超声波辅助提取玉木耳多糖及其抗氧化活性分析

以玉木耳为原料,考察液料比、超声功率、超声温度和超声时间对多糖得率的影响,在单因素试验基础上,通过响应面分析法优化提取工艺条件,采用傅里叶红外光谱(FT-IR)对多糖结构进行初步表征,并通过测定玉木耳多糖清除1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基能力、铁离子还原能力(FRAP 法)和氧自由基清除能力(ORAC 法)研究其体外抗氧化活性。结果表明,超声波辅助法提取玉木耳多糖最优工艺为:液料比40∶1 mL/g,超声功率210 W,超声温度62 ℃,超声时间29 min,此工艺条件下玉木耳多糖的得率为7.43%;玉木耳多糖显示出多糖的典型特征吸收峰,是以β-糖苷键为主的吡喃型多糖;玉木耳多糖清除DPPH自由基IC₅₀值为1.445 mg/mL,FRAP值和ORAC值分别为35.14±0.16 mmol Trolox g^-1和0.627 mg Trolox mg^-1,具有较好的抗氧化能力,可作为天然抗氧化剂应用。     

Improvement of an Effective Protocol for Directed Differentiation of Human Adipose Tissue-Derived Adult Mesenchymal Stem Cells to Corneal Endothelial Cells

Corneal disease affects 12.5 million individuals worldwide, with 2 million new cases each year. The standard treatment consists of a corneal transplantation from a human donor; however, the worldwide demand significantly exceeds the available supply. Lamellar endothelial keratoplasty, the replacement of only the endothelial layer of the cornea, can partially solve the problem. Progressive efforts have succeeded in expanding hCECs; however, the ability to expand hCECs is still limited, and new sources of CECs are being sought. Crucial advances have been achieved by the directed differentiation of embryonic or induced pluripotent stem cells, but these cells have disadvantages, such as the use of oncogenes, and are still difficult to establish. We aimed to transfer such knowledge to obtain hCECs from adipose tissue-derived adult mesenchymal stem cells (ADSC) by modifying four previously published procedures. We present several protocols capable of the directed differentiation of human ADSCs to hCECs. In our hands, the protocol by Ali et al. was the best adapted to such differentiation in terms of efficiency, time, and financial cost; however, the protocol by Wagoner et al. was the best for CEC marker expression. Our results broaden the type of cells of autologous extraocular origin that could be employed in the clinical setting for corneal endothelial deficiency.     

Artificial Tears: Biological Role of Their Ingredients in the Management of Dry Eye Disease

Dry eye disease (DED) is the most common ocular surface disease, characterized by insufficient production and/or instability of the tear film. Tear substitutes are usually the first line of treatment for patients with DED. Despite the large variety of tear substitutes available on the market, few studies have been performed to compare their performance. There is a need to better understand the specific mechanical and pharmacological roles of each ingredient composing the different formulations. In this review, we describe the main categories of ingredients composing tear substitutes (e.g., viscosity-enhancing agents, electrolytes, osmo-protectants, antioxidants, lipids, surfactants and preservatives) as well as their effects on the ocular surface, and we provide insight into how certain components of tear substitutes may promote corneal wound healing, and/or counteract inflammation. Based on these considerations, we propose an approach to select the most appropriate tear substitute formulations according to the predominant etiological causes of DED.     

Epithelial Cell-Derived Extracellular Vesicles Trigger the Differentiation of Two Epithelial Cell Lines

Extracellular vesicles (EVs), specifically exosomes, carry a cell-type dependent cargo that is transported to the recipient cell and translated in the presence of a required machinery. Differences in the cargo carried by the corneal and conjunctival-derived EVs could be the agent that triggers the transdifferentiation of these two cell populations. Therefore, this study investigates the role of EVs in triggering the plasticity of corneal and conjunctival epithelial cells and identifies prospective miRNA and genes responsible for maintaining ocular surface homeostasis. The EVs were extracted from the conditioned media (after starving) of corneal epithelial (hTCEpi) and conjunctival (HCjE-Gi) cell lines using ultracentrifugation. HCjE-Gi cells were cultured with hTCEpi-derived EVs and vice-versa. The EVs were characterized as exosomes using Nanosight and Flow cytometry. KRT3 and KRT12 were used as associated corneal markers, whereas KRT7 and KRT13 were used as associated conjunctival markers with ΔNp63 as a differentiation marker. Shift of these markers was an indication of transdifferentiation. The cargo of the extracted exosomes from both the cell types was explored using next-generation sequencing. The hTCEpi-derived EVs induced conjunctival epithelial cells to express the corneal-associated markers KRT3 and KRT12, losing their conjunctival phenotype at both the mRNA and protein level. Simultaneously, HCjE-Gi-derived EVs induced corneal epithelial cells to express the conjunctival associated markers KRT7 and KRT13, losing their corneal phenotype. This process of differentiation was accompanied by an intermediate step of cell de-differentiation showed by up-regulation in the expression of epithelial stem cell marker ΔNp63, also shown on the ex vivo human cadaveric donor corneas. miRNA molecules (total of 11 including precursor and mature) with significant differences in their relative abundance between the two populations (p < 0.05) were found and investigated. miR-9-5p expression was higher in HCjE-Gi cells and HCjE-Gi-derived EVs when compared to hTCEpi cells and hTCEPi-derived EVs (p < 0.001). The results suggest that EVs released by the two cell types have the ability to influence the transdifferentiation of human conjunctival and corneal epithelial cells. miR-9-5p could have a role in stem cell homeostasis and cell differentiation via HES-1 gene.     

