The characteristics of integrins expression in decidualized human endometrial stromal cell induced by 8-Br-cAMP in in vitro

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in the endometrium during the menstrual cycle and in early pregnancy. Specificity of integrins is known to be different in human endometrial stromal cells and decidual cells. These shifts of integrins suggested to play an important role in embryo implantation and can be modulated by progesterone, cAMP derivatives, and cytokines. The mechanisms of decidualization and its precise physiological role are still not clearly understood and in vitro systems could provide an alternative that overcomes limitations of studying such complex biological phenomena in vivo at the time of implantation. This study was undertaken to establish an in vitro model system for human decidualization using 8-bromo-cAMP and to investigate the characteristics of stromal integrin expression in vitro by 8-Br-cAMP. Endometrial stromal cells were isolated and cultured, and then were induced to decidualize by 0.5 mM 8-Br-cAMP for 15 days. Immunofluorescence staining and flow cytometric analyses of the integrin subunits (alpha1, alpha4, alpha5, alpha6, beta1 and alphavbeta3) were performed at day 9. In the presence of 8-Br-cAMP, the staining intensity of alphavbeta3 was significantly higher than control and measurements for alpha1, alpha4, alpha5, alpha6, and beta1 were similar. Immunofluorescent localization of the integrins reflected the differences obtained from the flow cytometric analyses described above. In summary, the expression of alphavbeta3 integrin increased in stromal cells in vitro decidualized by 8-Br-cAMP and this up-regulation of alphavbeta3 integrin expression during decidualization might influence on human implantation.     

Expression of 3beta-hydroxysteroid dehydrogenase and P450 side chain cleavage enzyme in the human uterine endometrium

The enzyme complex 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3beta-HSD) is involved in the biosynthesis of all classes of active steroids. The expression of 3b-HSD in human uterine endometrium during the menstrual cycle and decidua was examined in an effort to understand its role during ova implantation. 3beta-HSD was weakly expressed in the glandular epithelium of the proliferative phase and moderately expressed in the glandular epithelium of secretory phase of the endometrium. In the decidua of the ectopic pregnancy, 3beta-HSD was strongly expressed. The human uterine endometrial 3beta-HSD was identified as being the same type as the placental 3beta-HSD by RT-PCR and sequence analysis. In addition to the expression of 3beta-HSD, P450scc was expressed in the decidua of the ectopic pregnancy. These results suggest that pregnenolone might be synthesized from cholesterol by P450scc de novo and then, it is converted to progesterone by 3beta-HSD in the uterine endometrium. The data implies that the endometrial 3beta-HSD can use not only the out-coming pregnenolone from the adrenal gland but also the self- made pregnenolone to produce progesterone. The de novo synthesis of progesterone in the endometrium might be a crucial factor for implantation and maintenance of pregnancy.     

Proteomic characterization of early-stage differentiation of mouse embryonic stem cells into neural cells induced by all-trans retinoic acid in vitro

Embryonic stem (ES) cells are totipotent stem cells, which can differentiate into various kinds of cell types, including neurons. They are widely used as a model system for investigating mechanisms of differentiation events during early mouse development. In this study, proteomic techniques were used to approach the protein profile associated with the early-stage differentiation of ES cells into neuronal cells induced by all-trans retinoic acid (ATRA) in vitro. In comparison of the protein profile of parent ES cells with that of ES-derived neural-committed cells, which was induced by ATRA for four days, 24 differentially displayed protein spots were selected from two-dimensional electrophoresis (2-DE) gels for further protein identification by pepide mass fingerprinting (PMF). Nine proteins were known to being involved in the process of neural differentiation and/or neural survival. Of those, alpha-3/alpha-7 tubulin and vimentin were down-regulated, while cytokeratin 8, cytokeratin 18, G1/S-special cyclin D2, follistatin-related protein, NEL protein, platelet-activating factor acetylhydrolase IB alpha-subunit, and thioredoxin peroxidase 2 were upregulated during differentiation of ES cells to neural cells. Additionally, other 12 protein (five upregulated and seven downregulated) spots associated with ES cell differentiation into neuronal cells were not matched to known proteins so far, implicating that they might be novel proteins. The results above indicated that the molecular mechanisms of differentiation of ES cells to neural cells in vitro might be similar to those of other neural systems in vitro and identified that proteomic analysis is an effective strategy to comprehensively unravel the regulatory network of differentiation.     

