VEGF抗体及孕酮对无排卵性功能失调性子宫出血患者子宫内膜间质细胞MMP-9表达的影响

目的探讨孕激素、细胞因子和基质金属蛋白酶-9在更年期无排卵性子宫出血发病机制中的作用及相互关系。方法采集对照组和功血组患者子宫内膜各11例和16例，原代培养子宫内膜间质细胞。在对照组和功血组内设置空白对照组、孕酮组和VEGF抗体组，孕酮组分别加入孕酮10^-7moL／L（生理浓度），10^-5mol／L．（高浓度），各孵育24h。VEGF抗体组加入VEGF抗体10μl，分别孵育24h，48h，ELISA法检测MMP-9含量。结果功血组患者子宫内膜间质细胞MMP-9含量明显高于对照组，加入孕酮作用后，各组MMP-9含量均下降，功血组下降更明显，高浓度孕酮有更强的抑制作用（P〈0．05）。VEGF抗体作用后各组MMP-9含量均下降，以功血组明显（P〈0．05）。不同作用时间时MMP-9含量无显著影响，（P〉0．05）。结论功血患者子宫内膜间质细胞分泌较MMP-9较对照组高。VEGF抗体和孕酮均能下调MMP-9水平，提示VEGF和MMPs均参与了功能失调性子宫出血的病理过程。     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinase in uterine endometrial carcinoma and a correlation between expression of matrix metalloproteinase-7 and prognosis

Matrix metalloproteinases (MMPs) are associated with invasion and metastasis of several human malignant tumors, in particular MMP-7, which is mainly produced by the cancer cell itself. We examined the expression of MMP-2, 7 and 9, and tissue inhibitors of metalloproteinase (TIMP)-1 and 2 in uterine endometrial carcinoma, and compared the expression with clinicopathological characteristics in uterine endometrial carcinoma (UEC). A group of 256 patients with UEC received surgery at the Osaka City University Medical School Hospital, and 196 tumor samples were immunohistochemically stained to examine the expression of MMP-2, 7 and 9, and TIMP-1 and 2. Additionally, the invasion ability of cell stain established from UEC was examined using an in vitro invasion assay. The expression of MMP-2, 7 and 9, and TIMP-1 and 2 was observed in the cytoplasm, and the expression of MMP-2 and 7, and TIMP-1 and 2 was observed in stromal cells around the tumor cells. The expression of MMP-7 was significantly stronger in higher-grade than lower-grade tumors (P<0.05). The invasion assay showed that the invasion of cells derived from UECs was significantly inhibited by TIMP-1 and 2. The disease-free interval was significantly shorter when MMP-7 expression was intense. This increased expression of MMP-7 in high grade UECs may be associated with tumor invasion and metastasis, and MMP-7 could serve as a prognostic maker in UEC.     

Doxycycline alters the expression of inflammatory and immune-related cytokines and chemokines in human endometrial cells: implication in irregular uterine bleeding

BACKGROUND: Increased production of pro-inflammatory mediators is considered central in the manifestation of events leading to irregular uterine bleeding in progestin-only contraceptive users. Evidence suggests that in addition to its antimicrobial property, doxycycline (Dox) acts as an anti-inflammatory agent mainly through the suppression of pro-inflammatory mediators. METHODS: We tested this hypothesis in the endometrial environment using an in vitro model consisting of isolated human endometrial glandular epithelial and stromal cells and a human endometrial surface (HES) epithelial cell line cultured under defined conditions. RESULTS: We found that Dox at doses ranging from 1 to 100 microg/ml had a limited growth-inhibitory effect on these cells, whereas Dox in a dose-dependent manner inhibited the production of tumour necrosis factor-alpha (TNF-alpha). Using multiplex cytokine/chemokine protein analysis to test a broader range of Dox activity, we found that Dox at 25 microg/ml either alone or in the presence of 17beta-estradiol (E2), medroxyprogesterone acetate (MPA) and E2+MPA (10(-8) M) as well as TNF-alpha (25 ng/ml), representing the endometrial environment exposed to contraceptives as well as inflammatory conditions, respectively, altered the production of multiple cytokines and chemokines as compared with untreated controls. These actions of Dox occurred in cell-, ovarian steroid- and cytokine/chemokine-dependent manners. Although Dox reduced the regulatory action of steroids on the production of these cytokines/chemokines, it was less effective on TNF-alpha-treated cells. CONCLUSIONS: The results support the hypothesis that Dox, by modulating the endometrial expression of multiple inflammatory-related cytokines/chemokines in a cell- and cytokine/chemokine-dependent manner, may have a therapeutic potential in patients experiencing irregular uterine bleeding, in particular in progestin-dominant contraceptive users.     

