High glucose suppresses the corneal limbal stem cell migration and changes the cell phenotype / 眼科新进展

Objeetive To explore the influence of extracellular high glucose on the proliferation,migration and biomarkers of corneal limbal stem cells.Methods Establishment of a model of high glucose in cultured human limbal stem cells to observe and investigate the effects of extracellular high glucose on the proliferation and migration of corneal limbal stem cells by immunoflurescence,CCK-8 and Transwell assay,respectively.Totally 16 SPF rats were collected and induced diabetic model by streptozotocin as the high-glucose group,and the normal rats of the same age served as the control group.Corneal epidermises of rats in both groups were scraped to observe the repair of corneal epithelium.And the corneas were treated with HE staining and immunohistochemical staining to detect the expression of biomarkers of corneal limbal stem cells and the modality changes of cells.Results The proliferation rate of human limbal epithelial cells was significantly decreased when exposed to high glucose,and the rate at 24 h,48 h and 72 h was 0.728,0.345 and 0.395,respectively,which was markedly lower than that in the control group,with a significant difference (P ＜ 0.05);meanwhile the cell migration rate of the high-glucose group was 17.6％ at 48 h,which was significantly slower than that of the control group (100％).And the inhibition was accompanied by the decreased expression of β-catenin and vimentin.Furthermore,the expression levels of β-catenin and vimentin mRNA and protein were down-regulated,with abnormal location,in the high-glucose group.And diabetic rats had poor corneal epithelial healing.The epithelial layer became thinner and the structures were disorganized in diabetic rats through HE staining.The immunohistochemical assay revealed the expression of β-catenin and vimentin of cornea limbal stem cells was down-regulated in high-glocose group when compared with the control group.Conclusion High glucose can significantly inhibit the proliferation and migration of cornea limbal stem cells,and its main damage mechanism is correlated with the abnormalities of β-catenin and vimentin.

Pathological changes of the cornea in rabbits with hyphema and concurrent ocular hypertension / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the impact of hyphema secondary to high intraocular pressure on corneal pathology in rabbits.</p><p><b>METHODS</b>Thirty adult New Zealand rabbit were randomized into 3 equal groups, and in each rabbit, one eye served as the experimental eye with the other as the control eye. In the experimental eye, autoblood was injected into the anterior chamber to induce high intraocular pressure maintained for 3, 5, or 8 days. Only saline was injected into the control eye. After the injections, the cornea was observed with slit-lamp microscopy, and at 3, 5, or 8 days, the experimental and control eyes were taken from the 3 groups for microscopic examination of the corneas to detect the occurrence of cornea bloodstain with prolonged high intraocular pressure. Corneal edema, elastic fibers changes, growth of new blood vessels, changes of eosinophils, fibroblasts, lymphocytes and plasma cells, as well as the pathological changes of the corneal layers were observed and compared between the experimental and control eyes.</p><p><b>RESULTS</b>Maintenance of high intraocular pressure for 8 days resulted in the most severe corneal edema and thickening, and histopathologically, the corneal stroma showed widened space between the elastic fibers and obvious fiber distortion. Neovascularization was seen in the marginal cornea where eosinophil infiltration occurred with a small number of lymphocytes, plasma cells and fiber cells. All the three groups showed more obvious edema in the posterior than in the anterior cornea.</p><p><b>CONCLUSION</b>Prolonged hyphema with ocular hypertension results in aggravation of corneal edema, and corneal blood staining does not occur until 8 days of high intraocular pressure but corneal elastic fiber disruption can be seen, suggesting the impending irreversible pathological changes of cornea.</p>

Role of Toll-like receptor 2 in corneal graft rejection following penetrating keratoplasty / 南方医科大学学报

