Stem Cell 幹細胞

Gaucher disease causes pathologic skeletal changes that are not fully explained. Considering the important role of mesenchymal stromal cells (MSCs) in bone structural development and maintenance, we analyzed the cellular biochemistry of MSCs from an adult patient with Gaucher disease type 1 (N370S/L444P mutations). Gaucher MSCs possessed a low glucocerebrosidase activity and consequently had a 3-fold increase in cellular glucosylceramide. Gaucher MSCs have a typical MSC marker phenotype, normal osteocytic and adipocytic differentiation, growth, exogenous lactosylceramide trafficking, cholesterol content, lysosomal morphology, and total lysosomal content, and a marked increase in COX-2, prostaglandin E2, interleukin-8, and CCL2 production compared with normal controls. Transcriptome analysis on normal MSCs treated with the glucocerebrosidase inhibitor conduritol B epoxide showed an up-regulation of an array of inflammatory mediators, including CCL2, and other differentially regulated pathways. These cells also showed a decrease in sphingosine-1-phosphate. In conclusion, Gaucher disease MSCs display an altered secretome that could contribute to skeletal disease and immune disease manifestations in a manner distinct and additive to Gaucher macrophages themselves.

The use of herbal dietary supplements in the United States is rapidly growing, and it is crucial that the quality and safety of these preparations be ensured. To date, it is still a challenge to determine the mechanisms of toxicity induced by mixtures containing many chemical components, such as herbal dietary supplements. We previously proposed that analyses of the gene expression profiles using microarrays in the livers of rodents treated with herbal dietary supplements is a potentially practical approach for understanding the mechanism of toxicity. In this study, we utilized microarrays to analyze gene expression changes in the livers of male B6C3F1 mice administered Ginkgo biloba leaf extract (GBE) by gavage for 2 years, and to determine pathways and mechanisms associated with GBE treatments. Analysis of 31,802 genes revealed that there were 129, 289, and 2,011 genes significantly changed in the 200, 600, and 2,000 mg/kg treatment groups, respectively, when compared with control animals. Drug metabolizing genes were significantly altered in response to GBE treatments. Pathway and network analyses were applied to investigate the gene relationships, functional clustering, and mechanisms involved in GBE exposure. These analyses indicate alteration in the expression of genes coding for drug metabolizing enzymes, the NRF2-mediated oxidative stress response pathway, and the Myc gene-centered network named "cell cycle, cellular movement, and cancer" were found. These results indicate that Ginkgo biloba-related drug metabolizing enzymes may cause herb-drug interactions and contribute to hepatotoxicity. In addition, the outcomes of pathway and network analysis may be used to elucidate the toxic mechanisms of Ginkgo biloba.

The association of kava products with liver-related health risks has prompted regulatory action in many countries. We used a genome-wide gene expression approach to generate global gene expression profiles from the livers of male B6C3F1 mice administered kava extract by gavage for 14 weeks, and identified the differentially expressed drug metabolizing genes in response to kava treatments. Analyses of gene functions and pathways reveal that the levels of significant numbers of genes involving drug metabolism were changed and that the pathways involving xenobiotics metabolism, Nrf2-mediated oxidative stress response, mitochondrial functions and others, were altered. Our results indicate that kava extract can significantly modulate drug metabolizing enzymes, potentially leading to herb-drug interactions and hepatotoxicity.

To identify the differentially expressed gene profiles in myocardium of patients with heart failure using human whole genomic oligonucleotide microarray-assisted pathway analysis. Phalanx whole genomic oligonucleotide microarrays were used to detect the gene expression profiles of myocardium in four patients died of heart failure and 4 brain died patients without heart diseases. The microarray findings were confirmed by real-time quantitative reverse transcriptase-polymerase chain reaction. The genes with a threshold of 1.2 times fold-change were selected and BioCarta Pathway and KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathway databases were used to identify functionallyrelated gene pathways. A total of 2806 genes with differentially expression were detected between the failing and non-failing heart samples,expression changes of 399 genes were more than 2-folds. Eleven pathways were identified by BioCarta pathway database and sixteen pathways were identified by KEGG PATHWAY Database. Genomic microarray-assisted pathway analysis could help to identify gene expression profiles in failing heart.

