膀胱移行细胞癌免疫相关基因的研究

目的 探讨人膀胱移行细胞癌组织中免疫功能相关基因的表达变化。方法使用人肿瘤基因表达谱芯片检测11例膀胱移行细胞癌组织基因表达谱的变化，以寻找与免疫功能相关的差异表达的基因。结果以正常膀胱黏膜组织为对照，膀胱肿瘤组织中有87个基因表达明显下调，102个基因表达明显上调。其中与免疫功能相关的基因有17个，明显上调基因8个，明显下调基因9个。结论膀胱肿瘤的发生、发展与多种免疫功能相关基因的异常表达有关。     

Hypoxia and an angiogenic response in the partially obstructed rat bladder

Previous molecular and blood flow studies performed on animal models of partial bladder outlet obstruction (PBOO) caused us to propose that bladder hypoxia/ischemia was a significant effector of the cellular and functional changes that occur in the bladder as a result of this condition. To confirm the occurrence of hypoxia in the partially obstructed bladder, we obtained rat bladders at increasing intervals following PBOO and measured biomarkers of hypoxia (intracellular formation of hypoxyprobe-1 adducts and expression of hypoxia inducible factor-1 alpha [HIF-1 alpha] protein) and whether such hypoxia might elicit an angiogenic response in the tissue. Rats receiving PBOO or controls were treated with hypoxyprobe-1 at increasing intervals subsequent to surgery and their bladders were sectioned and immunostained using an antibody that detects hypoxyprobe-1 adducts. Control rat bladders were unstained, whereas intense, but regionally restricted, hypoxyprobe-1 immunostaining was detected in all obstructed bladders in a unique pattern that changed over time. Proteins were extracted from bladders removed from similarly treated rats and were analyzed for the expression of the HIF-1 alpha protein as well as for expression of angiogenic regulatory factors (vascular endothelial growth factor, angiopoietin-1, and endostatin) using Western blotting techniques. HIF-1 alpha protein was not expressed in control bladders, however, the protein was highly up-regulated over the 2-week period after PBOO. Likewise, the expression of vascular endothelial growth factor (a downstream target of HIF-1 alpha action) and angiopoietin-1 was also up-regulated in obstructed bladders confirming an angiogenic response to this hypoxia. Enigmatically, however, expression of the antiangiogenic molecule endostatin was also up-regulated by chronic PBOO. These results further support the concept that hypoxia is involved in the cellular remodeling as well as in the progressive functional impairment exhibited by the urinary bladder after PBOO.     

膀胱癌基因芯片生物信息分析及功能探讨

目的 从分子水平探讨膀胱癌的发病机制,为临床诊断及预后评估提供新思路。 方法 从基因芯片数据库中下载人膀胱癌的相关基因芯片数据GSE31189（包括52例尿路上皮癌和40例正常膀胱组织）,使用Morpheus（software.broadinstitute.org/morpheus/）在线工具分析癌症尿路上皮组织和正常尿路上皮组织的差异表达基因,然后使用生物技术信息基因云（GCBI,https://www.GCBI.com.cn)在线分析系统进行差异表达基因信号通路富集以及差异基因之间相互作用的分析,最后选择差异基因通过细胞计数试剂盒8（CCK-8）、侵袭实验和免疫印迹法初步验证其功能。 结果 按q值进行排序并筛选出差异最大的前20个基因,其中膀胱癌中上调基因18个,下调基因2个。基因分类（GO）分析发现,这些差异基因主要集中在炎症反应、免疫反应,负调控凋亡过程,来自RNA聚合酶Ⅱ启动子的转录负调控等多个生物学功能。信息通路（Pathway）分析结果显示,这些差异基因主要参与癌症中转录失调、代谢途径、核因子（NF）-κB信号通路等生物学过程。基因网络分析发现,CXC趋化因子受体4（CXCR4）、丝裂原活化蛋白激酶10（MAPK10）是这些基因网络的中心环节。初步体外实验表明,基质金属蛋白酶12（MMP-12）下调后,膀胱癌细胞的增殖明显减少,侵袭能力下降,细胞外调节蛋白激酶（ERK）磷酸化水平下降。 结论 通过多重生物信息学分析可以找出膀胱肿瘤中关键基因,CXCR4、MAPK10是膀胱基因网络中的关键环节,MMP-12在膀胱癌细胞中高表达,下调MMP-12可抑制膀胱癌细胞增殖和侵袭,它可能通过ERK途径发挥其生物学功能。     

