OBJECTIVETo investigate the inhibition effect of leukemic bone marrow stromal cells (BMSCs) on daunorubicin (DNR) induced apoptosis of human Jurkat cell line, and analyze the differentially expressed genes between Jurkat cells cocultured with leukemic BMSCs or without.

METHODSSuppression subtractive hybridization (SSH) was employed to establish subtracted cDNA library of differentially expressed genes in Jurkat cells cocultured with leukemic BMSCs and DNR. The cDNA fragments were sequenced and analyzed.

RESULTSThe differentially expressed gene cDNA library was successfully developed. Primary screening was done by reverse Northern hybridization. Thirty up-regulated and 22 down-regulated cDNA fragments were isolated and sequenced. Analysis and comparison were performed in GenBank using BLAST. These genes are related to cell cycle regulation, cell apoptosis and energy metabolism.

CONCLUSIONLeukemic BMSCs influence gene expression of Jurkat cells. The resulting differentially expressed genes might be associated with the protection of leukemic cells by BMSCs from injury.

Apoptosis ; drug effects ; Bone Marrow Cells ; drug effects ; metabolism ; pathology ; Cells, Cultured ; Coculture Techniques ; Daunorubicin ; pharmacology ; Gene Expression Profiling ; Gene Expression Regulation, Leukemic ; drug effects ; Gene Library ; Humans ; Jurkat Cells ; Stromal Cells ; drug effects ; metabolism ; pathology

OBJECTIVETo investigate the correlation of single nucleotide polymorphisms (SNP) of XRCC1 gene to hereditary susceptibility of colorectal cancer.

METHODSXRCC1 genotypes in 124 colorectal cancer patients and 214 matched healthy people as control were analyzed by SnaP Shot SNP-typing technique. Five different inheritance models including codominant, dominant, recessive, overdominant and log-additive were analyzed using logistic regression model. The haplotype distribution was estimated with phase and its correlation with the risk of colorectal cancer was evaluated.

RESULTSThe frequencies of mutant 25487G-A, 25489C-T and 1799782C-T alleles were 0.20, 0.11, 0.32 respectively in the patients, and 0.23, 0.13, 0.34 in the controls. There was no significant correlation of polymophisms of XRCC1 gene to the risk of colorectal cancer in 5 different inheritance models (P>0.05). GCT, GCC, ACC and GTC were the most common haplotypes and the odds ratios were 1, 1.35, 0.90 and 0.84 respectively. There was no significant difference of distribution between 2 groups in haplotypes.

CONCLUSIONPolymorphisms of XRCC1 gene, including rs25487, rs25489, rs1799782, are not associated with to the risk of colorectal cancer.

Colorectal Neoplasms ; genetics ; DNA-Binding Proteins ; genetics ; Female ; Genetic Predisposition to Disease ; Genotype ; Humans ; Logistic Models ; Male ; Middle Aged ; Models, Genetic ; Polymorphism, Single Nucleotide ; X-ray Repair Cross Complementing Protein 1

OBJECTIVETo detect the expression of CBX7 in human glioma and investigate the potential regulatory effect of abnormally expressed microRNAs on CBX7 expression.

METHODSSemi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot were applied to detect the expression pattern of CBX7 in 2 human normal brain tissues, 9 glioma tissues, and 3 glioma cell lines. Miranda algorithm and Ensemble Machine Learning algorithm were combined to predict miRNAs that target human CBX7. The expression of miR-9 in those tissues and cell lines were detected by real-time PCR. After miR-9 overexpression in 293ET and miR-9 knock-down in T98G, luciferase assay and Western blot were used to confirm the effect of miR-9 on CBX7 expression. MTT assay and flow cytometry were applied to detect the effect of miR-9 knock-down on T98G cells.

