Research On The Promoting Neural Differentiation Capacity Of Effective Compounds Of Chinese Materia Medica

Objective:The reporter cell line of mouse embryonic stem cells and human induced pluripotent stem cell line were used to test the neurodifferentiation effect of effective compounds of Chinese materia medica,and the effective compounds that could potentiate neurodifferentiation were screened to provide experimental data support for the development of promoting endogenous nerve regeneration drugs.Methods:1.Using induced pluripotent stem cell technology to reprogram human kidney epithelial cells isolated from the urine of healthy subjects and AD patients to obtain human induced pluripotent stem cell lines.The constructed human induced pluripotent stem cell lines were identified by alkaline phosphatase staining,immunofluorescence staining,in vivo teratoma formation experiments,and karyotype analysis.2.First,the mouse embryonic stem cell reporter cell line was used to test the efficacy of promoting neurodifferentiation of total 464 drugs come from the active compounds of traditional Chinese medicine and the natural product library.And the phenazopyridine hydrochloride was used as a positive control.By using the dual luciferase detecting kit to detect Renilla luciferase expression that reflects cell proliferation and firefly luciferase expression that reflects cell neural direction differentiation to.K-Means clustering was used to perform cluster analysis on the screening results,and the candidate drugs with stronger neurodifferentiation efficacy than the positive control drugs were screened for subsequent experiments.3.The constructed human induced pluripotent stem cell line was used to verify the neurodifferentiation efficacy of the screened drugs.The monolayer cell differentiation protocol was used to intervene drug differentiation for 7 days at a determined safe concentration of the drug.Cells were then passaged into poly-L-ornithine and laminin-coated culture plates.Stem cell markers Pax6,Nestin and early neuron marker Tuj1 were stained with immunofluorescence to quantitatively analyze the length of neurites and compare the efficacy of drugs in promoting neuronal differentiation.Results:1.Healthy subjects and AD patients derived induced pluripotent stem cells were positive for alkaline phosphatase staining.And immunofluorescence staining showed the expression of pluripotency markers Oct4,Nanog,and Tra-1-60.Teratoma could be formed after the cell transplantation,and the HE staining result show that there were representative cells and tissues of all of three germ layers.Karyotype identification showed the normal chromosome karyotypes.2.Through cluster analysis,the 464 drugs screened were divided into 4 categories,the first of which was to promote neural differentiation and had no effect on cell proliferation.9 of the screened drugs were more effective than the positive control drugs in promoting neurodifferentiation.3.Immunofluorescence staining showed that Nestin and Pax6 double-positive neural stem cells did not appear in the QCJ intervention group;rosette-like Nestin and Pax6 double-positive neural stem cells appeared in the other drug intervention groups.Quantitative analysis of neurite outgrowth showed that MBHS and DDG could significantly promote the differentiation of induced pluripotent stem cells towards mature neurons.Conclusions:Induced pluripotent stem cells derived from the renal epithelial cells comes from the urine of healthy subject and AD patients constructed in our laboratory have normal differentiation functions.By using mouse embryonic stem cells,we’ve screened nine drugs with strong neurodifferentiation efficacy.By using human induced pluripotent stem cells,2 of 9 drugs were verified having strong ability to promote the differentiation of induced pluripotent stem cells to mature neurons.