The Establishment Of IPSCs Model For Schizophrenia From Peripheral Blood Mononuclear Cells

BackgroundSchizophrenia(SZ)is a debilitating mental illness.It has neurotransmitter dysfunction,abnormal distribution of prefrontalcortical neurons and other physiological characteristics,which are closely related to neurons.A large number of autopsy data,animal models,and imaging models provide the basis for the pathological and physiological studies of the disease,but there is still a lack of appropriate models in clinical trials.The emergence of iPS technology provides a new cell disease model for the pathological study of SZ,which provides a basis for the in-depth study of its pathogenesis and clinical treatment.Objectives1.The optimal culture time and seeding density required for 293T cells with higher lentivirus packaging efficiency were obtained by single factor comparison,provides technical support for iPSCs reprogramming.2.PBMCs were reprogrammed by lentivirus-transfected OKSM transcription factor,constructive the schizophrenic i PSCs model,and then analysis the karyotype and the expression levels of inflammatory factors in i PSCs from SZ patients.Methods1.293T cells were seeded at the same density respectively and cultured for 19h,24h and 29h to package lentivirus and then 293T cells with different seeding densities were virus-packed after the same culture time(24h).By comparing the two variables,the optimal culture time and seeding density of the cells required for high-efficiency lentivirus packaging were obtained.2.The reprogramming factors C-MYC-IRES2-KLF4 and OCT4-IRES2-SOX2 were amplified by PCR using plasmid pEP4EO2SEM2K.Then the PCR products were respectively ligated into the lentivirus vector pLVX-IRES-mCherry.Finally,recombinant lentiviral plasmids pLVX-C-MYC-IRES2-KLF4 and pLVX-OCT4-IRES2-SOX2 were obtained by a series of enzyme digestion,ligation and transformation of E.coli.3.Lentivirus packaging and detection of efficiency.The above two recombinant plasmids were used as the target plasmid to bind the packaging plasmids pMD2.G and pSPAX2 to package lentivirus respectively by calcium phosphate transfection method,and then the lentivirus particles were collected by cryogenic ultracentrifugation.The 293T cells were infected with a concentration gradient from the lentivirus concentrate,and the infection efficiency of the lentivirus was detected by fluorescence microscopy and flow cytometry.4.The PBMCs of SZ patients were induced into iPSCs and identified.The two viral particles with appropriate titer were selected to induce PBMCs.After successful reprogramming,the pluripotency and differentiation abiliy of iPSCs in vitro can be detected by various techniques such as ALP staining,PCRand immunofluorescence.5.The analysis of karyotype about SZ-iPSCs model by Giemsa staining.6.The levels of inflammatory cytokines in iPSCs from SZ patients and normal individuals were compared by qRT-PCRResults1.It was found that inoculation of 5-7.5×10~6 cells at 24h after lentivirus packaging can result in a more efficient lentivirus with a 75cm~2 culture flask.2.In this experiment,the recombinant lentivirus plasmids pLVX-OCT4-IRES2-SOX2(referred to as OCT4/SOX2)and pLVX-C-MYC-IRES2-KLF4(referred to as C-MYC/KLF4)required for the induction of iPSCs were successfully constructed;The two plasmids successfully packaged two highly efficient lentiviruses when they were transfected into293T cells as the target plasmid respectively in combination with packaging plasmids.3.PBMCs from SZ patients were successfully reprogrammed into iPSCs by ALP staining,PCRand immunofluorescence with the pluripotency and differentiation abiliy of cell clones.4.It was found that there was no abnormal karyotype of iPSCs model from SZ patients,but the expression level of inflammatory factors was significantly higher than iPSCs of normal individuals.Conclusions1.The optimal culture time and seeding density required for 293T cells with higher lentivirus packaging efficiency were obtained by single factor comparison.2.The model of iPSCs derived from peripheral blood mononuclear cells in patients with schizophrenia was successfully established,which laid the foundation for the subsequent establishment of neuronal cells model of schizophrenia and the study of the pathogenesis of SZ.