硬脑膜细胞体外培养模型的建立

目的：建立兔硬脑膜缺损的体外培养模型,观察不同细胞外基质对其愈合过程的影响,研究硬脑膜修复的机制,为以后检测硬脑膜移植物提供理论基础。方法：兔硬脑膜体外培养,再用免疫细胞化学方法和扫描电镜进行观察。结果：预先铺过胶原的培养板中培养8～10d后有细胞从硬脑膜缺损的边缘逸出、增殖,13～15d硬脑膜缺损愈合;而以多聚赖氨酸及层粘连蛋白铺底的培养板中没有看到细胞逸出的现象。硬脑膜细胞对波形蛋白抗体呈强阳性反应,对第Ⅶ因子抗体则为阴性反应;在电镜下可以看到新生胶原的生成。结论：成功建立一种硬脑膜缺损的体外培养模型;成纤维细胞从硬脑膜创缘的逸出、增殖是硬脑膜缺损愈合的重要机制。     

甲真菌病体外模型研究

目的 构建一个简单、经济的新的甲真菌病体外模型.方法 制备猪甲板,在其腹侧面中央黏贴一个“O”型圈,将受试菌液加入“O”型圈内,将甲板放置培养皿中培养,用大体观察及组织病理的方法动态观察不同真菌侵袭甲板的情况.结果 用猪甲板制备甲真菌病体外模型,通过该模型观察了红色毛癣菌、须癣毛癣菌以及白念珠菌穿透甲板的能力.通过组织病理学方法观察到了真菌动态穿透甲板的过程.结论 该模型适用于评估真菌对甲板的致病作用.     

Establishment of an in vitro organoid model of dermal papilla of human hair follicle

Human hair dermal papilla (DP) cells are specialized mesenchymal cells that play a pivotal role in hair regeneration and hair cycle activation. The current study aimed to first develop three‐dimensional (3D) DP spheroids (DPS) with or without a silk–gelatin (SG) microenvironment, which showed enhanced DP‐specific gene expression, resulting in enhanced extracellular matrix (ECM) production compared with a monolayer culture. We tested the feasibility of using this DPS model for drug screening by using minoxidil, which is a standard drug for androgenic alopecia. Minoxidil‐treated DPS showed enhanced expression of growth factors and ECM proteins. Further, an attempt has been made to establish an in vitro 3D organoid model consisting of DPS encapsulated by SG hydrogel and hair follicle (HF) keratinocytes and stem cells. This HF organoid model showed the importance of structural features, cell–cell interaction, and hypoxia akin to in vivo HF. The study helped to elucidate the molecular mechanisms to stimulate cell proliferation, cell viability, and elevated expression of HF markers as well as epithelial–mesenchymal crosstalks, demonstrating high relevance to human HF biology. This simple in vitro DP organoid model system has the potential to provide significant insights into the underlying mechanisms of HF morphogenesis, distinct molecular signals relevant to different stages of the hair cycle, and hence can be used for controlled evaluation of the efficacy of new drug molecules.     

Characterization of the nuclear matrix proteins in a transgenic mouse model for prostate cancer

The nuclear matrix (NM) contains a number of proteins that have been found to be associated with transformation. We have previously identified changes in the NM associated with prostate cancer. In this study, we examine the molecular changes that are associated with prostate cancer development in transgenic adenocarcinoma of mouse prostate (TRAMP) model by studying the differences in the NM proteins (NMPs). We collected prostates from the TRAMP males at six critical time points: 6 weeks (puberty), 11 and 19 weeks (development of mild hyperplasia), 25 weeks (development of severe hyperplasia), 31 and 37 weeks (development of neoplasia). The nuclear matrices from the prostates collected at these time points were then isolated and the NMPs were characterized by high-resolution two-dimensional gel electrophoresis. We found three NMPs (E1A, E1B, and E1C) that were present in the 6-week-old prostate and two NMPs (E2A and E2B) that were present in the 11-week-old prostate. These NMPs were absent in the 31- and 37-week-old prostate. We also found five NMPs (E3A-E3E) that were present in the 31-week-old prostate, but absent in the earlier time points. In addition, three NMPs (Le1, Le2, Le3) were present at higher expression in the 6-, 11-, 19-, and 25-weeks old TRAMP prostates, but they were expressed lower during the development of neoplasia at 31- and 37-weeks old. Identification of these NMPs permits the development of novel markers that can characterize various stages of prostate cancer development as well as potentially therapeutic targets.     

