Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions

The extracellular matrix (ECM) contributes to the generation and dynamic of normal breast tissue, in particular to the generation of polarized acinar and ductal structures. In vitro 3D culture conditions, including variations in the composition of the ECM, have been shown to directly influence the formation and organization of acinus‐like and duct‐like structures. Furthermore, the density of the ECM appears to also play a role in the normal mammary tissue and tumour formation. Here we show that the density of the ECM directly influences the number, organization and function of breast acini. Briefly, non‐malignant human breast MCF10A cells were incubated in increasing densities of a Matrigel®–collagen I matrix. Elastic moduli near and distant to the acinus structures were measured by atomic force microscopy, and the number of acinus structures was determined. Immunochemistry was used to investigate the expression levels of E‐cadherin, laminin, matrix metalloproteinase‐14 and ß‐casein in MCF10A cells. The modulus of the ECM was significantly increased near the acinus structures and the number of acinus structures decreased with the increase in Matrigel–collagen I density. As evaluated by the expression of laminin, the organization of the acinus structures present was altered as the density of the ECM increased. Increases in both E‐cadherin and MMP14 expression by MCF10A cells as ECM density increased were also observed. In contrast, MCF10A cells expressed lower ß‐casein levels as the ECM density increased. Taken together, these observations highlight the key role of ECM density in modulating the number, organization and function of breast acini. Copyright © 2013 John Wiley & Sons, Ltd.     

层黏连蛋白对胰腺癌细胞内在性耐药的影响及其机制

  目的  探讨层黏连蛋白(laminin, LN)对胰腺癌细胞内在性耐药的影响, 并探讨其作用机制。  方法  选择胰腺癌细胞系AsPC-1, 检测LN对盐酸吉西他滨(健择, gemcitabine, Gem)的细胞毒性及对Gem诱导细胞凋亡能力的影响。通过Western blot等方法检测LN对下游信号通路的影响; 在AsPC-1中, 过表达黏着斑激酶相关非激酶(focal adhesion kinase-related non-kinase, FRNK)或应用PF-573, 228抑制黏着斑激酶(focal adhesion kinase, FAK)磷酸化与活性, 检测FAK及其下游信号的抑制对LN作用的影响。  结果  在胰腺癌细胞系AsPC-1中, LN处理使Gem的细胞毒性及Gem诱导细胞凋亡的能力显著降低。Gem作用72 h后, LN处理组细胞存活率为57.71%±6.08%, 形成的克隆数目为55.33±5.01;而在无LN包被组, 细胞存活率与形成的克隆数目分别为36.65%±4.14%及31.43±4.62(P均 < 0.05);LN处理组Annexin V标记阳性细胞百分数(41.00%±5.46%)较无LN处理组(55.70%±3.44%)显著下降(P < 0.05)。LN能够时间依赖性上调AsPC-1细胞的FAKTyr397位点及Akt磷酸化水平, LN还可以显著增加AsPC-1细胞的survivin蛋白表达水平及BAD Ser-136位点磷酸化水平。过表达FRNK或应用PF-573, 228能够显著抑制FAK磷酸化水平及其下游通路的活化, 并能够对抗LN对胰腺癌内在性耐药的效应。应用PF-228可以使LN处理后Gem诱导的AsPC-1细胞凋亡率从26.77%±0.49%升高至38.53%±2.83%(P < 0.05)。  结论  细胞外基质蛋白LN能够诱导胰腺癌细胞系对Gem产生内在性耐药。LN影响胰腺癌细胞内在性耐药的具体机制可能与FAK磷酸化及其下游PI3K-Akt通路活化、Bad Ser-136位点磷酸化与survivin表达水平改变有关, FAK靶向治疗与Gem联合在胰腺癌治疗中具有重要的潜在应用价值。     

The extracellular matrix in oral squamous cell carcinoma: Friend or foe?

Oral squamous cell carcinoma is a disfiguring, highly invasive and metastatic cancer. Despite advances in detection and therapy, many patients will continue to face a poor prognosis. It is well established that the predominate factor determining overall survival in patients with oral squamous cell carcinoma is lymph node involvement. Tumor growth and progression to invasive cancer requires tumor cell interactions with the extracellular matrix. An understanding of how the extracellular matrix influences tumor development and invasion is fundamental in the development of new prognostic indicators and treatment strategies for oral squamous cell carcinoma. In this review, we summarize how changes in the extracellular matrix contribute to oral cancer development.     