Superficial endometrial vascular fragility in Norplant users and in women with ovulatory dysfunctional uterine bleeding

The aim of this study was to develop an objective test for superficial endometrial vascular fragility at hysteroscopy, and to apply this test to women using Norplant((R)) for contraception and to women with ovulatory dysfunctional uterine bleeding (DUB). A prospective observational study was carried out in 34 Norplant users and 20 women with menorrhagia due to ovulatory DUB. Superficial endometrial vascular fragility was assessed at hysteroscopy by observing the source and extent of frank and subepithelial bleeding during controlled collapse and redistension of the uterine cavity. Superficial endometrial blood vessels were more fragile in Norplant users compared to women with DUB (chi(2) = 11.60, P = 0.02). Superficial endometrial petechiae (chi(2) = 37.9, P < 0.0001) and ecchymoses (chi(2) = 42.2, P = 0.0001) were more frequently observed in the Norplant users than in the menorrhagia group. In Norplant users, superficial endometrial vascular fragility was increased in those with frequent bleeding or spotting during the past 30 days (chi(2) = 6.15, P = 0.01), and in those who were examined during a bleeding episode (chi(2) = 5.3, P = 0.02). Fragility was increased in the menorrhagia group during the perimenstrual period (days 24 to 05; chi(2) = 12.83, P = 0.01). There was no obvious relationship between subepithelial bleeding and circulating concentrations of oestradiol and progesterone.     

EBP50抑制HeLa细胞增殖及基质金属蛋白酶表达

与膜-细胞骨架连接蛋白(ezrin-radixin-moesin,ERM)家族结合的磷酸化蛋白50(ERM-binding phosphoprotein-50,EBP50)与肿瘤细胞迁移和增殖有密切关系,但各个研究者对EBPSO在肿瘤中所起的具体作用尚存争议[1-2].     

成骨细胞旁分泌影响软骨细胞中MMPs与TIMPs的表达

背景：细胞之间体外共培养能最大限度的模拟体内真实的微环境,细胞划痕实验及炎症因子白细胞介素1β刺激后基质金属蛋白酶及基质金属蛋白酶抑制剂之间的平衡可能破坏,从而导致关节软骨细胞外基质的降解,软骨细胞功能的失调,关节软骨的退变。目的：在成骨细胞上清液与软骨细胞体外共培养下,观察炎症因子白细胞介素1β对体外培养的软骨细胞的迁移、基质金属蛋白酶及组织金属蛋白酶抑制剂表达的影响。方法：实验分为软骨细胞单培养组﹑软骨细胞与成骨细胞上清液共培养组和软骨细胞与成骨细胞上清液共培养+白细胞介素1β组,划痕实验观察3组24 h软骨细胞的迁移变化;半定量PCR实验分析以上3组24 h软骨细胞中基质金属蛋白酶1,2,3,9及组织金属蛋白酶抑制剂1,2,3,4的变化情况。结果与结论：与单培养组比较,共培养组和共培养+白细胞介素1β组细胞迁移率显著增加(P < 0.01);与单培养组比较,共培养组中基质金属蛋白酶1,2,3,9基因表达明显增高(P < 0.05),共培养+白细胞介素1β组基质金属蛋白酶1,3,9基因表达明显增高(P < 0.01);与单培养组比较,共培养组和共培养+白细胞介素1β组中组织金属蛋白酶抑制剂1基因表达明显升高(P < 0.01),组织金属蛋白酶抑制剂3,4基因表达明显下降(P < 0.05)。提示成骨细胞上清液与软骨细胞共培养促进软骨细胞的迁移,增强软骨细胞中基质金属蛋白酶1,2,3,9的基因表达且调节组织金属蛋白酶抑制剂家族的基因表达。白细胞介素1β抑制共培养的软骨细胞迁移及组织金属蛋白酶抑制剂家族的基因表达。        中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

