[ ABSTRACT] AIM:To study the process of promoting mouse embryonic stem cells ( ESC) to specify to definitive endoderm by up-regulating of Nodal signal pathway in order to find the best cultivated systems of differentiation of mouse ESC to definitive endoderm cells.METHODS:The cells were divided into different groups based on the culture medium:ESC group ( serum-free medium +LIF) , natural differentiation group ( serum-free medium) and activin A group ( serum-free medium +50μg/L activin A).The cells and the sterilized coverslips with cells were collected at 1, 3, 5 and 7 d of the cultivation.The proportion of CXCR4 +cells was detected by flow cytometry.The expression of CXCR4 was determined by immunocytochemical method, and the protein expression of OCT4 and CXCR4 was detected by Western blot.RE-SULTS:The proportion of CXCR4 +cells showed no dramatic change in ESC group along with the extending of cultivation day, while there were gradually increased in natural differentiation group and activin A group and the highest level was ob-served at 5 d.Among the 3 groups, the proportion of CXCR4 +cells at 5 d was the highest in activin A group.The brown or tan staining in the cells observed under microscope was considered as positive CXCR4 by immunocytochemistry.The pro-tein levels of OCT4 and CXCR4 in ESC group along with the extending of cultivation days was observed.The expression levels of OCT4 were gradually decreased in the cells in natural differentiation group and activin A group, while those of CX-CR4 were gradually increased with the highest level at 5 d.It was highest in the cells in activin A group.CONCLUSION:The proportion of definitive endoderm was the highest at 5 d of the induction during in vitro mouse ESC differentiation.Up-regulation of Nodal signal pathway by adding activin A at the early stage of mouse ESC differentiation promotes mouse ESC to specify to definitive endoderm with CXCR4 molecular marker.

The purpose of the present work was to investigate the innovative self-assembling system, "beads", prepared by continuously shaking alpha-cyclodextrin and soybean oil without the use of organic solvents and surfactants at room temperature. Berberine hydrochloride previously dissolved in soybean oil was chosen as a model drug to explore the shape, structure, drug loading and in vitro release of beads. The particle size and drug loading of berberine hydrochloride-loaded beads were (2.25 +/- 0.23) mm and (67.02 +/- 0.64) microg x g(-1), respectively. Confocal microscopy showed that the core-shell structure of beads could contain poorly water soluble drugs or lipophilic drugs in the lipid core. The drug release rate and cumulative releases of beads were both higher than those of raw medicine of berberine hydrochloride in simulated intestinal fluid. These results suggested that beads were the novel and potential lipid-based drug delivery system for lipophilic or poorly water soluble traditional Chinese medicine.
Berberine ; administration & dosage ; Drug Delivery Systems ; Particle Size ; Solubility ; Soybean Oil ; administration & dosage ; alpha-Cyclodextrins ; administration & dosage

AIM:To investigate the effect of microRNA-429 (miR-429) on the expression of occludin (Ocln) in intestinal epithelial cells ( IECs) and intestinal epithelial barrier function in diabetic mice.METHODS:Diabetes mel-litus mouse model was induced by intraperitoneal injection of streptozocin.The expression of miR-429 in IECs of diabetic mice was inhibited by antagomiRNA-429 injected via tail vein.The expression of miR-429 and mRNA expression of Ocln were detected by real-time PCR.The protein expression of Ocln was determined by Western blotting and immunohistochem-istry.The urinary lactulose/mannitol ratio was measured by gas chromatography.The plasma LPS concentrations in the mice were measured by chromogenic end-point TAL kit.RESULTS:The results of real-time PCR confirmed that the ex-pression of miR-429 in IECs of diabetic mice was remarkably inhibited by tail-vein administration of antagomiRNA-429, and resumed to similar level of normal mice on the 6th day after the administration.After suppressing the level of miR-429, the expression of Ocln in IECs of diabetic mice increased significantly (P<0.05), while the urinary lactulose/mannitol ra-tio and the plasma LPS concentration decreased obviously ( P<0.05 ) .CONCLUSION:AntagomiRNA-429 effectively suppresses miR-429 expression in IECs of diabetic mice, and then enhances the expression of Ocln and partially resumes the intestinal epithelial barrier function.

