与盆底韧带成纤维细胞间接共培养对大鼠骨髓间充质干细胞弹性蛋白、LOX及Fibulin-5mRNA表达的影响

目的:研究与盆底韧带成纤维细胞间接共培养对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)分化的影响,探索在体外条件下诱导BMSCs向韧带成纤维细胞分化微环境的创建并检测诱导分化细胞内弹性蛋白、LOX、Fibulin-5的表达。方法:选择7天SD大鼠,利用密度梯度离心法体外分离纯化大鼠骨髓间充质干细胞并对其进行鉴定;取大鼠盆底韧带组织,自行分离传代,获得大鼠盆底韧带成纤维细胞;取纯化的第四代盆底韧带成纤维细胞与纯化培养后的第四代骨髓间充质干细胞间接共培养;实时定量RT-PCR检测弹性蛋白、LOX及Fibulin-5在不同共培养天数BMSCs中mRNA的表达情况。结果:与相应天数阴性对照组相比,3天共培养组弹性蛋白、LOX、Fbulin-5 mRNA的表达量增加,但均无显著变化(P>0.05),6、12天共培养组mRNA的表达量均显著增高(P<0.05);与3天对照组相比,6天对照组和12天对照组的表达量均无显著变化(P>0.05);与3天共培养组相比,6天共培养组的表达量增高,但差异无统计学意义(P>0.05),12天共培养组的表达量均有显著增高(P<0.05)。结论:与盆底韧带成纤维细胞间接共培养可以促进BMSCs弹性蛋白、LOX、Fbulin-5mRNA的表达,这一结果为BMSCs作为韧带组织工程的种子细胞在体外诱导分化条件的探索提供了一条新的研究思路。     

Soluble and insoluble collagen and elastin in the rat hair cycle

Summary Soluble and insoluble collagen and elastin were chemically evaluated in the dorsal skin of rats during the third hair cycle.Both fractions of collagen remain unchanged during anagen, while they increase considerably in the first days of telogen to drop afterwards.Elastin content gradually increases throughout anagen and falls in telogen.The authors suggest that the increasing amounts of collagen in the first part of telogen may be responsible for the progressive block in the downwards movements of previously growing follicles into the dermis.Elastin data could be the result of a mechanical readjustment of skin to the expansion provoked by growing follicles.     

Changes of glycoprotein and collagen immunolocalization in the uterine artery wall of postmenopausal women with and without pelvic organ prolapse

Pelvic organ prolapse (POP) is accompanied by an altered composition of the extracellular matrix (ECM). However, it is unclear whether the changed ECM is the cause or the consequence of POP, as stretching of the tissue may have an effect on the composition of the ECM. To address this question, we analyzed the connective tissues of the uterine artery wall of postmenopausal women with and without POP. The uterine artery wall is stretched in patients with POP, but this stretching is unlikely to cause the POP. Twenty-one women (13 with POP and 8 without POP) hospitalized for hysterectomy were included in this study. Tissue samples from the uterine artery were analyzed for collagen (types I, III, IV, V and VI) and other ECM proteins (fibronectin, laminin, tenascin, vitronectin and elastin) using immunofluorescence microscopy. Results revealed that uterine artery samples of women with prolapse showed a significantly weaker immunoreactivity to type VI collagen, vitronectin and elastin and a stronger immunostaining for type III collagen and tenascin as compared to control samples.Our results suggest that the ECM may be altered in response to mechanical stretch. Changes in the ECM composition as observed in POP may not necessarily be the reason for the development of pelvic floor relaxation in postmenopausal women.     

