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发布于:2018-2-10 06:48:45  访问:5 次 回复:0 篇
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For flow-induced expression of lymphatic valve genes. (A and B) {Changes
P 0.01, P 0.001, calculated by Student‘s t test. (C) Western blot of FOXC2 molecular weight shift in siControl vs. siGATA2 LEC exposed to lymphatic shear for 24 hours or kept static. n = three experiments. (D ) Whole-mount staining for FOXC2 (green), GATA2 (red), and PROX1-GFP (blue) in E17.five mesenteric lymphatic vessels of Clec2+/+ and Clec2embryos. Scale bars: 100 m.imately three.five dynes/cm2 a minimum shear tension of roughly dyne/cm2, and an typical shear stress of 0.64 dynes/cm2 (15). To reproduce these forces in vitro, we exposed LEC for 48 hours to a pulsatile, reversing flow regimen with approximate maximum and minimum wall shear strain of three.25 dyne/cm2 and .45 dyne/cm2, respectively. Although there‘s a reversal element for a portion with the cycle, the net flow is forward, with an average of 0.67 dynes/cm2 (lymphatic shear pressure, Figure 5A). Dextran (five ) was added towards the cell culture media to raise the dynamic viscosity of the media to two.738 cP to be able to reach the preferred wall shear stress magnitude even though sustaining a low-flow rate equivalent to that observed in lymph in vivo. Gene expression was measured in LEC exposed to lymphatic fluid shear forces Omapatrilat site versus static control cells. The lymphatic fluid shear force regimen improved the expression of 4 genes -- FOXC2, CX37, ITGA9, and GATA2 -- which can be identified to become upregulated and play crucial roles in valve-forming LEC in vivo (refs. 6, 7, 10, 12, and Figure 5B). Substantially, LYVE1, a gene that is certainly downregulated in each collecting3002 jci.org Volume 125 Quantity 8 Augustvessels and lymphatic valves in vivo (32), is also downregulated by lymphatic flow in vitro. Interestingly, the expression of PROX1, a master regulatory transcription aspect needed for LEC specification and valve development (34, 35), was not Cetrorelix (Acetate) msds altered by lymphatic fluid shear forces in vitro (Figure 5B). Ultimately, the expression of KLF2, EFNB2, and NRP1 -- genes that are known to become regulated by higher shear strain in blood ECs (368) -- were also upregulated in LECs exposed to lymphatic fluid shear forces (Figure 5C), even though levels had been lower than have already been previously reported for BECs exposed to higher shear forces (36). Though a function for KLF2 in lymphatic vessel development has not been reported, loss of EFNB2 or NRP1 outcomes in lymphatic valve defects in vivo (13, 30), and SEMA3A-NRP1 has been shown to repel SMC recruitment at internet sites overlying lymphatic valves (30), a process that‘s defective in Clec2mice. Western blot evaluation confirmed the upregulation of GATA2, FOXC2, and CX37 proteins by lymphatic shear anxiety at the protein level (Figure 5D). Unexpectedly, the FOXC2 protein band detected right after shear strain exhibited a larger molecu-The Journal of Clinical Investigationlar weight, indicating posttranslational modification (Figure 5D). FOXC2 phosphorylation at several serine/threonine resides has been reported in LECs, and phosphorylation of FOXC2 regulates its capability to bind chromatin (39).For flow-induced expression of lymphatic valve genes. (A and B) Modifications in LEC valve gene expression induced by lymphatic fluid shear following siRNA knockdown of GATA2. Gene expression was measured in LEC exposed to lymphatic fluid shear forces versus static handle cells.
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