Experimental Studies Of Rat's Neural Stem Cells Induced And Differentiated From Bone Marrow Stromal Cells And Autologous Transplantation For The Treatment Of Temporal Lobe Epilepsy

Neural stem cells(NSCs) are a kind of cells that have the ability to differentiateinto neurons, astrocytes and oligodendrocytes. They can also self-renew and providea large number of brain cells. They are mainly located in embryonal brain andextensive areas around the ventricle of adult central nervous system. But some recentstudies show that bone marrow stromal cells(BMSCs) can differentiate into NSCsunder certain condition, which provides unlimited source for the use of NSCs. Thedevelopment and use of NSCs derived from BMSCs not only get rid of the limit anddanger of acquiring NSCs from brain tissue, but also avoid the exiting questions ofethics, immune and limited source. It will have a promising future in the treatment ofhead trauma and degenerative diseases of the central nervous system.Epilepsy is one of the common nervous system diseases. Its prevalence in peopleis about 5‰, while in our country about 7‰. Epilepsy is a chronic non-specificcerebral disease. It is a clinical brain dysfunction syndrome caused by various congenital or secondary factors. It results from sudden high-frequency over-dischargeof some neurons in the brain, characterized by frequent seizures. Temporal lobeepilepsy(TLE) is one of the most important types of focal epilepsy, clinicallycharacterized by special abnormality of smelling, conscious disturbance anddrunk-dreaming. The common pathology of TLE is hippocampal sclerosis, that is,neuronal loss and glial proliferation in some areas of the hippocampus. Althoughanterior temporal lobectomy or selective hippocampo-amydala-ectomy can cure somepatients with temporal lobe epilepsy, the operation itself can have some danger, suchas hemiplegia, hemiopalgia and memory destruction. And to patients with theepileptic lesions in both temporal lobes, it is impossible to resect the lesion. So, theexperimental studies on the repaired restoration of the hippocampal function throughneural stem cells(NSCs) transplantation can explore a new way to treat and reduce thefrequency and degree of seizure, which has promising significance.Part One. Experimental study of rat's neural stem cells induced anddifferentiated from bone marrow stromal cells in vitro1. ObjectivesTo explore the feasibility of acquiring rat's NSCs which are cultured,inducedand differentiated from BMSCs in vitro, and to comfirm the appropriate culturemethod and condition of BMSCs differentiating into NSCs, which will make a basefor it to be widely used in tissue-enginnering and clinical medicine.2. Materials and MethodsSD rat's bone marrow was acquired by puncturing its marrow cavity underaseptic condition. Density gradient centrifugation was used to acquire mononuclearcells with nucleus. Adherence technique was also used to acquire the bone marrowstromal cells. The cell density of the cultured cells was adujusted to 10⁶/ml, and thecells were inoculated in Petri dish, which was added with fetal calf serium(1%), LIF(10ng/ml) and bFGF(10ng/ml) respectively, and then cultured in 37℃, 5% CO₂and saturation wetness condition. The culturing medium was first changed 48 hourspost-inoculation in order to wip off the non-adherent cells. The medium was changedtwice per week. In this way, relatively purified BMSCs were obtained andsubcultured. In subculture, the bovine blood serum and RA (0.5ug/ml) were added inorder to induce BMSCs to differentiate into NSCs in one week. Under CK2 inversionphase difference microscope, the cells were continuously observed and taken photos.In various culturing stages, the cell samples were detected by immunocytochemistry(The first antibodies were rat anti-Nestin, rat anti-NeuN, rat anti-NSE and ratanti-GFAR The second antibodies were biotinylated goat anti-mouse IgG andstreptavidin peroxidase). The sample was fixed in 4% polymerisatum, flushed by0.01M PBS. Then the first antibody was added and incubated in 37℃for 60 minutes,and then the second antibody was added and incubated in room temperature for 10minutes. The streptavidin peroxidase was added for it to be incubated in roomtemperature for 10 minutes. And finally, it was stained by DAB for 5～15 minutes.The color of the positively stained cells was brown. The photos were taken withJapanese