Gene expression profile in labouring and non-labouring human placenta near term

The duration of pregnancy and initiation of labour are thought to be controlled by fetal, maternal and placental factors. The aim of this study was to investigate whether labour influences gene expression in placenta near term. Placental samples were obtained from 27 women after vaginal delivery (labouring) and 17 women after elective Caesarean section (non-labouring). All women were Caucasian and had uncomplicated pregnancies. For global gene expression analysis, 17 human oligo-arrays were used, representing 24 650 genes each. An empirical Bayes analysis was applied in order to find differentially expressed genes. About 8000 genes that were represented on the arrays met our quality criteria. Ninety two genes were down-regulated and 94 genes were up-regulated in labouring placentas compared to non-labouring placentas. However, none of these was differentially expressed at a significant level (>2.5-fold change and a P-value of <0.01). We conclude that gene expression in near term human placenta is not significantly altered by labour.     

Effect of Sustained Tension on Bladder Smooth Muscle Cells in Three-Dimensional Culture

Previous studies demonstrated that bladder cells respond to changes in their mechanical environments by exhibiting alterations in cellular functions, such as hypertrophy or fibrosis. In the present study, we hypothesize that changes in smooth muscle cell (SMC) behavior triggered by mechanical stimuli may represent a phenotypic shift between contractile and synthetic phenotypes. Using a custom-made device, rat bladder SMCs were cultured in three-dimensional (3-D) collagen gels and exposed to sustained tension. When compared to no-tension controls, SMCs exposed to tension exhibited significantly (p < 0.05) higher expression of alpha-smooth muscle actin (α-SMA), while cell population density was similar in both groups. In addition, both mean and median aspect ratios of SMCs in 3-D collagen constructs exposed to tension were significantly (p < 0.05) greater than those of cells cultured under no externally applied tension, indicating that there are more elongated, spindle-shaped cells in the tension group. These SMCs in 3-D cultures exposed to tension also exhibited cellular alignment along the direction of applied tension. Since contractile SMCs are known to exhibit greater expression of phenotypic marker proteins as well as a more elongated morphology, we concluded that sustained tension on cells is an important mechanical stimulus for maintenance of the contractile phenotype of bladder SMCs in vitro.     

肌层浸润性膀胱癌亚型的生物信息学分析

目的通过生物信息学分析研究两种膀胱癌亚型(基底样膀胱癌和管腔型膀胱癌)之间不同的分子调控机制和分子特性,为更准确地区分膀胱癌亚型和探索潜在的治疗靶点提供帮助。方法利用稳健的多芯片平均算法将由22个基底样膀胱癌和132管腔型膀胱癌样本组成的数据集进行标准化,并选择其中前1000个具有最高标准差的基因进行两种亚型的差异表达基因分析。将得到的差异表达基因进行GO功能注释和KEGG通路富集分析。此外,选择前100个差异表达基因构建蛋白质互作网络。结果得到基底样和管腔型膀胱癌差异表达基因共742,其中基底样亚型上调的基因405个,下调的基因337个。GO富集分析显示差异表达基因显著富集在细胞外区基质、趋化性、炎症等功能上,KEGG通路富集显示差异表达基因显著富集在细胞外基质受体相互作用的通路上。构建的蛋白质互作网络显示重要的hub蛋白质为LNX1、MSN和PPARG。结论本研究得到的基底样和管腔型膀胱癌亚型分子机制的区别主要体现在细胞外区域的分子作用机制、细胞趋化性和炎症反应等,基因LNX1、MSN和PPARG为区别两种膀胱癌亚型的特征基因。     

