An Experimental Study On Adipose Tissue-derived Stem Cells Transplantation For Treatment Of Brain Cold Injury

In the modern society,on one hand the incidence of the acute cerebral injury is almost increasing linearly as the rapid development of industry and transportation,and on the other hand,the rate of mutilation and mortality of the injury in Central Nervous System remains rather high, although modern scientific techniques have been developed increasingly than ever before,and the frontier equipments have been applied to the medical diagnosis and treatment.Confronted with such a troublesome reality,many countries throughout the world have already devoted a large amount of money and a large number of experts to the study in the repair of the injury in CNS.It is really difficult to repair the injury in CNS,but the discovery and study of the Stem cells brings a gleam of hope to this subject,but at present the study about the Stem cells still doesn't be applied to clinical practice,that means the study remains at the preclinical phase except the treatment of embryonic stem cell for Parkinson,and the treatment of the Stem cells for neurodegeneration,the latter remains advanced.As we all know,after the injury of central nerve it is an inclination in central nervous system to repair the injury by forming scar which is made up of colloid cells because of the death of neurocyte,the fracture of synaptic linkage and the apoptosis of neurocyte,so it is hard to keep the normal nervous function as before after such a scar-repair,and it will naturally and strongly affect the prognosis of the disease.Nowadays it is very popular to apply the Stem cells to the treatment of nervous injury, but it still developed slowly just for various reasons,such as the collection of the Stem cells and the problems in ethics and so on.And recently,Adipose-derived stem cells(ADSCs)which arises from the steatoblast have become a new orientation in the study of the Stem cells just because it is not only easy to collect but also could differentiate into diverse tissues such as fat,bone,cartilage,smooth muscle,cardiac muscle, endothelium,liver and neurocyte.The study below just shows you the effect of ADSCs in the treatment of the cry lesion of brain and wants to make solid foundation to the clinic appliance of ADSCs in the treatment of the injury in nervous system.Our study is composed of several parts,namely to cultivate ADSCs, to detect related marker,to observe the differentiation ability of ADSCs and induce the cells differentiate into neurocyte,to make the model of rat's brain cryoleision,and finally to observe whether it could protect the cerebral cryoleision and inhabit the apoptosis of neurocyte after ADSCs being transplanted to the injured tissue.Our study is composed of two parts as follow:PartⅠThe culture and oriented differentiation of ADCSs to neurocyteObjective:To detach the ADSCs from the rat's adipose tissue,to cultivate the cells in vitro and observe their formation,means of growth and biological markers on the cells' membrane,and to induce ADSCs to differentiate into neurocyte and observe their formation and specific markers.Methods:Firstly,to fetch the fatty tissue located at rat's inguinal groove and peritoneum in sterility,to detach ADSCs by mechanical way and collagenase and inoculate the detached cells in 15%FBS DMEM low Glucose culture-basement to purify and generate the cells,and then to observe the cells' formation continually with phase contrast microscope. As the cells have been transferred to the fifth generation,I will detect the antigenic marker of CD29,CD34,and CD44 by flow cytometry. Secondly,to induce ADSCs to differentiate into neurocyte by means of what is called two-step,to detect the expression of antigen NSE and GFAP by immumofluorescence method,and finally to reserve the ADSCs under the low-temperature circumstance.The revival cells can still be generating for a long time.Results:ADSCs can be extracted from the rat's fat tissue.They developed and stick to the plastic fast in plastic-stick growth mode into fusiform cells which can generation and proliferation in the serum included base stablely.CD34 negative while CD29 and CD44 express rate is higher than 90%in this kind of adipose-derived cells by flow cytometer.ADSCs differentiated into neuron-like cells that strongly express NSE by the combination use of induction agent cytokine bFGF andβ-mercaptoethanol while astrocyte special marker GFAP was not detected.Conclusions:1.We can extract a kind of cells from the rat's fat tissue that express some mesenchymal stem cells markers such as CD44,CD29 and has powerful proliferation ability in vitro.2.Adiposed-derived stem cells generated steadily and can be induced to neuron-like cells that express NSE antigen by neuronal induction technique.3.ADSCs can be reserved in low temperature condition and be revived in normal condition which can still generate steadily. PartⅡTreatment of brain cold injury animal model with ADSCsObjective:To investigate the influence to the brain cold injured rats when ADSCs was transplanted into the brain and to study the feasibility of transplantation of ADSCs as a new way to treat the nervous system diseases.Methods:Harvest and culture purified ADSCs from SD rats and these cells were labeled with Brdu for use.Rat's brain cold injured model was made by liquid nitrogen epidural freeze.All rats were divided into three group:the experiment group for 25 animals(brdu labeled ADSCs prepared before were transplanted into brian cold injury rats twice 12 and 24 hours after injury);the control group for 25 animals(DMEM base were transplanted into brain cold injury rats twice 12 and 24 after injury);Fake operation group for 5 animals(nothing transplanted and only craniotomy performed).NSS test were made to the animals 1,3,7,14and30 days after cell transplantation.Experimental rats were killed and brain tissue was made histological sections for immunofluorescence examination to find brdu positive cells.We still use Tunel staining to find apoptosis cells and RT-PCR to discover the expression rate of mRNA of VEGF and BDNF factors and Western blot to discover the expression rate of VEGF and BDNF protein.One-factor analysis of variance was used to statistics by SPSS 11.0 software.Results:We found brdu positive cells distributed in brain tissue of experimental group SD rats after transplantation and trends to migrate to the center of the injury area.NSS test was made to the all rats for all time point.The scores of experimental group rats and control group rats were obvious different from 3 to 14 days(P＜0.05).RT-PCR found that the mRNA of BDNF factor express of the experimental group was higher than that of the control group from 1-14 days(P＜0.05)while the expression of VEGF in the experimental group was higher than that in the control group for any observed time point(P＜0.05).Western blot found that the BDNF protein expression in experimental group was higher than that in the control group from 1-14 days(P＜0.05)while VEGF in the experimental group was higher than that in the control group for any observed time point(P＜0.05).Tunel staining found that the apoptosis cells were obvious decreased in the experimental group brain tissue than in the control troup for any observed time point(P＜0.05).Conclusions:1.We made the brain cold injury model successfully and simulated the brain edema and secondary brain injury.2.ADSCs can survive in the central nervous system and migrate and differentiate into neuron-like cells.3.ADSCs can depress apoptosis in the brain injury then promote nerve regeneration and protect the brain from injury by increase the expression of factors such as BDNF and VEGF.4.ADSCs transplantation may obvious improve the function of experimental rats.