Tobacco-specific Carcinogen 4- （Methylnitrosoamino） -1- （3-pyridyl） - 1-butanone（NNK） Activating ERK1/2 MAP Kinases and Stimulating Proliferation of Human Mammary Epithelial Cells

Cigarette smoking is correlated with the development of various cancers. 4- （Methylnitresoamino） -1- （3-pyridyl） - 1-butanone（NNK） is one of the major tobacco-specific carcinogens in the cigarette smoke, which increases the risk of breast cancer. In the present study, it was demonstrated that NNK rapidly activated ERK1 and ERK2 MAP kinases in human normal mammary epithelial cells. It was found that there are two different routes for the activation of ERK1/2 with NNK. One is from nicotinic receptor nAchR to MEK1/2, and the other is from tyrosine kinase containing receptor to MEK1/2. The tobacco-specific carcinogen NNK shows a strong proliferative effect on normal human mammary epithelial cells and cancer mammary epithelial cells.     

Tobacco-specific Carcinogen 4-(Methylnitrosoamino)-1-(3-pyridyl )-1-butanone(NNK) Activating ERK1/2 MAP Kinases and Stimulating Proliferation of Hmnan Mammary Epithelial Cells

Cigarette smoking is correlated with the development of various cancers. 4 - (Methylnitrosoamino) -1 - ( 3 -pyridyl) -1-butanone (NNK) is one of the major tobacco-specific carcinogens in the cigarette smoke, which increases the risk of breast cancer. In the present study, it was demonstrated that NNK rapidly activated ERK1 and ERK2 MAP kinases in human normal mammary epithelial cells. It was found that there are two different routes for the activation of ERK1/2with NNK. One is from nicotinic receptor nAchR to MEK1/2, and the other is from tyrosine kinase containing receptor to MEK1/2. The tobacco-specific carcinogen NNK shows a strong proliferative effect on normal human mammary epithelial cells and cancer mammary epithelial cells.     

Tyrosine nitration site specificity identified by LC/MS in nitrite-modified collagen type IV

Non-enzymatic nitrite induced collagen cross-linking results in changes reminiscent of age-related damage and parallels the well-known model system, non-enzymatic glycation. We have recently observed that nitrite modification of basement membrane proteins can induce deleterious effects on overlying retinal pigment epithelial cells in studies relevant to age-related macular degeneration. The present work was undertaken in order to confirm 3-nitro-tyrosine (3-NT) as a product of the reaction and to identify the site specificity of nitration in collagen IV, a major component of basement membranes. Human collagen type IV was modified via incubation with 200 mM NaNO(2) (pH=7.38) for one week at 37(o)C. The modified protein was prepared in 2 different ways, including acid hydrolysis and trypsin digestion for site specificity determination. The samples were analyzed by LC/MS using a C(12) RP column. Site specificity was determined from tandem MS/MS data utilizing TurboSEQUEST software and the Swiss-Prot sequence database. 3-NT was detected in protein digests and acid hydrolysates of nitrite modified collagen IV. Positive identification with standard 3-NT was confirmed by identical R(t), lambda(max)=279 nm and 355 nm, and m/z=227. Analyses of tryptic digests identified four sites of tyrosine nitration, alpha1(IV)Y348, alpha1(IV)Y534, alpha2(IV)Y327, and alpha2(IV)Y1081. These sites are located in the triple-helical region of the protein and provide clues regarding potential sites for nitrite modification in collagen type IV.     