The role of matrix metalloproteinases and leukocytes in abnormal uterine bleeding associated with progestin-only contraceptives

Progestin-only contraceptives are associated with menstrual bleeding disturbances; a major reason why these agents are discontinued. The pathogenesis of abnormal uterine bleeding associated with progestin-only contraceptives remains ill-defined. Matrix metalloproteinases (MMPs) and leukocytes are postulated to be involved in the process of normal menstruation. Immunolocalization of MMPs and leukocytes in (Norplant), and injectable depot medroxyprogesendometrium from women using the progestinterone acetate (DMPA), are widely used, safe and only contraceptives, Norplant or depot medroxyprogesterone acetate (DMPA) compared with normal controls, revealed foci of positive MMP-1 and -3 immunostaining in stromal cells and adjacent extracellular matrix, the presence of MMP-9 in various subtypes of leukocytes and alterations in mast cell phenotype. In women using progestin-only contraceptives, extent of endometrial MMP, neutrophil and eosinophil immunolocalization and the mast cell activation state was similar to or greater than that observed in perimenstrual control women. However, differences in MMP immunostaining were observed in endometrial samples from women using different progestin-only contraceptive agents; in particular, significantly higher MMP-1 immunostaining was observed associated with the use of Norplant compared with DMPA. No correlation was observed with the number of bleeding days recorded. These results suggest that MMP and leukocytes may be involved in endometrial breakdown in women using progestin-only contraceptives.     

Changes in endometrial blood vessels in the endometrium of women with hormone replacement therapy-related irregular bleeding

BACKGROUND: Irregular bleeding affects up to 60% of hormone replacement therapy (HRT) users. The mechanism of this bleeding is not understood. Reduced endometrial microvascular integrity appears to underlie breakthrough bleeding in pre-menopausal women and the aim of this study was to establish whether similar changes are seen in HRT users and hence to elucidate a possible mechanism of irregular bleeding. METHODS: Endometrium from 34 HRT users with amenorrhoea, irregular bleeding or regular bleeding was assessed for endometrial endothelial cell density (anti-CD34), number of blood vessels per mm(2), vascular basal lamina components (laminin, collagen IV and heparan sulphate proteoglycan) and in 32 subjects and 23 controls for perivascular smooth muscle alpha (SMA). Findings were compared with a control population of 29 post-menopausal women not using HRT, other sex steroids or tamoxifen and with no vaginal bleeding. Staining intensity was assessed in a blinded fashion in all immunohistochemical studies. RESULTS: Four significant differences in endometrial blood vessels were observed between HRT users and controls: (i) a significantly lower density of endometrial endothelial cells (EC staining for CD34) per mm(2) was present in HRT users compared with controls (P < 0.001); (ii) endothelial cells (EC) were predominantly organized within blood vessels (83%) in controls but in HRT users EC were dispersed in the tissues with only 29% in organized vessels (P <0.001); (iii) supportive perivascular cell SMA was significantly reduced in 23 post-menopausal HRT users compared with 23 post-menopausal controls (n = 29, P = 0.013) and (iv) an atrophic or inactive histological pattern of endometrium was more frequently seen in the controls (P < 0.001). CONCLUSIONS: These findings support the hypothesis that exposure to HRT profoundly alters endometrial blood vessels, reducing structural integrity thereby predisposing to irregular bleeding in HRT users.     

