Bv8 nucleic acids and polypeptides with mitogenic activity
A cell proliferation and endothelial cell technology, applied in the direction of organic active ingredients, medical preparations containing active ingredients, animal cells, etc., can solve the problem of unclear properties of local beneficial signals
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Embodiment 1
[0442] Example 1 Northern blot analysis
[0443] To elucidate the expression pattern of Bv8, Northern blot analysis was performed with RNA from various tissues of human, mouse and rat. Human Northern blot with 32 P-labeled human Bv8 cDNA-based DNA probe hybridization, mouse and rat Northern blots with 32 P-labeled murine Bv8 cDNA-based DNA probe hybridization.
[0444] Northern blot analysis was performed according to methods known in the art. For example, with 30-50ng human or mouse cDNA fragments, Redi-Prime II Kit (Amersham), 32 P-dCTP 3000 μCi / mmol (Amersham) was used to prepare cDNA probes. Probes were purified on Sephadex G50 spin columns (Pharmacia) and hybridized in ExpressHyb hybridization solution (Stratagene) at 68°C. In another example, blots were incubated with probes in hybridization buffer (5X SSPE; 2X Denhardt's solution; 100 mg / mL denatured and sheared salmon sperm DNA; 50% formamide; 2% SDS) at 42°C for 60 Hours. Blots were washed several times in 2X...
Embodiment 2
[0446] Example 2 Analysis of Cell Proliferation
[0447] To determine the response of specific cell types to Bv8, the proliferative responses of bovine adrenal cortical capillary endothelial cells (ACE) and bovine brain capillary endothelial cells (BBC) were analyzed.
[0448] Briefly, ACE (adrenocortical capillary endothelial cells) and BBC (bovine brain capillary endothelial cells) were cultured in low glucose DMEM supplemented with 10% calf serum. For cell proliferation analysis, 6000 cells were plated in each well of a 12-well plate containing the above-mentioned medium, no control ("C" in Figure 11), 10 ng / ml VEGF ("V" in Figure 11 ”), 50, 10 or 1 nM Bv8 (Fc-tagged recombinant protein). Total cell counts were obtained after 1 week with a Coulter counter. Randomly set the control condition to 1 and obtain the fold increase in cell number relative to it. Media and other cell culture reagents were obtained from Life Technologies, Inc. The performance of the assay can b...
Embodiment 3
[0450] Example 3 Cell Survival Analysis
[0451] The effect of Bv8 on endothelial cell survival was examined. will be approximately 2x10 5Bovine brain capillary (BBC) cells were plated in each well of a 6-well plate containing complete medium (see Example 2). The next day, the complete medium was aspirated, and the cells were cultured in a medium without any additives, or in a medium containing one of the following components: 2% FCS, 10% FCS, 20ng / ml VEGF ( Figure 12 "V"), 5nM Bv8, 25nM Bv8, 20ng / ml VEGF+25nM Bv8 ( Figure 12 "V+Bv8"), or 25nM EG-VEGF. After 48 hours of incubation, cells were trypsinized, removed and fixed in cold 70% ethanol for several hours. Cells were then stained with 5 μg / ml propidium iodide and 20 ng / ml RNase in PBS for 2-4 hours at room temperature. Cells in sub-G1 phase were determined by FACS analysis. The percentage of the cell population as Figure 12 The percentage of apoptotic cells on the vertical axis is plotted.
[0452] Such as ...
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