| BackgroundEpilepsy is a very common neurological disease, and it can occur over a wide age range. At present, prevalence estimates for epilepsy from survey in China about 0.5%, there are more than 6 million patients over the whole country. Now the main therapy to epilepsy is drug treatment and about 80 percent seizures can be controlled by the rational and scientific use of antiepileptics, and about 70 percent of epileptic patients can achieve complete control. But still about 20 percent of patients remain seizure after drug treatment, and most of them eventually turn into refractory epilepsy, that contains two reasons, one that many patients are unable to stand the high toxic and adverse effect of antiepileptics, the other one is that some patients still get seizures because the antiepileptics are not functioning properly in their bodies. Not only the pathological change cannot be reversed but also the natural process of epileptic development cannot be prohibited under drug treatment, because antiepileptics can only inhibit seizures. Moreover the serious ADR (Adverse Drug Reaction), prolonged use and expensive prices of antiepileptics bring great difficulties to the patients and their families. So there is a pressing need to find a safe, effective, and feasible method in the treatment of epilepsy. From a lot of research works we can see that seizures are associated with the distribution of GABA neurons degeneration and absence within epileptogenic focus. Due to the GABA neurons functional defect gives rise to the opposite or absolute scarcity of GABA neurotransmitter, thus the GABA neurotransmitter deficiency disturbs the balance between excitatory and inhibitory neurotransmitter, consequently the maladjustment of neurotransmitter causes neurons abnormal discharge. Many researchs have revealed that the transplantation of neural stem cells within epileptogenic focus was an effective way to treat epilepsy.But the NSCs have been limited in application because of their own disadvantages. Therefore with the changes of epileptic pathophysiology, we try to investigate a new potential way to treat patients by the transplantation of GAD67 modified bone mesenchymal stem cells within epileptogenic focus.ObjectiveTo construct human glutamic acid decarboxylase (GAD67) gene's recombinant eukaryotic expression vector pDC316-CMV-EGFP-GAD67 and to transfect it into bone marrow stromal cells of rat, investigate the efficiency of transfection and the expression of GAD67 gene in bone marrow stromal cells after transfecting .MethodsWith the technology of gene rearrangement, digested pCMV6-XL5-GAD67 plasmid respectively with Not I and Spe I, obtain the target sequence of human GAD67 gene, and then inserted it into cloning vector pDC316-CMV-EGFP obtaining recombinant eukaryotic expression vector pDC316-CMV-EGFP-GAD67, the recombinant was identified by restriction enzyme analysis and sequencing. To isolate and cultivate bone marrow stromal cells of rat by their adherence-dependent growth character, and transfect the recombinant pDC316-CMV-EGFP-GAD67 into them with lipofectamine2000. To observe the cells by fluorescence microscope, detect the efficiency of transfection by flow cytometry and detect the expression of GAD67 gene in bone marrow stromal cells by immunohistochemistry after transfecting. ResultsThe plasmid pDC316-CMV-EGFP-GAD67 was constructed successfully, containing 3.5 kbp fragments of human GAD67 cDNA, certified by restriction enzyme analysis and sequencing; Many green fluorocytes were shown under fluorescence microscope, the efficiency of transfection was 37.3% detected by flow cytometry, GAD67 was positive in tansfected cells detected by immunohistochemistry.ConclusionThe eukaryotic expression vector pDC316-CMV-EGFP-GAD67 can be successfully constructed and effectively transfected into bone marrow stromal cells with lipofectamine2000, this study provide an experimental basis for gene modified bone marrow stromal cells transplantation within epileptogenic focus in the therapy of epilepsy...
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