Human mesenchymal stem cells (hMSCs) can be genetically modified with viral vectors and hold promise as a cell source for regenerative medicine, yet how hMSCs respond to viral vector transduction remains poorly understood, leaving the safety concerns unaddressed. Here, we explored the responses of hMSCs against an emerging DNA viral vector, baculovirus (BV), and discovered that BV transduction perturbed the transcription of 816 genes associated with five signaling pathways. Surprisingly, Toll-like receptor-3 (TLR3), a receptor that generally recognizes double-stranded RNA, was apparently upregulated by BV transduction, as confirmed by microarray, PCR array, flow cytometry, and confocal microscopy. Cytokine array data showed that BV transduction triggered robust secretion of interleukin-6 (IL-6) and IL-8 but not of other inflammatory cytokines and beta interferon (IFN-beta). BV transduction activated the signaling molecules (e.g., Toll/interleukin-1 receptor domain-containing adaptor-inducing IFN-beta, NF-kappaB, and IFN regulatory factor 3) downstream of TLR3, while silencing the TLR3 gene with small interfering RNA considerably abolished cytokine expression and promoted cell migration. These data demonstrate, for the first time, that a DNA viral vector can activate the TLR3 pathway in hMSCs and lead to a cytokine expression profile distinct from that in immune cells. These findings underscore the importance of evaluating whether the TLR3 signaling cascade plays roles in the immune response provoked by other DNA vectors (e.g., adenovirus). Nonetheless, BV transduction barely disturbed surface marker expression and induced only transient and mild cytokine responses, thereby easing the safety concerns of using BV for hMSCs engineering.

Although EGb 761 exhibits neuroprotective effects and exerts beneficial effects on many neurological disorders, its mechanism on the neuronal functions is unclear so far. In this study, we used oligonucleotide microarray technique to investigate the effect of EGb 761 on the transcriptional profile of mouse genes. RNA samples were obtained from frontal cortex, straitum, and kidneys after the oral administration of EGb 761 for seven consecutive days. Our data showed that EGb 761 significantly altered the neuroactive ligand-receptor interaction pathway in frontal cortex but not in straitum and kidney. Then we analyzed 26 receptor genes that were significantly altered by EGb 761 in this pathway and found that EGb 761 treatment highly up-regulated the subgroup of dopamine receptors, especially dopamine receptor 1a (Drd1a), in frontal cortex. Quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemical staining confirmed the increased level of Drd1a expression after EGb 761 treatment. In summary, we investigated for the first time the overall effects of EGb 761 on the gene expression in brain using a powerful systemic biological technique. Our results suggested that EGb 761 altered unique pathways and regulated the expressions of some specific neuronal receptor genes exclusively in frontal cortex.

Hepatocellular carcinoma (HCC) is a major challenge because of its resistance to conventional cytotoxic chemotherapy and radiotherapy. Multi-targeted therapy might be a new option for HCC treatment. Our previous study showed that N-ras gene was activated in HCC and was inhibited by RNA interference. In the present study, we investigated the alternation of gene expression by microarray in N-Ras-siRNA-treated HepG2 cells. The results revealed that the EREG gene, encoding epiregulin, was dramatically up-regulated in response to silence of N-ras. We speculated that the up-regulation of epiregulin was involved in the compensatory mechanism of N-ras knockdown for cell growth. Therefore, we evaluated whether dual silence of N-ras and epiregulin display a greater suppression of cell growth. The results confirmed that dual knockdown of N-ras and epiregulin synergistically inhibited cell growth. Our results also showed that dual knockdown of N-ras and epiregulin significantly induced cell arrest at G0/G1 phase. Furthermore, Western blot assay showed that dual knockdown of N-ras and epiregulin markedly reduced the phosphorylations of ERK1/2, Akt and Rb, and inhibited the expression of cyclin D1. Our findings imply that multi-targeted silence of oncogenes might be an effective treatment for HCC.