Objective To comparatively study the characteristics of 3 kinds of culture substrates of human odontogenic induced pluripotent stem cells(iPSCs).Methods The human odontogenic iPSCs were cultured by 3 kinds of substrates:mouse embryonic fibroblasts(MEF),matrigel and recombinant human vitronectin(VTN-N).The iPSCs growth situation was compared among three groups.Results The preparation time of these 3 kinds of substrates was 14,3,1 hlespectively,and,the difference was statistically significant (P＜0.05).The iPSCs reprogramming time was (30± 1.6),(26 ± 2.1),(27 ± 1.4) d,lespectively,wht that in the MEF group significantly higer than in other two groups (P＜0.05).The reprogramming efficiencies were 0.3 ％ ± 0.03 ％,0.56 ％ ± 0.08 ％,0.7 ％ ± 0.02 ％ respectively (P＜ 0.05).Three kinds of substrate could better support iPSCs growth and make them to maintain un-differentiation status.Conclusion with no heterologous animal components,and the adrantaga of simple pleparation,oonfrollable standard and shorter gramming time is easy to prepare,the standard is controllable and the reprogramming time is shorter,which is an ideal substrate for supporting iPSCs growth.

Aim To develop a simple and rapid method to monitor insulin receptor kinase activity and provide a novel cell-based model for screening anti-diabetes drugs.Methods CHO cells were co-transfected by plasmids which respectively contained insulin receptor gene,STAT5b gene and luciferase gene driven by STAT5 response elements.The expression of exogenous gene in transfected cells was examined by RT-PCR.The transfected cells were treated by insulin,and then the concentration and time-dependent response of luciferase expression to insulin induction was examined.Moreover,the specificity was identified by AG1024 treatment and PTP1B gene transfection.Results Expressions of insulin receptor and STAT5b were detected in the transfected CHO cells.The expression of luciferase in transfected cells was induced by insulin in concentration and time-dependent way.The maximal induction fold was 6.25.Moreover,the inducible expression of luciferase by insulin could be specifically blocked by tyrphostin AG1024,an inhibitor of insulin receptor kinase,or co-transfected PTP1B gene.Conclusions The insulin receptor kinase activity can be detected by expression of reporter gene with high sensitivity and specificity in this cell model,and with potential value in high throughput screening for insulin receptor activators and sensitizers.

Objective To compare the growth characteristics,proliferation and osteo/odontogenic differentiation capability of stem cells from human dental pulp (dental pulp stem cells,DPSCs) and apical papilla (stem cells from apical papilla,SCAP) in vitro.Methods Human dental pulp and apical papilla tissues were separated from impacted third molars of young healthy donors at the age of root development and digested by collagenase type Ⅰ and dispase type Ⅱ to derive primitive DPSCs and SCAP.Cells were then induced for osteo/odontongenic differentiation by medium containing β-glycerophosphate,dexamethasone and KH2PO4.Flow cytometry was utilized to test the expression of specific markers of stem cells,including CD24,CD34,CD45,CD90,CD105,CD146,STRO-1 and OCT-4.AR-S was used to display the mineralization structure and RT-PCR was applied to analyze the expression of bone sialoprotein (BSP),osteocalcin (OCN) and dentine sialophosphoprotein (DSPP).Results Both DPSCs and SCAP were positive for CD90,CD105,CD146,STRO-1 and OCT-4,in percentages varying according to cell type,without expression of CD34 or CD45.Only SCAP expressed CD24 positively.Both cells formed organized mineralization structure after 2 weeks of induction in time-dependent manner,with more mineralization by SCAP and expressed differentiation markers,including BSP,OCN and DSPP.Conclusion Human DPSCs and SCAP possess the characteristics of MSCs and could be differentiated into odontonblast-like cells in vitro.Both cells are approachable stem cell sources for dental tissue engineering.

Objective To comparatively study the features of two reprogramming systems of induced pluripotent stem cells (iPSCs)from human dental origin.Methods Two kinds of reprogramming system,i.e.STEMCCA lentivirus /feed layer and Sen-dai virus /matrigel were used to induce human stem cells from apical papilla(SCAP)into iPSCs,respectively.The induction efficien-cies,workload of generating iPSCs,aneuploidy karyotype ratio,complexities of eliminating exogenous transcription factors and spe-cific markers expression were compared between these two systems.Results The STEMCCA reprogramming system required to prepare the feeder cell MEF.The reprogramming efficiency was 0.1%.Transcription gene-free iPSCs cells were obtained by the Cre-loxp enzyme digestion technique at the later stage.Sendai virus reprogramming system was feeder-free and the preparation of matrigel was quite simple with unified standard.The reprogramming efficiency was 0.7%,which was much higher than that of STEMCCA system(P <0.05).The exogenous virus and transgenes could be gradually eliminated after several passages of natural subclone.Conclusion The Sendai virus/matrigle reprogramming system is much more applicable for the induction of iPSCs from dental origin than the STEMCCA system.

