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Objective: To evaluate the effect of thiazovivin, a novel ROCK inhibitor, on the morphology and function of human corneal endothelial cells(HCECs)., Methods: The primary HCECs were identified by light microscopy and immunofluorescence staining of neuron-specific enolase. To screen the optimal concentration and action time of thiazovivin for maintaining the morphology and function of primary HCECs, Na (+)/K (+)-ATPase and N-cadherin were chosen as indicators, and the morphology and function of HCECs in various concentrations(0 μmol/L, 2 μmol/L, 4 μmol/L, and 6 μmol/L)for different durations(24 h and 48 h)were examined by immunofluorescence experiments. The effect of thiazovivin on the expression of ROCK was investigated by immunofluorescence and Western blot., Results: The primary HCECs cultured were hexagonal, closely packed, homogeneously and obviously stained by neuron-specific enolase. The immunofluorescence staining of Na(+)/K(+)-ATPase showed that when the primary HCECs cultured with various concentrations of thiazovivin(0, 2, 4, 6 μmol/L)for 24 h, the fluorescence were obvious, and the average absorbance values(A)were 1.27±0.08, 3.72±0.17, 21.07±4.67, 3.69±0.34, respectively. And the immunofluorescence staining of N-cadherin revealed that when the primary HCECs treated with 4 μmol/L thiazovivin for 24 h, the cell boundary was clear and the structure of the cells was intact. While the treating time of thiazovivin(4 μmol/L)on HCECs extended to 48 h, the immunofluorescence staining of Na(+)/K(+)-ATPase and N-cadherin showed that compared to HCECs treated with thiazovivin(4 μmol/L)for 24 h, the fluorescence intensity did not change significantly, but the cells arranged slightly untidy. In addition, the immunofluorescence staining of ROCK was weakened and the expression of ROCK was reduced by thiazovivin. Thiazovivin was effective for protecting the morphology and function of HCECs. An optimal improvement in the morphology, connection and function of HCECs was found when the primary HCECs were cultured with 4 μmol/L thiazovivin for 24 h. Moreover, the expression of ROCK protein could be significantly inhibited by thiazovivin. (Chin J Ophthalmol, 2016, 52: 686-692).

Objective: To investigate the effects of the small interfering RNA (siRNA) approach to silence the expression of connective tissue growth factor (CTGF) gene expression on the expression of the protein p27., Methods: Experimental research. The specific siRNA of CTGF was designed and synthesized, then transfected into cultured bovine corneal endothelial cells by liposomes. The blank control group was only treated with the culture medium and not be transfected with siRNA. Reverse transfection-polymerase chain reaction (RT-PCR) was applied to show the effect of gene-silencing, and western blot was used to observe the protein p27 expression of bovine corneal endothelial cells, which were treated with 1.00 µg/L transforming growth factor β(2) (TGF-β(2)). The results were analyzed by one-factor analysis of variance., Results: CTGF siRNA was successfully transfected into cultured bovine corneal endothelial cells and effectively silence the expression of CTGF mRNA. The CTGF mRNA expression decreased to 17.3%, 24.4% and 41.7% of control values at 24, 48 and 72 h after transfection, there was significant difference between these groups (F = 389.9, P < 0.05). Significant decrease of protein p27 expression was detected at 36 h after transfection (F = 299.3, P < 0.05)., Conclusion: CTGF-specific siRNA was able to inhibit the expression of CTGF mRNA effectively and down-regulate the expression of p27.

Purpose: To observe the effect of TGF-beta1 applied topically to the alkali-injured rabbit eye on corneal epithelial wound healing and expression of integrin beta1 and its therapeutic action on corneal alkali burns., Methods: Alkali burn was produced in 60 corneas from 30 rabbits. Two groups were randomly divided. One group was treated with TGF-beta1 solution (200 ng/ml) topically 3 times one day within the first 7 days, the other group was treated with phosphate-buffered saline (PBS) solution. The injured eyes were photographed after the fluorescence staining with a digital camera and the pictures were analyzed with computer-aided picture analysis system to calculate the rate of corneal epithelial healing. The expression of integrin beta1 was investigated in the point 6 h, 1 d, 3 d, 7 d, 14 d after the injury by means of immunohistochemical analysis., Results: On the 4th, 10th, 11th, 12th and 14th days after the burning,the rate of corneal epithelial healing of TGF-beta1 groups was markedly higher than that of the PBS group (P < 0.05). The expression of integrin beta1 in the cornea epithelial cells gradually increased during the wound healing. On the 7th and 14th days after the burning, the expression of integrin beta1 in the cornea epithelial cells of TGF-beta1 group was remarkably higher than that of the PBS group (P < 0.05)., Conclusions: TGF-beta1 could up-regulate integrin beta1 in vivo corneal alkali burn model, which could stimulate the cornea epithelial cells to migrate and adhere to the cornea stroma, that can reduce the cases of the epithelial cells'detachment from the cornea stroma and sustain the corneal reepithelization.