RESULTSNo obvious difference in the CBX7 mRNA level between normal and tumor tissues was observed, while the protein level of CBX7 was abrogated or markedly reduced in glioma tissues and cell lines. Several miRNAs including miR-9 may target CBX7 by bioinformatics prediction. MiR-9 was up-regulated in glioma tissues and cell lines. In 293ET cell, luciferase activity of CBX7-3'UTR reporter was decreased to 24% after miR-9 overexpression. After miR-9 knock-down in T98G cell, the luciferase activity was increased by 1.8 fold and there was no change of CBX7 mRNA, while the protein level of endogenous CBX7 was significantly increased. The number of survival T98G cells increased and cells in G1 phase decreased after miR-9 knock-down.

CONCLUSIONIn human glioma, CBX7 is down-regulated by the inhibition of miR-9 at posttranscriptional level.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Algorithms ; Blotting, Western ; Brain ; metabolism ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Child ; Female ; Flow Cytometry ; Glioma ; genetics ; metabolism ; Humans ; In Vitro Techniques ; Male ; MicroRNAs ; genetics ; physiology ; Middle Aged ; Polycomb Repressive Complex 1 ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

OBJECTIVETo evaluate the therapeutic effects of magnetically labeled mononuclear stem cells (MR-MNC) and mesenchymal stem cells (MR-MSC) transplantation in a swine acute myocardial infarction (AMI) model by MR imaging.

METHODSAMI model was established in swines by balloon occlusion of the left anterior descending coronary artery, 10(7) autologous MR-MSC (n = 7), MR-MNC (n = 6) or PBS (n = 6) were delivered via intracoronary infusion within 1 week after AMI [(4.8 +/- 1.3) days]. Changes of infarct size and cardiac function were assessed with the use of 3.0T MR scanner before AMI, at 1 and 8 weeks post AMI.

RESULTSMagnetically labeled stem cells could be identified in the region of AMI by cardiac MR imaging. Eight weeks post transplantation, infarct size was significantly reduced in MR-MSC transplantation group (8.5% +/- 0.5% vs. 24.7% +/- 3.1%, P < 0.05) and in MR-MNC transplantation (12.3% +/- 1.5% vs. 26.1% +/- 1.5%, P < 0.05) while infarct size remained unchanged in PBS group (P > 0.05) compared to values at 1 week post AMI, left ventricular ejection fraction (LVEF) was also significantly higher in MR-MSC transplantation group (56.9% +/- 1.3% vs. 40.7% +/- 2.0%, P < 0.05) and MR-MNC transplantation group (52.8% +/- 1.4% vs. 41.9% +/- 3.3%, P < 0.05) compared to LVEF at 1 week post AMI. LVEF increase was more significant in swines received MR-MSC transplantation than MR-MNC transplantation (16.2% +/- 1.2% vs. 10.9% +/- 3.0%, P < 0.05). Prussian blue staining identified stem cells in corresponding myocardial regions with as by MRI. Western blot analysis demonstrated that cardiac expressions of myosin heavy chain (MHC) in MR-MSC group (100.3 +/- 5.5) and in MR-MNCs group (95.5 +/- 4.2) were significantly higher than that in PBS group (75.7 +/- 5.7, P < 0.05), myocardial troponin T (cTNT) expression in MR-MSC group (124.0 +/- 5.8) and MR-MNC group (118.4 +/- 4.4) were also significantly higher than in PBS group (93.3 +/- 3.9, P < 0.05) while MMP2/TIMP1 ratios in MR-MSC group (0.6 +/- 0.1) and MR-MNC group (0.6 +/- 0.1) were significantly lower than that in PBS group (4.2 +/- 0.2, P < 0.05).

CONCLUSIONSMagnetically labeled MR-MSC and MR-MNC homed to heart post myocardial infarction and reduced infarct size, improved cardiac function. MR-MSC is superior to MR-MNC on improving cardiac function.

