1.Mechanism Of Doxycycline-induced Apoptosis In HeLa Cells 2.Therapeutic Effect Of GDNF Modified HAECs On The MCAO Model Of Rats

Doxycycline (Dc) has been demonstrated to inhibit cell growth and induce apoptosis in tumor cells, although its mechanism of action is not fully understood. The present study demonstrates that apoptosis can be induced in HeLa cells by Dc, and that process was concentration and time dependent. Western blot data demonstrated that cytochrome c (Cyt c), Smac (the second mitochondria-derived activator of caspase), calpain I, caspase-9, -3 and -8 were all involved in the apoptotic process, while the pan caspase inhibitor zVAD-fmk almost completely inhibited Dc-induced apoptosis. We further demonstrated that the release of mitochondrial proteins and the activation of calpain occurred upstream of the caspase cascade, in which caspase-9 was activated in response to the release of Cyt c, caspase-8 activation was caspase- and calpain-dependent, and caspase-3 was activated mainly by caspase-8 and -9. Caspase-8 played important roles in the activation of caspase-3 and induction of apoptosis, while the role of the caspase-9 was limited. Synovial fibroblast cell (SFB cell) is one of the most important inflammatory cells in rheumatoid arthritis (RA) and its hyperplasia could induce joints damage. Recent works have shown that Doxycycline (Dc) is a pluripotent drug that affects many mammalian cell functions including proliferation, apoptosis, and matrix remodeling. It has been reported that Dc has therapeutic function on rheumatoid arthritis. In this paper we focus our interest on the functions of Dc to induce SFB cells apoptosis. SFB cells separated from knee joints of male Lewis rats having RA (RA-SFB cells) were used. The apoptosis rate of Dc-induced RA-SFB cells is concentration- and time- dependent. Its apoptosis rate was significantly increased after treatment with Dc (45μg/ml) for 3 days. Transmission electron microscope observation showed that mitochondria were swelled and the critaes were dispeared, compared with control. More over, mitochiodria membrane potential was decreased significantly after treatement with Dc for 48 h. Western blot analysis showed that caspase-3, calpain I were activated and XIAP was partially degraded, during treatment with Dc. Human amniotic epithelial cells (hAECs), with the characteristics of both embryonic stem cell and pluripotent stem cell, have the potential to differentiate into various tissues. EGFP and GDNF were transferred into the hAECs by using lentivirus vector, respectively. Intracerebral graft of hAECs-EGFP and hAECs-GDNF into ischemic rats prepared by middle cerebral artery occlusion (MCAO) could significantly ameliorate behavioral dysfunction, and reduce the infarct volume. Furthermore, neuronal marker MAP2 and astrocytic marker GFAP were detected in the transplanted hAECs. Meanwhile, the activation of caspase-3 was prevented in the vicinity of graft area. In addition, the low antigenic nature of hAECs and the immuno-privileged characteristic of brain favor no immunosuppressor treatment during the transplantation therapy. Being easily obtained with less ethical arguments gives hAECs the advantage for cell therapy. As both hAECs and lentiviral vectors are low antigenic, lentiviral transfected hAECs can be used as an effective transfer vehicle for GDNF in ischemia treatment. Moreover, lentivirus modified hAECs may have the potential to be used for cell-mediated gene therapy in the future.