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BACKGROUND:Previous studies have demonstrated that normal rat liver homogenate supernatant can induce human umbilical cord mesenchymal stem cels to differentiate into hepatocyte-like cels with partial hepatocyte functions. However, whether fibrotic liver homogenate supernatant can work or how the inducing effect is remains unclear. OBJECTIVE:To investigate the differentiation potential of human umbilical cord mesenchymal stem cels into hepatocytes under the normal liver and fibrotic liver microenvironment in vitro. METHODS:Liver fibrosis was induced in the SD rats by repeated intraperitoneal injections of 3% thioacetamide at a dose of 200 mg/kg body mass, twice a week for 4 weeks, and then fibrotic liver tissues and normal liver tissues were used to prepare liver homogenate supernatants. Passage 3 human umbilical cord mesenchymal stem cels were used and divided into standard control group (cels were cultured in DMEM/F12 with 10% fetal bovine serum), fibrotic liver homogenate supernatants group (cels were cultured in DMEM/F12 with 10% fetal bovine serum and 50 g/L fibrotic liver homogenate supernatants), normal liver homogenate supernatants group (cels were cultured in DMEM/F12 with 10% fetal bovine serum and 100 g/L normal liver homogenate supernatants). The morphological changes of the cels in each group were recorded under inverted microscope; the protein levels of CK18, AFP, CYP3A4, CYP2E1, CYP2D6 and TPH2 were evaluated using western blot assay. Furthermore, the concentration of albumin in the cels was measured. RESULTS AND CONCLUSION:After a 7-day inducement, the stem cels in liver homogenate supernatants groups lost their fusiform shape and changed into hepatocyte-like cels with the morphous of round shape. Compared with the standard control group, the hepatocyte-like cels in the two liver homogenate supernatants groups exhibited human hepatocyte biomarkers, CK18 and AFP. The standard control group cels could express a little amount of CYP2E1, while cels in the two liver homogenate supernatants groups could express CYP3A4, CYP2E1, CYP2D6, TPH2. Compared with the standard control group, the expression level of CYP2E1 in the two liver homogenate supernatants groups increased significantly (P < 0.01), and however, the relative levels of CYP3A4, CYP2E1, CYP2D6, TPH2 in the two liver homogenate supernatants groups showed no statistical significance (P > 0.05). At the same time, compared with the standard control group, the concentration of albumin in the two liver homogenate supernatants groups markedly increased (P < 0.01), but there was no difference between the two liver homogenate supernatants groups (P > 0.05). Experimental findings demonstrated that both of normal liver tissue and fibrotic liver tissue microenvironments could induce human umbilical cord mesenchymal stem cels to differentiate into hepatocyte-like cels. To achieve the same effect, compared with normal liver tissue, fibrotic liver tissue required lower concentrations, suggesting that fibrotic liver tissue microenvironment may be more conducive to differentiation of umbilical cord mesenchymal stem cels into hepatocytes.

Objective To evaluate the effect of morphine on the proliferation and migration of human hepatocellular carcinoma (HCC) cells in an in vitro experiment.Methods The experiment was performed in 2 parts.Experiment Ⅰ Human HCC cells were inoculated in 6-well plates at a density of 2×105 cells/well (2 ml/well) and divided into 6 groups (n=12 each) using a random number table:control group (C group) and 10,25,50,100 and 200 ng/ml morphine groups (M1-M5 groups).Morphine at the final concentration of 10,25,50,100 and 200 ng/ml was added in M1-M5 groups,respectively.The equal volume of phosphate buffer solution was added in group C.The cells were cultured or incubated for 48 h.The expression of μ1-opioid receptor (MOR1) mRNA was measured by real-time polymerase chain reaction.The expression of MOR1 was detected by Western blot in C and M4 groups.Experiment Ⅱ Human HCC cells were inoculated in 96-well plates (1×103 cells/well) or in Transwell chambers(200 μl) and divided into 2 groups (n=9 each) using a random number table:control group (C group) and morphine group (M group).Morphine was added at the final concentration of 100 ng/ml in group M,and the equal volume of phosphate buffer solution (final volume 100 μl/well) was added in group C.The cells were cultured or incubated for 7 days.The cell proliferation was detected by methyl thiazolyl tetrazolium assay at 1-7 days of incubation or culture.The cell migration was determined by Transwell chamber assay at 30 h of incubation or culture.Results Experiment Ⅰ Compared with group C,the expression of MOR1 mRNA was significantly up-regulated in M1-M5 groups,and the expression of MOR1 was significantly up-regulated in group M4 (P<0.01).Compared with group M4,the expression of MOR1 mRNA was significantly down-regulated in M1-M3 and M5 groups (P<0.01).Experiment Ⅱ Compared with group C,the cell proliferation was significantly enhanced on 4th-7th days of incubation,and the number of cells passing through Transwell chambers was increased in group M (P<0.05 or 0.01).The cell proliferation was gradually enhanced on 4th-7th days of incubation in group M (P<0.05).Conclusion Morphine can promote the proliferation and migration of human HCC cells,and the mechanism is related to up-regulation of MOR1 expression in an in vitro experiment.

