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。     

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.     

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。     

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...     

子宫内膜间充质干细胞对小鼠模型薄型子宫内膜的修复作用

目的观察小鼠子宫内膜间充质干细胞对小鼠模型薄型子宫内膜的修复作用。方法选择8周龄动情期规律C57BL/6J小鼠共288只,随机分为空白对照组、模型对照组、干细胞移植组,每组96只。空白对照组不做任何处理;模型对照组宫腔注射95%乙醇损伤后立即原位注射PBS 0.1 ml,3 d后尾静脉注射PBS 1 ml;干细胞移植组宫腔注射95%乙醇损伤后立即原位注射子宫内膜间充质干细胞0.1 ml,3 d后尾静脉注射子宫内膜间充质干细胞1 ml,干细胞浓度为5×105/ml。每组根据取样时间(3 d、7 d、14 d、30 d)不同分为4小组,取样当日,半数小鼠子宫组织取样,4%多聚甲醛固定送检,进行子宫内膜厚度、HE染色、免疫组化等检测;另半数小鼠与确定有生殖能力C57BL/6J雄鼠合笼,进行生育力评估。结果干细胞移植组小鼠子宫内膜厚度在各时间点均显著厚于模型对照组(P<0.05),但与空白对照组仍有一定距离(P<0.05)。HE染色结果显示,干细胞移植后的受损子宫内膜上皮层及基质层均增厚,腺体增加。免疫组化结果显示,干细胞移植组角蛋白、波形蛋白、VEGF、ERα各项指标均有显著上升,与模型对照组有显著性差异(P<0.05)。生育力评估发现,模型对照组生育率接近于0(仅30 d亚组1例),而干细胞移植组生育率显著升高,30 d亚组生育率达50%,接近空白对照组(66.7%)。结论小鼠子宫内膜间充质干细胞对薄型子宫内膜有显著改善。     

血管内皮生长因子在子宫内膜的表达及调控

血管内皮生长因子 ( vascular endothelial growth factor,VEGF)为特异性促血管内皮细胞丝裂原并具促血管通透性作用。 VEGF及其受体在子宫内膜中表达 ,受雌孕激素、缺氧、c AMP的调节 ,其与生理性月经后的内膜修复、胚胎植入及子宫内膜癌、内膜异位症、子宫异常出血密切相关     

Norplant使用者子宫异常出血内膜血管变化的研究

目的　探讨 Norplant使用者子宫内膜出血病理变化的特点。　方法　应用组织学及免疫组织化学方法对正常子宫内膜和避孕药具 Norplant使用者子宫内膜进行组织形态学和血管内皮细胞 CD3 4和基膜层粘连蛋白 ( LN)的比较研究。　结果　避孕药具 Norplant引发的出血为弥漫性 ,内膜发育受抑制 ;出血部位的血管呈断裂像 ,血管内皮细胞中 CD3 4和血管基膜中 L N的阳性表达都明显减弱。　结论　 Norplant导致子宫出血的直接因素是毛细血管内皮细胞中的 CD3 4和基膜中 LN的含量减少     

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.     

去细胞羊膜载体复合自体子宫内膜干细胞治疗重度宫腔粘连1例报道

宫腔粘连又称Asherman综合征,是由于宫腔手术操作、宫腔内感染等原因导致子宫内膜肌层被破坏,从而出现以月经异常、不孕、反复流产等为临床表现的综合征。人工周期、节育器、球囊等传统治疗方法效果欠理想。本文报道了1例重度宫腔粘连患者使用了负载着自体子宫内膜干细胞与去细胞羊膜载体的宫内节育器(IUD)后,经体外受精-胚胎移植,成功获得临床妊娠。本病例提示,去细胞羊膜载体复合自体子宫内膜干细胞可以有效预防宫腔粘连复发,帮助子宫内膜重建和修复。     

孕酮维持下的小鼠子宫蜕膜化内膜的变化

目的在小鼠子宫诱导蜕膜化发生行经样变化模型中，血管反应是这一变化的重要表现之一。通过对蜕膜组织中血管结构的研究，期望对于血管结构在该模型的作用有进一步的认识。方法制备小鼠诱导蜕膜化模型并维持孕酮水平，诱导后96h处死并取子宫。对组织材料进行全程从系膜对侧向系膜侧连续系列石蜡切片并进行HE染色。对该系列染色切片等距取102张进行显微照相，所得图像结果进行计算机三维重组。通过CD31免疫组化判断空腔结构组成。结果子宫中的空腔结构由CD31阳性细胞组成。三维重组后获得了三种不同的空间结构及其相互之间空间关系。结论小鼠子宫内膜中空腔结构由内皮细胞构成，为扩张的静脉。通过三维重组清晰地定位了该结构在蜕膜整体组织的位置及其与其他成分的关系，为血管结构在月经出血和妊娠中的作用提供了有价值的信息。     

