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Objective To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients. Methods Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCATM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway. Results The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t =-10.688; REX1 mRNA (2-ΔΔCt):1.419±0.206 vs. 0.103±0.066, t =-14.245; OCT4 mRNA (2-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t =-9.028; Sox2 mRNA (2-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t =-12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC. Conclusion The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.
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Objective@#To recombine the induced pluripotent stem cells (iPSC) derived from the urine of septic encephalopathy (SE) patients, and provided a specificity cell model to explore the mechanism of the neuronal damage and treatment for SE patients.@*Methods@#Urine of SE patient was collected, and tubular epithelial cells were isolated and cultured from the urine. iPSC were derived from SE patient by introducing 4 transcription factors OCT4, Klf4, Sox2, c-Myc (OKSM) into patient-specific urine cells by Millipore's Human STEMCCATM Constitutive Polycistronic (OKSM) Lentivirus Kit. Colony morphology, alkaline phosphatase (AKP) activity, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction (RT-qPCR), and differentiation ability were used to identify the pluripetency of these iPSC lines. In addition, neurons were derived from these iPSC by inhibiting transforming growth factor-β (TGF-β) pathway.@*Results@#The SE-iPSC exhibited morphological and growth characteristics of human embryonic stem cell (hES), showed positivity for AKP by histochemical staining, and expressed embryonic stem cell (ESC) marker genes. There was a significant statistical difference in ESC-marker mRNA expression between the SE-iPSC and the urine cells [NANOG mRNA (2-ΔΔCt): 1.153±0.142 vs. 0.126±0.024, t = -10.688; REX1 mRNA (2-ΔΔCt): 1.419±0.206 vs. 0.103±0.066, t = -14.245; OCT4 mRNA (2-ΔΔCt): 1.233±0.176 vs. 0.201±0.022, t = -9.028; Sox2 mRNA (2-ΔΔCt): 1.334±0.119 vs. 0.159±0.017, t = -12.653, all P < 0.01]. Subcutaneous injection of iPSC into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers. Furthermore, neurons were successfully induced from SE-iPSC.@*Conclusion@#The SE patient-specific iPSC could be generated from urine cells and differentiated into neurons, furthermore, the SE-iPSC cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for SE.
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Objective To establish disease-associated or cell type relevant neuron model generated from patients with Critical Illness Polyneuropathy (CIP) by making CIP patient-derived induced pluripotent stem (iPS) cell lines and neurons to provide a cell-based disease model of CIP.Methods Skin tissue of CIP patient was obtained clinically,and specific skin fibroblasts were isolated and cultured.The iPS cells were derived from CIP patient by introducing 4 transcription factors,namely Oct4,Klf4,Sox2,c-Myc,into patient-specific fibroblast cells by Millipore's Human STEMCCA Constitutive Polycistronic (OKSM) Lentivirus Kit.Colony morphology,alkaline phosphatase (AP) activity,immunofluorescence staining,quantitative reverse transcription polymerase chain reaction (RT-PCR),and differentiation ability were used to identify the pluripotency of these iPS cell lines.In addition,neurons were derived from these iPS cells by inhibiting SMAD pathway.Results The CIP-iPS cells presenting morphological and growth characteristics of human embryonic stem cell (hES) showed the presence of alkaline phosphatase detected by histochemical staining,and the expression of ESC-marker genes.The relative expressions of endogenous pluripotency genes,namely Sox2,REX1,NANOG and OCT4,in iPS cell lines were significantly increased compared with their primary fibroblasts (t values were-9.020,-10.753,-13.295,-12.677,P＜0.01).Subcutaneous injection of iPS cells into NOD-SCID mice resulted in teratomas containing tissues from all the 3 germ layers.Furthermore,cholinergic neurons were successfully induced from CIP-iPS cells.Conclusion The CIP patient-specific iPS cell line and cholinergic neurons were successfully established.Furthermore,the CIP-iPS cell line can be used as models for further elucidating the cellular pathology and developing therapeutic strategies for Critical Illness Polyneuropathy.
