胃保护素对破骨细胞的作用.ppt

Ghrelin is an Osteoblast Mitogen and Increases Osteoclastic Bone Resorption In Vitro Ghrelin(Ghrelin(胃动素、胃肠动素胃动素、胃肠动素)是生长激素促分泌物质受体是生长激素促分泌物质受体(growth hormone secretagogue receptor,GHS-R)(growth hormone secretagogue receptor,GHS-R)的内源性的内源性配体配体.GhrelinGhrelin的结构：的结构：Ghrelin是由是由28个氨基酸组成的小分子多肽，它有两种形式：个氨基酸组成的小分子多肽，它有两种形式：Ghrelin-28和和des-Gln14-ghrelin,除了第除了第14位氨基酸位氨基酸Gln的缺的缺失失,des-Gln14-ghrelin生物活性与生物活性与Ghrelin相同。相同。Ghrelin N端第端第3位丝氨酸残基上的位丝氨酸残基上的n-辛酰基辛酰基（n-octanoyl group）对）对生物活性具有重要作用。人和大鼠的生物活性具有重要作用。人和大鼠的Ghrelin在进化上高度保在进化上高度保守，仅守，仅11位和位和12位上相差两个氨基酸。位上相差两个氨基酸。Background KnowledgeStructures of human and rat ghrelinsBoth human and rat ghrelins are 28-amino acid peptides,in which Ser3 is modified by a fatty acid,primarily n-octanoic acid.This modification is essential for ghrelins activity.K：赖氨酸，A丙氨酸;R:精氨酸，V:缬氨酸The structure of ghrelin receptor,GHSRGHSR is a seven transmembrane-domain G-protein coupled receptor.Two transcripts are produced from the human GHSR gene by alternative splicing:GHSR1a encodes the functional receptor,and GHSR1b,which is truncated after transmembrane domain 5,appears to be unable to stimulate intracellular signaling pathways.Signal transduction pathwayGH secretagogues(GHSs)stimulate GH release through the GHS receptor(GHS-R or ghrelin receptor)to increase intracellular Ca2(Ca2i)levels.GHSs are artificial molecules and do not exist in nature,so there is no structural homology between ghrelin and peptide GHSs.SummaryvGhrelin is released in response to fasting,such that circulating levels are highest immediately prior to meals.Ghrelin increased the bone-resorbing activity of rat osteoclasts,but did not alter osteoclast differentiation in a murine bone marrow assay nor bone resorption in ex vivo calvarial cultures.Ghrelin showed mitogenic activity in osteoblasts,with a strong effect in human cells and a weaker effect in rat osteoblasts.1.IntroductionBone turnover is acutely responsive to food intake,with bone resorption increased during fasting and suppressed during feeding.Numerous factors,such as nutritional content or size of meals,may play a role in this relationship and there is evidence that some gut hormones,such as glucagon-like peptide-2 3,4,also affect the normal postprandial reduction of bone resorption.A number of studies investigated the effects of ghrelin in skeletal tissue.A study of the expression of GHSR1a in human tissues demonstrated a weak positive signal in bone marrow。Osteoclast response to ghrelin has never been investigated previously.The aim of the current study was to further investigate the activity of ghrelin in bone cells,and in particular its possible role in the acute stimulation of bone resorption that occurs during fasting.Acylated ghrelin BSA(牛血清白蛋白)Gibco brand FBS(胎牛血清)and media were used Tritiated thymidine（氚化胸腺嘧啶）Tartrate-resistant acid phosphatase(TRAP)staining kit (抗酒石酸酸性磷酸酶染色试剂盒试)1,25-dihydroxyvitamin D3 osteoprotegerin(OPG)（骨保护素）salmon calcitonin（鲑鱼降钙素）MaterialsOsteoclast differentiation in mouse bone marrow cultures1,25V-D3(10nM)0,2,5d2,4dGhrelin C(0.1-10nM)7dTRAP stainingMNC48-well plates Mixed marrow cells The mixed population of cells from the bone marrow is cultured in the presence of 1,25-dihydroxyvitamin D3 that promotes osteoclast formationResult Osteoclast differentiation in mouse bone marrow cultures is not affected by ghrelin.OPG:Recombinant human osteoprotegerin(10 ng/mL)was included as a positive control for inhibition of osteoclastogenesis.Ghrelin increased the bone-resorbing activity of rat osteoclasts in vitroBovine bone slices Mature osteoclast Incubate for 24hGhrelin C:1nM/10nM fixed and stained for TRAPTRAP(+)CountScrubbingReflected light image of resorption pits formed by rat osteoclasts on bovine bone slices Stain and dry Resultsv Ghrelin at a concentration of 1 nM induced a 30%increase in the number of pits excavated by osteoclasts,whereas at 10 nM there was only a 15%increase which was not significantly different from the control.The potent inhibitor of osteoclast activity,salmon calcitonin(1010 M),was used as a positive control.vOsteoclast activity in mouse calvarial culture is not affected by ghrelin.Calvariae labeled in vivo with 45Ca were collected and incubated ex vivo with experimental substances orvehicle for 48 hours.Values are expressed as percent released 45Ca detected over total 45Ca injected(3 Ci).PTH:parathyroid hormone(positive control).Ghrelin is mitogenic to primary human osteoblasts.3H-thymidine incorporation was measured as an indicator of cell proliferation.Human primary osteoblasts incubated for 24 or 48 hours in the presence of 0.1 nM ghrelin showed a 2-fold increase in proliferation,as measured by thymidine incorporation.How to determine thymidine incorporation?vGhrelin weakly induced cell proliferation in primary rat osteoblasts,with a 1.1-1.2-fold stimulation of thymidine incorporation(a)and a similar,modest increase in cell numbers(b).Conclusion Ghrelin increased the bone-resorbing activity of rat osteoclasts,but did not alter osteoclast differentiation in a murine bone marrow assay nor bone resorption in ex vivo calvarial cultures.Ghrelin showed mitogenic activity in osteoblasts,with a strong effect in human cells and a weaker effect in rat osteoblasts.感谢亲观看此幻灯片，此课件部分内容来源于网络，感谢亲观看此幻灯片，此课件部分内容来源于网络，如有侵权请及时联系我们删除，谢谢配合！如有侵权请及时联系我们删除，谢谢配合！