The apoptotic cell volume and expression of Bcl-2 before and following the transplantation were observed in order to evaluate the mechanisms of the protective effect of the exogenous stem cell and the neurotrophic factor on brain ischemia. better than those transplanted with common MSCs. As compared with common MSCs transplantation, GDNF+MSCs transplantation was significantly more effective in reducing apoptotic cell volume and enhancing Bcl-2 expression, which was favorable for the ischemic brain injury.Conclusions:Transplanted GDNF modified MSCs can improve the nervous function and have a protective effect on the ischemic brain injury through reducing apoptotic cell volume and enhancing the expression of anti-apoptotic gene Bcl-2. Keywords:marrow stromal cells (MSCs), glial cell derived neurotrophic factor (GDNF), cerebral ischemia, SB 743921 Bcl-2, apoptosis == Introduction == As a principle component of non-hematopoietic system in bone marrow, marrow stromal cells (MSCs) (Shichinohe et al.,2008; Zacharek et al.,2010; Yilmaz et al.,2011) can express multiple neuron- and gliocyte-specific markers and grow morphological processes similar to that of neural cells when treated with inducersin vitro. This indicates the potential of MSCs SB 743921 to differentiate into neural cells and thus provides theoretical evidence for cell transplantation in treating ischemic brain injuries. Glial cell derived neurotrophic factor (GDNF) (Oppenheim et al.,1995) is a potent neurotrophic factor, playing an important role in the differentiation, development, growth, and survival of the cells in the central nervous system. In the present study, MSCs were Rabbit Polyclonal to 14-3-3 cultured using the adherence method. The expression vector was constructed after the GDNF gene was cloned from rats. The MSCs culturedin vitrowere transfected with cationic liposome-mediated eukaryotic expression pDNA3-GDNF. The expressed green fluorescent protein was considered the reporter gene to optimize the transfection condition. The transgenic MSCs obtained through screening were considered as the cell transplantation tracer. The focal brain ischemia model was established in rats by using the suture method and was transplanted with GDNF modified MSCs. The apoptotic cell volume and expression of Bcl-2 before and after the transplantation were observed in order to evaluate the mechanisms of the protective effect of the exogenous stem cell and the neurotrophic factor on brain ischemia. This will provide theoretical evidence for the clinical application of cell and gene therapy for ischemic cerebrovascular diseases. == Materials and methods == == Animals and reagents == Adult SpragueDawley (SD) rats, male and female, weighing between 250 g and 300 g, and four-week-old newborn rats were purchased fromLaboratory Animal Center of Chongqing Medical University. Rabbit anti-Bcl-2 antibody was purchased from Jingmei (Shanghai, China). Cationic liposome-mediated eukaryotic expression pDNA3-GDNF plasmid (constructed in our laboratory) carrying green fluorescent protein was used to transfect MSCs culturedin vitro. == Animal model == Right middle cerebral artery occlusion (MCAO) was induced with an intraluminal filament as previously reported (Longa et al.,1989). Transient right MCAO was induced for two hours by advancing a 40 mm length monofilament nylon suture with its tip rounded by heating near a flame to block the origin of the MCA. Reperfusion was performed by withdrawal of the suture two hours after MCAO. Animals subjected to sham surgery were treated similarly, except that the filament was not advanced to the origin of the MCA. Rats that failed to exhibit neurological abnormalities at two hours after stroke were excluded from this study. == Culturing and identification of MSCs from newborn rats and GDNF gene transfection == The MSCs from newborn rats were successfully cultured using the adherence method after serial passage. Four-week-old SD SB 743921 rats received the left hindquarter amputation. The rat bone marrow was harvested from femur and tibia that were just amputated. Flushed out the bone marrow, separated the mononuclear cell layer, add the culture liquid -Minimal Essential Medium (MEM) which contains 10% serum of fetal cow to the cell-floating liquid and plant them in the culture instruments in the condition of 37C and 5% CO2for the primary generation culture. The third and fourth generation cells grow well, and the amount cells more than primary generation. The GDNF gene expression vector was constructed after the gene was cloned from rats. The MSCs culturedin vitrowere transfected with pDNA3-GDNF which was expressed by eukaryocytes under the mediation of cationic liposome. The expressed green fluorescent protein was considered the reporter gene in order to optimize SB 743921 the transfection condition. The transgenic MSCs obtained through screening were considered the.