Lhx8 Funtion On Cholinergic And GABAergic Neurons In The Hippocampus Of Neonatal Rats

Objective:Lhx8 is a key factor for the determination of cholinergic and GABAergic neurons fate.While it is little known how Lhx8 affect the cholinergic and GABAergic neurons in neonatal rats,especially about the GABAergic neurons.Using the lentiviral vetors and Tet-on system,we study the role of overexpression Lhx8 in the cholinergic and GABAergic neurons.Method:According to Rattus norvegicus Lhx8?rLhx8?seqence in Gene Bank,NM₀01012219.1?1011bp?,the gene which was designed and synthesized containing restriction endonuclease digestion site of Eco RI and Not I by Biowit Technologies,was inserted into vetor plasmid pLVX-Tight-IRES-Zs GreenNeo and generated pLVX-Tight-rLhx8-IRES-Zs Green-Neo.And also the Kozak sequence was inserted to improve the expression level of rLhx8.At the same time,pLVX-Tet-On-Puro was designed and synthesized.We identified and amplified the plasmids.Human embroic kidney 293 T cells were useed for the lentivirus packaging.After 48 hours and 72 hours of transfection,the viral supernetant was collected and concentrated.For testing the virus,293 T cells was used.The lentivirus was microinjected into the brain.The 5mg/ml tetracyclinewas useed to induce the Lhx8 expression.After 48 h,72h and 96 h infection,the whole brains was separated for immunofluorescence experiments.Then total RNA and proteins were extracted from the hippocampus.Using RT-PCR,we detect the transcription of CHAT?choline acetyltransferase?m RNA,GAD65?glutamic acid decarboxylases 65?m RNA andLhx8 m RNA.The expression level of CHAT and GAD65 protein were assayed by western blot.After immunofluorescence for CHAT and GAD65,the sections were observed using an Olympus immunofluorescence microscope.Positive cells were counted in four randomly selected microscopic visual fields per well.Data from the experiments were subjected to Sthdent's test using Graph Pad,all data were represented as the mean ± SEM,and all experimental data were obtained from a minimum of three independent experiments.Results:By plasmid extraction kit instructions,the plasmids,pLP1,pLP2,pLP/VSVG,pLVX-Tight-IRES-Zs Green-Neo,pLVX-Tet-On-Puro as well as pLVXTight-rLhx8-IRES-Zs Green-Neo were correctly identified by restriction enzyme digestion for the amplification.Then the five plasmids were transfected nto 293 T cells line.Green fluorescent could be seen in 293 T cells,and success for cell transfection as well as lentivirul packaging.It also showed that lentivirus have the ability to infect 293 T cells when the 293 T cells were affected by lentivirus.The RT-PCR studies showed that the m RNA of GAD65 and CHAT were affected by the overexpression of rLhx8.Especially after the lentivirus was induced by tetracycline for 72 h,the level of rLhx8 m RNA were improved,comparing with the control groups.Following this phenomenon,the level of CHAT m RNA ware upregulated,while the GAD65 opposite.This situation went on when we observed after the injection of 96 h.At the same time,the immunofluorescence analysis and western blot presented the same thing.All these experiments indicated that rLhx8 affect the expression of cholinergic and GABAergic neurons in neonatal rats.Conclusion:After the overexpression of Lhx8 in the hippocampal of neonatal rats,cholinergic neurons marker were increased,while the GABAergic neurons were decreased.