Change Of Fibrosis On Cultured Human Tenon's Capsule Fibroblasts Transfected With Smad7 Vector

PrefaceGlaucoma filtration surgery is still the main therapy method for glaucoma. The healing process after glaucoma filtration is the main determinant of surgical failure and, even more important, the final intraocular pressure. Excessive scarring in the sclerostomy site or bleb connective tissue may reduce the efficiency of the aqueous draining, resulting in a renewed increase in intraocular pressure. Mitomycin C and 5-fluorouracil are now being widely used to suppress over-proliferation of subconjuntival fibroblasts during healing following filtration surgery, resulting in a satisfactory clinical outcome. However, administration of these drugs at the time of filtration surgery can cause late-onset post-operative complications. Therefore, continued research in the area of healing modification is necessary, which is leading to further advances in our understanding of healing processes and ultimately better outcomes for patients after glaucoma surgery.The transforming growth factor beta (TGF-β) superfamily, in particular, has been shown to play a pivotal role in scarring through the body, where it is believed to be a potent stimulator of scarring. TGF-β has also been implicated as a potent stimulant of the scarring process in the eye. Recently researches showed that the signal transduction of TGF-β was conducted mainly by Smad proteins. Smads relay the signal from the cell membrane to the nucleus, where they affect the transcription of target genes.Until now, nine Smad proteins have been found, and they were divided into 3 groups. â‘  Receptor-regulated (R-) Smad proteins including Smad1, Smad2, Smad5 and Smad8. â‘¡ Common-partner (Co-) Smad protein including Smad4. â‘¢ Inhibitory (I-) Smad proteins including Smad6 and Smad7.Previous researchs showed that the transient transfection of Smad7 to skin fibroblasts could inhibit the expression of α 2 - type I procollagen (COL1A2) gene. Transient gene transfer and expression of Smad7 prevents bleomycin-induced lungfibrosis in mice.The main purpose of the present study is to transfect the Smad7 cDNA to human Tenon's capsule fibroblasts (HTFs), to construct HTFs transfectant overexpressed Smad7 gene, to observe the effect of Smad7 gene to the expression of type I collagen, fibronectin, cellular proliferation, cell cycle and cell migration, and to observe the effect of Smad7 to the expression of Smad2 and phosphorylated Smad2.Part I Construction of HTFs Transfectants OverexpressingSmad7 GeneSection I Culture and Identification of human Tenon's capsule fibroblasts Objectives In vitro Culture and Identification of human Tenon's capsule fibroblasts.Methods The Tenon's capsule tissue was obtained during cataract surgery and cultured using method of explanting. DMEM contain 20% fetal bovine serum (FBS) was used for subculture in 5% CO₂ 37℃. Antibodies to Vimentin, cytokeration and fibroblast were used in immunocytofluorescense staining for identification of fibroblasts.Results The cultured cells were active growing, stable in manifestation, and confirmed to be fibroblast by positive staining with vimentin and fibroblast antibodies, but negative staining with cytokeratin antibody.Summary In vitro cultured HTFs cells successfully.Section II The Construstion of HTFs Transfectants Overexpressing Smad7 GeneObjectives To construct HTFs Transfectants Overexpressing Smad7 GeneMethods The plasmid was purified and identified. Cultured rat HTFs were transfected stably with recombinant plasmids of pCMV5-HA-Smad7 by Nucleofector? device. Screened by G418, the positive clones were constructed anddetermined by realtime RT-PCR, Western blot analysis respectively.Results After treatment with the enzymes of EcoRI and XbaI, the recombinant plasmid of pCMV5-HA-Smad7 was cut into two fragment, carrier and aim gene, whose lengths were 4.7kb and 2.8kb. Sequencing results showed complete coincidence to Smad7 in GeneBank. U-23 program was selected by 4:1 co-transfection of pCMV5-HA-Smad7 and pmaxGFP to HTFs. Realtime RT-PCR results showed that the expression of Smad7 mRNA in pCMV5-HA -Smad7- HTFs increased significantly compared with HTFs and pCMV5-HA -HTFs whether there was exogenous TGF-β₂ stimulation or not. Western blot results revealed that HA-tagged Smad7 could be detected in pCMV5-HA -Smad7- HTFs and not affected by exogenous TGF-β₂ stimulation.Summary Successfully transfecting plasmid containing Smad7 to HTFs by Nucleofector^TMdevice . Cell clones overexpressing Smad7 mRNA and HA-tagged Smad7 were established by G418 screening.Part II The Changes of Type I Collagen and Fibronectin Expression on HTFs by Smad7 Gene Transfection and Its Possible MechanismObjectives To investigate the influence of Smad7 gene transfection on the expression of Type I Collagen and Fibronectin on HTFs and the possible mechanism.Methods