腹腔液中层粘连蛋白含量与胃癌病理及腹膜转移相关因素的关系

目的探讨胃癌腹腔液中层粘连蛋白含量与其病理生物学行为及腹膜转移相关因素的关系.方法术中收集50例胃癌和10例胃良性病变的腹水或腹腔冲洗液,采用放射免疫技术检测上清液中层粘连蛋白、CEA蛋白(p-CEA)含量,同时进行腹腔冲洗细胞学(peritoneal lavage cytology,PLC)和病理学检查.结果层粘连蛋白含量与胃癌组织学类型、生长方式、浸润深度及淋巴结转移呈正相关.PLC和p-CEA阳性组层粘连蛋白含量均高于阴性(P＜0.05);肉眼腹膜转移的10例中层粘连蛋白均显著升高.全组层粘连蛋白含量升高者占68%,明显高于p-CEA(44%)和PLC(34%)的阳性率.结论腹腔液中的层粘连蛋白是反映胃癌生物学行为的分子标志物,与胃癌腹膜转移密切相关,其预测腹膜亚临床转移的灵敏性优于p-CEA和PLC.     

[Online First]Improvement of human embryonic stem cell-derived retinal pigment epithelium cell adhesion, maturation， and function through coating with truncated recombinant human vitronectin

AIM: To explore an xeno-free and defined coating substrate suitable for the culture of H9 human embryonic stem cell-derived retinal pigment epithelial (hES-RPE) cells in vitro, and compare the behaviors and functions of hES-RPE cells on two culture substrates, laminin521 (LN-521) and truncated recombinant human vitronectin (VTN-N). METHODS: hES-RPE cells were used in the experiment. The abilities of LN-521 and VTN-N at different concentrations to adhere to hES-RPE cells were compared with a high-content imaging system. Quantitative real-time polymerase chain reaction was used to evaluate RPE-specific gene expression levels midway (day 10) and at the end (day 20) of the time course. Cell polarity was observed by immunofluorescent staining for apical and basal markers of the RPE. The phagocytic ability of hES-RPE cells was identified by flow cytometry and immunofluorescence. RESULTS: The cell adhesion assay showed that the ability of LN-521 to adhere to hES-RPE cells was dose-dependent. With increasing coating concentration, an increasing number of cells attached to the surface of LN-521-coated wells. In contrast, VTN-N presented a strong adhesive ability even at a low concentration. The optimal concentration of LN-521 and VTN-N required to coat and adhesion to hES-RPE cells were 2 and 0.25 µg/cm2, respectively. Furthermore, both LN-521 and VTN-N could facilitate adoption of the desired cobblestone cellular morphology with tight junction and showed polarity by the hES-RPE cells. However, hES-RPE cells cultivated in VTN-N had a greater phagocytic ability, and it took less time for these hES-RPE cells to mature. CONCLUSION: VTN-N is a more suitable coating substrate for cultivating hES-RPE cells.     

层黏连蛋白对人翼状胬肉成纤维细胞生长的抑制作用

目的:探讨层黏连蛋白对体外培养的人翼状胬肉成纤维细胞增殖的影响.方法:体外培养的第3代翼状胬肉成纤维细胞,接种于层黏连蛋白包被培养皿(Ln组)和空白培养皿(对照组)中,倒置显微镜观察细胞生长状态,MTT法检测细胞增殖状态,作出生长曲线;免疫荧光法检测α-SMA在细胞内的表达;RT-PCR法检测TGF-β1和cytokeratin mRNA的表达水平.结果:Ln组成纤维细胞数量少于对照组;3～7 d,Ln组A570明显低于对照组(0.20 vs 0.28,0.22 vs 0.30,0.28 vs0.34,0.31 vs0.36,0.33 vs 0.36,P＜0.05);α-SMA在对照组细胞内表达良好,在Ln组的表达受到抑制;与对照组相比,Ln组TGF-β1 mRNA的表达下降,cytokeratin mRNA表达增加.结论:层黏连蛋白能抑制翼状胬肉中成纤维细胞的增殖.     

层粘连蛋白对人视网膜母细胞瘤株基质金属蛋白酶-2和基质金属蛋白酶-9的影响

目的研究层粘连蛋白(laminin,LN)对人视网膜母细胞瘤株(Hxo-Rb44)基质金属蛋白酶-2(matrix metallopro-teinase,MMP-2)和基质金属蛋白酶-9(matrix metalloproteinase,MMP-9)及其mRNA表达的影响。方法在体外培养的人视网膜母细胞瘤细胞株(Hxo-Rb44)内分别加入含0μg/ml、5μg/ml、10μg/ml、20μg/mlLN的无血清RPMI-1640培养基,培养24h后,分别利用免疫组化SP染色法和PT-PCR检测每个实验组Hxo-Rb44表达MMP-2,MMP-9及其mRNA的含量,统计学方法比较不同实验组Hxo-Rb44表达MMP-2,MMP-9及其mRNA的差异。结果视网膜母细胞瘤细胞经LN刺激后,细胞内MMP-2和MMP-9及其mRNA表达均增加,免疫组化结果显示5μg/ml、10μg/ml、20μg/mlLN处理组MMP-2和MMP-9的染色灰度值分别是178.1±11.7和167.0±15.7、147.9±13.4和140.3±12.0、114.4±13.6和104.0±10.6,和对照组相比,差异有显著性(P<0.05),且LN浓度越高,其表达越强。5μ/ml、10!g/ml、20!g/mlLN处理组的MMP-2/β-actin、MMP-9/β-actin灰度值比值分别是0.181±0.016和0.254±0.023、0.201±0.021和0.287±0.020、0.243±0.019和0.320±0.022,与对照组相比,差异均有显著性(P<0.05),且随着LN浓度增高而增高,呈明显的量效关系。结论LN可诱导视网膜母细胞瘤细胞中表达的MMP-2和MMP-9及其mRNA增加,并呈剂量依赖型。提示LN对视网膜母细胞瘤的侵袭和转移可能具有促进作用。     