Culturing precision-cut human prostate slices as an in vitro model of prostate pathobiology

Due to the complex morphology of the prostate, it was hypothesized that precision-cut tissue slices from human prostate would provide a unique in vitro model. Precision-cut slices were generated from zones of human prostate and their viability was assessed under conditions of different media for up to 120 h. Slices were also exposed to several concentrations of CdCl₂, which was used as a model toxicant. Maintenance of both stromal and epithelial cells was noted; however, there was a gradual loss of luminal epithelial cells when the medium was not supplemented with dihydrotestosterone (DHT). Minimal leakage of lactate dehydrogenase occurred throughout the incubation. Prostate-specific antigen (PSA) was detected in the medium at all time points, although the rates of secretion fell over time. There was a loss of PSA-positive cells when the medium was not supplemented with DHT, consistent with a loss of luminal cells, whereas PSA-positive cells were maintained in the DHT-supplemented media. A proliferation of basal cells was observed in the presence of media containing 10% fetal bovine serum. Exposure of slices to CdCl₂ demonstrated a dose-response effect ranging from proliferation to complete cellular necrosis. Given the retention of stromal-epithelial interactions and the use of acquired human tissue, prostate slices represent a unique in vitro model for investigating human prostate pathobiology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Neuroprotective effect of GMP in hippocampal slices submitted to an in vitro model of ischemia

1. Guanosine-5-monophosphate (GMP) was evaluated as a neuroprotective agent against the damage observed in rat hippocampal slices submitted to an in vitro model of ischemia with or without the presence of the ionotropic glutamate receptor agonist, Kainic acid (KA).2. Cellular injury was evaluated by MTT reduction, lactate dehydrogenase (LDH) release assay, and measurement of intracellular ATP levels.3. In slices submitted to ischemic conditions, 1 mM GMP partially prevented the decrease in cell viability induced by glucose and oxygen deprivation and the addition of KA.4. KA or N-methyl-D-aspartate (NMDA) receptor antagonists, -D-glutamylamino-methylsulfonate (GAMS) or (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801, 20 M) also prevented toxicity in hippocampal slices under ischemic conditions, respectively.5. The association of GMP with GAMS or MK-801 did not induce additional protection than that observed with GMP or that classical glutamate receptor antagonists alone.6. GMP, probably by interacting with ionotropic glutamate receptors, attenuated the damage caused by glucose and oxygen deprivation in hippocampal slices. This neuroprotective action of GMP in this model of excitotoxicity is of outstanding interest in the search for effective therapies against ischemic injury.     

Use of an in vitro tuberization system to study tuber protein gene expression

Summary Nodal cuttings from micropropagated potato plantlets give rise to microtubers when placed on Murashige and Skoog medium containing 6% sucrose and 2.5 mg/liter kinetin and incubated in the dark at 19°C. Microtubers produced from the cultivar Superior were shown to contain the same characteristic group of proteins as field-grown tubers. As with field-grown tubers, the 40 000-dalton major tuber glycoprotein, patatin, accumulated to high levels in microtubers, reaching 3.7±0.2 mg/g fresh weight after 90 d. Also in agreement with field-grown plants, stems and leaves of micropropagated plantlets did not contain detectable levels of patatin, but small amounts of an electrophoretically distinct form accumulated transiently in roots. Patatin mRNA is readily detectable in developing microtubers 15 d after transfer of the cuttings to inductive medium. Patatin mRNA was also present in roots, but as with field-grown plants, was 50- to 100-fold less abundant and could be distinguished from that in tubers by primer extension. Microtuber development and patatin accumulation were inhibited by gibberellic acid. This work was supported by grants 83-CRCR-1-1348 and 85-CRCR-I-1792 from the U.S. Department of Agriculture Competitive Grants program and with funds from the Texas Agricultural Experiment Station.     