Objective To investigate the value of DWI using 3.0 T MRI to predict response to radiotherapy(RT) and concurrent chemoradiotherapy(CCRT) in patients with advanced non-small cell lung cancer (NSCLC).Methods From January 2014 to May 2015, 40 patients with stageⅢ(Ⅲa orⅢb) NSCLC underwent DWI using 3.0 T MRI before RT/CCRT were enrolled. The imaging quality of diffusion-weighted images were evaluated on 3-level grades as good, moderate and non-diagnostic.The patients with good or moderate image quality were underwent DWI at 2 weeks after starting therapy(total dose of 20 Gy), and at the end of therapy (total dose of 60 Gy). Apparent diffusion coefficient(ADC) of lung cancer with good and moderate image quality were calculated by Funtool. The following quantitative parameters were recorded and calculated: the mean pretreatment ADC value(ADCpre), the mean mid-treatment ADC value (ADCmid), the mean post-treatment ADC value(ADCpost), the rate of changes inmean ADC value at 2 weeks post therapy (ΔADCmid) and the rate of changes inmean ADC value at the end of therapy(ΔADCpost). The patients were classified into response group and non-response group according to the tumor response, which was assessed with revised response evaluation criteria in solid tumors (RECIST1.1) after CCRT. The Mann-Whitney U test was used to compare parameters between the two groups.The relationship between these obtained parameters and tumor response was evaluated by Spearman correlation analysis. The value of parameters on predicting tumor response was calculated by receiver operating characteristic curve.Results 96.4%(80/83) DW images were graded as good or moderate image quality. The responders had lower median ADCpre[1.32 (0.77—1.96) × 10- 3 mm2/s] than non-responders[1.60(1.12—2.33) × 10- 3 mm2/s], which had statistically significant difference (Z=-2.934,P=0.003).Tumor regression rate after treatment had negative correlation with ADCpre(r=-0.386, P=0.018).The responders had increased ADC [ΔADCmid: 38.9%(12.8%—139.0%),ΔADCpost: 48.3% (25.6%—148.1%)] than non-responders [ΔADCmid: -2.5% (-15%—29.4%), ΔADCpost:14.2% (- 28.1% —71.3% )], which had statistically significant difference (Z=- 2.847, - 2.221, respectively;P<0.05). Tumor regression rate after treatment had positive correlation with ΔADCmid(r=0.637, P=0.001) and ΔADCpost(r=0.631, P=0.005).From ROC analysis,when setting threshold on pretreatment ADCpre=1.38 × 10-3 mm2/s, ΔADCmid=21.6%, ΔADCpost=38.8%, the area under curve was 0.782, 0.838 and 0.813.Conclusion The mean ADC value before RT/CCRT and its changes during treatment is likely to be a valuabletool for predicting the response after RT/CCRT in advanced NSCLC, which may be helpful to clinical decision on individualized therapy.

Objective The detection of malignant lymphoma with invasion in liver and spleen using PET/CT has not been well documented in the literature. This study aimed to investigate the usefulness of PET/CT in this regard and to compare it with plain CT. Methods Forty-one pathologically confirmed malignant lymphoma patients with liver and spleen invasion were recruited into this study. Among all patients, there were 38 non-Hodgkin's lymphoma (NHL), 2 Hodgkin's lymphoma (HL) and 1 gastric mucosa associated lymphoma. PET/CT imaging was recorded 1h after injection of 296～444 MBq 18F-fluorodeoxyglucose (FDG). Results (1) There were 30(30/41) patients with liver invasion, including hepatic nodules, mass and portal nodes. The mass was large to invade surrounding liver parenchyma. (2) There were 23(23/41) patients with spleen invasion. The spleen was enlarged and demonstrated diffused hyper-metabolism. (3) Other invasion included: lung (n=13), cortical bone and marrow (n=12), stomach (n=9), pleural (n=6), and subcutaneous soft tissue (n=5) and so on. Conclusion PET/CT could accurately diagnose the invasion in liver and spleen of malignant lymphoma, which was of potential role on the diagnosis and staging of lymphoma.

OBJECTIVETo assess the value of (18)F-FDG PET/CT in the diagnosis of solitary nodular-type bronchoalveolar carcinoma (BAC).

