| Epilepsy is one of the most common neural disorders in central nervous system andcharacterized by chronic and recurrent occurrence of abnormal discharging of neurons inthe brain. According to the reports made by World Health Organization (WHO), thenumber of the petients who are suffering from epilepsy was approximately fifty million toone hundred million, furthermore, there are about5percent of the world population has arisk of developing epilepsy. About thirty percents of patients with epilepsy show noresponse to anti-epilepsy drugs. However，surgery and electrical nerve stimulation therapywhich including Vagus Nerve Stimulation (VNS) and Deep Brain Stimulation (DBS) wasnot suitable for all the epilepsy patients, especially those who has no indications. So, it isnecessary to find a new treatment strategy which is more effective, safe and long-lasting.It is the key mechanism that the losing of GABA (Gamma-amino Butyric Acid)ergicneurons in brain. Studies showed that GABAergic neurons derived from differentcategories of stem cells including embryonic stem cells (ESCs)，neural stem cells (NSCs)and mesenchymal stem cells (MSCs) can inhibit epileptic seizures, prolong the survival of epileptic animal model. So, a lot of researchers are focusing on the treatment of epilepsybased on stem cells. However, the biggest problem is the lack of cell sources.Mesenchymal stem cells are considered to be a promising candidate for cell therapystrategy due to those features of having the potentiality of self-renewal and differentiationcapacity toward different cell linages. As a member of mesenchymal stem cells, bonemorrow mesenchymal stem cells (BMSCs), which are derived from bone marrow, arewidely distributed in the human body and easy to extract and separation. There were noimmune rejection and ethical problems when using of BMSCs. Researchs showed thatBMSCs grafting can decrease the spontaneous recurrent seizure (SRS) of epileptic animalmodel. It is possible to form the synaptic connection between grafted cells and hostneurons by transplantation of BMSCs. However, there are still few studies on the effectsof GABAergic neurons derived form BMSCs in epileptic animal. Previous studiesindicated that，by induction with chemical components such as potassium Chloride (Kcl)，β–Mercaptoethanol (BME) and retinoic acid (RA) or cytokines (bFGF) and so on，BMSCs can differentiate toward GABAergic neurons. But the application of theseinductions in vivo is hard to realize and may cause unexpexted side effects.Basic Helix-Loop-Helix (bHLH) gene plays an important role in the process ofproliferation and differentiation of stem cells. As a member of bHLH, mammalianachaete-scute homologue (Mash1) is an active transcription factor and not only intiate theprocess of neuronal differentiation of stem cells but also decide the formation of neuronalsubtypes which is closely related to the GABAergic differentiation. So, we hypothesizedthat over-expression of Mash1gene may improve the GABAergic differentiation ofBMSCs. Over-expression of Mash1in BMSCs was performed by transfection of lentivirusvector. After neuronal induction with compound culture medium, BMSCs differentiatedinto GABAergic-like cells. To verify the hypothesis, comparison between the efficiency ofthe GABAergic differentiation of BMSCs and Mash1over-expressed BMSCs wasperformed. Finaly, Mash1over-expressed BMSCs were transplanted into the brains ofepileptic rat model and the effects of seizures inhibition were observed.Part one: Extraction, isolation, culture and identification of rat BMSCs in vitro Objective: Set up an efficient approach of extraction, separation and identification of ratBMSCs.Methods: By sterile surgical operation, bone marrow was extracted from the bilateralfemur and tibia from2~3week-old rats. Separate the BMSCs with two ways: bonemarrow adherent separation method and Histopaque density gradient centrifugationseparation method. Identification of cell surface markers was carried out with flowcytometry and differentiation potentials towards fat cells, chondrocytes and osteoblastswere performed under conditioned medium. Growth curve was detected by MTT (3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium bromide).Results: BMSCs achieved by density gradient centrifugation separation method showedmore uniform cell morphology and less impurity cells compared with those obtainedthrough bone marrow adherent separation method. Growth curve (grew slow in Day1~3after incubation, and got into the logarithmic growth pahse in Day4~6and to Day7~9into the plateau), cell surface markers (low express CD34(1.79±0.23)%and high expressCD105(98.6±0.68)%, CD90(98.5±1.02)%and CD73(98.8±0.98)%as well) andpluripotency of the former meet the international standardized well.Conclusion: Density gradient centrifugation method was efficient and by this method wecan get BMSCs with high-purity, amplified fast and pluripotency effectively and easily.Part two: Over-expression of Mash1gene improves the GABAergic differentiation ofBMSCs in vitro.Objective: Study the effects of over-expression of Mash1in the process of GABAergicdifferentiation of BMSCs.Methods: Extract BMSCs from2~3week-old SD rat