Gene expression and chromosomal location for susceptibility to Sjögren's syndrome

Primary Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease affecting mainly the exocrine glands. Its physio-pathology is poorly understood and most of the knowledge has been related to the inflammatory component. The aim of this work was to evaluate gene expression profiling in fractions enriched in epithelial cells from labial salivary glands (LSGs) of patients with primary SS and identify chromosomal regions harboring susceptibility genes expressed in epithelial cells. A combined approach of gene expression and genome-wide association study was used. Enriched epithelial cell fractions were obtained from LSGs of patients and controls. Amplified total RNA was labeled and hybridized to 10K cDNA microarrays. Results were normalized and subjected to statistical and functional analysis. A genome-wide microsatellite screen at 10 cM resolution (393 markers) was performed. In salivary gland-epithelial cells from patients 528 genes were expressed differentially in comparison to controls. Pathways not previously linked to disease were found to be altered. Twenty-eight and 15 genes associated with apoptosis were up-regulated and down regulated, respectively. Interferon-related genes, most of which participated in interferon signaling, were also found to be up-regulated. From the genome-wide screen, 6 markers showed evidence of highly significant association with the disease. Of these, five loci harbor genes differentially expressed in patients LSG-epithelial cells. Our results show that in enriched gland-epithelial cells of pSS, both pro-apoptotic/anti-apoptotic and interferon signaling inhibition/stimulation balances may occur. Genes found over-expressed in epithelial cells are candidates for disease susceptibility.     

Altered urinary bladder function in mice lacking the vanilloid receptor TRPV1

In the urinary bladder, the capsaicin-gated ion channel TRPV1 is expressed both within afferent nerve terminals and within the epithelial cells that line the bladder lumen. To determine the significance of this expression pattern, we analyzed bladder function in mice lacking TRPV1. Compared with wild-type littermates, trpv1(-/-) mice had a higher frequency of low-amplitude, non-voiding bladder contractions. This alteration was accompanied by reductions in both spinal cord signaling and reflex voiding during bladder filling (under anesthesia). In vitro, stretch-evoked ATP release and membrane capacitance changes were diminished in bladders excised from trpv1(-/-) mice, as was hypoosmolality-evoked ATP release from cultured trpv1(-/-) urothelial cells. These findings indicate that TRPV1 participates in normal bladder function and is essential for normal mechanically evoked purinergic signaling by the urothelium.     

The effect of enzymatically degradable poly(ethylene glycol) hydrogels on smooth muscle cell phenotype

The formation of scar tissue due to dedifferentiation of smooth muscle cells (SMCs) is one of the major issues faced when engineering bladder tissue. Furthermore, cell sources for regenerating the SMC layer are also limiting. Here we explore if human mesenchymal stem cells (MCSs), cultured in enzymatically degradable poly(ethylene glycol) (PEG) hydrogel scaffolds can be differentiated into SMC-like cells. We explored the degree to which a less synthetic SMC phenotype can be achieved when primary human SMCs are cultured within these scaffolds, It was observed that when both MSCs and SMCs are cultured in the PEG hydrogel scaffolds, but not on traditional tissue culture plastic, they up-regulate markers associated with the less synthetic SMC phenotype, decreased expression of alpha(5) integrin and THY-1, and increased expression of alpha-smooth muscle actin (alphaSMA) and myosin. Furthermore, we show that MSCs and SMCs cultured in the PEG hydrogels are able to proliferate and express matrix metalloproteinases for up to 21d in culture, the duration of the study. This study addresses the importance of the cellular microenvironment on cell fate, and proposes synthetic instructive biomaterials as a means to direct cell differentiation and circumvent scar tissue formation during bladder reconstruction.     