METHODSThe clinical and radiographic data were analyzed retrospectively in 30 patients with pathologically confirmed solitary nodular-type BAC who underwent (18)F-FDG PET/CT examinations between August, 2005 and December, 2006. The morphological and radioactive findings of the lesions were reviewed, and the maximum standard uptake values (SUVmax) were measured. The diagnostic accuracy of PET, PET/CT, and HRCT were analyzed.

RESULTSThe (18)F-FDG SUV was markedly lower in BAC than in other well differentiated adenocarcinoma. In 19 of the BAC cases, PET showed a SUVmax of no less than 2.5, demonstrating positive changes. Of the total of 30 cases, 5 had ground glass opacity (GGO) changes, 3 exhibited mixed nodules with GGO changes around the lesions, and 22 cases presented with solid nodules. HRCT showed that BAC located often in the superior lobes of the bilateral lungs, mostly below the pleura in the surrounding lung field; the lesions were patchy or nodular with irregular shapes, showing lobulation in 22 cases, spiculation in 15 cases, pleural indentation in 21 cases, and vacuolar changes in 4 cases. The diagnostic accuracy of PET, PET/CT and HRCT for solitary nodular-type BAC was 36.67%, 93.33%, and 93.33%, respectively.

CONCLUSIONThe SUVmax of BAC provides only limited value for defining the nature of the lesions, but can serve as a general reference for assessing the disease activity. PET/CT, which allows both functional and imaging assessment, can be a valuable modality to reduce the misdiagnosis rate of BAC.

Carcinoma ; diagnosis ; Fluorodeoxyglucose F18 ; Humans ; Lung ; pathology ; Lung Neoplasms ; diagnosis ; Positron-Emission Tomography ; Retrospective Studies ; Solitary Pulmonary Nodule ; diagnosis ; Tomography, X-Ray Computed

OBJECTIVETo study the APC and E-cadherin gene promoter hypermethylation as tumor marker and to investigate the correlation of free tumor-related DNA in serum and tumor tissue with clinicopathological parameters. Their feasibility in early diagnosis, predicting therapeutic effect and monitoring recurrence was evaluated.

METHODS84 cases with operated breast cancer were recruited from March 2002 to August 2002 at Beijing Cancer Hospital. Aberrant methylation of E-cadherin and APC genes was detected in tumor tissues, adjacent normal tissues and peripheral blood serum by methylation-specific PCR (MSP). 10 cases with benign breast diseases were selected as control group.

RESULTSThe positive rate of promoter hypermethylation of E-cadherin and APC genes in tumor tissues was 52.4% and 45.2%, in the paired serum was 33.3% and 31.0%, respectively. Aberrant methylation of free DNA in serum presented the same alteration in tumor tissues. E-cadherin and APC hypermethylation in serum and tumor samples significantly correlated each other (E-cadherin P < 0.001; APC P = 0.002). The sensitivity of detection of free DNA methylation of E-cadherin and APC genes in serum was 63.6% and 63.2%, respectively. The specificity was 100% and 95.7%, respectively. There was no correlation for the aberrant methylation in cancer tissues and serum with the clinicopathological parameters of patients including age, tumor staging, tumor size, histological type and receptor. None of the aberrant methylation was found in adjacent normal tissues and control group serum.

CONCLUSIONThe same aberrant methylation in cancer tissues and serum, not correlating with tumor staging, can be detected in about one third of breast cancer patients. The aberrant methylation in serum can disappear after operation. The results imply that this approach may be feasible for early diagnosis, evaluation of therapeutic effects and monitoring recurrence of breast cancers.

Adult ; Aged ; Biomarkers, Tumor ; Breast Neoplasms ; blood ; genetics ; Cadherins ; blood ; genetics ; CpG Islands ; DNA Methylation ; DNA, Neoplasm ; blood ; genetics ; Female ; Genes, APC ; Genes, Tumor Suppressor ; Humans ; Middle Aged ; Promoter Regions, Genetic ; Sensitivity and Specificity ; Young Adult

OBJECTIVE: To explore the genetic basis for a child with infantile liver failure syndrome type 2 (ILFS type 2). METHODS: Clinical features of the child were analyzed. Next generation sequencing was also carried out for him. RESULTS: The child was found to harbor compound heterozygous variants of the NBAS gene, which included a novel nonsense c.2746A>T (p.R916X, 1456) variant in exon 24 and a missense c.3596G>A (p.C1199Y) mutation in exon 31, which has been associated with ILFS type 2. The two variants were respectively inherited from his father and mother. CONCLUSION The compound heterozygous variants of c.3596G>A and c.2746A>T of the NBAS gene probably underlay the ILFS type 2 in this child.
Child ; Exons/genetics* ; Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Liver Failure ; Male ; Mutation

OBJECTIVETo investigate the changes in peripheral blood Th17 and CD4(+)CD25(+) regulatory T (Treg) cells and their significance among children with hand, foot and mouth disease (HFMD).