Effect of Feixian Recipe on laminin, collagen I and III in rats with pulmonary fibrosis induced by bleomycin

The aim of this paper is to observe the effect of the Feixian Recipe on pulmonary fibrosis in rats. A rat model with pulmonary fibrosis was established by intratracheal injection of bleomycin. On days 14, 28 and 45, the contents of laminin, collagen I and collagen III in lung tissue homogenate in the model group, the sham operated group, the Feixian group and the prednisone group were measured. The contents of laminin and collagen I and III were decreased significantly by the Feixian Recipe. Feixian Recipe has a significant therapeutic effect on bleomycin-induced pulmonary fibrosis in rats. __________ Translated from Journal of Beijing University of Traditional Chinese Medicine, 2007, 30(9): 608–610 [译自: 北京中医药大学学报]     

Expression of dystroglycan and the laminin-alpha 2 chain in the rat peripheral nerve during development

In Schwann cells, the transmembrane glycoprotein beta-dystroglycan comprises the dystroglycan complex, together with the extracellular glycoprotein alpha-dystroglycan, which binds laminin-2 (alpha 2/beta 1/gamma 1), a major component of the Schwann cell basal lamina. To provide clues to the biological functions of the interaction of the dystroglycan complex with laminin-2 in peripheral nerves, we investigated the expression of beta-dystroglycan and the laminin-alpha 2 chain in rat sciatic nerve during development by immunoblot, immunofluorescence, and immunoelectron microscopic studies. The expression of beta-dystroglycan and the laminin-alpha 2 chain in the rat sciatic nerve was low and not confined to the Schwann cell outer membrane from embryonic day 18 to birth, when there was only an immature basal lamina assembly and no compact myelin formation by Schwann cells. However, the expression of these proteins increased markedly and became clearly localized to the Schwann cell outer membrane between birth and postnatal day 7, when both basal lamina assembly and compact myelin formation by Schwann cells progressed rapidly. From postnatal day 7 to adult, there was no remarkable change in the expression of these proteins. Our results support the hypothesis that the dystroglycan complex functions as an adhesion apparatus, binding the Schwann cell outer membrane with the basal lamina, and suggest that the dystroglycan complex plays a role in Schwann cell myelination through its interaction with laminin-2.     

Temporal progression of angiogenesis and basal lamina deposition after contusive spinal cord injury in the adult rat

After spinal cord injury (SCI), the absence of an adequate blood supply to injured tissues has been hypothesized to contribute to the lack of regeneration. In this study, blood vessel changes were examined in 28 adult female Fischer 344 rats at 1, 3, 7, 14, 28, and 60 days after a 12.5 g x cm NYU impactor injury at the T9 vertebral level. Laminin, collagen IV, endothelial barrier antigen (SMI71), and rat endothelial cell antigen (RECA-1) immunoreactivities were used to quantify blood vessel per area densities and diameters in ventral gray matter (VGM), ventral white matter (VWM), and dorsal columns (DC) at levels ranging 15 mm rostral and caudal to the epicenter. This study demonstrates an angiogenic response, defined as SMI71/RECA-1-immunopositive endothelial cells that colocalize with a robust deposition of basal lamina and basal lamina streamers, 7 days after injury within epicenter VGM. This angiogenesis diminishes concurrent with cystic cavity formation. GAP43- and neurofilament- (68 kDa and 210 kDa) immunopositive fiber outgrowth was associated with these new blood vessels by day 14. Between 28 and 60 days after injury, increases in SMI71-immunopositive blood vessel densities were observed in the remaining VWM and DC with a corresponding increase in vessel diameters up to 15 mm rostral and caudal to the epicenter. This second angiogenesis within VWM and DC, unlike the acute response observed in VGM, did not correspond to any previously described changes in locomotor behaviors in this model. We propose that therapies targeting angiogenic processes be directed at the interval between 3 and 7 days after SCI.