机械牵张应力刺激成骨细胞的差异蛋白质组学研究

目的应用蛋白质组学方法分析人成骨样细胞Saos-2在牵张应力作用下蛋白质表达的差异,为更全面地阐明成骨细胞对牵张应力的反应机制提供分子基础。方法将Saos-2分为加力组与对照组,采用Flexcell牵张应力加载系统,用12%大小的应力值进行力学刺激24 h后,应用蛋白质双向凝胶电泳分离蛋白样品,质谱技术鉴定差异表达蛋白点,并用生物信息学分析差异蛋白参与的生物学过程和主要功能。结果对照组和加力组分别得到(1031±41)和(928±25)个蛋白质点,质谱鉴定出肽酰脯氨酰异构酶A样3、线粒体ATP合成酶、抗氧化蛋白1、丝切蛋白1、蛋白磷酸酶1、延伸因子2等17个表达增高的差异蛋白质,涉及应激反应、能量代谢、细胞增殖、细胞骨架重建、信号转导及成骨分化等生物学功能。结论成骨细胞在机械牵张应力作用下蛋白表达发生显著变化,这些差异蛋白参与了成骨细胞力学反应机制的不同过程。     

基于双光子共聚焦成像的大鼠小梁网三维重建及有限元分析

青光眼是一组威胁视神经视觉功能、与眼压升高密切相关的眼病。目前认为临床上大部分眼压的升高与房水流出阻力增加有关,特别是小梁网流出阻力的增加。因此,研究高眼压下影响房水外流阻力的重要区域小梁网的形态学信息尤为重要。通过前房灌注的方法制造大鼠急性高眼压动物模型,将6只SD大鼠分成两组(A组和B组),B组大鼠处死后于左眼球加压60 mmHg处理,其余眼球均为未加压对照组,利用双光子共聚焦成像系统采集正常眼压和高眼压下小梁网组织的断层序列图,通过图像处理方法,定量分析眼压对小梁网组织孔隙率变化的影响。通过三维重建获取正常眼压下的小梁网结构模型,并利用有限元方法,探讨眼压对于小梁网组织形态结构的影响。结合实验数据与模拟计算的结果,综合分析眼压的变化对于小梁网外流阻力的影响。高眼压组数据显示,部分小梁与周围组织融合,胶原纤维出现塌缩,越是靠近前房的小梁组织损伤越严重。有限元分析结果表明,孔隙越多的区域变形越大,而且压力越大,小梁变形程度越明显。眼内压升高可能会引起房水外流通道结构发生异常,主要表现为小梁网胶原纤维发生塌缩。高眼压与正常眼压情况相比,小梁网区域外流阻力增大的可能性较大。     

大鼠小梁细胞的体外培养及弹性模量的测量

目的小梁网是由小梁薄片和其上的小梁细胞构成的网状结构,它对眼压和房水流出具有重要的调节作用,同时小梁细胞的力学特性和生物学特性与房水流出阻力密切相关。因此本研究主要探讨体外培养的大鼠小梁细胞的生物学特性并应用原子力显微镜测量其弹性模量,为今后建立高眼压动物模型并探究原发性开角型青光眼的发病机制提供理论依据。方法取3只SD大鼠双眼小梁网组织,应用消化法对小梁细胞进行体外混合培养。倒置相差显微镜和免疫组化SABC染色的方法确定小梁细胞并观察其生物学特性。应用原子力显微镜压痕方法测量细胞的弹性模量。结果大鼠小梁细胞10 d左右达到融合,细胞形态多样,免疫组化检测结果显示层粘连蛋白、纤维连接蛋白和神经元特异性烯醇化酶染色阳性。小梁细胞弹性模量为1. 02 kPa±0. 66 k Pa。结论消化法成功培养出大鼠小梁细胞,并利用原子力显微镜测得小梁细胞的弹性模量,为之后研究青光眼小梁细胞的特性奠定基础。     