The impact of fine particulate matter (PM) on various beneficial functions of human endometrial stem cells through its key regulator SERPINB2

Fine particulate matter (PM) has a small diameter but a large surface area; thus, it may have broad toxic effects that subsequently damage many tissues of the human body. Interestingly, many studies have suggested that the recent decline in female fertility could be associated with increased PM exposure. However, the precise mechanisms underlying the negative effects of PM exposure on female fertility are still a matter of debate. A previous study demonstrated that resident stem cell deficiency limits the cyclic regenerative capacity of the endometrium and subsequently increases the pregnancy failure rate. Therefore, we hypothesized that PM exposure induces endometrial tissue damage and subsequently reduces the pregnancy rate by inhibiting various beneficial functions of local endometrial stem cells. Consistent with our hypothesis, we showed for the first time that PM exposure significantly inhibits various beneficial functions of endometrial stem cells, such as their self-renewal, transdifferentiation, and migratory capacities, in vitro and in vivo through the PM target gene SERPINB2, which has recently been shown to be involved in multiple stem cell functions. In addition, the PM-induced inhibitory effects on the beneficial functions of endometrial stem cells were significantly diminished by SERPINB2 depletion. Our findings may facilitate the development of promising therapeutic strategies for improving reproductive outcomes in infertile women.Subject terms: Stem-cell research, Adult stem cells     

Tissue-Specific stem cell differentiation in an in vitro airway model

The respiratory tract is the primary site of exposure to airborne compounds, with the bronchial epithelium providing one of the first lines of defence. A growing need exists for an accurate in vitro model of the bronchial epithelium. Here, normal human bronchial epithelial (NHBE) cells cultured at an air/liquid interface create a fully differentiated, in-vivo-like model of the human bronchial epithelium. Developmental characterisation includes (i) trans-epithelial electrical resistance, (ii) morphology and (iii) bronchial cell specific stains/markers. It is concluded that the basal/progenitor cells create a pseudo-stratified, mucociliary NHBE model containing basal, serous, Clara, goblet and ciliated cells, reflective of the normal human bronchial epithelium (days 24-33 ALI culture).     

近红外光谱技术结合主成分分析法用于子宫内膜癌的诊断

应用近红外光谱技术结合化学计量学方法研究了子宫内膜癌组织近红外光谱特征提取和早期诊断的可行性. 测定了154 例子宫内膜组织切片的近红外光谱, 选取适宜的波段和光谱预处理方法进行主成分分析, 很好地区分了癌变、增生和正常子宫内膜组织切片, 并且分辨出处于不同分化期的组织切片, 为子宫内膜癌的早期诊断提供了可靠依据. 该法快速、简便, 有望发展成为一种新型的肿瘤无创诊断方法.     

Bionic antibacterial modification of IOL through SI-RAFT polymerization of P(TOEAC-co-MPC) brushes to prevent PCO and endophthalmitis

Various complications such as posterior capsule opacification (PCO) and endophthalmitis occur after intraocular lenses (IOLs) implantation in cataract surgery. It is urgent to construct antifouling and antibacterial IOLs to lower the incidence of PCO and endophthalmitis. Bionic zwitterionic polymer such as 2-methacryloyloxyethyl phosphorylcholine (MPC) shows excellent performance in resisting nonspecific proteins and bacterial adhesion. In this work, a novel bionic brushes coating containing MPC and N, N, N-trimethyl-2-((4-(2-(4-nonylphenoxy) ethoxy)-4-oxobut-2-enoyl) oxy) ethan-1-aminium chloride (TOEAC), a quaternary ammonium monomer, was prepared onto IOLs using reversible addition–fragmentation chain transfer (RAFT) polymerization method. The P(TOEAC-co-MPC) brushes exhibited excellent antifouling efficiency against bovine serum albumin, Staphylococcus aureus, and human lens epithelial cells. In addition, the P(TOEAC-co-MPC) brushes showed excellent antibacterial and antibiofilm abilities and good biocompatibility. An in vivo study confirmed that the P(TOEAC-co-MPC) brushes effectively prevented PCO and endophthalmitis. Consequently, the P(TOEAC-co-MPC) bionic brushes are promising for IOLs surface modification to resist postoperative complications for long-term implantation.     

