| Bleb scarring is the main cause of glaucoma filtration surgery failure. Abnormal subconjunctival thickening of the collagen and activated fibroblasts can be found in the connective tissue, and they will blocked the scleral tunnel under the conjunctival flap, and lead to the loss of the aqueous drainage function. Treatment with antimetabolites, such as mitomycin and5-fluorouracil, could decrease the scar formation and improve the success rate of surgery, but they are often associated with serious complications such as hypotony, bleb leakage etc. So explore a more safe and effective therapy agains the fibrosis after surgery is very important.Phenotypic Modulation is the basis of fibrosis which is mostly regulated by the TGF-β cytokines, leading to further migration, proliferation and synthesis of extracellular matrix. After the surgery or trauma, blood TGF-β level increases, resulting in human Tenon’s capsule fibroblasts (HTF) phenotype translate into myofibroblast (MF) and start the procedure of wound healing.MF plays an important role in different periods of wound healing, once the wound healed, MF should generally return to fibroblast (FB) or into the course of apoptosis, but Long-standing of MF will lead to excessive cell proliferation, increased synthesis of extracellular matrix and scar formation.More and more researches choose TGF-β as the target to inhibit the extent of scarring because of its importance in cell phenotypic change. However, the TGF-β signaling pathway is very complex and showing an interlacing network, so intervene at a lower level could not inhibit the phenotypic change significantly. Therefore, we considered to regulate the membrane receptors as a higher point to explore the MF phenotype transformation process and possible mechanisms of fibrosis.There are mainly three kinds of TGF-β receptors in mammal:TGF-βR Ⅰ (activin receptor-like kinase, ALK), TGF-βRⅡand TGF-βRⅢ in which type Ⅰ and Ⅱ receptors are involved in signal pathways of TGF-β. They combine TGF-β ligand in united forms, resulting in phosphorylation of type I receptor and activation of downstream signaling pathways. As ALK5is the main kind of TGF-βR Ⅰ in mammalian cells, it is considered to be the focus of the study of TGF-β receptors.In preliminary work, we choose an ALK5inhibitor SB-431542to study the role of ALK5. in scarring after glaucoma filtration surgery, which shown that local subconjunctival injection of SB-431542can inhibit the scar formation in scleral tunnel. We believe that ALK5may be the key point to the regulation of TGF-β induced scarring. By establishing a man-controled ALK5level of cell and animal scarring model after glaucoma filtration surgery, we hope to disscuss the interaction of TGF-β and ALK5, and then find the regulatory role of ALK5on phenotype change and fibrosis. Aim To provide more theoretical basis for the researches of scar formation after glaucoma filtration surgery.Purpose:To investigate the regulatory role of TGF-β1in the expression of activin receptor-like kinase5(ALK5) in cell and animal model of bleb scarring after glaucoma filtration surgery, and by establishing ALK5gene high expressed model by lentiviral transfection technology, to discuss the effect of ALK5level change in scar formation.Materials and methods:Part Ⅰ:Primary cultured Human tenon’s capsule fibroblast was identificated and induced by10μg/μl of TGF-β1. a-SM-actin and fibronectin was mesured by Western blot to certify the existence of cell phenotype transformation and fibrosis. ALK5mRNA levels was quantified by real-time PCR while Western Blot and immunocytochemistry were used to detect the expression changes of ALK5protein.Part Ⅱ:ALK5gene overexpressive lentivirus was thansfected into HTFs by Tronolab lentiviral vector system. Transfection efficiency and cell biological characteristics were observed by optical microscope. Cells were cultured with normal medium or10mM SB-431542, and then MTT was performed to observe the effect on the proliferation, Western Blot was applied to detect phenotypic change(a-SMA), fibrosis(FN) and apoptosis (Caspase-3).Part Ⅲ:1.24healthy New Zealand rabbits received filtration surgery on the right eyes under general anesthesia and were randomly divided into three groups (control group, ALK5group,0.5mM SB-431542group). Clinical examination and IOP was measured to evaluate the general appearance of treated eyes. Two rabbits in each group were sacrificed and the eyes were enucleated on day3,7,14and28. Paraffin section of the eyes were labeled with HE staining, a-SM-actin and ALK5were observed by immunohistochemical detection.2.30healthy New Zealand rabbits received filtration surgery on the right eyes under general anesthesia and were randomly divided into five groups (n=6):A:control group (NS+NS), B:negative control group (NS+0.5mM and SB-431542), C:high expression group (ALK5transfected+NS), D:high expression+low-dose group (ALK5transfected+0.5mM SB-431542), E:high expression+high-dose group (ALK5transfected+2.0mM SB-431542). Clinical examination and IOP was measured to evaluate the general appearance of treated eyes. All the rabbits were killed28days after the surgery, paraffin section of the eyes were labeled with HE staining and the pathological morphology was observed under a microscope, α-SMA, FN and ALK5were observed by immunohistochemical detection.Results:Part Ⅰ:After TGF-β1simulation, the protein expression of a-SM-actin and FN were up-regulated, respectively, to hint the HTF transdifferentiation into MF and produce sacrs. ALK5mRNA was up-regulated with a peak at12to24hours, protein expression was also up-regulated with the peak around24to36hours.Part Ⅱ:ALK5gene was transfected by lentivirus into HTFs. Over-expression of ALK5protein lead to a significantly increased cell proliferation accompanied by apoptosis while cultured with SB-431542can reverse the effection. MTT showed no significant changes in proliferation between the GFP transfected cells and non-transfected cells, ALK5transfected HTFs showed a significant increased proliferation rate while adding SB-431542will significant inhibit cell growth. The expression of a-SMA and FN were declined after transfected.Part III:The postoperative IOP was reduced, in the first day after the surgery, the mean IOP was low (5.63±1.73mmHg, P<0.05) and gradually increased with time. Till10days after surgery, the IOP gradually returned to pre-operative level (11.07±1.72mmHg, P>0.05). And a slight upward trend of IOP level was observed in28days (12.40±1.16mmHg, P<0.05). There was no statistically significant between the groups. Immunohistochemistry showed that ALK5was not obviously observed in subconjunctival tissue in3days. By day7, immunoreactivity increased concomitant with increased cellularity of the wound. By day14, Spindle-shaped fibroblasts, probably myofibroblasts were strongly immunoreactive around the wound. Till day28, the immunoreacticity was reduced but still performed. Histologic profiles revealed massive subconjunctival scarring with apparent collagen clumps in the control group. ALK5transfected group showed an extremely activated proliferation of the cells, accompanied by mild collagen deposition. Treatment with SB-431542showed only a mild fibrotic response with little deposition of collagen in the subconjunctival space.Conclusion:In the model of scaring in HTFs and rabbit glaucoma filtration surgery, through the regulation of TGF-(3, the expression of ALK5was increased. Transfection of ALK5gene into HTFs and subconjunctival tissue in rabbit eyes may promote stong fibroblast cell proliferation and apoptosis; however adding inhibitor SB-431542could suppress the effect of cell proliferation and scarring. It suggests that ALK5may play an important role in the regulation of bleb scarring in glaucoma filtration surgery. |
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