Observe The Fate Of The Newborn Inherent Neural Progenitor Cells And Transplant Genetically Engineered Cells In The Hippocampus After Pilocarpine-induced Status Epilepticus In Adult Rats.

Part I :Observe the fate of newborn inherent neural progenitor cells in the hippocampus after pilocarpine-induced status epilepticus in adult rats.Objective:Observe the fate of newborn inherent neural progenitor cells after pilocarpine-induced status epilepticus(SE) in adult rats,including migration, differentiation and apoptosis.Investigate neurogenesis and plasticity change occured after SE and the effect of the proliferation of newborn inherent neural progenitor cells onSE.Method:Status epilepticus(SE) was induced by lithium-pilocarpine injected, bromodeoxyuridine(BrdU) was injected at 3, 7, 14 and 28d after the first SE,the expression of polysiolylated neural cell adhesion molecule(PSA-NCAM) and PSA-NCAM/BrdU was observed in the hippocampus by immunohisochemistry and immunofluorescence staining;BrdU was injected at 14d after the first SE, BrdU/NeuN, BrdU/GFAP, BrdU/TUNEL positive cells was observated in the hippocampus by immunofluorescence and TUNEL staining at 5, 14 and 28d. Results:Compared with the controls,the number of PSA-NCAM-positive cells and PSA-NCAM/BrdU positive cells in the hippocampus increased at 3d after SE (p<0.05),reached maximum at 14d(P<0.01),and decreased markedly at 28d,but it was still higher compared with that of the controls(p<0.05).All positive cells in bilateral dentate gyrus(DG) was resemble, located in the subgranule zone(SGZ). BrdU was injected at 14d after the first SE, compared with the controls,BrdU positive cells which in the hippocampus increased markedly.Most of BrdU positive cells were differentiated into neurons(about 70%),with labeled time prolonged, the number of BrdU positive cells was decreased,a few of the TUNEL-positive cells appeared in BrdU positive cells,Condusions:These observations indicate that SE stimulate proliferation and migration of inherent neural progenitor cells .Most of these newborn inherent neural progenitor cells were differentiated into neurons,a few of these cells were changed into astrocytes. Neurogenesis and plasticity change of originally-existed inherent neural progenitor cells may occur after SE .But only a few of these cells were surviving and concerned with the structure reconstructed of the hippocampus.Part II:Construct genetically engineered cell lines to produce and release GABAObjective:Conctruct genetically Engineered cell lines to produce and release GABA through immortalized rat neural progenitor cells and astrocytes.Methods: Used immortalized rat neural progenitor cells and astrocytes as cell carriers,construct cell lines with the GABA-synthesizing enzyme GAD(65) cDNA.The GABA-producing cells express GAD65 with immunohisochemistry and Western-blot; The content of GABA was detected with Capillary Electropherograms;GABA current was recorded in cell lines using whole-cell voltage clamp technique.Result: Compared with the controls,the content of GAD65 in GABA-producing cells increased obviously by immunohisochemistry and Western-Blot.Using Capillary electropherograms,the content and secretion of GABA in GABA-producing cells was markly increased;GABA current was recorded in the GABA-producing cells by the whole cell voltage clamp technique.ConclusionsrUsing immortalized rat neural progenitor cells and astrocytes as cell carriers,the GABA-producing cells was engineered successfully,the cell lines can produce and release GABA.Part III :Transplant genetically engineered cell lines in the hippocampus in the epileptic rats model induced by pilocarpineObjective: Transplant genetically engineered immortalized neural progenitor cell lines can survive and differentiate in the hippocampus in the epileptic rats model induced by pilocarpine ,have the ability to suppress spontaneous seizures.Method: Labeled genetically engineered immortalized neural progenitor cell lines with SPIO, transplant genetically engineered cells in the right hippocampus in the epileptic rats model induced by pilocarpine.After transplantation, SPIO-labeled cells were tracked with 1.5T MRI scanner in vivo; Behavior were monitored for 6 days after surgery; The change examined with electroencephalogram recording; The survival and differentiation of transplanted cells was observed with immunohisochemistry.ResuIt:Animals that received genetically engineered GABA-producing cells had significantly fewer spontaneous seizures than did that the control group;Mortality of two groups was different; Epileptiform spikes was suppressed significantly in transplanted GABA-producing group in the electroencephalogram; 1.5 Tesia MRI in vivo displayed that remarkable low signal area on T2WI was seen in the right brain which transplanted with SPIO-labeled cells,no signal change in the left brain and the contral group without cells transplanted;By immunohisochemistry,we find some SV40Tag-positive cells in the right hippocampus transplanted with GABA-producing cells,at the same time show GAD65 positive.SV40Tag-positive cell can change into neurons and astrocytes in the hippocampus. Conclusion: These data demonstrate that genetically engineered cells could suppress spontaneous seizures in an accepted model of temporal lobe epilepsy.This is an important step toward defining a clinical potential for this approach in epilepsy. The fact of transplanted therapy may be possible.SummarizationObserved the fate of newborn inherent neural progenitor cells including migration , differentiation and apoptosis in an accepted model of temporal lobe epilepsy.SE stimulated the proliferation and migration of inherent neural stem cells , which most of these newborn cells were changed into neurons and a few of these cells were changed into astrocytes. Neurogenesis and plasticity changes of originally-existed neural stem cells may occur in SE. But only a few of these cells were survivingand concerned with structural recombination of hippocampus.Used immortalized rat neural progenitor cells and astrocytes as cell carriers, genetically engineered cell lines with the GABA-synthesizing enzyme GAD(65) cDNA, can produce and release GABA.Transplanted immortalized neural progenitor cells can produce and release GABA in the hippocampus in the epileptic rat model induced by pilocarpine, these cells could suppress spontaneous seizures in an accepted model of temporal lobe epilepsy.This is an important step toward defining a clinical potential for this approach in epilepsy. The fact of transplanted therapy may be possible.