Photophysical Studies of A2-E, Putative Precursor of Lipofuscin, in Human Retinal Pigment Epithelial Cells

With age, human retinal pigment epithelial cells accumulate lipofuscin that can absorb photons in the visible range leading to light-induced damage and impaired vision. A putative precursor of lipofuscin, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E- octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1 - cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2-E), has recently been isolated and characterized from aged human retinal pigment epithelial cells. We have found that A2-E inhibits the growth of human retinal pigment epithelial cells at concentrations greater than 1 microM. Time-resolved fluorescence measurements of 1 microM A2-E in solution, performed under 413 nm excitation, showed that fluorescence wave forms integrated across the spectrum (450-600 nm) were best-fitted with three decay times in the nanosecond and subnanosecond time scale: 6.6, 1.9 and 0.33 ns. Untreated retinal pigment epithelial cells were characterized by three fluorescence lifetimes: 6.3, 1.7 and 0.35 ns. In retinal pigment epithelial cells treated with 1 microM A2-E, the fluorescence decay was significantly faster, with the marked presence (approximately equal to 30%) of a fourth short lifetime (0.12 ns). These fluorescence decay times for A2-E bound to human retinal pigment epithelial cells are similar to those of lipofuscin granules isolated from aged human retinal pigment epithelial cells. This similarity supports the hypothesis that A2-E is a precursor of lipofuscin and suggests that A2-E may play a role in the overall light damage associated with age-related retinal diseases.     

Kiwifruit Actinidin: A Proper New Collagenase for Isolation of Cells from Different Tissues

Actinidin is a cysteine protease abundant in Kiwifruit. This enzyme is known as a meat-tenderizing protease. In this project, actinidin was purified from kiwifruit by salt precipitation and ion exchange chromatography. Collagenolytic effect of the purified enzyme was tested in four different buffer systems. Thereafter, the enzyme was used for isolation and culture of cells from three different tissues: endothelial cells from human umbilical vein, hepatocytes from rat liver, and thymic epithelial cells from rat thymus. Our results revealed that actinidin can hydrolyze collagen types I and II at neutral and alkaline buffers. Furthermore, actinidin compared with type II or IV collagenase isolated intact human umbilical vein endothelial cells, hepatocytes, and thymic epithelial cells with viability more than 90%. These results address a novel and valuable collagenase, which can be used efficiently for hydrolysis of collagen and isolation of different cell populations from various solid tissues.     

含苯并噻唑砌块的2,4,6-三取代嘧啶衍生物的合成及抗肿瘤活性评价(英文)

为了寻找高效的抗肿瘤药物,设计并合成了一系列含苯并噻唑砌块的2,4,6-三取代嘧啶衍生物.采用噻唑蓝(MTT)法对目标化合物在人类四种癌细胞[EC-109(人食管癌细胞)、MGC-803(人胃癌细胞)、PC-3(人前列腺癌细胞)、Hep G-2(人肝癌细胞)]、GES-1(人正常胃黏膜上皮细胞)和HEEC(人正常食管细胞)中进行抗肿瘤活性评价,结果显示部分化合物对MGC-803和PC-3细胞表现出中度至强效的抗肿瘤活性.其中2-(((4-(4-(吡啶-2-基)哌嗪-1-基)-6-(三氟甲基)嘧啶-2-基)硫基)甲基)苯并[d]噻唑(13h)和2-(((4-(4-(嘧啶-2-基)哌嗪-1-基)-6-(三-氟甲基)嘧啶-2-基)硫代)甲基)苯并[d]噻唑(13i)对PC-3表现出比较好的抗肿瘤活性, IC50值分别3.82和2.29μmol/L,且化合物13h和13i对GES-1的细胞增值毒性明显小于阳性对照5-氟尿嘧啶.     