Genetic variations in matrix metalloproteinases may be associated with increased risk of ulcerative colitis

Increased production of matrix metalloproteinases (MMPs) plays an important role in tissue damage in inflammatory bowel disease (IBD). Genetically encoded variation between individuals in MMP production may therefore contribute to disease onset, type, or severity. We undertook an extensive candidate gene single nucleotide polymorphism (SNP) study of MMP-1, -2, -3, -7, -8, -9, -10, -12, -13, and -14 and tissue inhibitor of metalloproteinases (TIMPs)-1, -3, and -4 in ulcerative colitis (UC). We identified tagging SNPs across these genes, and genotyped these SNPs in a Caucasian New Zealand dataset consisting of 419 UC patients and 907 controls. SNPs in a number of MMP genes were associated with UC. After correcting for multiple testing SNPs in MMP-3, MMP-8, MMP-10, and MMP-14 remained significant in their associations with UC. In a second study, using samples from a Dutch cohort, most of the significant findings in the New Zealand cohort were not replicated. However, data from an international meta-analysis provide some support for the initial findings. In conclusion, this study provides preliminary evidence to suggest that genetic variation in the MMPs may play a role in interindividual differences in UC susceptibility and clinical outcome. Further studies are needed in other cohorts to determine the robustness of these observations in different populations.     

Unstable pre-mutation may explain mosaic disease expression of incontinentia pigmenti in males

Mosiac skin lesions following the lines of Blaschko are found in boys affected by incontinentia pigmenti (IP). For an X-linked gene defect, this is rather surprising. To explain the mosaic disease expression of IP in males, we propose that the disease is caused by an unstable pre-mutation, which normally remains silent in males during early embryogenesis. Occasionally “silencing” is incomplete and gives rise to clinical manifest IP reflecting a mosaic state of alleles with the full and the pre-mutation in the same patient. This model can account for mother-to-son transmission of IP and for disparate phenotypes in monozygotic female twins. © 1994 Wiley-Liss, Inc.     

Bcl-2、Bax在功能失调性子宫出血子宫内膜中的表达

目的 研究凋亡调节蛋白Bcl-2、Bax在功能失调性子宫出血(功血)的子宫内膜中的表达.方法 采用免疫组织化学(免疫组化)方法,利用链霉素抗生物素蛋白--过氧化物酶法(SP)检测40例功血患者子宫内膜及40例子宫肌瘤标本的子宫内膜组织中Bcl-2、Bax基因的表达情况.结果 (1)Bel-2基因在正常月经周期子宫内膜组织的表达呈明显周期性变化,差异具有统计学意义(P<0.05).(2)Bcl-2基因随子宫内膜增生程度增加而表达逐渐增强,差异具有统计学意义(P<0.05).(3)Bax在正常月经周期子宫内膜组织的表达呈阳性.(4)Box随子宫内膜增生程度增加而表达逐渐减弱,差异具有统计学意义(P<0.05).结论 Bcl-2的过度表达和Bax的低表达可抑制子宫内膜细胞凋亡,使子宫内膜增生甚或不典型增生.与功能失调性子宫出血有密切关系.     

Multiple sclerosis: elevated expression of matrix metalloproteinases in blood monocytes

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the central nervous system (CNS) characterized by blood-brain barrier (BBB) breakdown. Disruptions of BBB continuity result in an influx of activated T cells and monocytes, and could contribute to lesion formation in the CNS. Matrix metalloproteinases (MMP) are enzymes implicated in BBB disruption, and in degradation of extracellular matrix proteins and myelin components. An imbalance in levels of MMP and tissue inhibitors of MMP (TIMP) has been implicated in the pathogenesis of MS. Since monocytes form a major cell population in acute MS lesions and may facilitate their entrance into the CNS by secretion of MMP, knowledge on MMP expression by blood monocytes could be useful to improve our understanding of the pathogenesis of MS. In the present study, we examined the expression of MMP-1, -3, -7, -9, -14 and TIMP-1 mRNA by blood monocytes in patients with MS using in situ hybridization. Levels of MMP-1, -3, -7, -9 and of TIMP-1 mRNA expressing monocytes were elevated in MS compared to controls, while those of MMP-14 did not differ. We therefore conclude that MS is associated with elevated levels of MMP and TIMP expressing blood monocytes that may contribute to MS pathogenesis.     

