Study On Lentiviral-mediated Smad7 Gene Interference Against Corneal Stromal Proliferation And Fibrosis

【Background and objective】More and more ophthalmologists prefer surface ablation for their patients in refractive surgery procedures as it is safer and less post-operative aberrations than LASIK. Surface ablation includes photorefractive keratectomy (PRK), the excimer laser subepithelial keratomileusis (LASEK) and epipolis excimer laser subepithelial keratomileusis (Epi-LASIK). However, haze is the common complication of surface ablation, which would result in regression and corneal stroma opacification. Haze is directly related to the healing process in the cornea and the unpredictable nature of the associated corneal cellular response. The corneal wound healing response is a remarkably complex cascade mediated by cytokines, growth factors, and chemokines. The cascade of responses to these cytokines leads to important changes in the stroma, including keratocyte apoptosis and necrosis, keratocyte activation, keratocyte proliferation, generation of myofibroblasts and production of altered extracellular matrix (ECM). One of the greatest challenges in reducing haze is to promote tissue repair via regeneration rather than fibrosis. It is believed that the choice between regeneration or fibrosis lies in the control of fibroblast activation, keratocyte proliferation, generation of myofibroblasts and production of extracellular matrix, which are most directly related to transforming growth factorβ(TGFβ). As an post-receptor inhibitory signal transduction factors of TGFP, Smad7 plays a key role in feedback or cross-talk control of TGFβsignaling. In this study, in order to evaluate whether Smad7 gene therapy could reduce fibrosis and haze, we first constructed lentiviral Smad7 recombinant(Lv-Smad7) to optimize the process of transmitting exogenous rat Smad7 gene. Transferring Lv-Smad7 into keratocytes and rat corneas, we evaluate the therapeutic effects of Lv-Smad7 gene delivery in cornea fibrosis by testing some indicators about proliferation and collagen synthesis in vitro and in vivo.[Methods]1. Construction and expression of lentiviral Smad7 recombinant encoding rat Smad7 geneSmad7 cDNA was obtained by extracting total RNA from rat cerebral cortex and kidney and reverse transcription PCR amplification. We inserted Smad7 cDNA into plasmid pcDNA-GFP Lentivector and test Samd7 gene sequence. Transferring plasmid-Smad7 into 293 cells, we evaluate the expression of Smad7 gene with GFP, real-time PCR and western blot analysis. Packed in 293TN cells, the recombinant of Ientiviral-Smad7 was generated. The recombinant of lentiviral-blank plasmid (Lv-plasmid) was constructed with the same method.2. The influence of Smad7 overexpression on the proliferative and fibrotic biological characters of keratocytes in vitroLv-Smad7 and Lv-plasmid were introduced into the cultured rat keratocytes respectively to establish Smad7-positive cell clones and blank plasmid ones. The keratocytes were divided into 4 groups. Group 1:keratocytes no treated; Group 2: TGFβ2 control (keratocytes supplemented with TGF(32, lOng/ml); Group 3: Lv-plasmid group (blank plasmid-positive cell clones, supplemented with TGFβ2, 10ng/ml); Group 4:Lv-Smad7 group (Smad7-positive cell clones, supplemented with TGFβ2, lOng/ml). After 2 hours, the expression of phosphorylated Smad2 (p-Smad2) in the cells were detected with western blot analysis. After 48 hours, the levels ofα-SMA,Ki67 mRNA and protein, and typeⅢcollagen (colⅢ) mRNA were detected with real-time PCR and western blot analysis methods. At the same time, the cell proliferation was determined by MTT.3. The therapeutic effect of Lv-Smad7 on corneal proliferation and fibrosis in vivo96 SD rats (96 eyes) were randomized to 4 groups. Group 1 (Sham-operated group) was served as a normal control, scraped corneal epithelium and not for PRK; Group 2 (PRK-operated group), group 3 (Lv-plasmid group) and group 4 (Lv-Smad7 group) were administered with PRK surgery; Group 3 was given Lv-plasmid solution in cornea and group 4 with Lv-Smad7 after PRK surgery,1/d for 7 days and then 1/w. The corneas were obtained at 1d,1w, 1m and 3m after PRK surgery, we evaluate the expression of Smad7 gene with real-time PCR and western blot analysis. Expression of TGFβ2,α-SMA, Ki67 and colⅢmRNA were evaluated by real-time PCR. At the same time, western blot analysis was used to determine the p-Smad2 levels in cornea.[Results]1. The construction of Lv-Smad7We had constructed a recombinant of Smad7 gene with Lentiviral vector. The recombinant molecular weight and gene sequence is correct by checking with enzyme cutting and gene sequencing test.1.0xl04ifu/μl titer of Lv-Smad7 and Lv-plasmid were obtained.2. The results of study in vitroCompared with other groups, the expression of Smad7 mRNA and protein in Lv-Smad7 group increased remarkably. TGFβ2 increased the expression of p-Smad2, a-SMA, Ki67 and colⅢ. Exogenous Smad7 expression suppressed the level of p-Smad2 protein in the cells. The expression ofα-SMA and Ki67 mRNA and protein was lower in Lv-Smad7 group than in Lv-plasmid group. Accordingly, the expression of colⅢal chain mRNA was lower in Lv-Smad7 group than in Lv-plasmid group. Smad7 overexpression decreased the proliferation rates of keratocytes significantly according to MTT method.3. The results of study in vivoThe expression of Smad7 mRNA and protein in Lv-Smad7 group increased remarkably. Compared with Sham-operated group, the levels of p-Smad2 and Ki67 increased at each time point, and ones of TGFβ2,α-SMA, and colIII raised at 1w, 1m and 3m in PRK-operated group. Exogenous Smad7 expression suppressed the level of p-Smad2 protein and Ki67 mRNA in cornea at each time point. Accordingly, the expression of TGFβ2,α-SMA and colⅢmRNA were lower in Lv-Smad7 group than in Lv-plasmid group at 1w, 1m and 3m.【Conclusion】1. A recombinant of Smad7 gene with Lentiviral vector can be constructed. Exogenous Smad7 expressed with high efficiency in vitro or in vivo.2. Smad7 gene delivery into keratocytes in vitro by Lv-Smad7 to block TGFβ1 signal transduction could inhibit the activation and proliferation of keratocytes, decrease the level of collagenⅢ, and reduce myofibroblast generation and keratocyte fibrosis.3. Smad7 gene delivery in vivo could down-regulate the expression of TGFβ2, a-SMA, Ki67, inhibit Smad2 phosphorylated, reduce ECM deposit, and thereby control corneal proliferation and fibrosis.4. The study suggested that Smad7 gene therapy may be a potential therapeutic method for haze after surface ablation, and even for corneal scar in other diseases.