Animals ; Disease Models, Animal ; Magnetic Resonance Imaging ; Male ; Mesenchymal Stem Cell Transplantation ; Myocardial Infarction ; therapy ; Swine ; Swine, Miniature ; Treatment Outcome

BACKGROUNDSuperparamagnetic iron oxide (SPIO) particles have shown much promise as a means to visualize labeled cells using molecular magnetic resonance imaging (MRI). Micrometer-sized superparamagnetic iron oxide (MPIO) particles and nanometer-sized ultrasmall superparamagnetic iron oxide (USPIO) are two kinds of SPIO widely used for monitoring stem cells migration. Here we compare the efficiency of two kinds of SPIO during the use of stem cells to treat acute myocardial infarction (AMI).

METHODSAn AMI model in swine was created by 60 minutes of balloon occlusion of the left anterior descending coronary artery. Two kinds of SPIO particles were used to track after intracoronary delivered 10(7) magnetically labeled mesenchymal stem cells (MR-MSCs). The distribution and migration of the MR-MSCs were assessed with the use of 3.0T MR scanner and then the results were confirmed by histological examination.

RESULTSMR-MSCs appeared as a local hypointense signal on T₂*-weighted MRI and there was a gradual loss of the signal intensity after intracoronary transplantation. All of the hypointense signals in the USPIO-labeled group were found on T₂*-weighted MRI, contrast to noise ratio (CNR) decreased in the MPIO-labeled group (16.07 ± 5.85 vs. 10.96 ± 1.34) and USPIO-labeled group (11.72 ± 1.27 vs. 10.03 ± 0.96) from 4 to 8 weeks after transplantation. However, the hypointense signals were not detected in MPIO-labeled group in two animals. MRI and the results were verified by histological examination.

CONCLUSIONSWe demonstrated that two kinds of SPIO particles in vitro have similar labeling efficiency and viability. USPIO is more suitable for labeling stem cells when they are transplanted via a coronary route.

Animals ; Cell Survival ; Contrast Media ; Ferric Compounds ; Magnetic Resonance Imaging ; methods ; Male ; Myocardial Infarction ; diagnosis ; pathology ; Stem Cells ; cytology ; Swine

BACKGROUNDMesenchymal stem cells (MSCs) transplantation provides a new approach for myocardial repair. However, many important fundamental questions about MSCs transplantation remain unanswered. There is an urgent need to identify MSCs from the beating heart and analyze the efficacy of this new approach. This study aimed to localize the magnetically labeled MSCs (MR-MSCs) and monitor the restorative effects of MR-MSCs with magnetic resonance (MR) imaging.

METHODSAcute myocardial infarction (AMI) was created in swine by a balloon occlusion of the left anterior descending coronary artery. Cells were delivered via intracoronary infusion after myocardial infarction. Infarct size change and cardiac function were assessed with 3.0T MR scanner. The results were then confirmed by histological and western blot analysis. All statistical procedures were performed with Systat (SPSS version 12.01).

RESULTSA total of 26 swine were divided into four groups (sham-operated group, n=6; AMI group with PBS transplantation, n=6; labeled MSCs group, n=7; unlabeled MSCs group, n=7). MSCs, MR-MSCs (10(7) cells) or PBS were delivered by intracoronary injection after MI and serial cardiac MR imaging studies were performed at 0, 4 and 8 weeks after transplantation. MR imaging demonstrated MI size decreased after MSCs transplantation in labeled and unlabeled groups, however, increases were seen in the AMI group at 8 weeks after MI. The left ventricular ejection fraction (LVEF) was slightly increased in the AMI group ((41.87+/-2.45)% vs (39.04+/-2.80)%, P>0.05), but significantly improved in the MR-MSCs group ((56.85+/-1.29)% vs (40.67+/-2.00)%, P<0.05) and unlabeled group ((55.38+/-1.07)% vs (41.78+/-2.08)%, P<0.05) at 8 weeks after treatment. MR-MSCs were further confirmed by Prussian blue and immunofluorescent staining. Western blot analysis demonstrated that there was an increased expression of cardiomyocyte markers such as myosin heavy chain and troponin T in the MSCs treatment groups and the ratio of matrix metalloproteinase 2 to tissue inhibitor of metalloproteinase 1 decreased in the labeled group and unlabeled group compared with the AMI group and sham-operated group.

