环氧合酶-2在小鼠月经样模型中对子宫内膜崩解剥脱的作用研究

目的探究环氧合酶-2(Cyclooxygenase-2,COX-2)在小鼠子宫内膜崩解过程中的表达及作用。方法首先通过生理性孕酮撤退的方法建立小鼠月经样模型(n=10),Western blot检测了COX-2在孕酮撤退后0 h、8 h、12 h、16 h和24 h的模型小鼠子宫内的表达情况。随后根据是否用COX-2选择性抑制剂Dup-697进行子宫内膜崩解干预实验,将40只小鼠分为干预组(Dup-697干预月经样模型小鼠,n=20)和对照组(月经样模型小鼠,n=20)。干预组在孕酮撤退前6 h以及撤退后0 h、6 h、12 h和18 h向小鼠腹腔注射剂量为12.5 mg/kg Dup-697,对照组小鼠在相同时间点腹腔注射等量生理盐水。孕酮撤退后0 h、8 h、12 h、16 h和24 h处死小鼠并分离子宫,观察子宫大体,通过HE染色与相对崩解面积的计算,比较小鼠子宫内膜的崩解程度。结果 Western blot结果显示,月经样模型小鼠子宫中的COX-2蛋白表达量从孕酮撤退后12 h开始显著升高,在24 h时达到最高峰值(P0.01)。子宫大体和组织形态学结果显示,与对照组相比,实验组小鼠子宫颜色较浅,子宫内膜相对崩解面积显著降低(P0.01)。结论 COX-2在小鼠月经样模型中能够促进子宫内膜的崩解剥脱。     

肿瘤坏死因子在小鼠药理性孕酮撤退月经样模型中的定位和表达

目的 在药理性撤退小鼠月经样模型中,观察孕酮撤退后,肿瘤坏死因子(TNF-α)的定位和表达情况.方法 卵巢切除的小鼠在诱导蜕膜化之后给予米非司酮即通过拮抗孕酮受体的方法(药理性撤退),导致孕酮撤退,并用免疫组化的方法观察米非司酮处理的不同时相,TNF-α在小鼠子宫内膜的定位;用实时PCR的方法观察TNF-α表达情况.结果 免疫组化结果显示米非司酮处理后0～8 h,TNF-α主要存在于腔周围的血管内皮细胞;12～16 h,主要存在于内流的白细胞;24～48 h,主要存在于崩解组织边缘的间质细胞区域和修复好腺上皮细胞周围的间质细胞.实时PCR结果显示TNF-α mRNA在孕酮撤退后24 h内都有表达,并且TNF-a表达量逐渐增高,在12h、16h达到峰值. 结论 在此小鼠月经样模型中,米非司酮处理即孕酮撤退后TNF-α在此模型子宫中定位呈区域性分布,并诱导TNF-α表达升高,可能参与了子宫内膜的崩解.     

HIF1A与VEGF在小鼠月经样模型的表达与共定位

目的在小鼠月经模型基础上,研究低氧诱导因子(HIF1A)与血管内皮生长因子(VEGF)蛋白及mRNA的表达与定位,探索HIF1A与VEGF在月经发生中可能的调控机制。方法建立小鼠月经样模型,假孕小鼠通过花生油子宫腔注射的方法诱导小鼠子宫内膜发生蜕膜化,切除卵巢致孕酮(P4)撤退以模拟月经的发生。在P4撤退后0、8、12、16、24h分别收集小鼠子宫;利用免疫组化与Western blot方法检测HIF1A、VEGF的定位与表达量,利用Real-time PCR检测二者mRNA相对表达量;分析HIF1A与VEGF的表达变化趋势与相关性。结果 P4撤退后,小鼠子宫内膜发生崩解出血,模拟月经发生。免疫组化结果显示,HIF1A蛋白在P4撤退后12、16h,内膜基质蜕膜细胞核中的表达量逐渐升高;HIF1A表达区域与VEGF表达的区域均集中在子宫内膜蜕膜化区域外周,即崩解组织与基底层交界区域。Western blot蛋白定量结果显示,细胞核中HIF1A蛋白表达量在P4撤退后8、12、16h显著增高,VEGF在细胞质中的表达量在P4撤退后0、8、12h较高。HIF1A与VEGF mRNA的表达变化趋势非常相似,即P4撤退后,逐渐升高,在12h达到峰值,随后逐渐下降。结论结果提示月经发生子宫内膜崩解中,HIF1A转录因子功能激活并调节VEGF mRNA的表达。     

