Three-dimensional cultures of human endometrial cells on Matrigel mimic in vivo morphology

Background  The regulation of endometrial physiology and morphogenesis by the paracrine effectors has been well established using in vivo studies. A more complete understanding of the endometrial function has been delayed due, in part, to a lack of appropriate culture models. In this study, we aimed to simulate the in vivo three-dimensional (3-D) growth pattern of endometrial cells using a 3-D in vitro culture system.

Methods  Isolated endometrial epithelial cells, stromal cells and RL95-2 cells were seeded into culture chambers coated with the extracellular matrix Matrigel and observed using light microscopy. Fluorescence staining and immunohistochemistry were used to assess the morphology.

Results  Depending on the culture conditions, epithelial cells and RL95-2 cells formed multicellular structures on Matrigel; stromal cells remained individually distinguishable or grew together to form 3-D lattice-like structures.

Conclusions  Matrigel provided a good microenvironment for culturing endometrial cells. The cells cultured in the Matrigel-coated chambers closely resembled those seen in vivo.

     

Up-regulation of T-lymphoma and metastasis gene 1 in gastric cancer and its involvement in cell invasion and migration

Background  T-lymphoma and metastasis gene 1 (Tiam1) produces a guanine nucleotide exchange factor (GNEF) that regulates guanosine triphosphatase, which transforms guanosine diphosphate to guanosine triphosphate. Recently published data indicate that Tiam1 was associated with gastric cancer. The aim of this study was to investigate biological effects and potential mechanisms of Tiam1 in gastric carcinoma.

Methods  We analyzed the expression of Tiam1 in 114 pair-matched gastric neoplastic and adjacent non-neoplastic tissues by quantitative real-time PCR. We investigated Tiam1 expression and its prognostic value for gastric cancer. Furthermore, the functions of Tiam1 over-expression were analyzed with stable-expression Tiam1 plasmid in human gastric cancer cell lines.

Results  Tiam1 expression was significantly associated with cell differentiation and lymphatic metastasis; expression of Tiam1 mRNA was up-regulated in gastric cancer compared to pair-matched adjacent non-tumor tissues. Analyses of surgical tissue samples and 5-year survival of gastric cancer patients showed that those with strong Tiam1 expression had significantly shorter overall survival time than those with negative Tiam1 expression. Ectopic expression of Tiam1 promoted cell growth, migration and invasion of gastric cancer cells in vitro.

Conclusions  In gastric cancer cells, Tiam1 affects multiple properties associated with acquisition of the metastatic phenotype, and may be a marker of gastric cancer progression and metastasis in a subset of cancer.

     

Characterization of Human Primary Chondrocytes of Osteoarthritic Cartilage at Varying Severity

Background  There is a difficulty in evaluating the in vivo functionality of individual chondrocytes, and there is much heterogeneity among cartilage affected by osteoarthritis (OA). In this study, in vitro cultured chondrocytes harvested from varying stages of degeneration were studied as a projective model to further understand the pathogenesis of osteoarthritis.

Methods  Cartilage of varying degeneration of end-stage OA was harvested, while cell yield and matrix glycosaminoglycan (GAG) content were measured. Cell morphology, proliferation, and gene expression of collagen type I, II, and X, aggrecan, matrix metalloproteinase 13 (MMP-13), and ADAMTS5 of the acquired chondrocytes were measured during subsequent in vitro culture.

Results  Both the number of cells and the GAG content increased with increasing severity of OA. Cell spreading area increased and gradually showed spindle-like morphology during in vitro culture. Gene expression of collagen type II, collagen type X as well as GAG decreased with severity of cartilage degeneration, while expression of collagen type I increased. Expression of MMP-13 increased with severity of cartilage degeneration, while expression of ADAMTS-5 remained stable. Expression of collagen type II, X, GAG, and MMP-13 substantially decreased with in vitro culture. Expression of collagen type I increased with in vitro cultures, while expression of ADAMTS 5 remained stable.

Conclusions  Expression of functional genes such as collagen type II and GAG decreased during severe degeneration of OA cartilage and in vitro dedifferentiation. Gene expression of collagen I and MMP-13 increased with severity of cartilage degeneration.

     

Dicycloplatin differentially inhibits proliferation of human aortic smooth muscle and endothelial cells: potential for use in drug-eluting stents

Background   Dicycloplatin is a relatively safe third generation platinum-complex anti-cancer drug. The present study focused on the effects of dicycloplatin on in vitro proliferation and apoptosis of human aortic smooth muscle cells (HASMC) and human aortic endothelial cells (HAEC).