Combination effect of PectaSol and Doxorubicin on viability, cell cycle arrest and apoptosis in DU-145 and LNCaP prostate cancer cell lines

The effect of PectaSol on Dox (Doxorubicin) cytotoxicity in terms of apoptosis and cell cycle changes in PCa (prostate cancer) cell lines (DU‐145 and LNCaP) has been investigated. Combination of PectaSol and Dox resulted in a viability of 29.4 and 32.6% (P<0.001) in DU‐145 and LNCaP cells. The IC₅₀ values decreased 1.5‐fold and 1.3‐fold in the DU‐145 and LNCaP cells respectively. In the DU‐145 cells, combination of PectaSol and Dox resulted in a reduction in p27 gene and protein expression (P<0.001). In LNCaP cells, this combination increased p53, p27 and Bcl‐2 expression. Treatment with both drugs in DU‐145 cells led to an increase in sub‐G₁ arrest (54.6% compared with 12.2% in Dox). In LNCaP cells, combination of the drugs led to an increased in G₂/M arrest (61.7% compared with 53.6% in Dox). Based on these findings, progressive cytotoxicity effect of Dox and PectaSol together rapidly induce cell death in DU‐145 through apoptosis and in LNCaP cells through cell cycle arrest (G₂/M arrest).     

Effects of C-xylopyranoside derivative on epithelial regeneration in an in vitro 3D oral mucosa model

Identifying substandard tissue-engineered oral mucosa grafts with a poor epithelium before clinical use is critical to ensure quality assurance/control in regenerative medicine, leading to success of grafting. This study investigated the effects of one of the C-xylopyranoside derivatives, β-D-xylopyranoside-n-propane-2-one (XPP), on oral epithelial regeneration. Using a three-dimensional oral mucosa model, we analyzed changes of the epithelial structure, glycosaminoglycan (GAG) synthesis, the expression levels of basement membrane zone markers, and substrates of Akt/mTOR signaling. Compared with the control, 2 mM XPP treatment increased the mean and minimal epithelial thickness, and reduced the variation of epithelial thickness. It also stimulated expressions of decorin and syndecan-1 with change of GAG amount and/or composition, and enhanced the expressions of integrin α6, CD44, and Akt/mTOR signaling substrates. These findings suggest that XPP supplementation contributes to consistent epithelial regeneration. Moreover, upregulation of those markers may play a role in increasing the quality of the oral mucosal epithelium.     

Development of an in vitro model of Toxoplasma gondii cyst formation

Abstract Toxoplasma gondii tissue cyst reactivation is a major pathogenic mechanism in ocular toxoplasmosis, disease associated with AIDS and organ transplantation. The mechanisms associated with cyst formation and reactivation have not been elucidated. The complexity of studying these issues in animal models has led to the development of in vitro tissue culture strategies for cyst formation. In the present study we have adopted the human embryonic lung fibroblast (HEL) as the host cell and have compared the cyst forming abilities of eight clinical isolates. We describe by transmission electron microscopy and quantitative light microscopy the development of cysts in vitro. The numbers of in vitro cysts increased with time for all isolates. Cyst cultures were stabilised by manipulation of the free parasite load, an observation not previously recorded. Thus, in this paper we describe a viable model for the analysis of the mechanisms of Toxoplasma cyst development.     

Studies on bioavailability of some bulk and trace elements in mexican tortilla using an in vitro model

An in vitro gut model was used to investigate the bioavailability of calcium, magnesium, cadmium, chromium, iron, manganese, and lead in the Mexican (maize) tortilla. The samples (4 g) were digested in quartz tubes using concentrated nitric acid (12 mL) at 65‡C (2 h) and then at 120‡C (4 h). Total element concentration was determined by atomic absorption spectrometry. Enzymatic hydrolysis was carried out in two steps: first, with pepsin in a hydrochloric acid medium (pH 1.8) and then after neutralization with sodium bicarbonate (pH 6.0), with the mixture of pancreatine, amylase, and bile salts extract. Elements under study were determined in the supernatant by atomic absorption spectrometry. The bioavailable fraction of each element in tortilla was evaluated as the percentage of total element content found in the solution after enzymolysis. The obtained results showed relatively low bioavailability of the selected elements (from 2% to 32%), which possibly may be ascribed to the presence of dietary fiber in tortilla.     