雌激素对小鼠子宫内膜崩解后修复的影响

目的 探讨假孕小鼠月经模型中,雌激素水平对子宫内膜崩解后修复再生进程的影响. 方法 对假孕小鼠一侧子宫人工诱导内膜间质细胞发生蜕膜化,并通过双侧卵巢切除的方式实现孕酮撤退,建立小鼠月经模型;将实验动物随机分为雌激素组以及溶剂组,在孕酮撤退后分别给以17β-雌二醇油溶液和溶剂;在孕酮撤退后24h及48 h检测各组子宫重量改变、并进行形态学观察和评级. 结果 比较孕酮撤退48h两组形态学评级,雌激素组子宫内膜修复率显著高于溶剂组(P＜0.01). 结论 在假孕小鼠月经模型中,溶剂组小鼠较雌激素组小鼠出现明显的修复延迟,说明雌激素水平对于子宫内膜崩解后修复进程有一定影响.     

孕酮回植激活Akt/GSK3β信号通路逆转月经发生

目的探讨在孕酮撤退月经模型小鼠月经发生的关键时期前,回植孕酮对蛋白激酶B/糖原合成激酶3β(Akt/GSK3β)信号通路的影响。方法 30只成熟去势雌性C57BL/6J小鼠在建成小鼠孕酮撤退月经模型的基础上,随机分为3组:孕酮撤退后12h组(12h组)、孕酮撤退后16h组(16h组),以及在孕酮撤退后12h回植孕酮皮埋管4h组(回植孕酮组),每组10只。通过qRT-PCR法检测子宫内膜组织中Akt、GSK3βmRNA表达,Western blotting和免疫组织化学检测总Akt(t-Akt)、磷酸化Akt(p-Akt)(Ser473)和p-GSK3β(Ser9)蛋白的表达。结果与12h组相比,回植孕酮组和16h组中Akt mRNA表达显著下降(P均0.05),而回植孕酮组和16h组无显著性差异(P0.05);12h组的GSK3βmRNA表达量显著高于回植孕酮组和16h组(P均0.05)。与12h组相比,p-Akt、p-GSK3β蛋白在回植孕酮组表达量显著增高(P均0.05);而16h组子宫内膜组织中p-Akt、p-GSK3β蛋白表达量亦显著低于12h组和回植孕酮组(P均0.05)。免疫组化结果显示,p-Akt和p-GSK3β蛋白在小鼠子宫组织中表达分布呈现相似的模式,主要广泛散在表达于蜕膜化区域的基质细胞及腺上皮和腔上皮细胞中,在蜕膜化区域周边稍有集中分布呈带的趋势。结论在月经发生关键时期之前回植孕酮可能通过非转录机制快速激活Akt/GSK3β信号通路及其下游级联反应,从而逆转月经发生。     

月经发生中低氧诱导因子-1A对VEGF的调节

目的在小鼠月经样模型和人蜕膜化子宫内膜基质细胞模拟月经发生基础上,探索低氧诱导因子-1A(HIF1A)对血管内皮生长因子(VEGF)的表达调控作用,以及月经发生中HIF1A对VEGF mRNA表达的调控机制。方法建立小鼠月经样模型,用ChIP方法检测HIF1A是否直接调控Vegf mRNA的表达;利用HIF1A抑制剂甲氧雌二醇(2ME)抑制HIF1A的功能,检测小鼠月经样模型中HIF1A、VEGF蛋白的表达量;人子宫内膜基质细胞体外诱导蜕膜化并模拟月经发生,检测HIF1A与VEGF mRNA的表达;用siRNA敲降的方法抑制HIF1A,检测VEGF mRNA的表达。结果小鼠月经样模型在孕酮撤退0h,HIF1A结合的Vegf promoter的相对百分比较低,随后在8h和12h逐渐上升,在12h达到峰值(P0.05);2ME抑制HIF1A入核的同时,抑制了VEGF的表达量(P0.05);人蜕膜化子宫内膜基质细胞中,孕酮撤退显著上调HIF1A和VEGF mRNA的表达量(P0.01);敲降HIF1A后,VEGF mRNA的上调受到明显的抑制(P0.01)。结论在月经发生中,孕酮撤退能够激活HIF1A转录因子的功能,HIF1A被激活后,结合于VEGF promoter区并启动VEGF mRNA的表达。     

