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Abstract #45947 Published in IGR 13-2
Inhibition of TGF beta 2 shRNA on human Tenon's capsular fibroblasts
Luo W; Sun L; Li J-MChinese Ophthalmic Research 2010; 28: 1150-1153
Background: Glaucoma is a kind of irreversible causing-blindness eye disease. Filtering surgery is the primary treatment of glaucoma, but the bleb scarring after surgery is a main reason of failure. A variety of factors play a role in wound healing, especially transforming growth factor-(beta)(2) (TGF-(beta)(2)). Objective: The aim of this experiment was to explore the suppressing effect of specific small hairpin RNA (shRNA) on TGF-(beta)(2) expression in human Tenon's capsule fibroblasts. Methods: Human Tenon's capsule tissue was obtained from strabismus children during the strabismus correct surgery and cultured in DMEM containing 10% fetal bovine serum using tissue expiant technique. Cultured human Tenon's capsule fibroblasts were identified using Vimentin antibody by immunochemistry. Specific shRNA expression vector was constructed according to the design principles of TGF-(beta)(2) mRNA human GeneBank siRNA synthesis and transfected into cultured human Tenon's capsule fibroblasts. The expression of TGF-(beta)(2) in the fibroblasts was detected at 24, 48 and 72 hours after shRNA transfection by ELISA, and the proliferation of human TCFs after shRNA transfection was assayed by methyl thiazolyl tetrazolium (MTT). Results: Cultured human Tenon's capsule fibroblasts showed the positive response for Vimentin. In the group of specific TGF-(beta)(2) shRNA transfection, the proliferation of human Tenon's capsular fibroblasts and the protein expression of TGF-(beta)(2) was inhibited. MTT assay showed that the proliferation value (A(570)) of Tenon's capsule fibroblasts for 24, 48, 72 hours were 0.542 6 (plus or minus) 0.0500, 0.521 0 (plus or minus) 0.0300, 0.405 5 (plus or minus) 0.0400 in sequence in shRNA group and significantly lower than those of negative control groups (0.565 5 (plus or minus) 0.0300, 0.537 8 (plus or minus) 0.0300, 0.526 8 (plus or minus) 0.0400), indicating the evident differences among different groups (F(group) = 54.691, P = 0.000) at various time points (F (time) = 66.888, P = 0.000). The expression levels of TGF-(beta)(2) protein in human Tenon's capsular fibroblasts at 24, 48, 72 hours were (543.58 (plus or minus) 25.32) ng/L, (400.26 (plus or minus) 30.74) ng/L and (202.72 (plus or minus) 23.24) ng/L in shRNA group and considerably lower than those in negative control group (659.78 (plus or minus) 20.95, 547.45 (plus or minus) 24.65, 442.87 (plus or minus) 17.43 ng/L) and those of non-transfection (721.75 (plus or minus) 30.19, 756.61 (plus or minus) 30.19, 787.60 (plus or minus) 18.37 ng/L), showing a significant differences among three groups (F(group) = 163.082, P = 0.000) at various time points (F(time) = 31.498, P = 0.000). Conclusion: The specific TGF-(beta)(2) shRNA can inhibit the expression of TGF-(beta)(2) in human Tenon's capsule fibroblasts in vitro. These results suggest the possible and potential application of TGF-(beta)(2) specific shRNA in the anterior segment surgery.
W. Luo. Department of Ophthalmology, Dalian Medical University, Dalian 116027, China.
Classification:
12.8.10 Woundhealing antifibrosis (Part of: 12 Surgical treatment > 12.8 Filtering surgery)
11.14 Investigational drugs; pharmacological experiments (Part of: 11 Medical treatment)
3.6 Cellular biology (Part of: 3 Laboratory methods)