Inhibition of corneal neovascularization by rapamycin

The purpose of this study was to determine whether rapamycin could inhibit corneal angiogenesis induced by basic fibroblast growth factor (bFGF). Using human dermal microvascular endothelial cells (HDMECs), we examined the effect of rapamycin on cell proliferation and migration, and the expression of vascular endothelial growth factor (VEGF). The rabbit's eye was implanted intrastromally into the superior cornea with pellet containing bFGF for the control group and pellet containing bFGF and rapamycin for the rapamycin group. Biomicrographically, corneal angiogenesis was evaluated for 10 days after pellet implantation. The neovascularized cornea also was examined histologically. bFGF induced corneal neovascularization was significantly reduced by treatment with rapamycin. Using in vitro model, rapamycin strongly inhibited bFGF induced proliferation, migration, and VEGF secretion of HDMECs. We could observe that the bFGF induced corneal angiogenesis was inhibited by rapamycin in a micropocket rabbit model. The score of neovascularization was significantly decreased in the rapamycin group than in the control group at 10 days after pellet implantation. Histologically, the cornea of rapamycin group also showed much less new vessels than that of control group. Collectively, rapamycin appears to inhibit bFGF induced angiogenesis in a rabbit corneal micropocket assay and may have therapeutic potential as an antiangiogenic agent.     

Scanning electron microscopy of the endometrium of mares infused with gentamicin.

Scanning electron microscopy (SEM) was used to study the endometrium of nine 1-year-old thoroughbred mares after twice intrauterine infusions of gentamicin, on 2 consecutive days. Five mares were infused on 2 consecutive days with 40 ml gentamicin (50 mg/ml) mixed with 80 ml of normal saline. Four mares served as controls and were infused with 120 ml of saline on 2 consecutive days. Endometrial biopsies were obtained from all mares 3 days after the second intrauterine infusion. Each biopsy was processed for SEM by standard methods. The endometrial epithelium of the gentamicin-infused mares had more cellular perforations than the saline-infused mares. The gentamicin-infused mares had less and shorter microvilli. The ciliated cells were fewer and some ciliated cells had disrupted and some had drooping cilia. The endometrial epithelium of the gentamicin-infused mares had a considerable number of endometrial cells that lost their luminal surfaces and some that lost their microvilli, compared to the saline-infused mares. We suggest that the information gathered in this pilot study should be used as basis for further investigation, on a larger scale basis, of the effects of repeated intrauterine infusion of gentamicin on the endometrial mucosa of mares.     

A Practical Approach for the In Vitro Safety and Efficacy Assessment of an Anti-Ageing Cosmetic Cream Enriched with Functional Compounds

(1) Background: Cosmeceuticals are topical products applied to human skin to prevent skin ageing and maintain a healthy skin appearance. Their effectiveness is closely linked to the compounds present in a final formulation. In this article, we propose a panel of in vitro tests to support the efficacy assessment of an anti-ageing cream enriched with functional compounds. (2) Methods: biocompatibility and the irritant effect were evaluated on reconstructed human epidermis (RHE) and corneal epithelium (HCE) 3D models. After a preliminary MTT assay, normal human dermal fibroblasts (NHDF) and keratinocytes (HaCaT) were used to evaluate the extracellular matrix (ECM) protein synthesis, and interleukin-6 (IL-6) and metalloproteinase-1 (MMP-1) production. (3) Results: data collected showed good biocompatibility and demonstrated the absence of the irritant effect in both 3D models. Therefore, we demonstrated a statistical increase in collagen and elastin productions in NHDF cells. In HaCaT cells, we highlighted an anti-inflammatory effect through a reduction in IL-6 levels in inflammatory stimulated conditions. Moreover, the reduction of MMP-1 production after UV-B radiation was demonstrated, showing significant photo-protection. (4) Conclusion: a multiple in vitro assays approach is proposed for the valid and practical assessment of the anti-ageing protection, anti-inflammatory and biocompatible claims that can be assigned to a cosmetic product containing functional compounds.     

Steroid oxidoreductase activity of alcohol dehydrogenases from horse, rat, and human liver.