时钟基因Cry1与Bmal1在小鼠子宫内膜生理性修复过程中的表达研究

目的 以小鼠月经模型为基础，探究时钟基因Cry1与Bmal1对小鼠月经期子宫内膜生理性修复过程的影响。方法 通过建立小鼠月经样生理性修复模型，利用苏木素伊红(HE)染色法进行组织形态学观察；利用免疫组织化学(IHC)染色法观察CRY1与BMAL1蛋白在小鼠月经样修复模型中的表达定位；进一步利用蛋白免疫印迹杂交(Western blot)检测CRY1与BMAL1蛋白的表达水平。结果 孕酮撤退后24～48 h子宫大体颜色逐渐变淡，子宫直径由粗逐渐变细。HE染色结果显示，孕酮撤退后24 h蜕膜化区域大部分细胞坏死崩解，且随着时间推移，子宫内膜厚度增加，子宫腔面出现再上皮化，腺体数量明显增多；48 h时，子宫内膜修复完整，基本恢复到正常子宫形态。IHC染色结果显示，CRY1和BMAL1蛋白的阳性染色定位于细胞质和细胞核中，主要集中在细胞核，且阳性信号随着孕酮撤退时间呈现一定差异。Western blot检测结果显示，孕酮撤退24 h后，子宫内膜CRY1蛋白表达显著降低(P<0.05),40 h时表达量相对更低，出现低谷。孕酮撤退后32 h子宫内膜BMAL1蛋白的表达量相对更低，显著低于2...     

白细胞在小鼠药理性孕酮撤退月经样改变模型中的作用

探讨小鼠药理性孕酮撤退月经样改变模型中白细胞的作用.方法 雌性去势NIH小鼠在蜕膜化后给予米非司酮,间隔8 h检测白细胞的数量和定位以及基质金属蛋白酶(MMP)-9的定位.结果 白细胞在双侧子宫角崩解前(给予米非司酮后16 h)和修复期(给予米非司酮后32 h)较多,其在月经中的作用可能依赖于蜕膜化状态.与生理性孕酮撤退月经模型存在差异,可能是由于内分泌环境造成的,源自嗜中性粒细胞的MMP-9定位存在区域性分布特征.结论 在此模型中,白细胞对子宫内膜崩解和修复重建起作用.     

缺血再灌注对小鼠蜕膜化子宫内膜的作用

目的以小鼠月经样模型为基础,探索缺血再灌注在月经发生中的作用。方法假孕NIH小鼠子宫诱导蜕膜化,完成蜕膜化后将小鼠分为对照组(n=8)、假手术组(n=8)与缺血再灌注组(n=10)。对照组小鼠无操作,假手术组仅实行开腹手术,而缺血再灌注组小鼠开腹后对单侧子宫角进行血管夹持手术阻断血流,30min后恢复血流,造成单侧子宫角内发生缺血再灌注,另一侧子宫角无操作。手术后观察阴道出血状况,且术后6h与24h取材进行大体观察与组织形态学观察,统计子宫内膜崩解的小鼠数量。结果处理后24h,对照组小鼠子宫内膜全部保持完好,无一例(0/8)发生内膜崩解;假手术组仅一例(1/8)发生内膜崩解;缺血再灌注组大部分小鼠(8/10)双侧子宫角的子宫内膜均发生崩解并伴随阴道出血现象。结论缺血再灌注能够直接导致绝大部分小鼠蜕膜化子宫内膜发生完全崩解。     

PGE2及cAMP在烧伤合并内毒素血症骨髓粒单系造血变化中作用的实验研究

目的　探讨前列腺素E2 (PGE2 )、环磷酸腺苷 (cAMP)在烧伤合并内毒素血症骨髓粒单系造血变化中的作用。　方法　 178只昆明种小鼠随机分为烧伤合并内毒素注射组、单纯烧伤组、单纯内毒素注射组和等渗盐水对照组。应用放射免疫分析和免疫组化法 ,动态检测各组小鼠致伤后 1周内 ,骨髓细胞环氧酶 2 (COX 2 )表达及PGE2 、cAMP含量 ,并对粒单系造血变化进行了动态观察。　结果　烧伤合并内毒素血症早期骨髓粒单系略呈增生反应 ,继而呈现造血抑制。骨髓细胞COX 2表达、骨髓细胞上清PGE2 和细胞内cAMP在 12h～ 5d显著升高 ,且cAMP与PGE2 呈显著正相关 (r=0 .978,P <0 .0 1) ,而与粒 巨噬细胞集落形成单位 (CFU GM)呈显著负相关 (r =- 0 971,P <0 .0 1)。　结论　PGE2 在烧伤合并内毒素血症骨髓粒单系抑制中起重要作用 ,抑制效应是通过其与特异性受体结合 ,激活腺苷酸环化酶 ,使骨髓细胞内cAMP升高     