METHODSEighty-nine children with HFMD, including 55 cases of common HFMD and 34 cases of severe HFMD, were included in the study; and 30 healthy children were selected as the control group. The percentages of Th17 and CD4(+)CD25(+) Treg cells in CD4(+) T cells in peripheral blood were determined by flow cytometry. The expression levels of interleukin (IL)-10, transforming growth factor-β (TGF-β), and IL-17 were measured by enzyme-linked immunosorbent assay.

RESULTSCompared with the control group, the cases of common HFMD and severe HFMD had significantly increased levels of Th17 cells and IL-17 (P<0.05) but significantly decreased levels of CD4(+)CD25(+) Treg cells, IL-10, and TGF-β (P<0.05). The severity of the HFMD was positively correlated with the levels of Th17 cells and IL-17 in peripheral blood but negatively correlated with the levels of CD4(+)CD25(+) Treg cells, IL-10, and TGF-β.

CONCLUSIONSChildren with HFMD have increased response of Th17 cells but decreased response of CD4(+)CD25(+) Treg cells in peripheral blood. Th17/CD4(+)CD25(+) Treg cell imbalance may play an important role in the pathogenesis of HFMD.

Child ; Child, Preschool ; Hand, Foot and Mouth Disease ; immunology ; Humans ; Infant ; Interleukin-10 ; blood ; Interleukin-17 ; blood ; T-Lymphocytes, Regulatory ; immunology ; Th17 Cells ; immunology ; Transforming Growth Factor beta ; blood

OBJECTIVETo investigate the effect of sumoylation of alpha-synuclein by SUMO-1 on the mitochondria subcellular localization of alpha-synuclein and its degradation via ubiquitin-proteasome system.

METHODSPrimers of wild-type, A53T pathogenic mutant and K96R mutant of human alpha-synuclein were designed to amplify the corresponding cDNAs without stop codon. The cDNAs were cloned into pGEM T-easy vector, analyzed by using enzyme mapping and DNA sequencing, and subcloned into pEGFP-N1 vector. The recombinant plasmids of pEGFP-alpha-synuclein-WT, pEGFP-alpha-synuclein-A53T and pEGFP-alpha-synuclein-K96R were transfected into HEK293 cells by lipofectamine method. The expression of the alpha-synuclein protein was measured by immunofluorescence and confocal microscope. Then mitochondria staining as well as immunofluorescence were utilized to investigate the effect of wild-type, A53T mutant and sumoylation of alpha-synuclein on mitochondria subcellular localization of alpha-synuclein. The effect of sumoylation of alpha-synuclein on its degradation via the ubiquitin-proteasome system in the cells was assayed by Western-blot.

RESULTSThe enzyme mapping suggested that the eukaryotic expression plasmids for human wild-type, A53T and K96R mutants of the alpha-synuclein gene were constructed successfully. By immunofluorescence and confocal microscope, it was observed that alpha-synuclein-WT and alpha-synuclein-A53T proteins aggregated in cytoplasm, and alpha-synuclein-K96R protein aggregation was decreased in cytoplasm of cultured cells. The alpha-synuclein proteins of wild-type, A53T and K96R mutants were co-localized with mitochondria. Western-blot analysis revealed that both wild-type and A53T mutant affected the amount of the ubiquitinated proteins.

CONCLUSIONNeither overexpression of wild-type and A53T pathogenic mutant alpha-synuclein, nor sumoylation of alpha-synuclein, affected the subcellular localization in the mitochondria. However, overexpression of wild-type and A53T mutant alpha-synuclein affected the amount of the ubiquitinated proteins.

Blotting, Western ; Cell Line ; Humans ; Mitochondria ; metabolism ; Proteasome Endopeptidase Complex ; metabolism ; SUMO-1 Protein ; metabolism ; Ubiquitin ; metabolism ; alpha-Synuclein ; metabolism