Screening Of Small Molecular Inhibitors Of KLF4 From Chinese Herb Medicine And Studying Of Their Function On Inducing Differentiation Of Cholinergic Neuron

Inducing differentiation of neural stem cells into neural cells is becoming one of the research hot spot in neuroscience.The construction of neuronal differentiation model not only simplify the research of molecular mechanism underlying neuronal differentiation,but also indicate new therapeutic methods for neurodegenerative disease.KLF4 is an important transcription factor which can modulate cellular developing and differentiation,it is also one of the four factors that can induce reprogramming of somatic cells.Previous research has demonstrated that,KLF4 is essential in maintaining self-renewing and differentiation potential of neural stem cells,but inhibiting its differentiation.Also,dysregulation of KLF4 will lead to abnormal development of neural system,indicating the fundamental function,of KLF4 in neural differentiation.Because of the important role of KLF4 in neural cell development,in order to find small molecules that can effectively inducing cell differentiate into neural cells,we target the promoter of KLF4 to conduct a screening of small molecules extracted from Chinese herb medicine.In this thesis,we find several small molecule compounds that can effectively inhibit the promoter activity of KLF4,and we also determined that these compounds can induce neural stem cell differentiated into cholinergic neuron,by using western blot and some other experimental methods.Main results and conclusions:1.Construction of KLF4 eukaryotic expression vector.Human KLF4 coding sequence was amplyfied by RT-PCR and ligated into pcDNA3 vector,then the construct was confirmed by gene sequecing.2.Construction of KLF4 promoter luciferase reporter vector.Huamn KLF4 promoter region was amplyfied by PCR and inserted into pGL3-basic vector,then the construct was confirmed by gene sequecing.Luciferase activity result revealed that the promoter fragment we identified had higher transcriptional activity.3.Screening of small molecular inhibitor of KLF4 expression from Chinese herb medicine.Using luciferase reporter system,Small molecular compounds extracted from Chinese herb in our lab were screened,we found that CA21,CA71,CA93 and CA95 can effectively inhibit KLF4 expression,results also corroborated at the protein level by western blot.4.The inhibition effect of CA 71 on the expression of KLF4,and its function on inducing neural ctem cells differentiated into cholinergic neuron cells.Results from western blot indicated that KLF4 inhibitor CA71 can promote neural ctem cells differentiated into cholinergic neuron cells,further results suggest that the underlying mechanisms may related to the down regulation of Wnt/?-catenin pathway.