角膜基质细胞的表型转化及其分子机制

角膜瘢痕是继发于多种角膜疾病的病理性改变,是许多角膜疾病造成视力不同程度损害甚至丧失的直接原因,也是影响角膜屈光手术效果的重要因素.对于角膜损伤愈合后的角膜瘢痕,目前临床上主要治疗方法是穿透性或者板层角膜移植术,但因为角膜供体材料缺乏和经济原因,大量的患者无法得到手术治疗而最终丧失视力.     

Corneal stromal repair and regeneration

The cornea is a specialized, transparent, avascular, immune-privileged, and heavily innervated tissue that affords 2/3rd of refraction to the eye. Ocular injuries, infections, and genetic factors affect corneal function and cause vision impairment. Presently, a variety of laser/non-laser surgeries, immunosuppressants, and/or corneal transplants are predominantly used to revive sight in human patients. The development of novel, precision-guided, and tissue-targeted non-surgical therapies promoting corneal repair and regeneration based on mechanistic understanding is of paramount importance to reduce the impact of global blindness. Research over the past decade revealed that modulation of pathological signaling pathways and factors by a variety of therapeutic delivery methods effectively treats corneal disorders including corneal scar/haze, inflammation, and angiogenesis in various pre-clinical animal models and are primed for human translation. This review discusses recent advances in the areas of corneal repair, restoration, and regeneration. Herein, we provide an overview of evolving approaches and therapeutic modalities that have shown great promise in reviving corneal transparency and function through the use of small drug molecules, gene therapy, nanomedicine, stem cells, trophic factors, exosomes, stromal equivalents, bioengineered stromal scaffolds, tissue adhesives, and 3D bioprinting.     

Análisis de superficie ocular en pacientes diagnosticados de ictiosis X

Seven patients (14 eyes) diagnosed with X-linked ichthyosis were studied using the Schirmer test, biomicroscopy, tonometry, endothelial count, optical coherence tomography, Pentacam®, ocular surface analyser, and confocal microscopy. The mean age was 33.83 ± 20.17 years (range: 7-64 years). The most frequent findings in biomicroscopy were Meibomian glands dysfunction (83.3%) and stromal corneal opacities (33%). The tear break-up time was found shortened in 25% of the eyes. Confocal microscopy (both eyes) revealed activated keratocytes with hyper-reflective particles inside them in the anterior stroma and outside them in the posterior stroma. It is believed that the inclusion of the use of confocal microscopy will help in a better understanding of the corneal pathology associated with ichthyosis X, as well as new characteristics of these patients.     

Corneal stromal bioequivalents secreted on patterned silk substrates

Emulating corneal stromal tissue is believed to be the most challenging step in bioengineering an artificial human cornea because of the difficulty in reproducing its highly ordered microstructure, the key to the robust biomechanical properties and optical transparency of this tissue. We conducted a comparative study to assess the feasibility of human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on groove-patterned silk substrates. In serum-free keratocyte differentiation medium, hCSSCs successfully differentiated into keratocytes secreting multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 90–100 μm thick, containing abundant cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. In contrast, hCFs tended to differentiate into myofibroblasts that deposited less organized collagen in a pattern resembling that of corneal scar tissue. RGD surface coupling coupling was an essential factor in enhancing cell attachment, orientation, proliferation, differentiation and ECM deposition on the silk substratum. These results demonstrated that an approach of combining hCSSCs with an RGD surface-coupled patterned silk film offers a powerful tool to develop highly ordered collagen fibril-based constructs for corneal regeneration and corneal stromal tissue repair.     

Corneal multiphoton microscopy and intratissue optical nanosurgery by nanojoule femtosecond near-infrared pulsed lasers

Multiphoton microscopy including multiphoton autofluorescence imaging (MAI) and second-harmonic generation (SHG) is being used as a novel diagnostic tool to perform tissue nonlinear optical tomography with submicron resolution. The three-dimensional corneal ultrastructure of whole depth has been viewed without any staining or mechanical slicing. Compared with photodisruptive surgical effects occurring at TW/cm2 light intensity, multiphoton imaging can be induced at MW-GW/ cm2 photon intensity. The intratissue surgical effect including nanojoule (nJ) femtosecond laser ablation and flap generation was induced through multiphoton nonlinear absorption at a wavelength of 800 nm and ascertained by the histological outcomes. More interesting, the multiphoton microscopy based on nonlinear absorption of femtosecond laser pulses at the wavelength of 715-930 nm emitted from solid-state Ti:sapphire system is acting as a precise non-invasive monitoring tool to determine the interest of region, to visualize and verify the outcomes in in vivo intrastromal laser nanosurgery. Overall, these data suggest that multiphoton microscopy is a highly sensitive and promising technique for studying the morphometric and biomechanical properties of biological tissues and that the nJ ultrashort Lasers can be used as a ultra-precise nanoscalpel for performing intratissue surgery.     

准分子激光原位角膜磨镶术后角膜细胞凋亡的研究

目的 了解准分子激光原位角膜磨镶术（laser in situ keratomileusis,LASIK）后早期角膜基质细胞凋亡的特点并探索LASIK术后角膜愈合反应程度与角膜刀的机械损伤、激光能量的相关性。方法 20只新西兰白兔,其中10只兔左眼为LASIK-4.0D组、右眼为LASIK-8.0D组、另10只兔左眼为单纯角膜瓣组、右眼作为空白对照组,在术后4小时处死兔取角膜制作病理切片,行透射电镜检查,用脱氧核糖核苷酸末端转移酶介导的缺口末端标法（terminal-deoxynucleotidyl transferase medi-ated dUTP nick end labeling,TUNEL）原位显示凋亡细胞,激光扫描共聚焦显微镜观察凋亡细胞形态,定量统计比较凋亡水平差别。结果 三个手术组中角膜基质中均出现凋亡细胞,主要分布于角膜瓣和基质床交接面两侧50μm范围内的基质层中;瓣缘的凋亡水平高于中央部。而空白对照组中,凋亡细胞仅出现在表层上皮,角膜基质细胞未见凋亡。三个手术组的TUNEL阳性细胞计数进行方差分析,三组之间差异无显著意义（F=1.008,P〉0.05）;每组不同个体的TUNEL阳性细胞计数存在明显的差异。结论 正常角膜基质细胞不发生凋亡。角膜基质细胞凋亡与角膜损伤有关。LASIK术后早期即可出现角膜基质细胞的凋亡表达,该表达与角膜瓣的制作有关,与激光切削的深度、能量无明显的相关性。