| ForewordGlaucoma is an ocular disease and the second leading worldwide cause of blindness,after cataract.Primary open-angle glaucoma(POAG) is one of the main and common types of glaucoma.Although the mechanism of POAG is unclear,it is currently tempting to argue that genetic factor is one of its main causes.In the whole field of glaucoma,there have been dozens of glaucoma-associated genes reported in succession,among which 15 chromosomal loci designated have been defined for POAG,but the candidate Disease-causing genes identified for POAG are still only MYOC / TIGR gene and OPNT gene.The role of Myocilin protein in POAG pathogenesis is still unclear.According to the present findings,we summarized its possible mechanism:The research on the expression of the normal and mutational MYOC / TIGR gene in ocular cells indicates that normal myocilin protein can be secreted,but almost any myocilin protein secretion expressed by various kinds of mutation form of MYOC/TIGR gene is not detected.The study that mutational myocilin protein deposited in endoplasmic reticulum is poisonous to the cells of human trabecular meshwork,shows that most mutations of MYOC / TIGR genes,such as Gly364Val,Gln368Stop,Lys423Glu etc.,cause the myocilin protein to stop secreting,and it have been determined that these mutational myocilin proteins accumulated in the endoplasmic reticulum lead to the weakening of cell proliferation and the subsequent dysfunction of trabecular meshwork cells until apoptosis,which is also considered as the cause of glaucoma.Because it is similar to the primary open-angle glaucoma in many aspects,such as pathophysiological process,the corticosteroid-induced glaucoma is likely to become the disease model for study of POAG.It has been established in the literature that there is similarity between the effects of dexamethasone on MYOC gene of trabecular meshwork cells in vitro and the ones in vivo.Therefore,we use dexamethasone to cultivate the trabecular meshwork cells of rat in vitro to simulate the pathogenesis process of POAG and determine the role ofMYOC gene in the pathogenesis process of POAGExperimental methods1.siRNA interference:According to the sequence of Myoc gene of rat(GenBank Accession Number:NM-030865),select two loci to design siRNA,the sequence is shown in Table 1.2.After siRNA interference,the quantitative analysis of MYOC gene:We used 15%FBS medium containing 10-7M dexamethasone(sigma) to cultivate the trabecular meshwork cells of the normal rat for 7 days,then carried out MYOC gene interference and tested the MYOC gene expression and the changes of the apoptosis rate of the trabecular meshwork cells.3.Data Analysis:All datas in experiments are treated by SPSS 11.5 Statistical Analysis Software.Experimental results1.The trabecular meshwork cells of rat cultivated by 15%FBS medium containing 10-7M dexamethasone:After the trabecular meshwork cells of rat are cultured by 10-7M dexamethasone medium for 7 days,the cell morphology is almost not changed,but the cell density is obviously lower,the change of apoptosis in the cell is seen,including karyopyknosis, Chromatin margination,cytoplasm condensation,organelle swelling,many microtubule-microfilaments are dissolved,perinuclear vacuolization region occur etc.2.siRNA interfered resulting picture:Using Real-time PCR method to do the relative quantitative analysis,and 18sRNA as a reference,the relative content ofMYOC gene of normal trabecular meshwork cells is about(1.87±0.09)×10-2pg(Mean±Std);the relative content of MYOC gene expression of trabecular meshwork cells cultivated in dexamethasone is about(28.93± 1.20)×10-2pg(Mean±Std);the content in the dexamethasone cultivating group is 15.47±0.70 times(Mean±Std ) of the one in the normal cell group;the comparison rate of MYOC gene expressive amount between the two groups of cells is p＜0.05,and the difference has the statistical significance.Using Real-time PCR method to do the relative quantitative analysisa,and 18sRNA as a reference,the interference rate available of Myoc-1242 and Myoc-1335 group are more than 80%,up to 85.8%,while all interference rates in other three groups are less than 75%,which can not reach the interference effect.Interference ratio =(mRNA amount in the comparison group-mRNA amount in the experimental group) / mRNA amount in the comparison group.The MYOC gene expression analysis results of trabecular meshwork cells cultivated in 10-7M dexamethasone after interference:the relative expression amount of MYOC gene cultivated in 10-7M dexamethasone in siRNA interference group is (5.22±0.66)×10-2pg(Mean±Std);The expression amount in control group is(28.32±0.70)×10-2pg(Mean±Std);The T test was done between the two groups of data, p＜0.05,and the difference has the statistical significance.3.The apoptosis test results of trabecular meshwork cells:The apoptosis rate of trabecular meshwork cell of normal 3rd generation of rat is 4.50±0.41%(Mean±Std%);The apoptosis rate of trabecular meshwork cell of rat culivated in 10-7M dexamethasone is 29.35±0.47%(Mean±Std%);The apoptosis rate of trabecular meshwork cell of rat cultured in dexamethasone after interference is 25.51±1.02%(Mean±Std%);The T test was done between the two groups of data, p＜0.05,and the difference has the statistical significance.ConclusionMYOC gene is acting in the pathogeneses of steroid-induced glaucoma and POAG by mediating the apoptosis of trabecular meshwork cells;In addition,the reduction of microfilaments-microtubules leads to the rearrangement of cytoskeletal proteins and the weakened migrating ability of trabecular meshwork cells,which decreases the outflow ability of aqueous humor of trabecular meshwork,increases intraocular pressure,eventually causes the glaucomatous changes of the optic nerve and visual fields.
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