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发布于:2018-5-8 09:52:57  访问:35 次 回复:0 篇
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Invasion in each of the shHSP27 constructs tested (Fig 2E). We
3A that the number of purchase APTO-253 metastases in knockdown mice was 30 of that of controls, while in overexpression mice it was 400 of controls, ANOVA p value = 0.023. Figure 2F depicts representative data from a 5-day MTT assay, showing no significant change in cell growth for HSP27 overexpression compared to VC cells, or for HSP27 knockdown compared to shCO cells. Taken together, these findings demonstrate that chronic alterations in HSP27 expression impact the invasive phenotype of human PCa cells in vitro. Surprisingly, HSP27 expression did not affect cell growth in our experimental model system.HSP27 Regulates Human Prostate Cancer MetastasisHSP27 expression is increased in primary PCa lesions [6, 10, 35], but its role in regulating their progression to distant metastasis remains unclear. We demonstrated above that chronic overexpression of HSP27 increases cell invasion in vitro. Given that cell invasion constitutes an initial step in the metastatic cascade, we hypothesized that HSP27 would increase invasion of cells out of the prostate gland and that this would result in increased formation of distant metastasis. To test this, we employed an orthotopic murine model of human PCa metastasis originally developed by Stephenson et. al. [36] and further refined and characterized by us to allow for quantification of distant organ metastasis [37, 38]. This model closely recapitulates human disease in that it requires cells to complete all of the steps in the metastatic cascade, including initial steps, such as invasion out of the prostate gland, as well as latter steps, involving formation of distant metastasis. There were four different cohorts of mice, 20 mice per cohort, and they were implanted with cell lines as follows: vector control (10 mice implanted with VC-1 cells, 10 with VC-2 cells), HSP27 overexpression (10 mice with HSP27WT-H1 cells, 10 with HSP27-WT-H2 cells), fpsyg.2016.01448 shRNA control (10 mice with shCO-1 cells, 10 with shCO-2 cells), and HSP27 knockdown (10 mice with shHSP27-www.impactjournals.com/oncotargetcells, 10 mice with shHSP27-2 cells). Six weeks after implanting cells into the prostate of 6-8 week old male Balb/c athymic mice, distant metastasis was quantified. Overexpression and knockdown mice by necessity had different controls, based upon the fact that each required transfection with different vector constructs. In order to examine the effect of HSP27 upon metastasis, and to do so across a range of HSP27 expression, we normalized metastasis in knockdown and overexpression mice to their relevant controls. It can be seen in Fig. 3A that the number of metastases in knockdown mice was 30 of that of controls, while in overexpression mice it was 400 of controls, ANOVA p value = 0.023. Comparisons between individual groups were also performed. We did so by determining the median value of metastasis in control mice, and then used Fisher‘s exact test to evaluate whether mice in the other groups had more or less metastasis than the median value. In this manner, knockdown mice were shown to have significantly less metastasis than controls (2-sided p value 0.03), fpsyg.2016.00135 overexpression mice exhibited a trend towards an increase compared to control (p value 0.28), and overexpression mice had significantly more metastasis compared to knockdown mice (p value 0.005).
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