Methods  Proliferation of HASMC and HAEC, DNA content, and cellular levels of proliferation- and apoptosis-related proteins were assessed using the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) (MTS) assay, flow cytometry and Western blotting assays, respectively.

Results  Dicycloplatin at 10 ng/ml significantly inhibited HASMC proliferation, however, 10 μg/ml were required to significantly inhibit HAEC proliferation. Cell cycle analysis showed that dicycloplatin was a non-specific inhibitor of the cell cycle. Although dicycloplatin significantly decreased proliferating cell nuclear antigen (PCNA) expression in HASMC at all concentrations tested, it did not significantly affect PCNA expression in HAEC; Bax and p53 protein expression was upregulated in dicycloplatin groups. 

Conclusions  Dicycloplatin at nanogram concentrations significantly inhibits HASMC proliferation, although the effect is relatively weaker than that of sirolimus. In contrast, the effect of dicycloplatin on inhibition of HAEC proliferation is much less pronounced than that on HASMC. The latter characteristics point to the potential for use of dicycloplatin in drug-eluting stents.

     

血红素氧合酶－1对肝硬化大鼠肺脏结构和功能的影响

目的：探讨血红素氧合酶－1/一氧化碳系统在实验大鼠肝肺综合征中的作用。 方法：35只SD大鼠随机分为肝硬化组、锌原卟啉组、钴原卟啉组和假手术组。胆总管结扎制备大鼠肝硬化模型。锌原卟啉组、钴原卟啉组模型制备同肝硬化组,仅在标本采集前24小时分别腹腔给予锌原卟啉组、钴原卟啉组30mg/Kg。测量平均动脉压、门静脉压,血气分析;病理观察肝硬化和肺血管扩张形成;应用免疫组化方法观察HO-1蛋白在肺内的表达,RT-PCR法检测肺组织中HO-1 mRNA的表达。 结果：病理显示手术组4周肝硬化形成;肺内血管扩张,肺泡间隔增加伴有肺不张。与假手术组相比,肝硬化组平均动脉压显著下降,门静脉压显著升高（P）;肺泡动脉血氧梯度增加（P）;动脉碳氧血红蛋白、肺组织HO-1蛋白及HO-1 mRNA显著增加（P< 0.05）。与肝硬化组相比,锌原卟啉组肺泡动脉血氧梯度下降、动脉碳氧血红蛋白、肺组织HO-1蛋白及HO-1 mRNA显著减少（P< 0.05）,肺内血管扩张缩小;钴原卟啉组肺泡动脉血氧梯度增高、动脉碳氧血红蛋白、肺组织HO-1蛋白及HO-1 mRNA显著增加（P< 0.05）,肺内血管扩张加重。 结论：肺内血红素氧合酶－1表达增加在肝肺综合征的发病中起着重要的作用。     

异丙酚对高糖诱导心肌细胞肥大时血红素加氧酶-1表达的影响

目的:观察血红素加氧酶-1在高糖诱导的心肌细胞肥大中的表达,以及异丙酚对其表达的影响。方法:体外培养乳鼠心肌细胞,分组给药后,用BCA法测心肌细胞的蛋白质含量;用计算机图像分析系统检测心肌细胞表面积;用DCFH-DA活性氧检测试剂盒检测细胞内氧自由基的产生;用RT-PCR和Western blot分别检测心肌细胞中HO-1mRNA及蛋白表达。结果:高糖组心肌细胞表面积、细胞蛋白含量及氧自由基产生均升高。50μmol/L浓度的异丙酚对高糖诱导的肥大心肌细胞表面积、细胞蛋白含量及氧自由基的产生均有显著的抑制作用,而给予HO-1抑制剂后其作用明显减弱。结论:HO-1可能参与了异丙酚抑制心肌细胞肥大的作用。     

血红素氧合酶-1对大鼠心肌缺血-再灌注损伤的保护作用研究

目的探讨血红素氧合酶-1（HO-1）对大鼠心肌缺血-再推注损伤的效果及作用机制。方法采用HO-1的诱导剂钴原卟啉（CoPP）和抑制剂锌原卟啉（ZnPP）分别进行干预处理后,建立大鼠的心肌缺血-再灌注损伤模型,观察大鼠再灌注后心肌形态变化;检测血红素氧合酶-1基因在大鼠心肌的表达情况;测定大鼠左心室心肌组织超氧化物歧化酶（SOD）活性及丙二醛（MDA）含量。结果再灌注前使用CoPP进行预处理。可以诱导HO-1蛋白的表达上调。HO-1蛋自表达上调可以减少缺血-再灌注后的心肌细胞坏死,提高心肌组织中SOD含量并降低MDA的含量。结论CoPP诱导的HO-1过表达可以抑制心肌缺血-再灌注损伤后的细胞坏死,从而减轻心肌的再灌注损伤,其主要机制与抗氧自由基有关。     