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Objective To determine kinetics of TNF-α and miR-146a (microRNA-146a)expressions in lipopolysaccharide (LPS)-induced NR8383 alveolar macrophages (AM) at different intervals and their relationships in order to explore regulatory effect and mechanism of miR-146a on alveolar macrophages inflammatory responses.Methods NR8383 alveolar macrophages were seeded in a 6-well plate,and stimulated with 1 μg/ml of LPS for 0 h,3 h,6 h and 12 h separately after 90 min.Cells were harvested and supernatant were collected 0 h,3 h,6 h and 12 h after incubation.The expressions of miR146a and TNF-α mRNA in cells were detected by real-time qPCR and the levels of TNF-α protein in the supematant of cells were assayed by enzyme-linked immunosorbent assay ( ELISA ).Pearson correlation analysis was used to analyze the correlation between miR-146a and TNF-α mRNA.Results ①The level of TNF-α protein in the supernatant of cell was significantly increased 3 h after LPS challenge ( 359.80 ±57.54) pg/ml (P ＜0.01 ),and peaked 12 h later (729.22 ±50.40) pg/ml (P＜0.01 ) ; ②the expression of TNF-α mRNA peaked 3 h after LPS challenge (67.48 ±24.52) fold,P ＜0.01 ),and then decreased gradually; ③the expression of miR-146a mRNA increased continuously until 6 h or 12 h after LPS challenge 6 h:(5.33 ±0.81) fold,12 h:(8.21 ±1.19) fold,(P＜0.01),and it showed an upward tendency;④ the expression of miR-146a mRNA was negatively correlated with TNF-α mRNA ( r =-0.895,P ＜0.01).Conclusions The miR-146a mRNA showed a negative correlation with TNF-α mRNA present in lipopolysaccharide-stimulated alveolar macrophages,suggesting miR-146a mRNA involved in regulating the inflammatory response of alveolar macrophages.
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Objective To investigate the effect of human interferon-beta (IFN-β) gene engineered human mesenchymal stem cells (hMSC) in the treatment of human prostate cancer xenograft in nude mice.Methods An adenovirus vector containing human IFN-β gene was constructed and transfected into hMSC in vitro.IFN-β-expressing mesenchymal stem cells (IFN-β-hMSC) were labeled with 4,6-diamidino-2-phenylindole (DAPI).The human prostate cancer cell line PC-3 were injected into the flank or axillary of severe combined immunodeficiency (SCID) mice subcutaneously to establish human prostate cancer xenograft models.IFN-β-hMSC were injected into the tail vein of mice bearing human prostate cancer xenografts.The tumors,livers,lungs,spleens and kidneys were harvested.Frozen sections and paraffin sections were used to observe the distribution of IFN-β-hMSC in vivo by fluorescence microscope.Mice were divided into seven groups of six animals randomly,IFN-β-hMSC (2 × 106,2 × 105 ) as treatment group,Ad-hMSC,unmodified hMSC,Ad-IFN-β,Recombinant IFN-β,and NS as control group.The weight of the tumor and the survival time of mice were observed to evaluate the experimental efficacies of IFN-β-hMSC in the treatment of prostate cancer. Results IFN-β-hMSC with blue nuclei were distributed extensively in the tumors,but no blue nucleus was seen in the livers,lungs,spleens and kidneys.After treating,the weights of the tumour masses from mice were (1.35 ±0.28) g,(1.43±0.41) g,(3.49 ±0.25)g,(3.58±0.30)g,(3.30 ±0.24) g,(3.32 ±0.25) g,(3.32 ±0.47) g in the IFN-β-hMSC (2 ×106),IFN-β-hMSC (2 ×105),Ad-hMSC,unmodified hMSC,Ad-IFN-β,Recombinant IFN-β,and NS group,the median survival time from mice were 91 d,87 d,57 d,59 d,62 d,61 d,61 d in the IFN-β-hMSC (2 × 106),IFN-β-hMSC (2 × 105),Ad-hMSC,unmodified hMSCs,Ad-IFN-β,Recombinant IFN-β,and NS group,respectively.Injection of IFN-β-MSC can significantly reduce tumor weight and increase animal survival compared with controls ( P ＜ 0.05 ). Conclusion IFN-β-hMSC can migrate to prostate cancer microenviroment in vivo,and injection of IFN-β-MSC can significantly reduce tumor weight and increase animal survival.