Alcohol dehydrogenase from horse (isoenzyme SS and ES, but not EE), rat and human liver were found to catalyze the NAD-dependent oxidation of 3beta-hydroxy groups in 5alpha- and 5beta-steroids of the C19, C21, and C24 series. The enzymes from horse and rat liver were more active on 5beta-than on 5alpha-steroids. This difference was most marked with the enzyme from rat liver, especially with 3beta-hydroxyandrostan-17-ones and 3beta-hydroxypregnan-20-ones as substrates. The Km of isoenzyme ES from horse liver was lower for 3beta-hydroxy-5alpha-cholanoic acid (0.4 muM) than for 3beta-hydroxy-5beta-cholanoic acid (0.9 muM). 3alpha-Hydroxysteroids were not substrates for the enzymes from horse and rat liver. Human liver alcohol dehydrogenase had low affinity for 3beta-hydroxy-5alpha (and 5beta)-cholanoic acids, but oxidation could be clearly demonstrated by gas chromatographic analysis of the products.     

Oxygen consumption rate of cells in 3D culture: the use of experiment and simulation to measure kinetic parameters and optimise culture conditions

Understanding the basal O(2) and nutrient requirements of cells is paramount when culturing cells in 3D tissue models. Any scaffold design will need to take such parameters into consideration, especially as the addition of cells introduces gradients of consumption of such molecules from the surface to the core of scaffolds. We have cultured two cell types in 3D native collagen type I scaffolds, and measured the O(2) tension at specific locations within the scaffold. By changing the density of cells, we have established O(2) consumption gradients within these scaffolds and using mathematical modeling have derived rates of consumption for O(2). For human dermal fibroblasts the average rate constant was 1.19 × 10(-17) mol cell(-1) s(-1), and for human bone marrow derived stromal cells the average rate constant was 7.91 × 10(-18) mol cell(-1) s(-1). These values are lower than previously published rates for similar cells cultured in 2D, but the values established in this current study are more representative of rates of consumption measured in vivo. These values will dictate 3D culture parameters, including maximum cell-seeding density and maximum size of the constructs, for long-term viability of tissue models.     

Anti-Fibrosis Effects of Magnesium Lithospermate B in Experimental Pulmonary Fibrosis: By Inhibiting TGF-βRI/Smad Signaling

Pulmonary fibrosis is a severe and irreversible interstitial pulmonary disease with high mortality and few treatments. Magnesium lithospermate B (MLB) is a hydrosoluble component of Salvia miltiorrhiza and has been reported to have antifibrotic effects in other forms of tissue fibrosis. In this research, we studied the effects of MLB on pulmonary fibrosis and the underlying mechanisms. Our results indicated that MLB treatment (50 mg/kg) for seven days could attenuate bleomycin (BLM)-induced pulmonary fibrosis by reducing the alveolar structure disruption and collagen deposition in the C57 mouse model. MLB was also found to inhibit transforming growth factor-beta (TGF-β)-stimulated myofibroblastic transdifferentiation of human lung fibroblast cell line (MRC-5) cells and collagen production by human type II alveolar epithelial cell line (A549) cells, mainly by decreasing the expression of TGF-β receptor I (TGF-βRI) and regulating the TGF-β/Smad pathway. Further studies confirmed that the molecular mechanisms of MLB in BLM-induced pulmonary fibrosis mice were similar to those observed in vitro. In summary, our results demonstrated that MLB could alleviate experimental pulmonary fibrosis both in vivo and in vitro, suggesting that MLB has great potential for pulmonary fibrosis treatment.     

Reconstruction of Rabbit Corneal Layer Composed of Corneal Fibroblasts and Corneal Epithelium on the Lyophilized Amniotic Membrane

Introduction Thehumancornea,animportantopticalcompo nentthatfocusesthelightontheiris,isatransparent avasculartissuethatalsoplaysaprotectiveroleforthe eye.Therefore,anyinjury,diseaseorcelldamagethat canleadtoopacificationofthecornea,canimpairthe vision,and…