Induction of heme oxygenase-1 in human hepatocytes to protect them from ethanol-induced cytotoxicity

We investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage. Methods Dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) ethanol exposure were used in the present study. HO-1 mRNA and protein expression were detected by PT-PCR and Western blot respectively. HO-1 activity was indicated by bilirubin and Fe^2 formation. Cytotoxicity was investigated by means of lactate dehydrogenate (LDH) and aspartate transaminase (AST) level in culture supematants, as well as the intracellular formation of malondialdehyde (MDA), cellular glutathione (GSH) status and CYP 2El activity. Results We first demonstrated a dose-dependent response between ethanol exposure and HO-1 mRNA and protein expression in human hepatocytes. We further observed a time-dependent increase of HO-1 mRNA expression using 100 mmol/L ethanol starting 30 minutes after ethanol exposure, reaching its maximum between 3 h and 9 h, Being similar to what had been demonstrated with the mRNA level,increased protein expression started at 6 h after ethanol exposure, and kept continuous elevated over 18 h. In addition, we found that ethanol exposure to hepatocytes markedly increased HO-1 enzyme activity in a time-dependent manner measured as bilirubin and Fez formation in human hepatocytes.Our results clearly showed that ethanol exposure caused a significant increase of LDH, AST, and MDA levels, while the antioxidant GSH was time-dependently reduced. Furthermore, we demonstrated that pre-administration of cobalt protoporphyrin (CoPP) induced HO-1 in human hepatocytes, and prevented an increase of MDA and a decrease of GSH. These effects could be partially reversed by zinc protoporphyfin (ZnPP), an antagonist of HO-1 induction. Conclusion HO-1 expression in cells or organs could lead to new strategies for better prevention and treatment of ethanol-induced oxidative damage in human liver.     

钴原卟啉诱导HO-1高表达抗大鼠心肌缺血/再灌注损伤的实验研究

目的 探讨血红素氧合酶-1(HO-1)对大鼠心肌缺血/再灌注损伤的保护效应及作用机制.方法 采用HO-1诱导剂钴原卟啉(CoPP) 和抑制剂锌原卟啉(ZnPP) 分别进行干预处理后建立大鼠心肌缺血/再灌注损伤模型,观察大鼠再灌注后I/R左心室心肌超微结构变化;检测HO-1蛋白在大鼠心肌的表达情况;测定大鼠左心室心肌组织超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量.结果 再灌注前使用CoPP 进行预处理可以诱导HO-1蛋白表达上调.HO-1蛋白表达上调可以减少缺血/再灌注后心肌细胞坏死,提高心肌组织中SOD含量,并降低MDA含量.结论 CoPP诱导的HO-1过表达可以抑制心肌缺血/再灌注损伤后细胞坏死,从而减轻心肌再灌注损伤,抗氧自由基是其机制之一.     

血红素加氧酶-1在肺早期缺血再灌注损伤中的表达及意义

目的研究血红素加氧酶-1（HO-1）与供肺缺血再灌注损伤之间的关系。方法采用改进的套管吻合技术，建立同系大鼠左肺原位再灌注损伤的动物模型。采用HO-1的诱导剂钴原卟啉（CoPP）和抑制剂锌原卟啉（ZnPP）分别进行干预处理后，运用免疫组织化学技术和RT-PCR技术分别检测HO-1蛋白在供肺组织中的表达以及供肺中HO-1mRNA的表达；运用TUNEL技术检测供肺组织中细胞凋亡。结果肺组织发生缺血再灌注时可诱导HO-1蛋白的表达，且随着再灌注时间的延长，表达逐步增多，再灌注8 h后达到高峰；再灌注前使用CoPP进行预处理，可以诱导HO-1蛋白的表达上调。HO-1蛋白表达上调可以降低肺缺血再灌注后细胞凋亡的发生率。结论肺缺血再灌注损伤可诱导HO-1表达上调，CoPP诱导的HO-1过表达可以抑制肺缺血再灌注损伤诱导的肺细胞凋亡，从而减轻供肺的再灌注损伤。     