BACKGROUND: The maximal problem of patient with serious hepatitis and surgical doctor is whether they can get donator and rational therapy timely. Looking for the suitable preoperative therapy method to enhance the success rate of operation and improve patient's prognosis is the focus of this domain.OBJECTIVE: To investigate the therapeutic effect of artificial liver support system (ALSS) combining with allotransplantation of the liver on patients with serious hepatitis.DESIGN: Retrospective case analysis. SETTING: Organ Transplantation Center, the Third Affiliated Hospital of Anhui Medical University.PARTICIPANTS: Five male patients with serious hepatitis who underwent allograft liver transplantation were selected from Organ Transplantation Center, the Third Affiliated Hospital of Anhui Medical University form June 2004 to May 2005. Their age ranged from 25 to 48 years. Inclusion criteria: The diagnosis was in accordance with phase standard established at the National Infectious Disease and Parasitology Academic Meeting in September 2000; all patients had signs of routine liver transplantation; their patients fiercely requested the operation.METHODS: Plasma exchange (PE) combined with continuous veno-venous hemofiltration (CVVH) technique was used in this study. Donor who supplied lives was from 20-38-year patients. All of them and their family agreed to donate their organ and signed the donate file before operation. All of 5 patients were used classical no-by-pass orthotopic liver transplantation (OLT). MAIN OUTCOME MEASURES: They were follow-up visited for 21-32 months for rechecking liver and kidney function,RESULTS: All of 5 patients' operation was succeeded. One continued coma postoperative and his serum creatinine and urea nitrogen raised up progressively and complicated by pulmonary infection 1 week after operation and died 2 weeks after operation although given medical treatment hemodialysis positively. The rest recovered well. All of them discharged one month after operation smoothly.CONCLUSION: Allotransplantation of the liver is an utilizable method to treat serious hepatitis. ALSS can be used as an effective method of supportive treatment preoperatively.

OBJECTIVETo compare characterization of microRNAs (miRNAs) expression profiles of induced pluripotent stem cells (iPSCs) reprogrammed from human dental pulp stem cells (DPSCs) and stem cells from apical papilla (SCAP) and screen-specific microRNA.

METHODSHuman DPSCs and SCAP were reprogrammed into iPSCs using a Sendai virus vector. Total RNA of human DPSCs-iPSCs and SCAP-iPSCs were extracted. miRNAs were labeled and hybridized. Slides were scanned, and images were imported into GenePix Pro 6.0 for grid alignment and data extraction. Significant differentially expressed miRNAs between the two groups were identified using fold change and P-value and were analyzed.

RESULTSBoth human DPSCs and SCAP were successfully reprogrammed into iPSCs. Among miRNA genes analyzed by miRNA microarray, 68 were differentially expressed by more than 10-fold in DPSCs-iPSCs; 37 of these genes were up-regulated, and 31 were down-regulated. In SCAP-iPSCs, 107 genes were differentially expressed by more than 10-fold; 68 were up-regulated, and 39 were down-regulated. In both cells, only miR-302e was up-regulated, whereas 9 miRNAs were down-regulated: miR-29b-3p, miR-181b-5p, miR-4328, miR-22-5p, miR-145-5p, miR-4324, let-7b-5p, miR-181a-5p, and miR-27b-3p.

CONCLUSIONSMultiple miRNAs participated in reprogramming of human DPSCs and SCAP into iPSCs. Most miRNAs are related to cell cycle, transforming growth factor-β signaling pathways and epithelial-mesenchymal transition.

Cell Cycle ; Cell Division ; Dental Pulp ; Down-Regulation ; Electrodes ; Epithelial Cells ; Humans ; Induced Pluripotent Stem Cells ; MicroRNAs ; Taste Buds ; Up-Regulation

Objective To compare the reprogramming effects and biological characteristics of two types of human odontogen-ic induced pluripotent stem cells(iPSCs).Methods Human dental pulp stem cells(DPSCs)and stem cells from apical papilla (SCAP)were isolated and primarily cultured.The Sendai reprogramming system was utilized to induce DPSCs and SCAP into iP-SCs.The morphology,reprogramming efficiency,reprogramming and time were compared between human DPSCs-iPSCs and SCAP-iPSCs.The SeV and exogenous transcriptional gene expression were detected by RT-PCR.Results Human DPSCs and SCAP were reprogrammed as iPSCs with classical ES-like clonal morphology.The reprogramming efficiencies were(0.68 ± 0.02)% and(0.7 ± 0.01)% respectively,the difference was not statistically significant(P>0.05).The reprogramming time was(26.0 ± 2.1)d and (27.0 ± 1.4)d respectively,the difference was not statistically significant(P>0.05).The RT-PCR results showed that no expres-sion of exogenous virus or transcriptional gene sequence in both iPSCs.Conclusion Human DPSCs and SCAP can be reprogrammed as virus-free and transgene-free iPSCs,which are the ideal sources of iPSCs.

