflected tissue, between the placenta and the cervical region, was cyclically stretched with a custom built inflation device. Samples were mounted on an in-situ stretching device and imaged with a multiphoton microscope at different deformation levels. Microstructural parameters such as thickness and collagen orientation were determined. Image entropy was evaluated for the spongy layer.

Results

The spongy layer consistently shows an altered collagen structure in the cervical and cycled tissue compared with the reflected membrane, corresponding to a significantly higher image entropy. An increased thickness of collagenous layers was found in cervical and stretched samples in comparison to the reflected tissue. Significant collagen fibre alignment was found to occur already at moderate deformation in all samples.

Conclusions

For the first time, second harmonic generation microscopy has been used to visualize the microstructure of fetal membranes. Repeated mechanical loading was shown to affect the integrity of the amnion–chorion interface which might indicate an increased risk of premature rupture of fetal membrane. Moreover, mechanical loading might contribute to morphological alterations of the fetal membrane over the cervical region.     

Tensile strain increased COX-2 expression and PGE2 release leading to weakening of the human amniotic membrane

IntroductionThere is evidence that premature rupture of the fetal membrane at term/preterm is a result of stretch and tissue weakening due to enhanced prostaglandin E₂ (PGE₂) production. However, the effect of tensile strain on inflammatory mediators and the stretch sensitive protein connexin-43 (Cx43) has not been examined. We determined whether the inflammatory environment influenced tissue composition and response of the tissue to tensile strain.MethodsHuman amniotic membranes isolated from the cervix (CAM) or placenta regions (PAM) were examined by second harmonic generation to identify collagen orientation and subjected to tensile testing to failure. In separate experiments, specimens were subjected to cyclic tensile strain (2%, 1 Hz) for 24 h. Specimens were examined for Cx43 by immunofluorescence confocal microscopy and expression of COX-2 and Cx43 by RT-qPCR. PGE₂, collagen, elastin and glycosaminoglycan (GAG) levels were analysed by biochemical assay.ResultsValues for tensile strength were significantly higher in PAM than CAM with mechanical parameters dependent on collagen orientation. Gene expression for Cx43 and COX-2 was enhanced by tensile strain leading to increased PGE₂ release and GAG levels in PAM and CAM when compared to unstrained controls. In contrast, collagen and elastin content was reduced by tensile strain in PAM and CAM.DiscussionFibre orientation has a significant effect on amniotic strength. Tensile strain increased Cx43/COX-2 expression and PGE₂ release resulting in tissue softening mediated by enhanced GAG levels and a reduction in collagen/elastin content.ConclusionA combination of inflammatory and mechanical factors may disrupt amniotic membrane biomechanics and matrix composition.     

Fetal membrane healing after spontaneous and iatrogenic membrane rupture: a review of current evidence

In view of the important protective role of the fetal membranes, wound sealing, tissue regeneration, or wound healing could be life saving in cases of preterm premature rupture of the membranes. Although many investigators are studying the causes of preterm premature rupture of membranes, the emphasis has not been on the wound healing capacity of the fetal membranes. In this review, the relevant literature on the pathophysiologic condition that leads to preterm premature rupture of membranes will be summarized to emphasize a continuum of events between rupture and repair. We will present the current knowledge on fetal membrane wound healing and discuss the clinical implications of these findings. We will critically discuss recent experimental interventions in women to seal or heal the fetal membranes after preterm premature rupture of membranes.