血红素氧化酶-1在鼠异种肝移植中的抗排斥作用及其机制研究

目的探讨血红素氧化酶1(HO1)在异种肝移植中的作用及其机制方法采用豚鼠对大鼠肝移植模型,实验动物分为3组,每组10对鼠。A组供体鼠术前24h腹腔注射5ml/kg生理盐水作为对照;B组供体术前24h腹腔注射HO1诱导剂钴原卟啉(CoPP),C组供体注射CoPP的同时给予HO1抑制剂锌原卟啉(ZnPP)。每组受体均接受眼镜蛇毒因子(CVF)。分别以RTPCR和western印迹法观察HO1mRNA及蛋白表达。通过凝胶电泳迁移率改变法(EMSA)检测移植肝核转录因子(NF)κB的活化,免疫组化观察E选择素表达,来评价HO1对内皮细胞活化的抑制作用。并比较各组受体鼠存活时间。结果各组均未发生超急性排斥反应。CoPP预处理可以诱导HO1mRNA和蛋白过度表达,进而显著抑制移植肝内NFκB的活化和内皮细胞E选择素表达;B组与(0.112±0.039)A组(0.211±0.030)、C组(0.283±0.075)相比差异有统计学意义(P<0.05)。HO1过度表达组与其它组相比,移植肝内皮细胞肿胀减轻,浸润细胞数减少,肝脏功能改善,而且受体鼠存活时间显著延长;B组(15.5h±3.8h)比A组(7.3h±2.1h)差异有统计学意义(P<0.01)。这种保护效应可被同时给予HO1抑制剂ZnPP所取消;B组比C组(6.7h±2.9h)差异有统计学意义(P<0.01)。结论HO1通过抑制内皮细胞活化,延缓异种移植肝排斥反应发生。     

血红素氧合酶-1对肝脏缺血再灌注损伤的保护作用

目的:探讨血红素氧合酶-1(HO-1)对肝脏缺血再灌注损伤的保护作用机制。方法:SD大鼠30只,建立二套袖法原位肝移植模型。随机分为3组:对照组,无任何处理;CoPP处理组,供肝收获前24h供体腹腔注射HO-1诱导剂CoPP5mg/kg。;ZnPP处理组,腹腔注射抑制剂ZnPP20mg/kg。移植后第三天检测肝脏移植物肝功能,免疫组织化学方法检测肝脏移植物HO-1的表达情况。TUNEL法检测凋亡变化。以Suzuki标准评分来反映肝脏移植物的缺血再灌注损伤程度。结果:①CoPP组ALT,AST较对照组和ZnPP组明显降低(P<0.01)。②HO-1表达:CoPP组明显高于其他组(P<0.05)。③移植物凋亡阳性细胞百分率:CoPP组明显低于对照组和ZnPP组(P<0.05)。④肝脏移植物病理变化:CoPP组和与对照组,ZnPP组比较,炎症细胞浸润少,肝细胞损伤轻。肝脏移植物Suzuki评分,CoPP组(0.76±0.59)明显低于对照组(1.32±0.74)与ZnPP组(1.80±0.70),有显著统计学差异(P<0.05)。结论:HO-1对肝脏移植物缺血再灌注损伤具有保护作用,其机制可能与抗炎症与抗凋亡有关。     

姜黄素对铜绿假单胞菌诱导人肺上皮细胞产生细胞因子的影响及其分子机制研究

目的 探讨姜黄素对铜绿假单胞菌（pseudomonas aeruginosa,Pa）感染的肺上皮细胞的体外抗炎效应.方法 体外培养肺上皮细胞系NCI-H292,加入姜黄素和培养的Pa感染不同时间.Western blot检测和实时定量PCR分别检测NCI-H292细胞中红素氧合酶1（Heme oxygenase-1,HO-1）mRNA和蛋白表达情况;ELISA检测IL-1β和IL-8的产生.同时采用RNA干扰HO-1表达后,观察对Pa诱导IL-1β和IL-8产生的影响.结果 Pa感染NCI-H292细胞后,IL-1β和IL-8含量分别为（87.52±7.96）pg/mL和（205.63 ±34.25） pg/mL,50 μg/mL姜黄素孵育8h后,其含量分别降至（15.78±10.74）pg/mL和（49.82±14.28）pg/mL,处理前后差异有统计学意义（P＜0.05）.同时,姜黄素能在转录翻译水平分别调控HO-1 mRNA和蛋白表达,其中50 μg/mL姜黄素能将HO-1 mRNA增高19倍,蛋白表达水平提高4.8倍（P＜0.05）.HO-1 siRNA沉默HO-1表达后,与Pa组相比,IL-1β和IL-8分泌进一步增高.结论 姜黄素可能通过上调HO-1的表达而发挥对肺上皮细胞的抗炎作用.