Effect Of ADSC Transplantation In Cerebral Hemorrhage Model On Neural Cells Apoptosis

ObjectiveStem cells are a category of cells in vivo which are capable of self-renewal and possess multi-directional differentiation potency. With the development of the study on tissue engineering and gene therapy, to search suitable seeding cells and cell replacement therapy have come more and more into the focus of life science research. The discovery of embryo stem cells and neural stem cells established a utility path for nerve restore. It's well known that cell replacement therapy (cell transplantation) adopted to central nervous system degenerative diseases is positive. Not only embry stem cells but also neural stem cells are restricted their development and application by their shortcomings.Bone marrow stromal cells derive from mesenchyme multipotent stem cells which are capable of self-renewal and possess multi -directional differention potency. The research on MSCs celluar transplantation has become profound more and more with each passing day, and has been applied to treat myocardium infarction. However in order to harvest the MSCs, it need draw off bone marrow or recipe ribs after anesthetize. The MSCs development is limited because patients have to suffer pain.Adipose tissue origins from mesoderm as same as bone marrow. Adipose tissue-dirived stromal cells are rich in adipose tissue and is capable of multi-directional differentiation. The source of adipose tissue is plentiful. ADSCs can autoimmune hepatitis cellular transplantation, having no immuno-rejection reaction and avoiding the propagation disease. Adipose tissue is a ideal resource of stem cells bioengineering research.In this experimentation, we have disassociated, cultivated, induced and differentiated ADSCs originated from SD rat's inguinal groove. By observing ADSCs bionomics characteristic, and the effect of ADSCs transplantation in cerebral hemorrhage model on neural cells apoptosis.MethodsThe healthy adult Sprague-Dawley rats were sacrificed under overdose anesthesia. The raw adipose tissue in inguinal groove of rat was obtained on the asepsis condition. To isolate stromal cells, samples were washed extensively with equal volumes of phosphate-buffered saline(PBS), and digested at 37°C for 30minutes with 0.075% collagenase. Enzyme activity was neutralized with L-DMEM, containing 10%FBS and centrifuged at 1200 rpm for 10 minutes to obtain a high-density cell pellet. The stromal cell pellet was resuspended in L-DMEN medium containing 10%FBS, and incubated overnight at 37°C /5% CO₂. Medium was replaced first at 24 hours and then every second-third day thereafter.For adipogenic differentiation, ADSCs were induced by passing cells at a 1:10 dilution in control medium and supplemented 10ng/ml insulin and 10^-9M dexamethasone.The intracerebral hemotoma model of SD rat's right cauate nucleus was maden fixed with stereotaxic apparatus. ADSCs were transplanted into right Ventricle, about 10⁶cells/10ul.Scores of neurological deficits were used to assess neurological functionTunel stain of brain tissue pathological section:Sprague-Dawley rats were euthanized at 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks after they were transplanted into ADSC. These animals were subjected to infuse into 4% paraformal dehydeond. The brain specimens were maken into frozen section the terminal deoxynucleotidyl transferrase-mediated duTP nick end labeling(TUNEL) staining was used to observe the apoptotic neurons in the cerebral hemorrhage model.ResultAdipose tissue was digested by collagenase and subcultured in 50ml culture dish. After 24 hours the seeding cells grew in the culture dish .After 2-4 days it was thus clear that these cells spread out. Most of them were fusiform shape, with ellipse nucleus. The primary cells were growth to at least 80% confluence at 7 days. The passaging cell got purification by frequently replacing medium and passaging. The nucleuses of ADSCs were prunosus and cytoplasms were pink by Giemsa stain.Under the adipogenic inducation media condition in the media at 2-4 day ADSCs were visualized by the presence of highly refractive intracellular lipid droplets in phase contrast microscopy of staining by oil-Red O.After transplantation in lateral cerebral ventricle, in ADSC transplantation group neurological functional recovery were improved significantly compared with untreated group and PBS group at the same time point. In the cerebral hemorrhage model after transplantation the numble of neural apoptosis cell obviously was significantly fewer compared with untreated group and control group.Conclusions1 .The adipose tissue from S-D rat's inguinal groove can isolate and culture the adipose tissue-devived mesenchymal stem cells. ADSCs can proliferate in vitro in L-DMEM medium containing 10% FBS. ADSCs can be induced into adipose cell in adipogenic inducation media.2. Neurological functional recovery was improved significantly after ADSC transplantion.3. The number of neuron apoptosis cells was fewer after ADSCs transplantation.4. ADSC transplantation in cerebral hemorrhage model may contribute to that it can decrease Tunel positive neurons and protect brain.