The Influence Of Th17 Cell On The Collagen Synthesis Of Fibroblast Clones Derived From Patients With Systemic Scleroderma

Part I Increased Th17 and Treg cell and related cytokines in systemic sclerosisObjective To investigate Th17 and Treg cell in the peripheral blood of patients with systemic sclerosis(SSc), and detect variation between before- and after- treatment.Methods Thirty-five patients with SSc and twenty-two normal people were included for blood collection. Samples were processed to detect Th17 and Treg by flow cytometry. After one month-treatment, ten of the patients were collected to detect Th17 and Treg for a second time. Twenty patients with SSc and sixteen normal people were enrolled to detect IL-17、IL-21、IL-22 production by ELISA, related gene expression by real-time reverse transcription-polymerase chain reaction.Results Both the percentage of circulating Th17 cell and the ability to produce IL-17 were elevated in diffuse SSc patients. Th17 increased in SSc patients, and decreased following certain treatment(P>0.05). While Treg cell was reduced in SSc patients and with there is no significant difference after treatment(P<0.05). Conclusion The expansion of Th17 and the depletion of Treg subpopulations may be linked to the pathogenesis of SSc.Part II PBMC from SSc patients promotes collagen synthesis of fibroblast clones and the intevention of IL-17 blocking antibodyObjective To isolate dermal fibroblast clones from patients of SSc, and pick up the high amount collagen-producing subpopulations. Investigate how PBMC influence the collagen synthesis of cell clones.Methods By means of modified limiting dilution cloning, single fibroblast cells from sclerotic skin were isolated and cultured. Then the clones were separated into different subgroups by the amount of collagen produced. PBMC of SSc patients and normal people were isolated and stimulated with PMA and Ionomycin. After coculture of supernate with cell clones, ELISA and real-time PCR were used to detect the expression of collagen type Ⅰ and type Ⅲ.Results Three fibroblast cell lines and 32 fibroblast clones were obtained. Eight clones with highest COL1A1 mRNA levels were chosen for the following tests. Fibroblast clones cocultured with PBMC from SSc patients produced higher levels of collagen(P>0.05). While the treatment with antibody to IL-17 suppressed the production of collagen type I and type III(P>0.05).Conclusion PBMC supernate from SSc patients can promote collagen synthesis of Fb clones. Antagonism of Th17 by IL-17-blocking antibody may delay the course of SSc. Part Ⅲ Th17 and Treg cell from SSc patients influence collagen synthesis of fibroblast clones Objective To investigate the role of Th17 and Treg cell from SSc peripheral blood in the collagen synthesis of Fb clones. Methods Five SSc patients and five normal controls were included to isolate Thl7 and Treg by flow cytometry. After coculture of supernate with cell clones, ELISA and real-time PCR were used to detect the expression of collagen type I and type III. Results Fibroblast clones cocultured with Th17 cell from progressive SSc patients produced higher levels of collagen(P>0.05). While there was no significant difference between Treg cell from both SSc patients and normal controls(P<0.05).Conclusion Th17 cell from SSc patients can promotes collagen gene expression in fibroblast clones.Part Ⅳ Increased IL-17 promotes collagen synthesis in SSc patientsObjective To investigate the influence of IL-17 on the collagen synthesis of Fb clones, and to explore the possible mechanisms.Methods After cocultured with IL-17, the proliferation of Fb clones were detected by MTT assay, type I procollagen、IL-17R、ICAM-1 mRNA by real-time PCR.Results Compared with null groups, Fb clones cocultured with IL-17 showed faster growth rates and shorter doubling time. Meanwhile, they produced higher levels of type Ⅰ procollagen、IL-17R and ICAM-1(P>0.05). The promotion can partly be supressed with anti-IL-17 antibody(P>0.05).Conclusion IL-17 is related to the proliferation and collagen synthesis of Fb clones, antagonism of IL-17 may serve as a promising target in SSc treatment.