钙敏感受体在高糖诱导的人眼小梁网细胞损伤中的作用

目的：观察钙敏感受体（CaSR）在高糖（HG）诱导的人眼小梁网细胞（HTMC）损伤中的作用和机制。方法：将HTMC随机分为：对照（control）组（10%胎牛血清-DMEM）、HG组（10%胎牛血清-DMEM+50 mmol/L葡萄糖）、HG+CaSR激动剂NPS R568组（10%胎牛血清-DMEM+50 mmol/L葡萄糖+10μmol/L NPS R568）和HG+CaSR抑制剂Calhex231组（10%胎牛血清-DMEM+50 mmol/L葡萄糖+3μmol/L Calhex231）,培养72 h。CCK-8法测量不同浓度的葡萄糖刺激下HTMC活力;试剂盒测定超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量;Western blot检测CaSR、SOD2、凋亡相关蛋白（cleaved caspase-3和Bcl-2）及自噬相关蛋白（ATG7、P62、LC3-I和LC3-Ⅱ）表达;免疫荧光染色观察CaSR细胞定位和表达。结果：葡萄糖浓度为50 mmol/L时,HTMC相对活力由1.00±0.03下降至0.60±0.07(P<0.05);与control组相比,HG使H...     

小梁网弹性变化与青光眼发病的关系

青光眼是一种以眼内压异常升高为主要危险因素的致盲性眼病,小梁网组织作为房水流出的主要通道,对于调节眼内压十分重要。研究表明,青光眼患者小梁网组织弹性较正常人小梁网组织明显升高,眼内压升高可能与小梁网弹性增加之间存在关联。本文在简要叙述小梁网细胞特性的基础上,着重对小梁网组织弹性与青光眼的关系、细胞外基质弹性对小梁网细胞的影响进行综述,为研究青光眼的发病机制以及预防和治疗提供参考。     

基于原子力显微镜压痕技术的大鼠小梁网组织弹性模量测定

目的 探索基于原子力显微镜(atomic force microscope,AFM)压痕技术确定大鼠小梁网组织弹性模量的方法,为揭示小梁网组织力学特性与小梁网通道房水外流阻力之间的关系等研究奠定基础.方法 首先利用大鼠眼球组织切片获取大鼠小梁网组织与毗邻组织之间的距离信息和结构信息;其次利用AFM压痕实验获取角膜-角巩膜缘-小梁网-巩膜方向的弹性模量与相对位置的关系;最后综合对比以确定小梁网组织测试区域.结果 探索了一种基于原子力显微镜压痕实验定位小梁网组织并获取该组织弹性模量的方法,利用该方法获取了大鼠眼球鼻侧、颞侧、上侧、下侧4个位置处的小梁网组织弹性模量范围为300～600 Pa.结论 本研究给出了一种基于AFM压痕技术确定大鼠小梁网组织弹性模量的方法,该方法提供了AFM测试环境下小梁网组织区域的位置参考,优势在于可快速定位到大鼠小梁网组织区域.     

大鼠小梁网组织表观弹性模量与组织弹性模量关系的数值模拟

背景：小梁网的组织力学特性与房水流出阻力密切相关。考虑到小梁网组织的多孔结构特点,利用原子力显微镜压痕技术可获取组织的表观弹性模量,但该方法无法获取组织弹性模量信息,因此,小梁网组织表观弹性模量与组织弹性模量之间的关系缺少定量研究。目的：通过有限元模拟进一步分析小梁网表观弹性模量和组织弹性模量的关系。方法：基于大鼠小梁网组织的双光子共聚焦图像构建该组织的三维结构模型,利用有限元分析模拟压痕实验,获取不同位置处组织表观弹性模量,模拟整体压入实验获取组织的弹性模量。结果与结论：实验获取了基于原子力显微镜压痕实验的一阶Ogden模型参数范围,模拟原子力显微镜压痕实验获取的小梁网组织表观弹性模量平均值介于当压缩比为1%-2%时组织的弹性模量范围之内。实验量化了小梁网组织表观弹性模量和弹性模量的关系,为该组织的力学特性的研究提供了方法学参考。     