CONCLUSIONTransplanted MR-MSCs can regenerate new myocardium and prevent remolding in an MI model at 2-month follow-up and represent a preferred method to better understand the mechanisms of stem cell therapy in future clinical studies.

Animals ; Blotting, Western ; Cell Survival ; Disease Models, Animal ; Magnetic Resonance Imaging ; Magnetics ; Mesenchymal Stem Cell Transplantation ; Myocardial Infarction ; physiopathology ; therapy ; Swine ; Ventricular Function, Left

Objective:To discuss the effect of Sishenwan on phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin（PI3K/Akt/mTOR） signaling pathway related genes and proteins in colon tissue and interleukin-1β（IL-1β） and interleukin-10（IL-10） expression levels in serum of ulcerative colitis （UC） model rats with spleen kidney Yang deficiency. Method:The 120 SPF Wistar rats were randomly divided into normal group and model group after 7 days of adaptive feeding in SPF laboratory. The model group were given dinitrobenzene sulfonate （DNBS）/ethanol solution enema+hydrocortisone subcutaneously injection+senna leaf gavage to establish UC model of spleen kidney yang deficiency. The rats who successfully established the model were randomly divided into five groups：model group， mesalazine （0.36 g·kg^-1） group， and Sishenwan high， medium and low dose （3.2，1.6，0.8 g·kg^-1） groups， the volume of which was 10 mL·kg^-1. The model group and the blank group were given distilled water of the same volume. Once a day for 21 days. Observe the general conditions of the rats daily， and record the weight， fecal traits and occult blood of the mice for disease activity index （DAI） scoring.Take the rat colon tissue to observe the gross morphology and colon injury， and score the colon mucosal injury index （CMDI）. Hematoxylin-eosin （HE） staining was used to observe pathological changes. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）was used to detect the expression level of PI3K， Akt， mTOR mRNA in colon tissue. The levels of IL-1β and IL-10 in serum were detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the expression level of PI3K， phosphorylation （p）-PI3K， Akt， p-Akt， mTOR， p-mTOR protein in colon tissue. Result:Compared with blank group， the general survival status of the rats in model group was relatively poor， the DAI score and the CMDI index were significantly increased （P<0.01）. The intestinal mucosa partially disappears， the glands disappear， and a large number of inflammatory cells infiltrate and gather in the mucosal layer and the base layer in the pathological sections of the model group. The expression levels of PI3K， Akt， and mTOR mRNA were significantly increased （P<0.01）. The IL-1β content was significantly increased and the IL-10 content was significantly decreased （P<0.01）. The expression levels of p-PI3K，p-Akt and p-mTOR protein were significantly increased （P<0.01）. Compared with the model group， the DAI scores of Sishenwan high and medium dose groups and mesalazine group decreased （P<0.05）. The CMDI index of mesalazine group and the high， middle and low dose groups of Sishenwan was significantly reduced （P<0.01）. Pathological sections of rats showed that the inflammatory cells in the drug group decreased， and the mucosal layer structure returned to normal to varying degrees. The mesalazine group and the Sishenwan medium-dose group had the best effects， and the mucosal structure was close to the blank control group. The expression levels of PI3K， Akt， mTOR mRNA in the high and medium dose groups of Sishenwan， mesalazine group and Akt mRNA in low dose group of Sishenwan were significantly reduced （P<0.05，P<0.01）. The expression levels of PI3K and mTOR mRNA in low-dose group of Sishenwan decreased， but the difference was not statistically significant. The IL-1β content was significantly reduced and the IL-10 content was significantly increased in high， medium dose groups of Sishenwan and mesalazine groups （P<0.05，P<0.01）. The level of IL-1β decreased and the level of IL-10 increased in the low-dose group of Sishenwan， but the difference was not statistically significant. The expression levels of p-PI3K， p-Akt and p-mTOR protein in the high， medium， and low dose groups of Sishenwan and mesalazine group decreased to varying degrees， and the differences were statistically significant（P<0.05，P<0.01）. Conclusion:Sishenwan has the effect of improving the general condition and intestinal mucosal damage of ulcerative colitis model rats with spleen and kidney Yang deficiency. The mechanism may be related to the inhibition of PI3K/Akt/mTOR signaling pathway.