卵泡内环境对猪卵泡卵体外成熟和发育的影响

研究卵泡内环境对猪卵母细胞体外成熟、受精及受精卵体外发育的影响。主要结果如下：直径≥5mm、4－4.9mm、3－3.9mm和2－2.9mm的卵泡卵母细胞体外成熟率分别为90.5%、89.7%、85.4%和67.4%,体外受精后,卵母细胞的发育能力随卵泡直径的增大而增强,直径≥5mm和4－4.9mm卵泡卵的2－细胞、3－4－细胞发育率显著高于直径2－2.9mm的卵泡卵（P＜0.05或0.01）。体外成熟培养36h、42h和48h,直径2－2.9mm卵泡卵的体外成熟率,体外受精后的卵裂率差异不显著（P＞0.05）。在体外成熟培养液中添加5％或15％的不同直径卵泡的卵泡液,各组间卵母细胞的体外成熟率,受精卵的体外发育率均无显著差异,结果表明：卵泡大小对猪卵母细胞体外成熟、受精及受精卵体外发育有重要影响。     

The role of the disintegrin metalloproteinase ADAM15 in prostate cancer progression

The metalloproteinase ADAM15 is a multi‐domain disintegrin protease that is upregulated in a variety of human cancers. ADAM15 mRNA and protein levels are increased in prostate cancer and its expression is significantly increased during metastatic progression. It is likely that ADAM15 supports disease progression differentially through the action of its various functional domains. ADAM15 may downregulate adhesion of tumor cells to the extracellular matrix, reduce cell–cell adhesion, and promote metastasis through the activity of its disintegrin and metalloproteinase domains. Additionally, ADAM15 can influence cell signaling by shedding membrane‐bound growth factors and other proteins that interact with receptor tyrosine kinases, leading to receptor activation. There is also evidence supporting a role for ADAM15 in angiogenesis and angioinvasion of tumor cells, which are critical for unrestrained tumor growth and metastatic spread. Given its diverse functions, ADAM15 may represent a pivotal regulatory component of tumor progression, an important target for therapeutic intervention, or emerge as a biomarker of disease progression. J. Cell. Biochem. 106: 967–974, 2009. © 2009 Wiley‐Liss, Inc.     

An in vitro approach to the study of target organ toxicity of drugs and chemicals

Summary A major goal of our laboratory has been the development of primary culture systems that retain differentiated fucntions and responses characteristic of intact tissues in vivo. Specifically, we have developed cellular models of primary cultures of rat heart, liver, and kidney cells to explore the mechanisms by which drugs or chemicals may be toxic to key organs of the body and to develop new techniques by which xenobiotics may be evaluated or identified as potential toxicants to living systems. The purpose of this paper is to describe our rationale and approach to the study of target organ toxicology with in vitro cellular systems.     

Human breast cancer decellularized scaffolds promote epithelial-to-mesenchymal transitions and stemness of breast cancer cells in vitro

Breast cancer, with unsatisfactory survival rates, is the leading cause of cancer-related death in women worldwide. Recent advances in the genetic basis of breast cancer have benefitted the development of gene-based medicines and therapies. Tissue engineering technologies, including tissue decellularizations and reconstructions, are potential therapeutic alternatives for cancer research and tissue regeneration. In our study, human breast cancer biopsies were decellularized by a detergent technique, with sodium lauryl ether sulfate (SLES) solution, for the first time. And the decellularization process was optimized to maximally maintain tissue microarchitectures and extracellular matrix (ECM) components with minimal DNA compounds preserved. Histology analysis and DNA quantification results confirmed the decellularization effect with maximal genetic compounds removal. Quantification, immunofluorescence, and histology analyses demonstrated better preservation of ECM components in 0.5% SLES-treated scaffolds. Scaffolds seeded with MCF-7 cells demonstrated the process of cell recellularization in vitro, with increased cell migration, proliferation, and epithelial-to-mesenchymal transition (EMT) process. When treated with 5-fluorouracil, the expressions of stem cell markers, including Oct4, Sox2, and CD49F, were maximally maintained in the recellularized scaffold with decreased apoptosis rates compared with monolayer cells. These results showed that the decellularized breast scaffold model with SLES treatments would help to simulate the pathogenesis of breast cancer in vitro. And we hope that this model could further accelerate the development of effective therapies for breast cancer and benefit drug screenings.     