Induced pluripotent stem cells(iPSCs)are obtained by introducing exogenous genes or adding chemicals to the culture medium to induce somatic cell differentiation.Similarly to embryonic stem cells,iPSCs have the ability to differentiate into all three embryonic cell lines.iPSCs can differentiate into cardiac muscle cells through two-dimensional differentiation methods such as monolayer cell culture and co-culture,or through embryoid body and scaffold-based three-dimensional differentiation methods.In addition,the process of iPSCs differentiation into cardiac muscle cells also requires activation or inhibition of specific signaling pathways,such as Wnt,BMP,Notch signaling pathways to mimic the development of the heart in vivo.In recent years,suspension culturing in bioreactors has been shown to produce large number of iPSCs derived cardiac muscle cells(iPSC-CMs).Before transplantation,it is necessary to purify iPSC-CMs through metabolic regulation or cell sorting to eliminate undifferentiated iPSCs,which may lead to teratoma formation.The transplantation methods for iPSC-CMs are mainly injection of cell suspension and transplantation of cell patches into the infarcted myocardium.Animal studies have shown that transplantation of iPSC-CMs into the infarcted myocardium can improve cardiac function.This article reviews the progress in preclinical studies on iPSC-CMs therapy for acute myocardial infarction and discusses the limitations and challenges of its clinical application to provide references for further clinical research and application.

Objective:To investigate neuroprotective effect of alpinetin on ischemic stroke(IS)rats by regulating Shh/Gli1 signaling pathway.Methods:Fifteen rats were randomly collected as control group,remaining rats were used to construct an IS model.Rats that successfully modeling were randomly grouped into model group,alpinetin group(5 mg/kg),Shh/Gli1 signaling pathway inhibitor cycloparamide group(15 mg/kg)and alpinetin+cycloparamide group(5 mg/kg alpinetin+15 mg/kg cycloparamide),with 15 rats in each group,and administered once a day for two consecutive weeks,control group and model group were given equal amounts of physiological saline.Zea-Longa scoring method was applied to evaluate neural function;ELISA was applied to detect inflammatory factors levels;mass of brain tissue was weighed and water content of brain tissue was calculated;TTC staining was applied to measure volume of cerebral infarction;HE staining was applied to observe nerve cell damage;TUNEL staining was applied to detect neuronal apoptosis;qRT-PCR and Western blot were used to detect mRNA and protein expressions of Shh and Gli1.Results:There were no abnormalities in hippocampal tissue of control group,while hippocampal tissue structure of model group rats was abnor-mal,with disordered cell arrangement and nuclear pyknosis of nerve cells,cell damage rate,Zea-Longa score,infarct volume,IL-1β,IL-6,TNF-α,brain tissue water content,and cell apoptosis rate in model group were obviously higher than control group(P<0.05),mRNA and protein levels of Shh and Gli1 were obviously decreased(P<0.05);compared with model group,cells in alpinetin group were arranged more neatly,and phenomenon of neuronal cell nucleus pyknosis was improved,cell damage rate,Zea-Longa score,infarct volume,IL-1β,IL-6,TNF-α,brain tissue water content,and cell apoptosis rate were obviously decreased(P<0.05),mRNA and protein levels of Shh and Gli1 were obviously increased(P<0.05),trend of above indicators in cyclophosphamide group was opposite,ciclopramide reversed neuroprotective effect of alpinetin on IS rats.Conclusion:Alpinetin may exert neuroprotective effects on IS rats by activating Shh/Gli1 signaling pathway.

AIM: To study the pharmacokinetics (PK) of pazopanib tablets and explore the genetic mechanism of individual differences in drug metabolism primarily. METHODS: Fourteen healthy male subjects were respectively administrated with a single dose pazopanib tablet (200 mg) orally on the day of dosing, and their blood samples were collected from baseline to 96 hours. The serum concentration of pazopanib was measured by LC-MS/MS, the parameters of PK were calculated by winnonlin 6.3 software, and the gene polymorphism of cytochrome P450 3A4 (CYP3A4) was determined by snapshot method. RESULTS: The range of C