| Background and objective:Primary open-angle glaucoma (POAG) is a major of blinding eye diseases that is late-onset, progressive and irreversible. At present its pathogenesis is not fully clear, but generally think elevated intraocular pressure is one of the main risk factors. Therefore, lop reduction has been the principal means of glaucoma treatment. Today most of IOP reduction drugs in curative effect is not ideal, and have certain side effects. Once suffering from glaucoma requires medication for a long time, so there are problems like high cost and poor treatment adherence et al. Moreover, in terms of mechanism in drug action, in addition to cholinergic agonist (as pilocarpine), other effects in medicine did not focus on the main exhaust passage of the aqueous humor or Trabecular channels. Therefore, there are many problems in the current IOP reduction treatment, the introduction of gene therapy has a special significance. This topic in the applicants on the basis of previous work, the auxiliary repressor of TGF-beta/Smad signaling pathways c-Ski gene into anterior chamber of rats, exploring c-ski gene expression on aqueous humor outflow of rat and the influence of intraocular pressure, possible mechanism and side effects of genetically modified expression. The research has not been reported at home and abroad. The project is expected to clarify in c-Ski the feasibility of gene therapy for glaucoma, for the majority of patients with glaucoma treatment brings new hope! Research purposes include:1. To build lentivirus expression vector of c-Ski gene, compound and obtain virus in 293TN cell;2. Lentivirus transduce trabecular cells, and to explore the c-Ski expression at a cellular level and their possible mechanism with the effects of intraocular pressure;3. The virus import anterior chamber of rats in one time, to observe the effects of c-Ski expression on intraocular pressure, intraocular toxicity reaction, et al and discussed the mechanism of c-Ski on intraocular pressure.Methods:1. To build lentivirus vector (HIV-c-Ski/GFP), expressing c-Ski and GFP gene at the same time and confirmed that the purpose protein expression in cell level; the lentivirus vector(HIV-GFP) only expressing GFP gene by contrast, the same below.2. HIV-c-Ski/GFP transduction cultured human trabecular mesh work cells(TMCs); to observe time of transgenic expression and the influence of cell morphology, extracellular matrix, the actin cytoskeleton and cellular connection.3. Experiments using SD rats. HIV-c-Ski/GFP transduce rats, compared with the control, to detect genetically modified expression on the influence of intraocular pressure, genetically modified products in expression of trabecular channel and its effect on the morphology and ultrastructure of trabecula, meanwhile watch the possible side effects such as inflammation, et al.Results:1. The pCDFl-MCS2-EFl-copGFP-c-Ski plasmid was constructed and confirmed by sequencing. Recombinant lentiviruses FHV-c-Ski and HIV-GFP were produced in 239TN cells and the titer of both viruses was 3.4×108TU/ml.2. Western blot results showed that its Ski have both expressed in HIV-c-Ski, FIV-GFP trabecular cells and untreated. Ski expression enhance in HIV-c-Ski trabecular cells, confirmed transduction is successful. Immunofluorescence, according to the results compared with FIV-GFP group and blank cells, actin skeleton is degraded in HIV-c-Ski human trabecular cells, beta catenin expression is weaken.3. The baseline IOP values of treated group and control group were 13.02±0.48 mmHg and 13.05±0.52 mmHg (measured between 10:00 and 11:00 AM), respectively, with no significant difference (P> 0.05) (n=12). IOP values of treated group decreased significantly compared with control group at 11 days post-injection (p.i.) (P< 0.05). Compared with the control group and its baseline IOP values, IOP values of treated group reducted significantly at 13 days p.i. (P< 0.05). IOP values of treated group were stable at a relative low level till the rats were sacrificed at 21 days. No significant intraocular toxic reation of c-Ski was found in 12 included rats.4. Immunocytochemistry results showed:the anterior chamber of rats are injected into virus after 4 weeks, the experimental group expression of extracellular matrix(ECM) and the cell actin protein skeleton in trabecular meshwork both decreased.Conclusion:1. Lentiviral-c-Ski expression system was constructed and pseudo-lentivirus HIV-c-Ski was produced in 239TN cells for gene therapy.2. Virus transduce trabecular cells, expressed c-Ski in trabecular cells, and confirmed by immunocytochemistry and Western blot, c-Ski genes can cause the degradation of human trabecular cells actin skeleton and reduction of cell connection protein beta-catenin expression.3. The expression in Lentiviral-c-Ski of ECM and actin protein is weakened in the trabecular meshwork of rats.4. Intravitreal injection of HIV-c-Ski resulted in IOP reduction in rat eyes. The mechanism of c-Ski-induced IOP reduction may involve c-Ski gene expression through trabecular channels inhibited TGF-beta/Smad signaling pathways, actin and ECM degradation. |
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