胃相关性疾病伴肠上皮化生差异表达基因及中药预测的生物信息学分析

目的：通过生物信息学方法筛选胃相关性疾病伴肠上皮化生（IM）的关键基因与通路,探讨其发病机制及潜在治疗靶点,进而预测治疗IM的中药。方法：从公共基因芯片数据库（GEO）数据库中下载包含IM患者的胃黏膜基因表达谱数据,利用Rstudio3.5.2筛选出IM组织与正常胃黏膜组织的差异表达基因（DEGs）;使用DAVID 6.8数据库对DEGs进行GO和KEGG富集分析;基于STRING数据库和Cytoscape 3.6.1软件构建蛋白相互作用（PPI）网络,明确关键基因及核心功能模块;通过将关键基因与医学本体信息检索平台（Coremine Medical）相对应,筛选治疗IM的中药。结果：纳入2个包含IM的基因芯片数据集GSE78523和GSE60427,将2个数据集中IM相关的DEGs取交集获得135个基因,其中上调基因90个、下调基因45个。GO分析结果显示,DEGs主要涉及消化、细胞增殖的调控、细胞间黏附、钠离子跨膜转运、钾离子转运、胆囊收缩素信号通路、单核细胞趋化性、白细胞迁移、细胞外泌体等功能。KEGG通路富集结果显示DEGs显著富集于胃酸分泌、氮代谢、肾素-血管紧张素系统、蛋白...     

基于双光子共聚焦成像的急性高眼压大鼠小梁网孔隙率变化研究

目的获取不同眼压下小梁网组织深层结构信息,为小梁网房水外排通道生理功能的探索奠定组织形态学基础。方法将4只SD大鼠分成A、B两组每组2只,处死后于左眼球分别加压40mm Hg(A组)、加压60 mm Hg(B组),维持24 h。右眼均为未加压对照组,利用双光子共聚焦成像系统采集每只眼球的小梁网组织形态图:从眼底剖开眼球后照射前房角小梁网处,每2μm采集图像,直至图像模糊停止。结果未加压的对照组眼球小梁网处胶原纤维排列较为规则,孔隙明显,小梁网与周围组织界限分明。加压40 mm Hg的A组眼球小梁网胶原纤维出现了部分塌缩,小梁网与周围组织出现融合,偶尔可见一些孔隙,胶原纤维排列呈无序状态。加压60 mm Hg的B组眼球小梁网胶原纤维断裂较明显,临管区被挤压到完全塌陷,与周围组织已无法分辨。A、B两组动物的小梁网均表现出骨架断裂,组织变薄,逐渐与周围组织融为一体,以及出现远端的巩膜静脉塌缩的现象。与A组眼球相比,B组眼球的葡萄膜小梁网孔隙率略有增加。结论急性眼内压升高可能引发房水外排通道结构异常,主要表现为前房角小梁网组织压缩、巩膜静脉塌陷。这一解剖结构异常造成房水排出困难,从而又加剧了眼内压的升高。     

肾脏纤维化大鼠模型尿液中差异表达基因筛选及生物信息学分析

目的研究大鼠肾脏纤维化(RF)模型尿液中差异表达的基因,并对其进行生物信息学分析。方法将大鼠分为对照组和纤维化模型组,单侧输尿管结扎术建立大鼠肾脏纤维化模型,收集尿液。提取总RNA,构建测序文库并进行转录组测序。对差异表达的mRNA进行了GO分析和KEGG分析,并对miRNA的前体和lncRNA的家族进行预测和分类。结果肾脏纤维化模型组的尿液和对照组相比,得到813条表达上调转录本数据以及213条表达下调的转录本数据。结论利用转录组高通量测序结合相关生物信息学分析,可为肾脏纤维化诊断靶点提供新的可能。     

Pathology of the trabecular meshwork

We demonstrated pathological changes in the trabecular meshwork in primary open-angle glaucoma (POAG) and various types of secondary open-angle glaucoma eyes. In POAG eyes, a sheath material of the elastic-like fiber was found to be significantly greater in the juxtacanalicular tissue. In secondary open-angle glaucoma eyes, several kinds of changes responsible for increased resistance of aqueous outflow were observed in or around the trabecular meshwork. The inner wall endothelial cells of Schlemm's canal were partially or extensively absent in capsular glaucoma and amyloid glaucoma eyes.     