力生长因子E肽与应力刺激对成骨细胞基因表达的影响

目的应用基因芯片技术分析在力生长因子E肽(MGF-Ct24E)和应力作用下成骨细胞基因表达的差异。方法体外培养原代成骨细胞,分别对细胞施加周期性拉伸刺激(应变12%,频率0.5 Hz)和MGF-Ct24E(50 mg/L)直接作用,基因芯片技术分析力学刺激和MGF-Ct24E作用对原代成骨细胞基因表达谱的影响,并用定量PCR验证基因芯片实验结果。结果与对照组相比,力学加载组共发现差异表达基因1 866个,其中上调基因1 113个,下调基因753个;MGF-Ct24E处理组共发现差异表达基因1 178个,其中上调基因796个,下调基因382个。GO分析发现两者的差异基因表达谱具有一致性,并且差异表达基因主要涉及细胞增殖与分化调节、细胞对应力刺激的响应和力学转导通路等。定量PCR实验结果验证的差异表达基因与芯片实验结果一致。结论基因表达谱分析显示应力刺激和MGF-Ct24E作用对成骨细胞的基因表达具有类似的调控效应,为后续使用MGF-Ct24E治疗骨修复以弥补应力刺激不足的研究提供了新的思路。     

In vivo gene expression analysis identifies genes required for enhanced colonization of the mouse urinary tract by uropathogenic Escherichia coli strain CFT073 dsdA

Deletional inactivation of the gene encoding d-serine deaminase, dsdA, in uropathogenic Escherichia coli strain CFT073 results in a hypermotile strain with a hypercolonization phenotype in the bladder and kidneys of mice in a model of urinary tract infection (UTI). The in vivo gene expression profiles of CFT073 and CFT073 dsdA were compared by isolating RNA directly from the urine of mice challenged with each strain individually. Hybridization of cDNAs derived from these samples to CFT073-specific microarrays allowed identification of genes that were up- or down-regulated in the dsdA deletion strain during UTI. Up-regulated genes included the known d-serine-responsive gene dsdX, suggesting in vivo intracellular accumulation of d-serine by CFT073 dsdA. Genes encoding F1C fimbriae, both copies of P fimbriae, hemolysin, OmpF, a dipeptide transporter DppA, a heat shock chaperone IbpB, and clusters of open reading frames with unknown functions were also up-regulated. To determine the role of these genes as well as motility in the hypercolonization phenotype, mutants were constructed in the CFT073 dsdA background and tested in competition against the wild type in the murine model of UTI. Strains with deletions of one or both of the two P fimbrial operons, hlyA, fliC, ibpB, c0468, locus c3566 to c3568, or c2485 to c2490 colonized mouse bladders and kidneys at levels indistinguishable from wild type. CFT073 dsdA c2398 and CFT073 dsdA focA maintained a hypercolonization phenotype. A CFT073 dsdA dppA mutant was attenuated 10- to 50-fold in its colonization ability compared to CFT073. Our results support a role for d-serine catabolism and signaling in global virulence gene regulation of uropathogenic E. coli.     

基于癌症基因组图谱筛查卵巢浆液性囊腺癌相关基因

目的:利用全基因组表达谱芯片筛查与卵巢浆液性囊腺癌发生相关的基因,对在卵巢浆液性囊腺癌发生过程中可能参与的基因间的信号转导通路进行分析。方法:选取癌症基因组图谱(TCGA)数据库中卵巢浆液性囊腺癌的Affymetrix Gene Chip Human Exon 1.0 ST Array数据共16张,分别为卵巢浆液性囊腺癌组8张和正常组8张,筛选出差异表达基因,并进行基因本体(gene ontology,GO)分析和信号通路分析,构建卵巢浆液性囊腺癌相关基因间的信号转导通路,分析网络中具有重要作用的基因。结果:共筛选出1 144个在卵巢癌中差异表达的基因,其中表达上调的基因有747个,表达下调的基因有397个。GO分析得到上调差异基因的显著性功能分析结果362项,下调差异基因的显著性功能分析结果 160项(P0.05)。其中包括与肿瘤发生相关的基因功能有细胞周期、DNA复制、细胞增殖、细胞凋亡、细胞黏附等。信号通路分析得到45个显著上调信号通路和14个显著下调信号通路(P0.05)。其中参与肿瘤发生相关的信号通路主要有细胞周期、P53信号通路、DNA复制、肿瘤中的信号通路、PI3K-Akt信号通路、ECM-receptor信号通路、细胞黏附因子、细胞凋亡等。挑选显著性基因功能和信号通路分析的交集基因229个,构建显著性GO与信号通路基因间信号转导网络。分析发现CDK1、PLK1、MCM3和PGK1这4个基因在卵巢癌的基因调控网络中具有重要作用。结论:卵巢浆液性囊腺癌中有大量差异表达基因,差异表达的基因在多个与肿瘤发生密切相关的信号通路中发挥重要的调控作用。     