子宫主韧带中弹性蛋白、赖氨酰氧化酶表达与盆底器官膨出的关系

目的：检测女性盆底器官膨出（POP）患者子宫主韧带中弹性蛋白（elastin）和赖氨酰氧化酶（LOX）表达,探讨两种基因与POP发生发展的关系。方法：选择因子宫脱垂而行全子宫切除术的患者60例,同期选择因妇科良性疾病、无压力性尿失禁（SUI）或POP行全子宫切除术患者60例作为对照。于手术中取子宫主韧带,采用逆转录-聚合酶链式反应（RT-PCR）法测定elastin、LOX mRNA的表达,免疫蛋白印迹法（Western blotting）检测两种蛋白表达。结果：无论绝经与否,POP组子宫主韧带中elastin、LOX mRNA及其蛋白表达明显低于对照组,POP组中绝经后患者elastin、LOX mRNA及其蛋白表达明显低于绝经前患者,差异显著（P<0.05）;子宫主韧带elastin mRNA及蛋白表达变化与LOX相应水平呈明显的直线正相关（r＝0.9959,r＝0.9708,P<0.05）。结论:POP患者子宫主韧带elastin表达减弱影响弹性纤维形成, LOX表达减弱可能通过影响胶原与弹性纤维的交联亦使盆腔支持结构稳定性降低,从而导致POP的发生;绝经后女性体内雌激素水平的降低可能通过影响LOX的表达,进而参与POP的发生。     

Lax eyelid syndrome (LES), obstructive sleep apnea (OSA), and ocular surface inflammation

Purpose

Lax eyelid syndrome (LES) is defined as the association of distensible “floppy” eyelids and chronic papillary conjunctivitis. LES is also found in patients with obstructive sleep apnea (OSA) who have systemic elevation of inflammatory markers, including matrix metalloproteinases (MMP). Locally elevated MMP levels have also been demonstrated co-localized with elastin loss in eyelids of patients with LES, accounting for their “floppiness.” The purpose of this study was to investigate tear film MMP levels and determine their association with eyelid laxity and OSA. We also evaluated 3 previous grading systems to determine the severity of lid laxity and introduced a new “laxometer” device.

Dermal carbonyl modification is related to the yellowish color change of photo-aged Japanese facial skin

Background

The photo-aged facial skin is characterized by various unique features such as dark spots, wrinkles, and sagging. Elderly people, particularly Asians, tend to show a yellowish skin color change with photo-aging. However, there has been no analytical study conducted on this unique skin color change of the aged facial skin.

Objective

The purpose of the present study is to examine whether the carbonyl modification in the dermal protein is involved in the yellowish color change that occurs in the photo-aged skin.

Methods

Normal skin samples excised from the face, abdomen and buttock of variously aged Japanese were separated into the epidermal and the dermal portions. These skin samples were histologically examined for carbonyl modification. Moreover, an in vitro constructed dermis model composed of a contracted collagen gel was treated with acrolein or 4-hydroxynonenal. All these samples were also studied colorimetrically.

Results

The dermal samples obtained from the photo-aged facial skin exhibited an appearance of yellowish color, whereas neither the facial epidermis nor the dermis obtained from the abdomen or buttock showed such a yellowish discoloration. The upper layer of the dermis that revealed the yellowish color showed elastosis whose elastic fibers were found to colocalize with carbonyl protein as detected by a labeled hydrazide, as well as by an immunohistochemical examination using the antibody against acrolein adduct. Experimental induction of carbonyl modification in a dermis model in vitro by a long-term treatment with acrolein or 4-hydroxynonenal was found to show the appearance of the yellowish change which was also proven by an increase in b* value of colorimetry. It was more pronounced than that induced by glycation.

Conclusion

Our present results strongly suggest that carbonyl modification of the dermal protein is involved in the production of the yellowish color change that is noted in the photo-aged facial skin.     

Differential elastin and tenascin immunolabeling in the uterosacral ligaments in postmenopausal women with and without pelvic organ prolapse

Connective tissue, consisting mainly of collagen and structural glycoproteins, is an important part of the supportive structures of the genitourinary region. Relatively few data have been published with respect to the role of elastin and glycoproteins in pelvic organ prolapse (POP). Connective tissue of the uterosacral ligament in postmenopausal women with and without genital prolapse was compared. Fifty-nine consecutive women referred for hysterectomy were included in the study. The patients had POP or benign gynecological disease (e.g. myoma of the uterus). Tissue samples from the uterosacral ligament were investigated for localization and distribution of tenascin and elastin using immunofluorescence microscopy. Tissue samples of women with prolapse showed a significantly (p<0.001) weaker immunofluorescent labeling of tenascin compared to samples taken from women without prolapse. Tenascin was detectable in tissues of all women with POP, whereas its immunolabeling was decreased in the uterosacral ligament in women without POP. Intact elastin fibers were observed in tissues of all women without POP, whereas elastin was undetectable or sometimes fragmented in the uterosacral ligament in women with POP. Greater amounts of tenascin and lesser amounts of elastin were therefore found in patients with POP. These results suggest that an altered turnover of connective tissue in the uterosacral ligament might be responsible for the presence of pelvic floor relaxation in postmenopausal women. These data indicate a complex architecture of the extracellular matrix in the uterosacral ligaments, with marked differences in tenascin and elastin expression between postmenopausal women with or without POP.     