SIRT2 Regulates LPS-Induced Renal Tubular CXCL2 and CCL2 Expression

Sirtuin 2 (SIRT2), a NAD⁺-dependent histone deacetylase, is involved in carcinogenesis and genomic instability and modulates proinflammatory immune responses. However, its role in renal inflammatory injury has not been demonstrated. In this study, we explored the expression patterns of CXCL2 and CCL2 in kidney tissue from Sirt2^−/− and Sirt2^+/+ mice and in mouse proximal tubular epithelial (MPT) cells. CXCL2 and CCL2 were significantly downregulated at both the mRNA and the protein levels in kidneys of LPS-treated Sirt2^−/− mice compared with those of LPS-treated Sirt2^+/+ mice. Furthermore, SIRT2 deficiency ameliorated LPS-induced infiltration of neutrophils and macrophages, acute tubular injury, and decrease of renal function. Supporting these observations, CXCL2 and CCL2 expression levels were lower in MPT cells treated with SIRT2-siRNA than in cells treated with control-siRNA, and adenovirus-mediated overexpression of SIRT2 in MPT cells significantly increased the LPS-induced expression of CXCL2 and CCL2 at the mRNA and protein levels. In addition, SIRT2 interacted with mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), and SIRT2-knockdown increased the acetylation of MKP-1 and suppressed the phosphorylation of p38 MAPK and c-Jun N-terminal kinase in LPS-treated MPT cells. SIRT2 also regulated p65 binding to the promoters of CXCL2 and CCL2. Taken together, these findings indicate that SIRT2 is associated with expression of renal CXCL2 and CCL2 and that regulation of SIRT2 might be an important therapeutic target for renal inflammatory injury.     

Multidomain synthetic peptide B2A2 synergistically enhances BMP-2 in vitro.

A multidomain, synthetic peptide designated B2A2 synergizes the activity of BMP-2. B2A2 interacts with BMP receptor isoforms, potentiating the action of BMP-2 in activating alkaline phosphatase and triggering Smad and MAPK signaling. B2A2's design permits its delivery as a local surface coating as well as a soluble co-factor, thus broadening potential bioengineering applications. INTRODUCTION: BMP-2 induces osteogenic differentiation and accelerates bone repair. Although BMP-2 inhibitors have been discovered, no BMP-2 mimetics or enhancers that function in the physiological range have yet been found. Here we report that a synthetic peptide designated B2A2, consisting of (1) a BMP receptor-targeting sequence, (2) a hydrophobic spacer, and (3) a heparin-binding sequence, is a positive modulator of recombinant BMP-2. MATERIALS AND METHODS: Cultures of mesenchymal cell lines C2C12 and C3H10T1/2 were given B2A2, recombinant BMP-2, or both. Alkaline phosphatase (ALP) activity was assayed by conversion of paranitrophenol phosphate (PNPP). Signaling through Smad and MAP kinase pathways was monitored by Western blot. Receptor binding was assessed by incubating immobilized B2A2 with soluble recombinant receptor-Fc chimeras and detecting bound receptor by anti-Fc antibody ELISA. Surface coating of medical device materials was done by first dip-coating with silyl-heparin, followed by B2A2. RESULTS AND CONCLUSIONS: Treatment of cells with B2A2 alone marginally increased ALP activity. However, B2A2 plus BMP-2 resulted in 5- to 40-fold augmentation of ALP compared with BMP-2 alone in C3H10T1/2 or C2C12 cells, respectively. This synergistic enhancement was observed over a broad concentration range (4-1000 ng/ml BMP-2). B2A2 interacted directly with BMP receptor isoforms (preferentially to BMPR-Ib and ActivinR-II). In cells, B2A2 + BMP-2 led to a repression of MAP kinase and an increase of Smad activation, consistent with known activation pathways of BMP-2. B2A2 was ineffective when paired with other cytokine/growth factors (basic fibroblast growth factor [FGF-2], TGF-beta1, vascular endothelial growth factor [VEGF]). Simultaneous co-administration was not strictly required. Pulse-chase experiments revealed that temporal separations up to 1 h were still effective. B2A2 was also effective when delivered in a polystyrene- or stainless steel-coated surface through a heparin platform (silyl-heparin) while BMP-2 was added exogenously in solution. These results suggest that B2A2 might promote aggregation of receptor subunits, enabling BMP-2 to activate signaling pathways at effectively lower concentrations. Synthetic multidomain constructs like B2A2 may be useful to accelerate bone repair/deposition through augmentation of endogenous levels of BMP-2 or through local BMP-2 contained in artificial or engineered matrices.     

