Screening Of Compounds With Neural Stem Cell Differentiation Promoting Action And Dami Cell Proliferation Inhibition Based On Gene Expression Data

Drug screening is generally based on the mechanism of drugs and the target structure of the drugs,and used cell and anima models to determine the effect and mechanism.With the large-scale application of high-throughput sequencing and gene chip,and the popularization of high-throughput synthetic chemistry methods,drug screening has entered a high-throughput era.More and more new targets and potential targets have been discovered,in case of the perfection of the genome database and the analysis of the database by the bioinformatics automation operating system.In this study,different cell models were used to obtained gene differential expression data by high-throughput transcriptome sequencing.Bioinformatics analysis in combination with gene-drug interaction database and gene expression phenotyping annotation database was used to screen gene-specific targeting compounds corresponding to cell model,which was also referenced drug compound database.Subsequently we verifed the effect of compounds on intervening cell models using cytological methods,thereby established a target compound screening and activity verification research system based on gene expression data.According to different cell models,our study was divided into the following two parts.1.Screening and research based on gene expression of compounds with neural stem cell differentiation promoting actionStem cells are kinds of self-replicating and multidirectional cells.Studies on the differentiation of bone marrow mesenchymal stem cells(MSCs)into neural stem cells(NSCs)have become the frontier field in stem cells.Transplantation of NSCs is one important method for future treatment for neurological diseases.NSCs differentiation is closely associated with the cellular environment and cytokine stimulation.Specificexpression-genes of NSCs play a key role in their function of maturation and subsequent differentiation into nerve cells.Therefore,study on the specificexpression-genes of NSCs and their impact on the regulation of NSCs differentiation is of great significance for guiding the clinical applications of NSCs treatment of neurological diseases.On the basis of in vitro separation and culture of human adult bone marrow MSCs as well as inductive experiments of NSCs,the present project examined the cell transcriptome before and after induction by using second generation sequencing technology.Bioinformatics analysis proved changes in gene expression during induction in the process and clustering of differentially expressed genes.The intervention compounds targeting the key genes was also looked for through gene-drug database and bioinformatics analysis,cell experiments were conducted to verify the effect of targeted compounds on the differentiation of neural stem cells.(1)Special culture medium induction of MSCs was used,so that MSCs was transformed to NSCs.Surface specific antigen detection of the prepared cells found surface signs of NSCs,and morphological observation showed the NSCs characteristics.(2)Next generation sequencing was used to investigate the changes of cell transcriptome before and after differentiation.From data analysis we found that a total of 3685 genes in the whole genome of the cell had expression changes before and after differentiation.There are 254 genes related to cell development,109 genes related to nervous system development and 113 genes related to cell differentiation.(3)Through gene expression phenotype annotation database and biological information analysis,we found that genes with differential expression before and after differentiation were concentrated in the following pathways,including MAPK signaling pathway,Wnt signaling pathway,Jak-STAT signaling pathway,Focal adhesion,Regulation of actin cytoskeleton,Neuroactive ligand-receptor,which are the hot pathways in studies of NSCs,and also the main regulation pathways reflecting the differentiation from NSCs to nerve cells.(4)It was shown from the analysis of expression specific-genes of NSCs and Protein-Protein Interaction Network expression of key pathways that the expression specific-proteins formed the protein-protein interaction network with MAPK-JAK2-STAST-AGTR1-TSPO-PPARA as the center,reflecting that these proteins played a dominant role in the regulation of NSCs differentiation.Through the gene-drug database and bioinformatics analysis,of the 3685 genes with differential expression before and after cell differentiation,468 genes had matching targeted drugs,162 genes which had corresponding targeted drugs were from the expression specific NSCs and core pathways.After literature retrieval and compound screening,we selected the TSPO gene specific activation drug-etifoxine for the intervention experiment of NSCs differentiation.The results showed that the single use of etifoxine promoted further differentiation of NSCs into nerve cells(differentiation rate 12.482±0.018%,P<0.05);induction with a combination of etifoxine and bFGF had significant promotion effect(differentiation rate 38.096±0.040,P<0.05),suggesting that etifoxine promoted the differentiation of NSCs to nerve cells via activation of the TSPO gene in mitochondria.2.Screening and research based on gene expression of compounds with Dami cell proliferation inhibition promoting actionLeukemia is a disease of malignant proliferation of hematopoietic cells.The patient hematopoietic system produces a lot of immature leukocytes,the cells lose cycle regulation,and the numbers of normal functional platelets.The transplantation technology of hematopoietic stem cells for clinical leukemia treatment makes it possible to cure the disease.However,successful transplantation rate is only around 50%,and there are many issues present in the technology such as matching and donating problems,so not all patients are able to get smooth transplantation.Currently,chemotherapy is still the main clinical treatment for leukemia.Using the sequencing data of leukemia patient peripheral blood transcriptome established earlier,relying on the drugs database and NCBI database,using gene-drug interaction to select amifostine as the candidate drug,and taking acute megakaryocytic leukemia Dami cell line and bone mesenchymal stem cells(BMSCs)as the experimental objects.The study tested and proved the impact of amifostine on the bioactivities of Dami cell line and BMSCs,and discussed the mechanism of drug intervention on leukemia Dami cell lines based on the changes of relevant genes before and after drug intervention on Dami cell line and the network interaction.(1)The compound amifostine [2-(3-aminopropyl)aminoethyl phosphorothioate] we found through databases,is a broad spectrum cell protectant for treatment from nuclear radiation and chemical weapon injuries.(2)In vitro cytological experiments proved that amifostine had effect on inhibiting growth of Dami cells and promoting their apoptosis while having no significant effect on BMSCs.Amifostine was able to stop the proliferation of Dami cells at G2 and reduce the cell ratio of GO/G1,indicating that amifostine was able to inhibit the growth of Dami cells.(3)According to the examination on whole genome expression difference of Dami cell line before and after amifostine intervention,it was found that amifostine up-regulated expression of CASP3 gene and down-regulated expression of BCL2 gene.(4)For the proteins encoded by 1519 genes with the most significant difference,the specific-expression-protein formed a network structure centered on CCND1-PSMA2-ACAT2 protein network,which may be the protein regulation network for amifostine intervention on the proliferation of Dami cells.