Research Into The Biologic Characteristics And Transplanting For Treatment Of Cerebral Ischemia-reperfusion Injury In Rats On Mesencmal Stem Cells

Afunction induced by nerve injury, no matter congenital of postnatal, will bring enormous agony and burden to patient, family and society, such as inflammation, wound, tumor or degenerative disease. Accordingly, repairing the nervous system after damaged is a topic to be solved urgently in the neural scientific research. The cell transplantation is a hot spot constantly in recent years. However, NSCs is stratum profundum tissue in the organism, and obtaining self-NSCs will cause original injury of nerve. So it is very difficult to get enough cells for treatment. Using foetal NSCs will cause some problems in the aspect of the ethics and law. There are some problems of immunological rejection and potential virulence gene if using variant NSCs. The reasons above-mentioned restrict clinical practice of NSCs consumedly. On the other hand, the stem cells differentiated from bone marrow haematogenesis stem cell, MSCs have powerful reproductive activity and multi-directional differentiation potentiality.In the compatible living condition, not only can MSCs differentiate mesoblastic mesenchymal tissue, such as bone tissue, cartilaginous tissue, but also have conspicuous plasticity, and keep the potentiality of differentiating to endoderm of ectoderm. It can differentiate Hepar cells, lung cells, enteric epithelium, neurocytes, and so on. The empirical study discovered that MSCs can be induced into neurocytes by using different method. It can be confirmed by empirical studies that MSCs transplanted into the rats injured their pars encephalica can differentiate into neurocytes, improve the cerebral nerves function, and protect brain.The advantage of MSCs is as following: plentiful source, drawing the materials conveniently, abstracting, depurating and cultivating easily, quick amplification in vitro under suitable culture condition, and fairly feeble immunogenicity.MSCs have overcome the ethics question compared with nerve stem cell and embryo's stem cell. So MSCs are thought highly more and more by researcher. In our experiments, MSCs were separated, depurated and amplificated in vitro on the basis of above-mentioned research and were induced to transform into the neurocytes. In our experiment, cerebral ischemia-reperfusion injury and mechanical injury rat models were established. MSCs were transplanted into rats via vein or injected partly. Through this experiment, the migration, distribution, establishment and therapeutic efficacy of MSCs in the model rats brain were observed. Protective function to brain and mechanism of MSCs was revealed further, and provided the experiment basis for treating the brain blood vessel disease by MSCs.1. Research approach(1) MSCs were separated, cultivated and identified in vitroAccording to the character of sticking to the wall rapidly, MSCs were separated, purified and amplified. MSCs morphology variation, cell cycle and growth characteristics were observed and identified though the inverted microscope, MTT method and FACs.(2) The neural differentiation and identity of MSCs in vitroMSCs were induced in vitro in the nutritive medium comprised with DMEM, 20% of the FBS, 10ng/ml bFGF, 0.5mmol/L IBMX, etc. And the expression of NSE in the neuron-type cells was detected by using immunohistochemistry method, in order to determine if the transforming was successful.(3) Preparation and identified of the hemiplegia and brain damage models in ratsRats cerebral ischemia-reperfusion injury model were established with suture emboli method. The right side limbs of the model rats paralyzed, and the fore-limbs were serious. Rat right forelimb flexing, endoduction while holding it by the tail, and turn-take or topple over to the right side accept or walk inside.(4) The migration, distribution, establishment and therapeutic efficacy of MSCsMSCs and the neuron-type cells were marked by the fluorescence mark of DAPI via vein and or injected partly. The cells concentration is 1×105个/ml. The brain tissue was get after injection 1d, 5d and 10d to observe the migration, distribution, establishment and therapeutic efficacy of MSCs in model rat.