A diagnostically useful histopathologic feature of endometrial polyp: the long axis of endometrial glands arranged parallel to surface epithelium

We describe a histologic feature useful in the diagnosis of endometrial polyps, namely, the parallel arrangement of the endometrial glands' long axis to the surface epithelium (PGE). Polyps that are removed intact are usually easy to diagnose because of the polypoid appearance plus surface epithelium on all sides. In addition, there are thick-walled blood vessels and irregularly shaped glands. Rarely are all or even some of the characteristic features easy to identify in biopsies and curettage specimens. We evaluated 76 cases of grossly identifiable polyps from hysterectomy or polypectomy (working group) for various histologic features and tested the validity of those findings with another 75 specimens (test group), which had been diagnosed as polyps in another institute by separate investigators. The frequency of the various histologic features in the polyps varied depending on the menstrual state, shape, and histologic types of the polyps. PGE was found in 80% (40 of 50 polyps) of premenopausal women as the most common histologic feature, but it was less common in postmenopausal women (42%, 11 of 26) (P = 0.001). All functional polyps (100%, 19 of 19), many of hyperplastic polyp (65%, 20 of 31), and some fibrous polyps (38%, 8 of 21) showed the change. In premenopausal women, incidence of PGE was significantly different by the polyp's shape or histologic types (P < 0.05), but not in postmenopausal women. PGE was not identified in any of the 56 normal background endometria, even though the surface was often undulated. Almost identical findings were observed in the series from the test group, confirming the validity of this new finding. In summary, PGE, if present, is a useful histologic finding in facilitating the diagnosis of endometrial polyp in curettage specimens.     

Immunohistochemical expression of nestin in adenohypophysial vessels during development of pituitary infarction

OBJECTIVES: The aim of this work was to investigate the immunohistochemical expression of nestin, a member of the intermediate filament family, in adenohypophysial vasculature during development and progression of pituitary infarction. METHODS: Forty-five nontumorous adenohypophyses and 34 pituitary adenomas of various types, all exhibiting acute or healing infarcts, were examined immunohistochemically using the streptavidin-biotin-peroxidase complex method. RESULTS: In both adenohypophyses and pituitary adenomas without infarction, nestin was expressed in only a few capillaries and endothelial cells. In acute infarcts without a vascular response, no nestin was demonstrable within necrotic capillaries (50 cases). In organizing infarcts, newly formed vessels spreading into necrotic zones showed nestin expression in all capillaries and practically every endothelial cell (25 cases). In the hypocellular, fibrotic scar phase, only a few vessels (4) were apparent, and immunoreactivity was focal and mild. CONCLUSIONS: Nestin is strongly expressed in newly formed capillaries and is downregulated when infarcts transform to fibrous tissue. Nestin expression may provide valuable insight into the process of pituitary angiogenesis.     

Donor islet endothelial cells participate in formation of functional vessels within pancreatic islet grafts

Pancreatic islet transplantation has emerged as a therapy for type 1 diabetes and is today performed using both freshly isolated and cultured islets. Islet blood vessels are disrupted during islet isolation; therefore, proper revascularization of the transplanted islets is of great importance for islet graft function and survival. We have studied intraislet endothelial cells after islet isolation, during islet culture, and following islet transplantation. By isolating islets from the transgenic Tie2-GFP (green fluorescent protein) mouse, characterized by an endothelial cell-specific expression of GFP, living endothelial cells could be studied in intact islets utilizing two-photon laser-scanning microscopy (TPLSM). Intraislet endothelial cells were found to survive islet transplantation but to rapidly disappear during islet culture. By transplanting freshly isolated Tie2-GFP islets and applying a novel ex vivo model for simultaneous perfusion and TPLSM imaging of the graft-bearing kidneys, GFP fluorescent endothelial cells were found to extensively contribute to vessels within the islet graft vasculature. Real-time imaging of the flow through the islet graft vasculature confirmed that the donor-derived vessels were functionally integrated. Hence, intraislet endothelial cells have the capability of participating in revascularization of pancreatic islets subsequent to transplantation. Therefore, preservation of intraislet endothelial cell mass may improve long-term graft function.     

