| The prevalence of epilepsy in our country is about seven out of one hundred thousandincluding intractable epilepsy around 20% to 25%.One of the most common types of intractable epilepsy is temporal lobe epilepsy,accounting for about 60%. A lot of study shows that 65 percent to 70 percent of the temporal lobe epilepsy patients exist side hippocampal sclerosis, pathology performance for missing hippocanlpal neurons and hyperplasia of glial cells. Although the hippocampus and the amygdala selectively removed can make part of epileptic patients recover, but surgery itself can cause hemiplegia, hemianopsia, and memory impairment. And it is unfavorable to make bilateral resection for the patients with bilateral hippocampal sclerosis. Therefore, cell transplantation has important clinical significance in the hippocampus restoration and reconstruction.Neural stem cells (NSCs) which can differentiate into neurons, oligodendrocytes, and astrocytes have the ability to automatically update themselves and provide enough nerve cells. Since the 1990s, with the success of NSCs successively separated and cultured from the central nervous system of embryonic and adult mammals,the traditional view that "central nervous system after the damage non-renewable" was broken and finding a new treatment in curing the diseases of the central nervous system, all of which have become a hot trend in stem cells study recently. NSCs and gama aminobutyric acid(GABA)ergic neurons transplantation have proved effective in the treatment of temporal lobe epilepsy in numerous experiments. But the reports and experimental studies comparing the effect of the two haven't seen yet. In this paper, three aspects of establishing rats temporal lobe epilepsy model,isolation and culture of NSCs, induced differentiation of NSCs and cell transplantation were researched as following:1. Establishment of temporal lobe epilepsy model in rats.Objective To find a proper dose for establishing the epilepsy model in rats. Methods Eighty SD rats, male, were randomly divided into 4 groups. Then KA 0.4μl, 0.7μl, 1.0μl and saline 1.0μl were respectively injected into the right hippocampus of rats in the groups. The concentration of KA was 0.5μg/μl. After the injection, all rats'behaviors were recorded for 4 hours according to Racine's classification standard. The rat that with 3 consecutive seizures of GradeⅣ～Ⅴ,defined as a successful epilepsy model. Results Rats in saline group,all survived,no seizure observed.KA 0.4μl group,1 rat died,1 rat reached seizures of gradeⅤ,6 rats reached seizures of gradeⅣ,9 rats reached seizures of gradeⅢ,3 rats reached seizures of gradeⅡ.KA 0.7μl group,2 rats died,15 rats reached seizures of gradeⅤ,and 3 rats reached seizures of gradeⅣ.KA 1.0μl group,14 rats died,and 6 rats reached seizures of gradeⅤ.Conclusion KA 0.7μl group has the best effect for establishing epilepsy models in rats.2. Isolation,culture , differentiation of NSCs and cell transplantation.Objective To obtain purified NSCs,explore the differentiation of NSCs into GABAergic neurons and comparison the cure effect of cell transplantation in KA rats. Methods 1) Isolation and culture of NSCs: Pregnant 12d SD rat after anesthesia and disinfection were placed in sterile petri dish inside a laminar flow cabinet. Remove embryos and put them into serumfree DMEM. Microscopically carefully peel to obtain the cerebral tissue which were cut into 1mm3 pieces and were digested by trypsin for 10～15min.Then the trypsin was neutralized by DMEM which contain 10% fetal bovine serum(FBS).After filtered by 100 mesh strainer, centrifuged for 5min, washed 3 times in serum-free DMEM, NSCs nutrient-containing medium was added. Then Adjust cell concentration and transfer them to the culture bottle and got the primary cultured cells. Static cultured for 2～3 days to change the first half of the amount of medium,and the Normal exchange of medium later for about every 3 days.7 days later the cells were passaged to obtain the first generation of the cells,and so did the later passage. Take the 3rd generation of NSCs and immunohistochemistry technique was used to identify the anti-nestin. 2) Induce rat NSCs differentiate into GABAergic neurons in vitro: Add GABAergic neurons directional differentiation medium to the 3rd generation of NSCs and change the half of the amount of medium for every 2～3 days. Observe the morphological change of the induced cells and analyzed the differentiated cells with Immunofluorescence 7days later by identifying anti-NF200,anti-GAD67 and anti-GFAP.3) NSCs and GABAergic neurons were respectively transplanted into the right hippocampus of KA rats 4 days after the models of temporal lobe epilepsy were established.Then the cure effects including ethology and pathology were observed 4,8 and 12 weeks after the cell transplan- tation.Pathological methods including Nissl dyeing,HE dyeing and immune fluorescence staining. Results We can see a lot of single cells round and of the same size with big cell nucleus,less cytoplasm and high refractive index 2 hours after isolation.Then the cells become more and more,6～7 days later a cloning ball with hundreds of cells were proliferated and show nestin positive.The cells'morphology began to change after induced for 2～3 days, began to appear bulge and processes following the differentiation completed, showing a typical neuron-like cell morphology. Immunofluorescence manifested that some cells show GAD67 positive,some show NF200 positive and some show GFAP positive. There is an ethological improvement in the two cell transplantation groups 8w after transplantation,but there are no statistical differences between the two groups.The number of pyramidal neurons of field CA3 of hippocampus counted most both in NSCs transplantation group and GABAergic neurons transplantation group 8 weeks after the transplantation, P<0.05.And there are statistical differences among the 4 groups 8 weeks after the cell transplantation, P < 0.05. Conclusion Obtained NSCs and NSCs had the trend of differentiating into GABAergic neurons in vitro. CA3 pyramidal neurons of the two grafted groups both counted most 8 weeks after the transplantation. And it seems that NSCs transplantation group has a better cure effect than GABAergic neurons transplantation group. |
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