Amniotic membrane covering promotes healing of cornea epithelium and improves visual acuity after debridement for fungal keratitis

AIM:To investigate the effect of amniotic membrane covering(AMC) on the healing of cornea epithelium and visual acuity for fungal keratitis after debridement.METHODS:Twenty fungal keratitis patients were divided into two groups randomly, the AMC group and the control group, ten patients each group. Both debridement of the infected cornea tissue and standard anti-fungus drugs treatments were given to every patients, monolayer amniotic membrane were sutured to the surface of the entire cornea and bulbar conjunctiva with 10-0 nylon suture for patients in the AMC group.The diameter of the ulcer was determined with slit lamp microscope and the depth of the infiltration was determined with anterior segment optical coherence tomography. Uncorrected visual acuity(UCVA) was tested before surgery and three month after healing of the epithelial layer. The healing time of the cornea epithelium, visual acuity(VA) was compared between the two groups using t- test.RESULTS:There was no statistical difference of the diameter of the ulcer, depth of the infiltration, height of the hypopyon and VA between the two groups beforesurgery(P 】0.05). The average healing time of the AMC group was 6.89 ±2.98 d, which was statistically shorter than that of the control group(10.23±2.78d)(P 【0.05).The average UCVA of the AMC group was 0.138 ±0.083,which was statistically better than that of the control group(0.053±0.068)(P 【0.05).CONCLUSION:AMC surgery could promote healing of cornea epithelium after debridement for fungal keratitis and lead to better VA outcome.

Promotion of minTBP-1-PRGDN on the attachment,proliferation and collagen I synthesis of human keratocyte on titanium

AIM: To investigate the influence of minTBP-1-PRGDN on the attachment,proliferation and collagen I synthesis of human keratocyte on titanium(Ti) surface. · METHODS: The chimeric peptide RKLPDAPRGDN(minTBP-1-PRGDN) was synthesized by connecting RKLPDA(minTBP-1) to the N-terminal of PRGDN,the influence of minTBP-1-PRGDN on the attachment,proliferation and collagen I synthesis of human keratocyte on Ti surface were tested using PRGDN and minTBP-1 as controls. The keratocytes attached to the surface of Ti were either stained with FITC-labeled phalloidin and viewed with fluorescence microscope or quantified with alamar Blue method. The proliferation of keratocytes on Ti were quantified with 3-(4,5-dim-ethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptaking methods. The secretion of type I collagen was determined using an ELISA kit. ·RESULTS: The results showed that minTBP-1-PRGDN at a concentration of 100ng/mL was the most potent peptide to enhance the attachment of human keratocytes to the surface of Ti(1.40±0.03 folds,P =0.003),to promote the proliferation(1.26 ±0.05 folds,P =0.014) and the synthesis of type I collagen(1.530 ±0.128,P =0.008). MinTBP-1 at the same concentration could only promote the attachment(1.13±0.04 folds,P =0.020) and proliferation(1.15±0.06 folds,P =0.021),while PRGDN had no significant influence(P 】0.05). ·CONCLUSION: Our data show that the novel chimericpeptide minTBP-1-PRGDN could promote the attachment,proliferation and type I collagen synthesis of human keratocytes on the surface of titanium.

Gene transfer to human trabecular meshwork cells in vitro and ex vivo using HIV-based lentivirus

AIM: To investigate whether the enhanced green fluorescent protein(EGFP) reporter gene could be transferred into human trabecular meshwork(HTM) cells by a HIV-based lentivirus both in vitro and ex vivo.METHODS: The HIV-based lentivirus that contains an EF1-α promoter driving EGFP expression cassette was constructed following the standard molecular cloning methods. The cultured HTM cells were transduced at a range of multiplicity of infection(MOI) with HIV-based lentivirus. EGFP positive cell populations were detected by flow cytometry. Human anterior eye segments were cultured with perfusion culture system and transfected by HIV-based lentivirus with a 1 ×108transducing unit(TU) virus in perfusion liquid. The intraocular pressure was recorded every 8h for 21 d. The expression of EGFP in the anterior segment of the human eye was detected by fluorescence microscopy. Furthermore, the distribution of EGFP expression was confirmed by anti-EGFP immunohistochemical staining.RESULTS: The HIV-based lentivirus which contains an EF1-α promoter driving EGFP expression cassette was constructed successfully. After HTM cells were transduced with HIV-based lentivirus containing EGFP in vitro, the ratio of EGFP positive cells to the total cell number reached 92.3%, with the MOI of 15. After the lentivirus containing EGFP were used to transduce human anterior eye segments, the EGFP could be directly detected by fluorescence microscopy in vivo.Immunohistochemistry staining revealed that 88.19%EGFP-positive trabecular meshwork(TM) cells were observed in the human anterior segment. Nevertheless,the intraocular pressure in the lentivirus-transduced group kept constant when compared with control group(P 】0.05).CONCLUSION: EGFP gene could be efficiently transferred into HTM cells both in vitro and ex vivo by using HIV-based lentivirus.