PGD2-CRTH2 Pathway Promotes Tubulointerstitial Fibrosis

Urinary excretion of lipocalin-type PGD₂ synthase (L-PGDS), which converts PG H₂ to PGD₂, increases in early diabetic nephropathy. In addition, L-PGDS expression in the tubular epithelium increases in adriamycin-induced nephropathy, suggesting that locally produced L-PGDS may promote the development of CKD. In this study, we found that L-PGDS–derived PGD₂ contributes to the progression of renal fibrosis via CRTH2-mediated activation of Th2 lymphocytes. In a mouse model, the tubular epithelium synthesized L-PGDS de novo after unilateral ureteral obstruction (UUO). L-PGDS-knockout mice and CRTH2-knockout mice both exhibited less renal fibrosis, reduced infiltration of Th2 lymphocytes into the cortex, and decreased production of the Th2 cytokines IL-4 and IL-13. Furthermore, oral administration of a CRTH2 antagonist, beginning 3 days after UUO, suppressed the progression of renal fibrosis. Ablation of IL-4 and IL-13 also ameliorated renal fibrosis in the UUO kidney. Taken together, these data suggest that blocking the activation of CRTH2 by PGD₂ might be a strategy to slow the progression of renal fibrosis in CKD.Kidney failure is a public health problem worldwide, with increasing incidence and prevalence, high costs, and poor outcomes. CKD is generally progressive, incurable, and ultimately fatal, although some patients resolve with little or no sequelae. Because current treatment is basically limited to slowing the progression to ESRD using angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor blockers, more efficient therapies with different or additional modes of action are clearly needed.Regardless of disease etiology, tubulointerstitial fibrosis is the common pathway leading to ESRD in many kidney diseases and is regarded as a prognostic factor for renal function.^1–3 It is noteworthy that some clinical trials are proving that antifibrotic therapies, such as pirfenidone against diabetic nephropathy,⁴ are also effective for CKD. Therefore, elucidating the etiological mechanism underlying renal fibrosis and developing novel therapeutic strategies remains a serious, unmet medical need.Lipocalin-type PGD₂ synthase (L-PGDS) is a secretary protein of the lipocalin superfamily that converts PG H₂, a common precursor of prostanoids, to PGD₂. Because the urinary excretion of L-PGDS increases in the early stage of diabetic nephropathy,^5,6 as well as in patients with essential hypertension without any apparent renal injury,⁷ urinary L-PGDS may be an early diagnostic marker of renal injury in these patients. There is evidence indicating that, in the monkey kidney, L-PGDS is synthesized de novo in the loop of Henle, podocytes, and Bowman’s capsule of the glomeruli.⁸ Furthermore, L-PGDS gene expression in the tubular epithelium was increased in adriamycin-induced nephropathy.⁹ These findings suggest that, under conditions of tubulointerstitial stress, locally produced L-PGDS may be involved in the development of CKD. However, the precise pathophysiological significance of L-PGDS in the kidney remains to be determined.PGD₂ interacts with two receptors, the prostanoid DP₁ receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Activation of the DP₁ receptor by PGD₂ has been shown to produce vasodilation¹⁰ and bronchodilation.¹¹ Furthermore, the DP₁ receptor is expressed by certain leukocyte populations,^12,13 including dendritic cells, where it controls various functions, including cytokine production. CRTH2 was originally identified as an orphan receptor expressed by Th2 lymphocytes. CRTH2 is not structurally related to the DP₁ receptor and belongs to the family of chemokine receptors. Activation of CRTH2 by PGD₂ plays an important role in allergic inflammation via the recruitment of Th2 lymphocytes and other leukocytes¹⁴ and, perhaps more importantly, by driving the production of the Th2 cytokines IL-4, IL-5, and IL-13.¹⁵