| Abstract |
Recurrent miscarriages are a major concern in family programming affecting psychologically, socially and economically the couple. This term defines three or more consecutive pregnancy failures until the 10-12th week of gestation in the absence of any pathologic, anatomical or genetic cause. Internationally, the maternal anti-paternal immunization protocols and the intravenous IgG treatments (IVIG) constitute one of the suggested protocols on that matter, which however are still under investigation. The IVIG treatment, which is used for a variety of other cases like autoimmune diseases, neurologic disorders etc. has been shown to increase maternal immunostimulation during the first weeks of gestation without however knowing the exact mechanism. The fact that trophoblasts express Fc receptors (FcγR) leads to the hypothesis that the Fc portion of IgG is responsible for the stimulatory effect of IVIG and fetus protection in the cases of recurrent abortions.
In the current essay we managed to isolate IgG from mouse serum and using limited trypsinolysis to obtain a 70kDa molecule corresponding to the Fc fragment as tested by ELISA. The Fc fragment, when used in radioactive thymidine experiments, increased proliferation of spleen macrophages as well as 12th day of gestation placental cells. ELISA experiments using supernatants from the placental cell culture showed a significant increase of IL–3, IL–15 and GM–CSF cytokine production, which are known to protect fetus during pregnancy.
The fact that an effective in vivo administration requires high concentrations of IgG proteins, led us to the production of recombinant Fc fragment. IgG gene was isolated from the N22 cell line using specific primers for the CH2 domain, which binds to the FcγRs. RNA extraction followed by RT – PCR experiments produced the expected product which should be cloned. Furthermore, cloning of the CH2 – Hinge region in the presence of His – Tag and purification by affinity chromatography was achieved.
The in vitro results presented here show that the isolated Fc fragment has a positive effect on the trophoblast proliferation. The goal thereafter will be the construction the recombinant CH2 – Hinge fragment for the production of larger concentrations, which will allow the confirmation of the initial hypothesis in vivo
|
Απομόνωση του Fc τμήματος της ανοσοσφαιρίνης Γ (IgG) και μελέτη του ρόλου του στην ενεργοποίση μακροφάγων και τροφοβλαστών
