The Production Of Recombinant Human Anti-CTGF ScFv Dimer And Its Application In Proliferation Of ASMCs And Chemotherapy-Induced Pulmonary Fibrosis

Pulmonary fibrosis caused by chemotherapy is known as irreversible damage to lung mesenchyme which is characterized of abnormal proliferation of fibroblast, excessive deposition of extracellular matrix, diffused or localized fibroblast foci formation. Connective tissue growth factor(CTGF), a downstream effector of TGF-β, is a versatile molecule involved in cell proliferation, migration, differentiation, ECM production, et al. CTGF seems to be a key factor during fibrosis pathogenesis and inhibition of CTGF expression may be a potential target for pulmonary fibrosis related disease.Part 1 Preparation of recombinant human anti-CTGF scFv dimerObjective:To prepare the anti-CTGF scFv dimer from E.coli cells with a high purity and a good immunoreactivity.Methods:1. A pET28a-scFv-matrilin plasmid was constructed; 2. IPTG was used to induce the expression of the fusion protein after the pET28a-scFv-matrilin plasmid was transformed into E.coli BL21 (DE3). The fusion protein was retrieved by Nickel-affinity chromatography. The protein purity was evaluated using SDS-PAGE, Native-PAGE and Western blot; 3. ELISA was used to evaluate the immunoreactivity of the scFv dimer against human CTGF.Results:1. Soluble expression of the fusion protein was detected after induced by IPTG; 2. The fusion protein was obtained with a purity over 90% after purification and the results from SDS-PAGE, Native-PAGE and Western-blot indicated that the scFv proteins existed as dimer; 3. The immunoreactivity of the produced scFv was detected with ELISA.Conclusion:The scFv was successfully expressed as soluble proteins and existed as dimer. The final product was obtained with high purity and demonstrated a good immunoreactivity with CTGF.Part 2 Effects of the recombinant human anti-CTGF scFv dimer in airway smooth muscle cell proliferation and the underlying mechanismObjective:To investigate the effects of scFv dimer on CTGF-induced ASMCs proliferation and the underlying mechanism.Methods:1. ASM cells was incubated at different CTGF concentrations (0、5、10、 20、30、40、50 ng/ml) for 1,3 or 5 days, and the proliferation of the ASM cells were evaluated with MTT assay; 2. The ASMs were pre-incubated with CTGF (20 ng/ml) for 3 days followed by an incubation with PBS, anti-CTGF scFv monomer or dimer, anti-CTGF mAb or LY294002. MTT assay was applied to measure the cellular proliferation from different groups; 3. Expression levels of p-Akt/Akt and p-mTOR/mTOR in human ASM cells were determined by Western blot analysis.Results:1. An apparent promotion in cellular proliferation had been observed when 20 ng/ml of CTGF was applied for 3 days (P<0.05); 2. CTGF-induced ASMCs proliferation was prohibited by scFv dimer treatment; 3. The p-Akt/Akt and p-mTOR/mTOR ratios increased after treated with CTGF, and this increase was prohibited after treated with scFv dimer, anti-CTGF mAb or LY294002.Conclusion:The CTGF induced ASMCs proliferation could be prohibited by scFv dimer treatment via inhibiting PI3K/Akt/mTOR pathway.Part 3 Roles of the recombinant human anti-CTGF scFv dimer on differentiation of fibroblast into myofibroblastObjective:To investigate the effect of scFv dimer on human lung fibroblast cells (HLEFs) differentiation.Methods:1. HLEFs was incubated at different CTGF concentrations (0、10、20、 30、40、50、60 ng/ml) for 12,24 or 36 h and the CCK8 assay was used to measure the cell proliferation; 2. After incubated with 50 ng/ml CTGF for 12 h, the HLEF cells were added with scFv or anti-CTGF mAb, and the cellular proliferation was evaluated with CCK8 assay; 3. RT-PCR was used to determine the α-SMA and FN mRNA level in HLEF cells from different groups; 4. Expression levels of α-SMA in HLEF cells were determined by Western blot analysis.Results:1. Cell proliferation of the ASMs after treated with 50 ng/ml CTGF was significantly enhanced after 12 h (P<0.05); 2. The scFv dimer prohibited the CTGF induced proliferation (P<0.05); 3. The a-SMA and FN mRNA level were significantly increased after CTGF stimulation (P<0.05). The scFv dimer and mAb downregulated the mRNA levels (P<0.05). In addition, the scFv monomer was less effective than the dimer; 4. The protein level of a-SMA significantly increased in the CTGF group (P<0.05) and was apparently downregulated after applying the scFv dimer or mAb (P<0.05). The scFv monomer had a weaker effect on the protein expressions.Conclusion:ScFv dimer could inhibit the differentiation of fibroblast into myofibroblast via downregulating the a-SMA protein expression induced by CTGF.Part 4 Effects of the recombinant human anti-CTGF scFv dimer on chemotherapeutic drug-induced pulmonary fibrosisObjective:To investigate the effects of scFv dimer on pulmonary fibrosis and the underlying mechanismMethods:1. The mouse pulmonary fibrosis model was established by injecting Bleomycin (BLM) into the mouse trachea and the anti-CTGF scFv monomer or dimer was administrated afterwards; 2. Mice were randomly divided into 4 groups:group A (NS+NS), group B (BLM+NS), group C (BLM+scFv monomer) and group D (BLM+scFv dimer); 3. Bronchoalveolar lavage fluid (BALF) was collected at the 7th,14th and 28th after molding for cellular counting and classification; 4. Pulmonary tissues were sampled for Hydroxyproline and TGF-β1 analysis; 5. HE and Masson stain were used to evaluate the level of alveolitis and fibrosis; 6. The CTGF and a-SMA mRNA level from pulmonary tissues were determined by RT-PCR; 7. Western blot were used to determine the levels of PI3K、p-Akt and p-mTOR.Results:1. Mouse model of pulmonary fibrosis were constructed successfully; 2. The number of total cells and macrophages, lymphocytes, neutrophile granulocyte in BALF from bleomycin group B were higher than the other groups (P<0.05), and the cytometry of group D were significantly lower compared with group B and group C (P<0.05); 3. HE stain and Masson stain showed that the level of alveolitis and fibrosis in group B were significantly increased (P<0.05) while lower in group D (P<0.05); 4. The hydroxyproline content and TGF-β1 level were significantly reduced in group D compared with group B and C (P<0.05); 5. The CTGF and a-SMA mRNA level were significantly higher in group B (P<0.05) and significantly lower in group D compared with group B and C (P<0.05); 6. The protein levels of PI3K, p-Akt and p-mTOR in group B were higher than the other groups (P<0.05), and significantly reduced in group D compared with group B and C (P<0.05).Conclusion:The scFv dimer could attenuate the bleomycin-induced pulmonary fibrosis in mice probably through downregulating the PI3K/Akt/mTOR pathway.