Realtime RT-PCR analysis was adopted to detect the expressions of mRNA of COL 1A2, fibronectin and Smad2 on normal and tranfected HTFs with or without exogenous TGF-β₂ stimulation. Radio immunoassay was used to detect the influence of Smad7 gene on concentration of carboxyterminal propeptide of type I procollagen (PICP) in culture medium. Western blot was used to detect the expression of Smad2 and p-Smad2 protein. Immunocytofluorscense was used to detect the p-Smad2 expression.Results Exogenous TGF-β₂ could time-dependently promote the expression of COL1 A2 mRNA in HTFs and pCMV5-HA-HTFs. In pCMV5-HA-Smad7- HTFs, the COL1 A2 mRNA expression was significantly inhibited compared to control groups inall time intervals. However, it was not changed by exogenous TGF-β₂ in pCMV5-HA-Smad7- HTFs. Exogenous TGF-β₂ could time-dependently increase fibronectin mRNA expression. But the inter-groups variation did not exist in every time intervals. The PICP concentrations in the medium were increased to 2.1, 2.0 and 1.6 folds respectively after stimulating for 72h by exogenous TGF-β₂. In pCMV5-HA -Smad7-HTFs, the extent of increasing was significantly less than the control groups, whether stimulated by TGF-β₂ or not. The expressions of Smad2 mRNA were increased to 2.7,2.4 and 2.3 folds respectively after stimulating by exogenous TGF-β₂. There was not difference in three groups. The p-Smad2 protein couldn't be detected without exogenous TGF-β₂. After 30min of TGF-β₂ stimulating the p-Smad2 expression elevated significantly in all groups. The p-Smad2 expression in pCMV5-HA-Smad7-HTFs took 56.01% and 53.48% of two control groups. In immunocytofluorescense assay, the p-Smad2 positive cells were 4.01 ±2.35%, 3.12± 1.78% and 3.45 ± 2.33% respectively in HTFs, pCMV5-HA-HTFs and pCMV5-HA-Smad7-HTFs groups. After the stimulation of exogenous TGF-β₂ for 30min, the p-Smad2 positive cells became 85.97±6.34%, 90.67±8.25% and 26.67± 8.42%.Summary Exogenous TGF-β₂ can promote the expression of Coll A2, FN mRNA. Smad7 gene can inhibit the expression of Coll A2 mRNA and PICP and antagonize the increasing effect of Exogenous TGF-β₂ but has no effect on fibronectin expression. Exogenous TGF-β₂ can promote the expression of Smad2 mRNA. In the early phase of TGF-β₂ stimulation, there is no significant increscent in Smad2 protein expression but the p-Smad2 protein expression increased. Smad7 gene has no effect on Smad2 expression and can antagonize the increasing effect of Exogenous TGF-β₂ on p-Smad2 protein.Part III The Effects of Smad7 on Cell Proliferation, Cell cycle and Migration of HTFsObjectives BrdU assay, FACS and Transwells chamber were adopted to determinate the Smad7 gene transfection on cell proliferation, cell cycle and migration of HTFs.Methods DMEM contain 10μM of BrdU was added to HTFs, with or withoutexogenous TGF-β₂. After 1h, immunocytofluorscense was used to detect the BrdU positive cells. Collecting HTFs, staining with PI, using FACS to determine the cell cycle. 10⁵ HTFs were seeded into upper chamber of Transwells, cultured for 16h, to determine the migrated cells.Results Exogenous TGF-β₂ could decrease the BrdU positive cells to 73.8% and 73.1% in HTFs and pCMV5-HA-HTFs. In pCMV5-HA-Smad7- HTFs, there is no difference in BrdU positive cells. The BrdU positive cells of pCMV5-HA-Smad7-HTFs were significantly more than that of HTFs and pCMV5-HA-HTFs. After stimulated by TGF-β₂, the G0/G1 phase didn't change in HTFs and pCMV5-HA-HTFs but significantly decreased in S phase cells. In pCMV5-HA-Smad7-HTFs, cell proliferation was not inhibited by TGF-β₂ and S phase cells increase clearly compared to control groups. GO/G1 phase cells decreased clearly. Transwells showed that exogenous TGF-β₂ could slightly enhance the migration, but not statistics significant.Summary Exogenous TGF-β₂ can decrease the BrdU positive cells and S phase cells to inhibit HTFs proliferation. Smad7 gene can antagonize the proliferation inhibiting effect of exogenous TGF-β₂ but has no effects on cell migration.Conclusions1. Successfully establish HTFs transfectants overexpressed Smad7 mRNA and protein.2. Exogenous TGF-β₂ can up-regulate the expression of type I collagen and fibronectin. Smad7 gene can inhibit the expression of type I collagen, and antagonize the increscent effect of TGF-β₂. Smad7 has no effect on fibronectin expression.3. Exogenous TGF-β₂ can phosphorate Smad2 rapidly, and increase the Smad2 protein expression. Smad7 gene can inhibit the Smad2 phosphoration induced by TGF-β₂. It is possibly the mechanism of inhibitory effect on type I collagen. Smad7 gene has on effect on Smad2 expression up-regulating induced by TGF-β₂.4. Exogenous TGF-β₂ can decrease the BrdU positive cells and S phase cells to inhibit HTFs proliferation. Smad7 gene can antagonize the proliferation inhibitingeffect of exogenous TGF-β₂. 5. Smad7 gene has no effects on cell migration.