and proliferate them. The passage3BMSCs were used in this experiment. Preparate equipped Mash1gene lentiviral vecterand transfect BMSCs. GABAergic differentiation of Mash1+-BMSCs and BMSCs withcompound induction medium (1×B27+1mMBt2cAMP+1μMATRA+Neurobasal) in vitro.Immunocytochemistry, PCR detection and Western blot analysis were performed to verifythe differentiation trend. Furthermore, the electrophysiological properties of differentiatedcells were tested by whole-cell patch-clamp detection. Results: The cell surface molecular markers and differentiation potential of the passage3BMSCs used in the experiment fit the international standard well. Western blot analysisshowed that the level of Mash1in the lentiviral transfected (multiplicity of infection, MOI=100) BMSCs was increased significantly after transfection. Mash1+-BMSCs groupshowed more neuron-like cells compared to control groups during the process ofGABAergic differentiation. Western blot and RT-PCR analysis showed that cells in Mash1+-BMSCs group expressed higher level of NeuN levels and GAD67than control cells afterdifferentiation. After GABAergic differentiation, immunofluorescence analysis showedthat cells in Mash1+-BMSCs group (71.6±2.8)%represented higher level of GAD67immunofluorescence positive rate compared to those cells in BMSCs group (61.4±5.3)%,Lv-con-BMSCs group (58.6±5.4)%and M-con-BMSCs group (0). In addition, cells inMash1+-BMSCs group (72.0±2.0)%represented higher level of GABAimmunofluorescence positive rate compared to those cells in BMSCs group (61.4±2.9)%,Lv-con-BMSCs group (62.4±5.5)%and M-con-BMSCs group (0). Whole-cellpatch-clamp detectation indicated that differentiated cells possess functionalcharacteristics like action potentials and spontaneous inhibitory postsynaptic currents.Conclusion: Lentivirus-mediated gene overexpression has a good effect; Mash1overexpression can promote BMSCs differentiate into GABAergic neurons withelectrophysiological activity.Part three: Study on the effects of treatment of Mash1gene over-expressed BMSCs inepileptic rat model.Objective: Investigate the therapeutic effects of Mash1gene over-expressed BMSCs intreating epilepsy rat model.Methods: After having been trained in morris water maze and getting spatial memory,60SD rats were used to preparate pilocarpine-induced epilepsy model. The rats that reachedRacine Ⅴ level were used in the experiment. The cells (Mash1+-BMSCs and BMSCs)used for transplantation were labeled with BrdU in vitro. Mash1+-BMSCs (M-BMSCsgroup)，BMSCs (BMSCs group) or rat fibroblast cells (positive control group)(5×105/rat)were injected into the right lateral ventricle of rats with epilepsy by stereotactic microinjection. Rats in different time points (7,14,21,28days after cell transplantation)were tested by functional detection (water maze test, spontaneous recurrent seizures (SRS)frequency monitoring), electrophysiology (EEG) and immunohistochemistry (Nisslstaining, paraffin immunity staining, DAB (diaminobenzidine) staining) to study thechanging of spatial memory, brain electrical activity and the level of neuronaldifferentiated cells after cell transplantation.Results: Cell transplantation can protect the spatial memory of epileptic rats anginst theinjury of epilepsy and the protective effects began to appear scine Day7. The protectiveeffect of Mash1+-BMSCs in M-BMSCs group appeared earlier, more obviously (escapelatency time recover to normal levels) in comparison to BMSCs group (P<0.05). Celltransplantation therapy can not only reduce the mortality of epileptic rats (although therewere no statistical difference between the groups of data, the numbers of deaths inM-BMSCs group and BMSCs group are lower than that in the positive control group), andcan but also reduce the occurrence of SRS (although statistical analysis showed nodifferences, the numbers of deaths in M-BMSCs group and BMSCs group are really lowerthan that in the positive control group). Also in the EEG testing, recovery of function hadbeen observed. After cell transplantation, compared with the positive control group, thenumber of spikes and spike-low waves in M-BMSCs group and BMSCs group weredecreased significantly (P<0.05) and the former had better effect. Immunofluorescenceanalysis showed that more NeuN+/BrdU+cells were detected in the parahippocampalcortex of rats brain in M-BMSCs group than that in the BMSCs group and moreGAD67+/BrdU+cells were detected in M-BMSCs group than that in the BMSCs. Nisslstaining analysis indicated that the density of vacuoles nucleated cells (neurons) inparahippocampal cortex in the rats of M-BMSCs group were significantly higher than thatin the control group (Day14and Day28, P<0.05). Locating of transplanted cells wasperformed by DAB staining and the results showed that transplanted cells are widelydistributed in the regions of the hippocampus and parahippocampal cortex whichsuggesting a structural basis for the transplanted cells to promote functional recovery inrats with epilepsy. Conclusion: Cell transplantation therapy using the Mash1over-expressed BMSCs canpromote functional recovery in epileptic rat model and superior to BMSCs transplantation. |
PDF Full Text Request