Olympus microscope.3. Results48 hours after the culture, the division and proliferation of the adherent cells inculture could be seen, which could form cell clones like islands. When beingcontinuously cultured and given appropriately differentiated condition, these cellscould differentiate into poly-form cells with long and thin apophysis. When beingevaluated by immunocytochemistry the cultured cells, even without induction anddifferentiation condition, could be Nestin positive in the earlier one week. It wasindicated that these cells had some characteristics of stem cell. When these cells wereinduced and differentiated, some cells could be NeuN, NSE and GFAP positive. And with the time going on, the positive cells slowly increased, which indicated that theyhad the tendency to differentiate into neuron and glial-like cells.4. ConclusionsBMSCs derived from rat's bone marrow can transdifferentiate into NSCs if thereis a suitable culture condition, and these cells have the ability of proliferation anddifferentiation. The NSCs derived from BMSCs can differentiate into the cells withthe characteristics of nervous system cells. These cells can express NeuN, NSE andGFAP respectively.Part Two: Experimental study on autologous transplantation of rat's bonemarrow-derived neural stem cells for the treatment of temporal lobe epilepsy1. ObjectivesTo explore the integrated and repaired effect of bone marrow-derived neuralstem cell after its autologous transplantation into the hippocampus of KA rats, and toprovide the theoretical basis of stem cell for the treatment of temporal lobeepilepsy(TLE).2. Materials and MethodsThe bone marrow stromal cells(BMSCs) of rats were isolated, and under specificcondition the BMSCs were cultured to induce and to differentiate into neural stemcells(NSCs). And the NSCs were labeled with Feridex. 60 male SD rats wererandomly divided into three groups: control group, graft group and non-graft group,each group containing 20 rats. Then the rat models of temporal lobe epilepsy wereestablished, and the NSCs were autologously transplanted into the right hippocampalCA3 area of KA rats. While the same amount of normal saline was injected into theright hippocampal CA3 area of the control group and non-graft group. The changes ofEEG, MRI and pathology were analyzed 1, 2, 4, 8, 16 weeks after the transplantationrespectively. 3. ResultsThe rats injected into kanate acid all exhibited typical symptoms of statusepilepticus. And after a short period of silence, the rats began to have recurrentseizures, similar as amygdala kindling seizure. Compared with the non-graft group,the EEG magnitude of the graft group decreased significantly, the highest level morethan 40% after 8 weeks' transplantation. MRI testing showed that low signal regionwas a little localized 1 and 2 weeks after the transplantation, while it spread morewidely with the time going on, and the low signal change slowly increased 4, 8, 16weeks after the transplantation. There was significant difference in the number ofhippocampal CA3 pyramid cells between the graft group and non-graft/controlgroup(P＜0.01).There was also significant difference between different phases in thegraft group(P＜0.01). The cell number was the largest till 8 weeks after thetransplantation, and then it began to decrease. In the non-graft group, with the timegoing on, the number of hippocampal CA3 region slowly decreased. There was alsosignificant difference in Timm staining score of the injured hippocampus between thenon-graft/control and graft group(P＜0.01). There was also significant differencebetween different phases in the graft group(P＜0.01). The Timm staining score was thelowest till 8 weeks after the transplantation, and then it began to increase. Theelectromicroscope testing showed that in the graft group the neuleus of the CA3pyramidal cells was obvious, the mitochindal body was swelling, and there werefewer synaptic bodies. While in the non-graft group, the free nuleus of the CA3pyramidal cells dereased, the synaptic menbrance were combined, and there weremore synaptic bodies.4. ConclusionsWhen the bone marrow-derived NSCs are autologously transplanted into the KArats, they can suppress seizure attacks of the rat model, integrate into the host and have a remarkable repaired effect on the hippocampus. But the study on its actualprinciple is still to be researched on.