The Role Of CTGF ASODN To The Expressing Of CTGF And Fibronetin In The HTF Induced By TGF-β₂

PART 1 Transforming growth factor-β2 ?induces the human Tenon's capsule fibroblasts to proliferate, express the CTGF and synthesize the FibronectinObjective : To investigate the role of connective tissue growth factor as a downstream mediator of TGF-β2 in the scarring after glaucoma filtering surgery ,through observing the influence of transforming growth factor-β2 to the proliferation of human Tenon's capsule fibroblasts (HTF)and the expression of CTGF and fibronectin .Methods : The fibroblasts were isolated from human Tenon's capsule and then cultivated in vitro. The cells from the third to fifth generation were used in our experiment. (1)Human Tenon's capsule fibroblast cells were identified by the method of SP. (2)The different concentration TGF-β2(0,0.5,1, 2, 4,5,10ng/ml)was added into the HTF,then the proliferation of HTF was detected using MTT colorimetry after incubated for 24h. (3)After treated by different concentration of TGF-β(20,2,4,5 ng/ml)based on the result of MTT , the expression of CTGF and fibronectin was detected by RT-PCR and immunocytochemistry assay respectively.Results:MTT method:The induction of TGF-β2 (2ug/l, 4ug/l,5ug/l) showed a promotion on the proliferation of HTF and the expression of CTGF and fibronectin (P<0.01) ,while 0.5 ug/l and 1 ug/l group vs normal group,5 ug/l group vs 10 ug/l group there was no significant difference on the proliferation of HTF ( P > 0.05). Conclusions : TGF-β2 can enhance the proliferation of the HTF and induce the expression of CTGF and Fn in a dose-dependent manner,inferring that CTGF may be an important cytokine in scarring after glaucoma filtering surgery as a downstream mediator of TGF-β2 action.PART 2 The role of CTGF ASODN to the expressing of CTGF and Fibronetin in the HTF induced by TGF-β2Objective:To explore whether the ASODN (antisense oligonucleotides) of CTGF-a downstream mediator of TGF-β2 could inhibit the role of TGF-β2 that enhancing the proliferation of the HTF and inducing the expression of CTGF and Fn, and then to investigate the special therapy of the sccaring after glaucoma filtering surgery by inhibiting the expressing of CTGF,that just is our objective.Methods (1) The activity of HTF treated by different concentration of DOTAP(2.5,5,10ul/ml)was detected using MTT colorimetry. (2)Using DOTAP encapsulating the CTGF ASODN and the right oligonucleotides to form DOTAP-AS and the DOTAP- right oligonucleotides compound;(3)Make groups: N:the blank group(only added into the DMEM without blood serum); T:treated by TGF-β2;A:the therapy group (TGF-β2+ DOTAP-AS);S:the contrast group with right oligonucleotide(TGF-β2 +DOTAP- right oligonucleotide);D:the contrast group without oligonucleotide(TGF-β2+DOTAP)。The concentration of TGF-β2 in each group is 5 ng/ml,the ultimate concentration of DOTAP and ASODN is 5ul/ml, 1.6ug/ml separately;(4) The expression of CTGF was detected by RT-PCR and Western blotting assays, and the synthesis of fibronectin was detected by RT-PCR and immunocytochemistry assays.Results: The influence to the HTF's proliferation by DOTAP(2.5μl/ ml ,5μl/ ml ,10μl/ ml) have no significant diversity( P > 0. 05) . TGF-β2 (5ug/ ml) can induce , but DOTAP-AS (include DOTAP5μl/ml,AS1.6μg/ml)can inhibit the CTGF and Fn expression of HTF.Conclusions DOTAP-AS can inhibit the proliferation of HTF and the CTGF and Fn expression of HTF induced by TGF-β2。...