Objective: To establish and optimize a TaqMan-probe quantitative real-time PCR (qPCR) assay for the detection of 7 important Rickettsiales pathogens and simultaneous identification of the infection types. Methods: Based on the ompB gene of Rickettsia prowazekii, Rickettsia mooseri and spotted fever group rickettsiae, the groEL gene of Orientia tsutsugamushi, the 16S rRNA of Ehrlichia chaffeensis, the gltA gene of Anaplasma phagocytophilum and the com1 gene of Coxiella burnetii, we synthesized primers and TaqMan-probes and optimized the reaction system and reaction process to same solution. The sensitivity, specificity and reproducibility of this assay were evaluated and the assay was used for the detection of simulated and actual samples. Results: The Ct value of the standard curves of the 7 pathogens showed a good linear relationship with the number of DNA copies (all R² >0.990 0), the minimum detection limit was 10 copies/μl, showing good specificity. In the 96 tick nucleic acid extracts, Coxiella burnetii was detected in 1 sampleand spotted fever group Rickettsiae was detected in 3 samples. In the 80 blood samples from patients with undefined febrile illness, Orientia tsutsugamushi was detected in 1 sample and spotted fever group rickettsiae was detected in 2 samples. Conclusions: In this study, based on the established TaqMan-probe qPCR assay, the reaction system and reaction condition of the 7 important pathogens of Rickettsiales were optimized to the same solution. This method overcomes the shortcomings of using different reaction systems and reaction conditions for different pathogens, which can precisely identify the species of 7 important pathogens of Rickettsiales in clinical sample detections and is important for the infection type identification and laboratory detection time reduction to facilitate precise treatment of the patients.
Humans ; Rickettsiales ; Real-Time Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Reproducibility of Results ; Orientia tsutsugamushi ; Spotted Fever Group Rickettsiosis

BACKGROUND: Human infections with zoonotic coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV, have raised great public health concern globally. Here, we report a novel bat-origin CoV causing severe and fatal pneumonia in humans. METHODS: We collected clinical data and bronchoalveolar lavage (BAL) specimens from five patients with severe pneumonia from Wuhan Jinyintan Hospital, Hubei province, China. Nucleic acids of the BAL were extracted and subjected to next-generation sequencing. Virus isolation was carried out, and maximum-likelihood phylogenetic trees were constructed. RESULTS: Five patients hospitalized from December 18 to December 29, 2019 presented with fever, cough, and dyspnea accompanied by complications of acute respiratory distress syndrome. Chest radiography revealed diffuse opacities and consolidation. One of these patients died. Sequence results revealed the presence of a previously unknown β-CoV strain in all five patients, with 99.8% to 99.9% nucleotide identities among the isolates. These isolates showed 79.0% nucleotide identity with the sequence of SARS-CoV (GenBank NC_004718) and 51.8% identity with the sequence of MERS-CoV (GenBank NC_019843). The virus is phylogenetically closest to a bat SARS-like CoV (SL-ZC45, GenBank MG772933) with 87.6% to 87.7% nucleotide identity, but is in a separate clade. Moreover, these viruses have a single intact open reading frame gene 8, as a further indicator of bat-origin CoVs. However, the amino acid sequence of the tentative receptor-binding domain resembles that of SARS-CoV, indicating that these viruses might use the same receptor. CONCLUSION A novel bat-borne CoV was identified that is associated with severe and fatal respiratory disease in humans.
Adult ; Aged ; Betacoronavirus ; genetics ; isolation & purification ; Coronavirus Infections ; diagnostic imaging ; therapy ; virology ; Female ; Humans ; Male ; Middle Aged ; Pandemics ; Pneumonia, Viral ; diagnostic imaging ; therapy ; virology ; Tomography, X-Ray ; Treatment Outcome