Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis¹. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity² and geometry³ of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments⁴. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).     

卵巢上皮癌中ING4 基因启动子的甲基化状态及其临床意义

目的：探讨卵巢上皮癌中ING4 基因启动子的甲基化状态及其临床意义。方法：收集2005 年7 月至2012 年6 月哈尔滨医科 大学附属第一医院行全面分期手术并经病理检查确诊的150 例卵巢上皮癌组织标本,并以同期因子宫肌瘤或子宫腺肌症行子宫 全切除术或次全切除术并经病理检查确诊为正常卵巢组织的150 例标本作为对照组。采用甲基化特异性PCR（MSP）技术检测卵 巢上皮癌组织与正常卵巢组织中ING4 基因启动子的甲基化状态,蛋白印迹法检测ING4 蛋白的表达,并分析ING4 基因启动子 的甲基化状态与卵巢上皮癌临床病例特征的关系。结果：卵巢上皮癌组织中ING4 基因启动子的甲基化阳性率为42.7%（64/150）, 明显高于正常卵巢组织（4%,6/150）,差异有统计学意义（P<0.05）。ING4 基因启动子甲基化阳性的卵巢上皮癌组织中ING4蛋白 表达阴性或弱阳性;ING4 基因启动子甲基化阴性的卵巢上皮癌和正常卵巢组织中ING4 蛋白表达阳性;在64 例ING4 基因启动 子甲基化的卵巢上皮癌组织中,ING4 蛋白表达强度与ING4 基因启动子的甲基化程度呈负相关（r=-0.435,P<0.05）。卵巢上皮癌 组织中,ING4 基因甲基化的阳性率随着手术病理分期和组织学分级的增加而增加（P<0.05）;卵巢透明细胞癌（55.6%,10/18）和卵 巢子宫内膜样癌（59.3%,16/27）中ING4 基因甲基化的阳性率显著高于浆液性囊腺癌（33.9%,20/59）和粘液性囊腺癌（39.1%, 18/46）（P<0.05）;ING4基因启动子的甲基化状态与患者的年龄、有无腹水及淋巴结转移均无显著相关性（P>0.05）。结论：ING4 基 因启动子的甲基化可能促进了其在卵巢上皮癌组织中的表达失活,进而促进了卵巢上皮癌的生长和分化。     

N-Acetylcysteine ameliorates lipopolysaccharide-induced organ damage in conscious rats

Lipopolysaccharide is strongly associated with septic shock, leading to multiple organ failure. It can activate monocytes and macrophages to release proinflammatory mediators such as tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and nitric oxide (NO). The present experiments were designed to induce endotoxin shock by an intravenous injection ofKlebsiella pneumoniae lipopolysaccharide (LPS, 10 mg/kg) in conscious rats. Arterial pressure and heart rate (HR) were continuously monitored for 48 h after LPS administration. N-Acetyl-cysteine was used to study its effects on organ damage. Biochemical substances were measured to reflect organ functions. Biochemical factors included blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), aspartate transferase (GOT), alanine transferase (GPT), TNF-, IL-1, methyl guanidine (MG), and nitrites/nitrates. LPS caused significant increases in blood BUN, Cre, LDH, CPK, GOT, GPT, TNF-, IL-1, MG levels, and HR, as well as a decrease in mean arterial pressure and an elevation of nitrites/nitrates. N-Acetylcysteine suppressed the release of TNF-, IL-1, and MG, but enhanced NO production. These actions ameliorate LPS-induced organ damage in conscious rats. The beneficial effects may suggest a potential chemopreventive effect of this compound in sepsis prevention and treatment.     

Tumor extracellular acidity-activated nanoparticles as drug delivery systems for enhanced cancer therapy

pH-responsive nanoparticles (NPs) are currently under intense development as drug delivery systems for cancer therapy. Among various pH-responsiveness, NPs that are designed to target slightly acidic extracellular pH environment (pH_e) of solid tumors provide a new paradigm of tumor targeted drug delivery. Compared to conventional specific surface targeting approaches, the pH_e-targeting strategy is considered to be more general due to the common occurrence of acidic microenvironment in solid tumors. This review mainly focuses on the design and applications of pH_e-activated NPs, with special emphasis on pH_e-activated surface charge reversal NPs, for drug and siRNA delivery to tumors. The novel development of NPs described here offers great potential for achieving better therapeutic effects in cancer treatment.     

不同转移潜能肿瘤细胞肝素酶的表达

目的研究肝素酶(Heparanase,Hpa)表达水平与人类肿瘤转移的相关性。方法利用半定量RT-PCR、免疫组织化学(S-P法)和Westernblot检测2组4种不同转移潜能的人类肿瘤细胞系中HpamRNA和蛋白的表达水平。结果HpamRNA和蛋白相对表达量在高转移潜能人类肺癌细胞(0·757±0·033,0·670±0·020)、乳腺癌细胞(0·617±0·024,0·661±0·013)中明显高于相应的低转移潜能肺癌细胞(0·518±0·012,0·406±0·012)、乳腺癌细胞(0·170±0·016,0·227±0·011)。结论在所研究的人类肿瘤中,HpamRNA和蛋白的表达水平与肿瘤的转移能力呈正相关。     

Integrin-binding Protein Nischarin Interacts with Tumor Suppressor Liver Kinase B1 (LKB1) to Regulate Cell Migration of Breast Epithelial Cells

Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44–436 of LKB1. Time lapse microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression. Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.