脓毒症大鼠心脏基质金属蛋白酶及其抑制物基因的表达变化

目的：探讨脓毒症大鼠心脏中基质金属蛋白酶（MMP）及其组织抑制物（TIMP）的基因表达谱变化及其与脓毒症导致的心脏损伤的关系。方法：雄性3月龄Wistar大鼠20只，随机均分为2组，分别以盲肠结扎针刺法建立脓毒症动物模型或行假手术作为对照组。术后24 h快速摘取心脏，以Langendorff 离体鼠心灌注法测量大鼠心功能参数，其后做心脏病理检查，并以寡核苷酸基因芯片检测基质金属蛋白酶及其抑制物基因在2组大鼠心脏组织中的表达差异。结果：脓毒症大鼠心脏无明显病理改变，但心功能明显下降，表明大鼠心脏处于抑制状态；基因芯片结果显示：20个MMP基因中14个表达上调3倍以上，包括胶原酶MMP8、明胶酶（MMP2和MMP9）、基质溶解素（MMP3、MMP7及MMP10）和膜型金属蛋白酶（MMP15、MMP17和MMP24）等；4个TIMP基因中仅TIMP3基因表达上调。结论：脓毒症时心脏组织中多种MMP基因表达上调、MMP/TIMP基因表达失衡，可能导致心肌抑制和ECM重构，是脓毒症诱发心脏损伤的重要分子机制。     

The matrix metalloproteinases as pharmacological target in osteoarthritis: statins may be of therapeutic benefit

Osteoarthritis (OA) is a common joint disease; however, current pharmacologic agents for OA are only symptomatic and they can not prevent the disease progression. Matrix metalloproteinases (MMPs) produced by chondrocytes play an important role in the development of cartilage destruction in OA, and agents that can target against MMPs activity may be of therapeutical value. There were reports that statins can inhibit the secretion of MMPs in vitro and in vivo, which were believed to account for the plaque stabilizing effects of statins in the treatment of atherosclerosis. We based our hypothesis on that atherosclerosis possesses some aspects that are similar to that of osteoarthritis, such as inflammation and matrix degradation. Since statins have displayed great benefits in modifying the progression of atherosclerosis via anti-inflammatory and matrix-stabilizing mechanisms, it is conceivable that statins may also prevent the disease progression of osteoarthritis. Further work are needed to verify if statins can protect cartilage from destruction through inhibition of MMP secretion by chondrocytes, and their potential to be used as therapeutic agents in OA should be investigated.     

Calretinin and CD34 immunoreactivity of the endometrial stroma in normal endometrium and change of the immunoreactivity in dysfunctional uterine bleeding with evidence of 'disordered endometrial stroma'

AIMS: We investigated the pattern of reactivity of calretinin and CD34 in normal and pathological endometria. METHODS: Various endometrial tissues were submitted for calretinin and CD34 immunostaining. RESULTS: Calretinin reactivity was limited to the endometrial stromal cells (ESC) of the superficial zone of the functionalis layer (FL) in the proliferative phase, and was extensive in all stages of the secretory phase. The ESC of the post-menopausal, ectopic, hyperplastic or neoplastic endometria showed negative or focal weak reactivity for calretinin. In dysfunctional uterine bleeding (DUB) with a normal or an abnormal histopathological appearance on routine stain, there were varying degrees of focal to extensive decreases in calretinin reactivity. The foci of negative calretinin reactivity in the FL displayed varying reactivity for CD34 and appeared to be continuous with the basalis layer (BL). Endometrial polyps were often reactive for CD34, but not reactive for calretinin. CONCLUSIONS: Immunostaining for calretinin and CD34 is helpful in the diagnosis of endometrial polyp and hyperplasia. In DUB, with or without abnormal histopathological findings, there were alterations of the zonal pattern of calretinin reactivity in the FL. This alteration appears to be an expansion of the stroma of the BL into the FL, resulting in a 'disordered endometrial stroma'.