Gene expression profile analysis of Porphyromonas gingivalis during invasion of human coronary artery endothelial cells

Microarrays were used to identify genes of Porphyromonas gingivalis W83 differentially expressed during invasion of primary human coronary artery endothelial cells. Analyses of microarray images indicated that 62 genes were differentially regulated. Of these, 11 genes were up-regulated and 51 were down-regulated. The differential expression of 16 selected genes was confirmed by real-time PCR.     

The influence of RGD-bearing hydrogels on the re-expression of contractile vascular smooth muscle cell phenotype

This study reports on the ability of poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with pendant integrin-binding GRGDSP peptides (RGD-gels) to support the re-differentiation of cultured vascular smooth muscle cells (SMCs) toward a contractile phenotype. Human coronary artery SMCs were seeded on RGD-gels, hydrogels with other extracellular matrix derived peptides, fibronectin (FN) and laminin (LN). Differentiation was induced on RGD-gels with low serum medium containing soluble heparin, and the differentiation status was monitored by mRNA expression, protein expression, and intracellular protein organization of the contractile smooth muscle markers, smooth muscle α-actin, calponin, and SM-22α. RGD-gels supported a rapid induction (2.7- to 25-fold up-regulation) of SMC marker gene mRNA, with expression levels that were equivalent to FN and LN controls. Marker protein levels mirrored the changes in mRNA expression, with levels on RGD-gels indistinguishable from FN and LN controls. Furthermore, these markers co-localized in stress fibers within SMCs on RGD-gels suggesting the recapitulation of a contractile apparatus within the cells. These results indicate that SMCs cultured on RGD-bearing hydrogels can re-differentiate toward a contractile phenotype suggesting this material is an excellent candidate for further development as a bioactive scaffold that regulates SMC phenotype.     

Regeneration of native-like neo-urinary tissue from nonbladder cell sources

Urinary pathology requiring urinary diversion, partial or full bladder replacement, is a significant clinical problem affecting ~14,000 individuals annually in the United States alone. The use of gastrointestinal tissue for urinary diversion or bladder reconstruction/replacement surgeries is frequently associated with complications. To try and alleviate or reduce the frequency of these complications, tissue engineering and regenerative medicine strategies have been developed using bio-absorbable materials seeded with cells derived from the bladder. However, bladder-sourced cells may not always be suitable for such applications, especially in patients with bladder cancer. In this study, we describe the isolation and characterization of smooth muscle cells (SMCs) from porcine adipose and peripheral blood that are phenotypically and functionally indistinguishable from bladder-derived SMCs. In a preclinical Good Laboratory Practice study, we demonstrate that autologous adipose- and peripheral blood-derived SMCs may be used to seed synthetic, biodegradable tubular scaffold structures and that implantation of these seeded scaffolds into a porcine cystectomy model leads to successful de novo regeneration of a tubular neo-organ composed of urinary-like neo-tissue that is histologically identical to native bladder. The ability to create urologic structures de novo from scaffolds seeded by autologous adipose- or peripheral blood-derived SMCs will greatly facilitate the translation of urologic tissue engineering technologies into clinical practice.