<p><b>OBJECTIVE</b>To gain insight into the role of Toll-like receptor 2 (TLR2) in graft rejection following penetrating keratoplasty, and investigate the expression of TLR2 mRNA in the corneal graft.</p><p><b>METHODS</b>Penetrating keratoplasty was performed in 3 groups of rats for orthotopic autologous corneal transplantation (group A), allograft corneal transplantation (group B), or allograft corneal transplantation with hormone treatment (C). The transparency and neovascularization of the cornea were observed using a slit-lamp microscope and scored according to the rejection index, with normal cornea serving as the control. The corneal tissues were sampled at 5, 7, and 9 days after the transplantation for histopathological examination and detection of TLR2 mRNA expression using RT-PCR.</p><p><b>RESULTS</b>With the passage of time, edema, opacities and neovascularization of the corneal graft occurred after the operation in all the groups. Seven days after the operation, the rejection index of group B, but not that of groups A and C, met the diagnostic criteria for graft rejection with also support by histopathological evidence. The expression of TLR2 mRNA was detected in normal corneas and augmented in the corneal grafts in the 3 transplantation groups. TLR2 mRNA expression in group B was significantly higher than that of group A, and the expression in group C decreased significantly in comparison with that in group B (P<0.05).</p><p><b>CONCLUSION</b>As the recognition receptors of native immune system, TLR2 in the rejected corneal grafts may recognize the allograft antigen and play a role in acute graft rejection after penetrating keratoplasty.</p>

Recombinant type 1 adeno-associated virus-mediated transfection of ECV304 cells with enhanced green fluorescent protein gene in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the feasibility of recombinant type 1 adeno-associated virus (rAAV1) as a vector for gene therapy of corneal neovascularization.</p><p><b>METHODS</b>The rAAV1 vector carrying enhanced green fluorescence protein (EGFP) gene (rAAV1-EGFP) was transfected into ECV304 cells at different multiplicities of infection (MOI=5 x 10(3), 5 x 10(4), 5 x 10(5)). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage determined by flow cytometry. MTT assay was used to assess the proliferation of the transfected cells.</p><p><b>RESULTS</b>The cells with rAAV1-EGFP transfection at MOI of 5 x 10(5) began to exhibit GFP expression 2 days after transfection, and the fluorescence intensity increased with the MOI used for transfection. GFP expression reached the maximum on day 7, at the point of which the transduction efficiency of rAAV1-EGFP in ECV304 cells was 45.90%, 58.56% and 68.31% corresponding to MOIs of 5 x 10(3), 5 x 10(4), and 5 x 10(5), respectively. MTT assay did not reveal significant difference in the absorbance between the transfected cells and the control cells at 72 and 96 h after transfection.</p><p><b>CONCLUSION</b>arAAV1-EGFP gene can be stably and efficiently expressed in ECV304 cells without causing cell growth inhibition, suggesting the potential of rAAV1 as a safe and efficient vector for gene therapy of corneal neovascularization.</p>

Survival,Proliferation and Differentiation of Human Mesenchymal Stem Cells Derived from Umbilical Cord Transplanted to Hypoxic-Ischemic Encephalopathy Rat Models / 实用儿科临床杂志

Objective To transplant the umbilical cord mesenchymal stem cells(UCMSCs) derived from human umbilical cord into cisterna magna of hypoxic-ischemic encephalopathy(HIE) rat model,and to observe their survival,proliferation and differentiation in the rat brain.Methods UCMSCs were isolated from human umbilical cord of babies delivered after full-term normal cesarean section,and labeled by bromodeoxyuridine(BrdU).Pregnant rats were randomly divided into experimental group(n=6) and control group(n=1).HIE models were built by ligating both sides of the uterine arteries of full-pregnant rats(21 days) in experimental group rats for 15 minutes.The neonatal rats in experimental group were divided into stem cells group(n=24) and PBS group(n=19) at random.The labeled UCMSCs were injected into cisterna magna of the rats in stem cells group,while PBS was injected into the rats of PBS group.In 1,2,3 and 4 weeks after transplantation,the brain tissue section slides were immunohistochemically stained with antibodies against BrdU,Nestin,neuron specific enolase(NSE) and glial fibrillary acidic protein(GFAP),and thionin.Control group with normal delivery was tested as concurrent control.Results At 1 week after transplantation,BrdU,Nestin,NSE and GFAP positive cells were found in the hippocampal dentate gyrus of the rats in stem cells group rats.The number of BrdU-positive and Nestin-positive cells increased(Pa0.05).The NSE-positive and GFAP-positive cells gradually increased from 1-4 weeks post transplantation and comparisons between groups had statistical significance(Pa