Objective: To evaluate the changes of porcine corneal endothelium, the morphology, histology, ultrastructure, enzymes activity and metabolism of the cornea induced by organ culture with two different media containing fetal bovine serum (FBS) or human umbilical cord serum (HCS)., Methods: Fifty pairs of porcine corneas were preserved at 31 degrees C for 7, 14, 21, 28 days. One cornea of each pair was cultivated in medium I containing 10% FBS (group 1); the other one was stored in medium II containing 10% HCS (group 2). Thirteen fresh porcine corneas served as controls. All stored corneas were dehydrated for 24 hours. Twelve corneas from each group were evaluated each week, including the morphology, histology and enzyme histochemical staining of the cornea. Scanning electron microscopy was performed on one cornea from each group at 14 and 28 days and compared with the fresh cornea. pH value, glucose and lactate concentration of the culture media before and after culture were examined. Microbiological evaluation was also performed., Results: Endothelium evaluation did not differ statistically between the two groups of porcine corneas. The morphological endothelium study showed some alterations such as pleomorphism. After 28 days of cultivation, the mean cell losses of endothelium were 10.98% and 10.85% in medium I and medium II stored corneas, respectively. There were no statistical differences of the histology, ultrastructure and enzymes activity of corneas between the two groups. The histological study showed corneal swelling and epithelial sloughing after preservation. Scanning electron microscopy showed an intact endothelial layer in all corneas. Enzyme histochemical staining showed vigorous enzyme activity in the corneal epithelium and endothelium. Enzyme activity in stroma decreased with preservation time. Corneas showed good glucose metabolism. Incidence of contamination was 6% for storage medium., Conclusions: The corneal endothelium can maintain a good viability for 4 weeks in these two organ culture media. HCS can replace FBS in the organ culture medium.

Objective: To transfer the gene encoding mammalian aFGF to rabbit corneal endothelial cell and determine transfection efficiency and the effect of Ad-aFGF on proliferation of RCEC., Methods: Replication-defective adenovirus was used to deliver the LacZ reporter gene to RCEC to determine transfection efficiency by X-gal staining. A cDNA encoding mammalian aFGF was cloned into an adenoviral vector that was used to transfect RCEC. The expression of aFGF gene mRNA and protein were demonstrated by Reverse transcription PCR and Western blot respectively. The effect of Ad-aFGF on proliferation of RCEC was determined by MTT assay., Results: RT-PCR and Western blot from RCEC infected with Ad-aFGF for 48 h detected a specific cDNA amplification and protein expression of aFGF gene, and no positive result in control cells. Consistently, RCEC infected by MOI 20 of Ad-aFGF demonstrated an enhanced RCEC proliferation compared to controls (F = 217.107, P < 0.05)., Conclusion: Adenoviral-mediated expression of aFGF can stimulate the proliferation of RCEC.

Objective: To observe the indication, surgical principle, clinical results and complications of microkeratome-assisted deep lamellar endothelial keratoplasty (DLEK)., Methods: DLEK was performed in six patients with bullous keratopathy. One of the six patients underwent DLEK combined with vitrectomy under temporary artificial corneal and intraocular foreign-body removal. Another underwent DLEK combined with vitrectomy and posterior chamber intraocular lens implantation. The patients were followed up for 6 approximately 9 months., Results: The postoperative best-corrected visual acuity showed clear improvement in 5 cases. Endothelial cell density averaged (2481 +/- 212) cells/mm(2). Pachymetry averaged (549 +/- 61) micro m. Astigmatism averaged (2.04 +/- 1.19) D. No serious complication was found., Conclusion: DLEK is an alternative choice for PKP. It may offer further advantages over conventional PKP surgery, and could serve as a surgical technique in endothelial keratoplasty. A long-term follow-up in a larger patient population is needed to confirm our initial results.

Objective: To investigate the water permeability (P(f)) of the cultured bovine corneal epithelial cells (CBCEC) and whether aquaporins (AQPs) were present., Methods: Laser light-scattering system was used to determine the volume change of CBCEC from isotonic status rapidly transferred to hypotonic one, and the P(f) value was calculated. Messenger RNA obtained from CBCEC was injected into Xenopus laevis oocytes. After four days, the P(f) was determined by monitoring volume changes of oocytes upon hypoosmotic challenge (180 mOsm/L changed to 15 mOsm/L)., Results: CBCEC challenged with 10% hypoosmotic solution at 37 degrees C swelled rapidly; the cellular P(f) was 72 microm/s. In the oocytes injected with mRNA from CBCEC, the P(f) increased to 76 microm/s. This increase was inhibited (by 89%) by 0.3 mmol/L HgCl(2)., Conclusion: Our results suggest that CBCEC express a typical mercurial-sensitive functional water channel in good numbers, judging from the comparatively high P(f) we measured. These begin to reopen the question of the presence and extent of an epithelial role in control of corneal hydration.

Using ConA-HRP and RCAI-HRP as probes, the distribution and changes of glycosides in mouse cornea were studied during pre- and postnatal development. Mannose residues were distributed mainly in stroma and endothelium, sialic acid residues in epithelium and galactose residues in both epithelium and stroma. Mannose residues in stroma showed an increased density toward endothelium before and after birth. Sialic acid and galactose residues were concentrated gradually at the corneal epithelial surface in accompanied with the development of cornea. The embryonic day 13 was the starting day to synthesize glycoconjugates from fibroblasts of mouse cornea.