Testosterone induces vascular endothelial growth factor synthesis in the ventral prostate in castrated rats

PURPOSE: Recent studies suggest that the vasculature is important for the control of prostate growth. Castration induces an involution of the prostate gland and its vasculature. Replacement of testosterone stimulates endothelial cell proliferation and normalizes vascular volumes and blood flow several days before organ regrowth. Antiangiogenesis treatment inhibits the growth of prostate tumors. Understanding the regulation of the prostate vasculature may therefore provide important knowledge of the mechanisms responsible for the growth of non-malignant and malignant prostate tissue. Castration induced regression and testosterone stimulated regrowth of the prostatic vasculature have here been used to study the involvement of the angiogenic factor vascular endothelial growth factor (VEGF) and its receptors flt-1 and flk-1/KDR in the regulation of the prostatic vasculature. MATERIALS AND METHODS: VEGF, flt-1, and flk-1/KDR levels were quantified in the rat ventral prostate following castration and testosterone replacement. Methods used were competitive RT-PCR, Western blot and immunohistochemistry. RESULTS: VEGF mRNA and protein levels were significantly decreased by castration and testosterone treatment induced VEGF synthesis in the rat ventral prostate epithelium. Flt-1 and flk-1/KDR receptor levels were unaffected by castration and testosterone treatment. CONCLUSIONS: Castration down regulates VEGF and testosterone induces VEGF synthesis in epithelial cells in the rat ventral prostate.     

Evidence of increased endothelial cell turnover in brain arteriovenous malformations.

OBJECTIVE: We hypothesized that human brain arteriovenous malformations (BAVMs) are nonstatic vascular lesions with active angiogenesis or vascular remodeling. To test this hypothesis, we assessed endothelial cell turnover in BAVMs. METHODS: We identified nonresting endothelial cells by use of immunohistochemistry for the Ki-67 antigen. From archived paraffin blocks, we selected BAVM vessels without intravascular thrombosis or embolic material in areas nonadjacent to the nidus edge. For controls, we used 50- to 100-microm diameter cortical vessels from temporal lobe cortex removed for epilepsy treatment. The Ki-67 index was calculated as a percentage of Ki-67-positive endothelial cells. The data were analyzed by the nonparametric Mann-Whitney test and reported as mean +/- standard deviation. RESULTS: Thirty-seven specimens that met the above criteria were selected. There were 26 +/- 15 vessels counted in each BAVM specimen versus 18 +/- 5 in each control cortex (n = 5). The mean Ki-67 index was higher for BAVM vessels than control cortical vessels (0.7 +/- 0.6 versus 0.1 +/- 0.2%; P = 0.005), which represented an approximately seven-fold increase in the number of nonresting endothelial cells. In the BAVM group, there was a trend for younger patients to have a wider variation and higher Ki-67 index than older patients; no trend was evident in the control group. CONCLUSION: Compared with control vessels, BAVM vessels have higher endothelial cell turnover, which suggests the presence of active angiogenesis or vascular remodeling in BAVMs.     

二至天癸颗粒对克罗米芬诱导排卵小鼠子宫内膜整合素β3表达的影响

目的 探讨二至天癸颗粒对克罗米芬（CC）诱导排卵小鼠子宫内膜整合素β3表达的影响.方法 40只生育期排卵障碍模型小鼠分中药组及CC组两组,每组20只,中药组予二至天癸颗粒加CC,CC组单纯予CC,20只非模型鼠为空白组.观察三组胚泡着床率、子宫内膜形态学变化,并采用免疫组化方法测定子宫内膜整合素β3的表达.结果与CC组比较,中药组模型小鼠恢复动情周期数明显增多（P〈0.05）;与CC组比较,中药组孕4 d小鼠子宫内膜形态学改变较优,适于着床;与CC组比较,中药组孕4 d小鼠子宫内膜腔上皮、腺上皮及间质三种组织中整合素β3的表达水平均较强,且均具有统计学意义;中药组与空白组小鼠比较,着床胚泡数、着床率无显著性差异（P〉0.05）;整合素β3的表达在腺上皮及间质积分无显著性差异（P〉0.05）,在腔上皮空白组表达较中药组强,且具有显著性差异（P〈0.05）.三组小鼠整合素β3在腔上皮、腺上皮中的表达均强于在间质中的表达,具有统计学意义.结论二至天癸颗粒提高CC诱导排卵小鼠子宫内膜腔上皮及腺上皮细胞中整合素β3的表达.     