Elastic Fibers Enhance the Mechanical Integrity of the Human Lumbar Anulus Fibrosus in the Radial Direction

The anulus fibrosus of the human lumbar intervertebral disc has a complex, hierarchical structure comprised of collagens, proteoglycans, and elastic fibers. Recent histological studies have suggested that the elastic fiber network may play an important functional role. In this study, it was hypothesized that elastic fibers enhance the mechanical integrity of the extracellular matrix in the radial orientation, perpendicular to the plane containing the collagen fibers. Using a combination of biochemically verified enzymatic treatments and biomechanical tests, it was demonstrated that degradation of elastic fibers resulted in a significant reduction in both the initial modulus and the ultimate modulus, and a significant increase in the extensibility, of radially oriented anulus fibrosus specimens. Separate treatments and mechanical tests were used to account for any changes attributable to non-specific degradation of glycosaminoglycans. Additionally, histological assessments provided a unique perspective on structural changes in the elastic fiber network in radially oriented specimens subjected to tensile deformations. The results of this study demonstrate that elastic fibers play an important and unique role in the mechanical properties of the anulus fibrosus, and provide the basis for the development of improved material models to describe intervertebral disc mechanical behavior.     

The time course of elastin fiber degeneration in a rat aneurysm model

Previous findings vary regarding the timing and cause of elastin fiber degeneration in the elastase-induced rat abdominal aortic aneurysm model. We examined the timing and cause of elastin fiber degeneration after elastase infusion using two different elastase infusion times. Twenty-four Sprague-Dawley rats were divided into two groups. The infrarenal abdominal aorta was infused with 15 U of elastase for 15 min (n = 12, 15-min infusion group) or 30 min (n = 12, 30-min infusion group). In each group, three rats were killed immediately and 1, 3, and 7 days after infusion, and then the aortas were excised for a histologic examination. Elastin fibers did not stain, even immediately after elastase infusion, in the 30-min infusion group. The degeneration of elastin fibers did not progress in the 15-min infusion group during the period of observation. Inflammatory cells infiltrated mainly to the adventitia near regions where the degeneration of elastin fibers spread totally through the aortic media. Elastin fibers degenerate immediately after elastase infusion and thus seem to degenerate not due to endogenous proteinases that are produced by the infiltrating cells, but due to the exogenously infused elastase itself. Inflammatory cell infiltration was thus found to be a result of the degeneration of elastin fibers in this model. Received: September 9, 1999 / Accepted: March 24, 2000     

Laser induced fluorescence spectroscopy of normal and atherosclerotic human aorta using 306-310 nm excitation

Ultraviolet excited laser induced fluorescence (LIF) was studied in normal and atherosclerotic human arterial wall in vitro. Using excitation wavelengths from 306 to 310 nm, two distinct emission bands were observed in the LIF of both normal and pathologic aorta: a short wavelength band, peaking at 340 nm emission, which was attributed to tryptophan; and a long wavelength band, peaking at 380 nm emission, which was assigned to a combination of collagen and elastin. The intensity of the short wavelength band was quite sensitive to the choice of excitation wavelength, while the long wavelength band was not, so that the relative contributions of the bands could be controlled by the precise choice of excitation wavelength. A valley in the spectra at 418 nm was attributed to fluorescence reabsorption by oxy-hemoglobin. By using 308 nm excitation to observe emission simultaneously from both the short and long wavelength bands, normal and atherosclerotic aorta were spectrally distinct. Two LIF emission intensity ratios were defined to characterize both the relative tryptophan fluorescence content as well as the ratio of elastin to collagen fluorescence in each spectrum. The differences in these two emission ratios among the various histologic tissue types correlated qualitatively with the histologic and biochemical compositions of these tissues. By combining these parameters in a binary classification scheme, normal and atherosclerotic aorta were correctly distinguished in 56 of 60 total cases. Furthermore, atherosclerotic plaques, atheromatous plaques, and exposed calcifications could be classified individually with sensitivities/predictive values of 90%/90%, 100%/75%, and 82%/82%, respectively.