The Ability of Barley Powdery Mildew to Grow in vitro

A technique was developed for the in vitro culture of Blumeria graminis f. sp. hordei , an obligate biotrophic pathogen of barley. Optimal growth occurred at pH 5.6 on a medium containing 39 gl^–1 potato dextrose agar, 40 gl^–1 shredded fresh barley leaves, 20 gl^–1 sucrose, 13 mgl^–1 kanamycin and 80 mgl^–1 benzimidazole. At 20°C (90% relative humidity), conidia germinated 48 h after inoculation, producing an average colony diameter of 1 cm after 10 days. However, numerous colonies were present on the medium after 15 days. Light microscopy showed that there was a positive relationship between the amount of leaf in the medium and fungus growth. The fungus retained its virulence during 60 days of storage in vitro , and was able to infect barley. This is a useful and novel technique that could be beneficial in barley pathology breeding programs.     

An in vitro model for the growth and analysis of chronic wound MRSA biofilms

Aims: To develop an in vitro model (Colony/drip‐flow reactor – C/DFR) for the growth and analysis of methicillin‐resistant Staphylococcus aureus (MRSA) biofilms. Methods and Results: Using the C/DFR model, biofilms were grown on the top of polycarbonate filter membranes inoculated with a clinical isolate of MRSA, placed on absorbent pads in the DFR and harvested after 72 h. The biofilms varied from 256 to 308 μm in thickness with a repeatability standard deviation of 0·22. Testing of antimicrobial agents was also performed where C/DFR biofilms were grown in parallel with conventional colony biofilms. A saline solution (control), 1% silver sulfadiazine solution, and 0·25% Dakin’s solution were used to treat the biofilms for 15 min. Microscopic evaluation of biofilm morphology and thickness was conducted. The Dakins solution in both models produced statistically significantly higher log reductions than silver sulfadiazine treatment. Conclusions: The C/DFR biofilms were thick and repeatable and exhibited higher resistance to Dakins solution than the treated colony biofilms. Significance and Impact of the Study: The C/DFR can be used as a tool for examining complex biofilm physiology as well as for performing comparative experiments that test wound care products and novel antimicrobials.     

Orai1 contributes to the establishment of an apoptosis-resistant phenotype in prostate cancer cells

The molecular nature of calcium (Ca²⁺)-dependent mechanisms and the ion channels having a major role in the apoptosis of cancer cells remain a subject of debate. Here, we show that the recently identified Orai1 protein represents the major molecular component of endogenous store-operated Ca²⁺ entry (SOCE) in human prostate cancer (PCa) cells, and constitutes the principal source of Ca²⁺ influx used by the cell to trigger apoptosis. The downregulation of Orai1, and consequently SOCE, protects the cells from diverse apoptosis-inducing pathways, such as those induced by thapsigargin (Tg), tumor necrosis factor α, and cisplatin/oxaliplatin. The transfection of functional Orai1 mutants, such as R91W, a selectivity mutant, and L273S, a coiled-coil mutant, into the cells significantly decreased both SOCE and the rate of Tg-induced apoptosis. This suggests that the functional coupling of STIM1 to Orai1, as well as Orai1 Ca²⁺-selectivity as a channel, is required for its pro-apoptotic effects. We have also shown that the apoptosis resistance of androgen-independent PCa cells is associated with the downregulation of Orai1 expression as well as SOCE. Orai1 rescue, following Orai1 transfection of steroid-deprived cells, re-established the store-operated channel current and restored the normal rate of apoptosis. Thus, Orai1 has a pivotal role in the triggering of apoptosis, irrespective of apoptosis-inducing stimuli, and in the establishment of an apoptosis-resistant phenotype in PCa cells.