The Experimental Study On Adipose-derived Stem Cells Transplantation For Intracerebral Hemorrhage

Intracerebral hemorrhage is defined as spontaneous haemorrhage in brain tissue not caused by injury of central nervous system,which covers about 21%～48%of stroke.30%～50%patients die of it in one month and neural function disturbances are remained in over 30% survivors.By now therapeutic measures are not abundant,surgical therapy includes aspiration of haematoma,neural endoscope measures, clearance of haematoma by operation and so on.The aims of all the therapeutic measures are to decrease effects of the occupancy and degradation products of haematoma.While the effectivenesses are not satisfactory.The appearance of stem cell bring hopes to patients with central nervous system injury.Recently,there are many reports about amendment of neural functions by stem cells transplantation,such as neural stem cells,embryonic stem cells,bone marrow stromal cells and so on.However,the restrictions about ethics and collection of cells made the study develop slowly and hard to be used clinically.Recent years, ADSCs(adipose-derived stem cells) have become hot spots of stem cells research.Above-mentioned restrictions not existing make ADSCs have broader perspective.So we transplanted ADSCs into rats model of intracerebral hemorrhage and observed the changes of some indexs to offer theary foundation for clinical application. PartⅠThe study on biological character of ADSCsObjective:To isolate,cultiviate,induce differentiation and identify ADSCs,observe the biological character in vitro,thus to build a foundation for applied research.Methods:Cells isolated from rats adipose of groin were cultured in medium which contained blood serum. When bespread the culture flask,cells were collected and inoculate into the mediums of neural stem cells and adipose cells.The changes of cells were observe and the differentiated cells were evaluated by immunohistochemistry and immunofluorescence.At the same time,the test of reserve ADSCs under the low-temperature condition and others were carried out.Results:The adpose tissue of rats groin contains lots of ADSCs.The cells are poorly differentiated under electron microscope. The appearance of ADSCs changed when culivated in the the mediums of neural stem cells and adipose cells medium.The differentiated cells present Nestine,GFAP,NSE and oil red O positive.The anabiosised reserved ADSCs still have activity.Conclusions:ADSCs have the ability to proliferation and self- refreshment,can differentiate into neural cells through NSCs and adipose cells.ADSCs can be cultivate long term in vitro,and it's character of stem cells still remain after being anabiosised. PartⅡEffects of adipose-derived stem cells transplantation on motor function in intracerebral hemorrhage modelObjective:To explore the effects of adipose-derived stem cells (ADSCs) transplantation on motor function in rats with intracerebral hemorrhage and the reasons.Methods:ADSCs were cultured, proliferated and labelled in vitro.After the rats of ADSCs-transplanted group underwent intracerebral hemorrhage for 24 hours,ADSCs were stereotaxically transplanted into the right ventricular(PBS-transplanted group and model group were designed to be contrasted).The motor function were evaluated at 3,7,14,21,28 d after transplantation.Every group was enthanized on time and brain sections were made.Some sections were used in immunohistochemistry to observe the distribution of transplanted cells,and others were used in immunohistochemistry for apoptosis and angiogenesis detection.Results:The transplanted cells migrated to vicinities of hemorrhage focus gradually from ventricular and the hole of needle.The ADSCs-transplanted group has better motor function at 7,14,21,28 d,fewer apoptotic cells at 3,7,14,21,28 d and more angiogenesis at 7,14,21,28 d after transplantation.Conclusions: ADSCs transplantation can improve the motor function by reducing apoptotic cells and increaseing angiogenesis in intracerebral hemorrhage rats. PartⅢTo explore the reasons of ADSCs transplantation improving CNS function on the level of molecular organismObjective:To explore some reasons of ADSCs transplantation improving the motor function of intracerebral hemorrhage rats on the level of molecular organism.Methods:ELISA was used to detect whether ADSCs can excrete BDNF and VEGF.The changes of BDNF and VEGF mRNA and protein in brain tissue of two groups were detected by RT-PCR and Western-blot.Results:ADSCs can excrete BDNF and VEGF in vitro.BDNF,VEGF mRNA and protein express higher in the brain tissue of ADSCs-transplanted group at 3,7, 14,21,28 d after transplantation.Conclusions:ADSCs can protect, nuish neural cells and can increase angiogenesis,reduce damage by some dissoluble cytokine such as VEGF and BDNF.