The development of human trabecular meshwork

The fetal development of the trabecular meshwork (TM) of the human eye was quantitatively studied. Anterior chamber angle tissues from developing embryos (20, 26, 32, 36, 40 weeks) and that of a 2-year-old were studied under both the light and electron microscopes. From the 20th fetal week onward, the progressive thickening of the trabecular meshwork was achieved mainly through the widening of intertrabecular spaces, whereas the thickness and number of trabeculae were almost constant. Significant alterations in the number of TM cells did not occur with development. However, a decrease in TM cellularity occurred through the widening of intertrabecular spaces. These findings suggest that all these changes support the expected increase in physiologic function to accomodate the increase in the out-flow of aqueous humor at this stage of development.     

肾上腺素抑制培养牛眼小梁细胞AQP1表达

目的测定肾上腺素对体外培养牛眼小梁细胞水通道蛋白-1(aquaporin1,AQP1)表达的影响,探讨肾上腺素降低眼压的机制。方法将传三代的牛眼小梁细胞随机分为对照和肾上腺素处理组。将不同浓度的肾上腺素(10-4、10-5和10-6mol/L)分别加入培养液持续培养14天后,免疫细胞化学方法检测牛眼小梁细胞AQP1表达水平,计算机图像分析软件测量细胞表面积。结果发现经不同浓度肾上腺素处理后的小梁细胞AQP1表达量吸光度值A和细胞表面积显著低于对照组(P<0.05)。结论肾上腺素对牛眼小梁细胞AQP1表达的抑制可能参与了肾上腺素降眼压作用。     

Localization of sialic acid in the human trabecular meshwork

The localization of sialic acid in the trabecular meshworks of the normal human primary open-angle (POAG), congenital and juvenile glaucomatous eyes was investigated using both an electron microscope and lectins specifically binding with sialic acid. Sialic acid was always localized in fine fibrils lying underneath the canal wall and around collagen fibrils of the whole trabecular tissue. In both congenital and juvenile glaucoma, sialic acid was detected not only in the fine fibrils but also in the basement membrane-like materials in compact tissue present 3 μm underneath the canal wall. Sialated glycoprotein appears to play an important role in the pathogenesis of both diseases.     

The effect of dexamethasone on organ-cultured human trabecular meshwork

The effect of dexamethasone (DEX) on the organ-cultured human trabecular meshwork (HTM) was studied with morphometric, autoradiographical, biochemical and immunohistochemical techniques. In approximately 36% of the cases examined, a statistically significant increase in extracellular materials in the endothelial meshwork was found with occurrence of vacuolated trabecular cells. Silver grains showing the presence of the proteins and glycoproteins labeled with³H-methionine were observed in vacuoles of the trabecular cells. In addition, elastin was identified on the surface of the vacuolated trabecular cells. The alterations of the proteins/glycoproteins and elastin in the HTM induced by DEX treatment may play a role in the reduction in aqueous outflow facility.     

Identification and characterization of mesenchymal stem cells derived from the trabecular meshwork of the human eye

Mesenchymal stem cells (MSC) have been isolated from several adult human tissues. Their propensity to differentiate into cell types of connective tissue, such as osteocytes, chondrocytes, and adipocytes, suggests that MSC may function as a reserve of progenitor cells that repair and maintain healthy adult tissues. Dysfunction of the trabecular meshwork (TM), a connective tissue at the anterior region of the human eye that regulates intraocular pressure, plays a major role in the pathogenesis of glaucoma. The mechanobiology and pharmacological aspects of the TM tissue have been relatively well studied in disease states. Less well understood is if there are progenitor cells within the TM that contribute to maintenance of this tissue. In this study, we have identified and characterized an expandable population of cells that have stem cell-like properties. In particular, these cells express the markers CD73, CD90, and CD105, which are typically associated with MSC. Thus, we have named these cells TM-MSC. As further evidence that these cells are MSC, they were differentiated in vitro into adipocytes, osteocytes, and chondrocytes. Through genomic characterization, we show that TM-MSC have gene expression patterns most similar to MSC derived from other tissues. TM-MSC express genes found on adult TM tissue, suggesting that TM-MSC are progenitor cells that serve to maintain a healthy TM.