化学-物理法制备多孔纳米羟基磷灰石/Ⅰ型胶原/聚乙烯醇复合水凝胶及其性能

以纳米羟基磷灰石(n-HA)、Ⅰ型胶原(Ⅰ-Col)、聚乙烯醇(PVA)为原料,采用化学-物理交联法制备人工角膜支架材料。通过对比测试其含水率、孔隙率、力学拉伸性能、扫描电镜及红外光谱,探讨了戊二醛加入浓度和冷冻解冻次数对材料性能的影响。结果表明当m(PVA)∶m(n-HA+Ⅰ-Col)=7∶1,戊二醛浓度为0.04mol/L时,复合水凝胶材料具有较好的综合性能:含水率为70.41%;孔隙率为58.95%;拉伸强度为3.87 MPa;红外分析表明原材料与交联剂之间产生了交联反应;扫描电镜分析表明经冷冻解冻物理交联5次,该复合水凝胶呈均匀三维多孔结构;生物相容性实验测得兔角膜上皮细胞增殖良好,毒性为Ⅰ级,达到国际要求,属于合格医用材料。     

Tm激光与角膜作用的热效应模拟

激光角膜热成形技术(LTK)在矫正远视临床中占有重要的应用前景。为了了解铥激光与角膜组织热相互作用中激光参数和角膜光学及热物性参数对温度场分布的影响,选取50W/cm2,55W/cm2,60W/cm2三种功率密度,1s、2s、3s三种作用时间,用有限元方法模拟了激光作用下角膜组织内温度场的分布。模拟结果表明激光有效穿透深度与激光功率密度和时间都是正向相关,而与能量密度的正向相关性较差;激光作用时间越长,角膜组织中的温度梯度越小。模拟得到激光功率密度50W/cm2作用时间1s适合于LTK。     

一种用于激光屈光术的新型激光光斑调制系统

激光光调调制装置包括－高分辨率的微镜阵列,后者由许多独立编址并且可移动的微镜构成。微镜在打开位置反射部分光束到角膜,在关闭位置反射光束偏离角膜。针对一系列的预定模式进行编程后,计算机可控制某些微镜移动到打开位置,而其它的微镜处于关闭位置。每个消融模式叠加起来构成矫正角膜折射率偏差的形成。并且由于微镜阵列的高分辨率,此形线非常平滑,这对减轻激光屈光术后的角膜浑浊程度是有利的。     

308nm紫外激光对兔眼角膜损伤的延迟反应和损伤阈值研究

308 nm 紫外激光照射青紫蓝灰兔眼所引起的角膜损伤,观察到损伤呈两种反应:瞬时反应和延迟反应,并测量了延迟反应的剂盐水平。统计了受照24小时内角膜发生的损伤点,经概率统计分析,获得角膜瞬时损伤阈值 ED_(50)=0.485J/cm~2(95%置信限为0.349～0.608J/cm~2)。     

Low Energy Blue Pulsed Light-Activated Injectable Materials for Restoring Thinning Corneas

Many alternatives to human donor corneas are being developed to meet the global shortage of donated tissues. However, corneal transplantation remains the gold standard for diseases resulting in thinning corneas. In this study, transparent low energy photoactivated extracellular matrix-mimicking materials are developed for intrastromal injection to restore stromal thickness. The injectable biomaterials are comprised of short peptides and glycosaminoglycans (chondroitin, hyaluronic acid) that assemble into a hydrogel when pulsed with low-energy blue light. The dosage of pulsed-blue light needed for material activation is minimal at 8.5 mW cm⁻², thus circumventing any blue light cytotoxicity. Intrastromal injection of these light-activated biomaterials in rat corneas shows that two iterations of the formulations remain stable in situ without stimulating significant inflammation or neovascularization. The use of low light intensities and the ability of the developed materials to stably rebuild and change the curvature of the cornea tissue make these formulations attractive for clinical translation.     

视线追踪系统中特征参数提取方法研究

为了提取人眼的高精度亚像素特征参数,利用亮瞳现象,提出了一种基于多通道图像的高精度亚像素特征参数提取方法。该方法首先通过差分图像滤波获得瞳孔区域,进而检测瞳孔区域的边缘,并在眼睛区域附近基于灰度,搜索角膜反射区域; 然后求取其质心用于定位角膜反射区域中心,并对瞳孔边缘做滤波,以消除角膜反射对瞳孔边缘轮廓的影响,进而利用椭圆拟合来定位瞳孔中心;最后提取包括人眼特征和人脸位置的多个参数,另外,还建立了一个多特征参数提取的流程,为下一步的视线估计提供了参数依据。 实验结果及视线追踪系统最终的视线估计结果证明,该方法是有效的。