Lhx8 Regulates The Development Of Hippocampal Neurons In Neonatal Rats Through Arx

Objective:To explore the mechanism of Lhx8 regulates cholinergic neurons and GABAergic neurons in neonatal rats,and to provide a basis for further clarifying the neurologic diseases such as Alzheimer's disease,vascular dementia,depression and epilepsy.Method:According to the prediction of bioinformatics software,a Arx oligonucleotide probe combined with Lhx8 protein was designed to synthesize a double strand,using biotin to mark the probe 3,,EMSA was performed to detect whether transcription factor Lhx8 could bind to Arx gene.Empty vector plasmid was named Lenti-CON(p LVX-Tight-IRES-Zs Green-Neo).r Lhx8 c DNA was inserted into vector plasmid p LVX-Tight-IRES-Zs Green-Neo and generated Lhx8 overexpression lentiviral vector plasmid,was named Lenti-Lhx8(p LVX-Tight-r Lhx8-IRES-Zs Green-Neo).Using the restriction endonuclease Bam HI,the Arx sh RNA gene fragment was linked into the p LVX-Tight-IRES-Zs Green-Neo plasmid and generated the lentivirus vector plasmid that interfered with the Arx gene expression,was named Lenti-Arx sh RNA(p LVX-Tight-Arxsh RNA-IRES-Zs Green-Neo).The Arx sh RNA gene fragment was linked into the p LVX-Tight-r Lhx8-IRES-Zs Green-Neo plasmid and generated the Lhx8 overexpression and interfered with the Arx gene expression lentivirus vector plasmid,was named Lenti-Lhx8/ Arx sh RNA(p LVX-Tightr Lhx8-Arxsh RNA-IRES-Zs Green-Neo).The vector plasmids,p LP1,p LP2,p LP/VSVG,and Tet-on regulatory plasmid were co-transfected into 293 T cells for packaging lentivirus.Four lentiviruses of Lenti-CON,Lenti-Lhx8,Lenti-Arx sh RNA and Lenti-Lhx8/ Arx sh RNA were used to inject the lateral ventricle of the neonatal rats.Intraperitoneal injection of doxycycline 2.7mg/kg(Doxitard,DOX)induced the expression of Lhx8 gene and / or Arx small interfering RNA.After 48 h,72h and 96 h infection,the total RNA was extracted from the rat's left hippocampus for PCR.The total protein of the rat's right hippocampus was extracted for Western Blot,and the whole brain of the rat was separated for immunofluorescence experiments.Results:1,EMSA experiment showed that Lhx8 protein could be combined with Arx DNA probe;2,Lhx8 c DNA and Arx hairpin structure were built in the same vector,which could realize the overexpression of Lhx8 gene and the low expression of Arx gene.3.Successfully packaged lentivirus carriers Lenti-CON,Lenti-Lhx8,Lenti-Arx sh RNA and Lenti-Lhx8/ Arx sh RNA.It also showed that lentivirus have the ability to infect 293 T cells after concentration and purification.4,After the lentivirus was induced by DOX for 48 h,the results of RT-PCR,Western Blot and immunofluorescence showed that there was no difference among the various indexes.5,After the lentivirus was induced by DOX for 72 h and 96 h,RT-PCR results showed that overexpression of Lhx8 reduced the expression of Arx m RNA,the overexpression of Lhx8 increased the m RNA expression of CHAT,and reduced the expression of GAD65 m RNA.Interference Arx did not affect the expression of CHAT m RNA,but reduced the expression of GAD65 m RNA,and there was no difference in GAD65 m RNA expression between the overexpressed Lhx8 group and the overexpressed Lhx8 at the same time interference Arx group.Western Blot and immunofluorescence results showed that over expression of Lhx8 increased the expression of CHAT protein,reduced the expression of GAD65 protein,and interfered Arx,resulting in the decrease of GAD65 protein expression.Conclusion:Lhx8,as a transcriptional regulator,can combine with Arx gene to inhibit the expression of Arx gene,and the overexpression of Lhx8 gene can promote the differentiation of cholinergic neurons in the hippocampus of newborn rats and inhibit the differentiation of GABA energy neurons.The Arx gene has a positive effect on the differentiation of GABA energy neurons in the hippocampus of neonatal rats,and has no effect on the differentiation of cholinergic neurons.