(5) Repairs function on brain damage of MSCs and neuron-like cellsThe HE dyeing of brain tissue paraffin section was prepared after the injecting 1d, 5d and 10d. 6 corona-blades of brain were fetched to observe pathological change and necrosis size.2. The results(1) The morphological character of MSCs after separating, cultivating and identifying in vitroAdherent cells were observed after cultivated 3d. The cells were circle, anomal-morph, polygon and little excrescent. After 7d, suspension dells and circle cells is decreased obviously. And a small quantity fusiform shape cells appeared. After 2nd～3rd generations, the thin and long fusiform shape cells occupied ascendant, fusiform cells show clone. There are some applan cells with excrescent. The cell shape of 6th～7th generations have no marked change, but big and applan cells increasing and fusiform cells decreasing. The cells growth appeared catabiosis without any growth factor. The cell cycle of 3rd generation was measured. The result shows that 85.87% cells occupied G0-G1 period. So the cells detected have the MSCs character.(2) The neural differentiation and identity of MSCs in vitroThe morphologic change of MSCs start at 30～40min viewing with the inverted microscope. Cytoplasm of the applan cells, fusiform cells recovery, concentrated to cell nucleus and become water-drop-like cell body. Little ecphyma protruded from cellular membrane. After 1～3d, cell ecphyma elongated and diacaustic of the round cells strengthened. Other ecphyma was similar to the pseudopodium of neuro-like cell. There are many ecphyma, little cytoplasm and round or oval-shape anachromasis cell nucleus in the differentiated cell. Neuron-like cell was identified by NSE immunohisto- chemistry method. The masculine cells were brown.(3) Preparation and identified of the hemiplegia and brain damage models in ratsRats cerebral ischemia-reperfusion injury model were established with suture emboli method. The right side limbs of the model rats paralyzed, and the fore-limbs were serious. Rat right forelimb flexing, endoduction while holding it by the tail, and turn-take or topple over to the right side accept or walk inside. (4) The migration, distribution, establishment and therapeutic efficacy of MSCsFrom the distal end of the injury brain tissue macroaxis some labeled Neuron -like cells were injected and infused via vein. The result shows that marking cells appeared in the injury brain tissue at 1st day, and the blood vessel of injury tissue. At 5th day it can be seen at vasa sanguinea of the injury and surrounding tissue. At 10th day the marker cells can disperse all of injury tissue.(5) Repairs function on brain damage of MSCs and neuron-like cellsIt is thus evident from the HE drum dyeing that 24 hours after 2 hour-reperfusion injury with suture emboli method, neurological deficit, infarction and abnormal cell can not be observed in the rats of the control group, but can be viewed at model group, PBS control group and MSCs therapeutics group rats. Obvious infarcts focus, nerve fiber rarefaction, distinct interstitial edema and inflammatory cell infiltration. 5 days after cerebral ischemia- reperfusion, the brain injury was still very severity of model group and PBS control group. But the symptom of MSCs therapeutics group gradually lessened. And 10 days after cerebral ischemia-reperfusion, the quantity of degenerated and necrosis nerve cell lessened obviously, and interstitial edema relieved. Many white districts appeared in the model group, PBS control group and MSCs therapeutics group concentrating at left ischemic region. 10 days later, different appeared in these 3 groups. Little change occurred in the model group and PBS control group, but MSCs therapeutics group changed obviously, such as white region decreasing and red region increased.3 The result of study verifies (1) Established hemiplegia and brain damage model rat successful(2) The MSCs from rat were isolated by its characteristics of growing adherence to the plastic surface with tissue culture. And it can be cultured in a long-term without obviously changing its morphology. There is still an obstacle to distinguish the cells from its morphology. Immunlolgical cell phenotype assay is helpful to recognize MSCs. When MSCs were plated at different density, the yield of cells is different. Culturing in low density can produce more cells.(3) MSCs quickly differentiated to neuron-like cells under the special culture and inducing factor. Immunohistochemistry stain showed that the cells expressed neuron specific proteins.(4) MSCs expended in culture have been introduced into the rat systemic circulation via injection into the vein and were found to migrate into the brain tissue. Vigorous generation and cell differentiation can promote repair of tissue injury no matter MSCs-transplanting or neuron-like-transplanting.On the whole, the study of MSCs provided the experiment basis for treating the brain blood vessel disease by MSCs. And MSCs can be hoped to become the seed cell to treat cerebrovascular disease and brain tissue damage. But it should be studied further that if these cells can establish synapsis connection and how to participate in the neurofunctional re-establishing of host.