040 The Function of ‘Choke Vessels’ to the Blood Flow: Angiographic and Laser Flow‐Graphic Study on the Rat Flap Model

Aim: The ‘choke vessels’, defined by Taylor 1], are reduced‐caliber anastomosing vessels which are normally the barrier in the vasculature. They also have capacity to dilate and increase the blood flow. The aim of this study is to investigate the anatomical and dynamic function of choke vessels. Study design: The 3‐territory bipedicled flaps were elevated on the back of the Wistar rats 2]. The flaps were directly sutured back in place in the revascularized group (10 animals), and the plastic sheets were underlaid in the non‐revascularized group (10 animals). On the day 3, each flap was converted to the mono‐pedicled flap and was scanned by the laser flow‐graphy and injected for the angiography. Results: All flaps survived completely. The revascularized flaps showed evenly dilated choke vessels in the 2 choke zones of the flaps and the blood flow from the vascular pedicle covered more than 2 territories. The non‐revascularized flaps showed more remarkable but uneven dilatation of choke vessels between 2 choke zones. The blood flow in non‐revascularized flaps, however, reached only 1 territory, blocked by the first choke zone which dilated less than the other zone. Conclusions: By dilating, the choke vessels are activated and can have the adverse function of regulating the blood flow, namely, suppression and escalation of the blood flow.     

Photodynamic endometrial ablation: morphological study

BACKGROUND AND OBJECTIVES: Endometriumablation provides an alternative treatment modality to hysterectomy for dysfunctional uterine bleeding (DUB). Its feasibility was demonstrated in animal studies. The purpose of this study was the evaluation of histological changes following photodynamic endometrial ablation in patients. STUDY DESIGN/MATERIALS AND METHODS: 5-Aminolevulinic acid (ALA) was topically applied in three premenopausal and one postmenopausal patient 4-6 hours before laser-illumination. ALA-concentration: 100-400 g/ml, NaOH-titration (pH 5.5). Volume applied, 1.5-2.0 ml; intrauterine balloon-lightdiffuser; wavelength, 635 nm; lightdose, 160 J/cm(2). Hysterectomy was performed 3-152 days after photodynamic ablation. Histological examination was carried out on endometrium sections stained with hematoxylin and eosin (H&E). RESULTS: Necrosis including the full thickness of the endometrium was found 3 days after photodynamic endometrial ablation. Specimens collected after 35 and 152 days did not exhibit fibrosis or adhesions. Foci of preserved endometrium were detected in all patients. CONCLUSIONS: Photodynamic endometrial ablation is selective and does not cause endometrial fibrosis or adhesions.     

去内皮、冻干辐照后异体小血管的组织结构、物理性能及抗原性变化

目的:研究去内皮、冻干辐照后同种异体小血管的组织结构、物理性能及抗原性的变化，旨在了解处理后的异体血管是否适用于血管移植。方法:将血管随机分为A，B两组；A组为对照组，B组为去内皮冻干辐照处理组。光镜下观察每组血管的组织结构，电镜下观察血管去内皮化情况，测出血管张力在达到15，30，45，60，90，120N及180N时的扩张直径、伸展长度及血管在横向和纵向被拉断时所能承受的最大拉力，并计算出血管在该张力下的横向和纵向变化率；采用免疫组化SP法检测处理前后主要组织相容性复合体（MHC）-Ⅱ类抗原的表达水平。结果:处理组血管组织结构保持良好，两组血管在不同张力下的横向及纵向变化率差异无统计学意义（P＞0.05）。处理组血管在横向上被拉断时所能承受的最大张力和在纵向上被拉断时所能承受的最大张力显著大于对照组（P＜0.01及P＜0.05）。处理组MHC-Ⅱ类抗原水平明显低于对照组（P＜0.01）。结论:经去内皮、冻干辐照处理后异体小血管组织结构未发生改变，且在正常生理范围内保留一定的血管物理性能；MHC-Ⅱ类抗原水平下降明显。提示，经处理的血管基本符合血管移植的要求，是小口径同种异体血管移植的理想材料。