Objective To detect the expression of chemokine receptors CXCR3 mRNA in the peripheral blood mononuclear cells (PBNCs) of patients with Rheumatoid Arthritis (RA) and to analyze the relationship between the expression and the disease activity. Methods mRNA was extracted from PBNCs and the expression of CXCR3 mRNA was detected by real-time fluorescence quantitative PCR (RFQ-PCR) in 51 RA patients and 32 controls. T-test, x2-test, ANOVA were used for statistial analysis. Results Comparison between the two groups had shown that the expression levels of CXCR3 mRNA in clinical active RA group were higher than those of the RA patients in remission and healthy controls (P<0.05). The expression levels of CXCR3 mRNA were positively correlated with serum levels of ESR and CRP in clinical active RA group (r=0.824, r=0.765, P<0.05). In addition, RF titer, APF, AKA, and anti-CCP had no significant correlation with the expression levels of CXCR3 mRNA in RA patients (P>0.05). Conclusion RFQ-PCR is a sensitive,reproducible and practical test. The mRNA expressions of CXCR3 are significantly elevated in RA patients,which suggest that CXCR3 may be involved in the pathogenesis of RA. The mRNA expressions of CXCR3 in active RA patients are higher than those of RA patients in remission. These results indicate that CXCR3 may play an important role in the pathogenesis and progression of RA, and CXCR3 may be considered as an indicator for disease activity, therapeutic efficacy and prognosis of RA.

There is growing interest in biodiesel and this results in the accumulation of glycerol. The exploitation and application of glycerol has attracted more and more attention. In the current study, glycerol was biotransformed to produce 3-hydroxypropionaldehyde by genetic engineering bacteria. It is known that 3-hydroxypopionaldehyde has been widely used as an important intermediate for chemicals, effective antimicrobial agent, and fix agent for tissues. A pair of primers was designed on the basis of the sequence of both NH2-terminus and the amino acid sequence of glycerol dehydratase reported by NCBI, and a fragment about 1.6 kb was obtained by PCR amplification using the total genome DNA of Lactobacillus reuteri as template, then the fragment was cloned to the pMD18-T vector and sequenced. Two specific primers were designed according to the obtained sequence, and a fragment with length of 1674 bp was amplified using PCR with these two specific primers. Consequently, the resulting products were digested with EcoR I and Hind III and ligated using T4 DNA ligase to the pET28b vector digested with the same enzymes. The recombinant plasmid, named pET28b-dhaB, was transformed into E. coli BL21. The positive clones were induced with IPTG and the expression products were further analyzed by SDS-PAGE, indicating that protein with a molecule weight of around 65 kD was obtained. Furthermore, the glycerol dehydratase activity was evaluated and compared with the wild type strain as well.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glyceraldehyde ; analogs & derivatives ; chemistry ; metabolism ; Hydro-Lyases ; biosynthesis ; genetics ; Lactobacillus reuteri ; enzymology ; genetics ; Propane ; chemistry ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Objective To analyze the impac factors of serum N?terminal pro?brain natriuretic peptide (NT?proBNP) in patients with renal failure in non?dialysis phase, and to determine the cut?off point of as a diagnostic values in these patients with heart failure (HF). Methods Cross?sectional study was applied. Clinical data of 145 patients (37 cases of CKD4, 89 cases of CKD5, and 19 cases of acute renal injury (AKI) with renal failure in non?dialysis phase were collected. Comparison between groups and lineal regression analysis were utilized to investigate the impact factors of NT?proBNP, and the receiver operating characteristic curve (ROC curve) to select a better cut?off point of diagnosis in these patients with HF. Results (1) Compared with patients without HF, patients with HF had significantly higher edema, cardiac troponin I, serum phosphorus concentration, and left atrial diameter (LA), while ALB and left ventricular ejection fraction (LVEF) were decreased (P<0.05). (2) The NT?proBNP was divided into 4 groups with four points: First groups of 36 cases, NT?proBNP 1 ?862 ng/L, second groups 37 cases, 866?2670 ng/L, third groups 37 cases, 2790?20 000 ng/L, fourth groups 35 cases, 20 900?35 000 ng/L. With the increase of NT?proBNP levels, the occurrence of AKI and CKD4 decreased gradually while the occurrence of CKD and edema were significantly increased (P<0.01). Systolic blood pressure, troponin I, uric acid, serum phosphorus, parathyroid hormone, 24 hours urine protein, LA, interventricular septum thickness (IVS), left ventricular posterior wall thickness (LVPW) level gradually increased. Hb, ALB, calcium, CO2, eGFR, LVEF significantly decreased (P<0.01). The serum NT?proBNP of patients with HF was significantly higher than that of patients without HF (19 150 ng/L vs 1530 ng/L, P<0.01). The serum NT?proBNP of patients with edema was significantly higher than that in patients without edema (5460 ng/L vs 1630 ng/L, P<0.01). (3) Single factor linear regression analysis indicated that higher NT?proBNP was positive correlated with HF, edema, cardiac troponin I, uric acid, serum phosphorus, LA, IVS and LVPW (P<0.05), while negative correlated with Hb, eGFR, ALB, serum calcium, CO2, LVEF (P<0.05), and not correlated with eGFR, uric acid, serum calcium (P>0.05). (4) The best cut?off point of NT?proBNP predicting HF in patients with renal failure in non?dialysis phase was 3805 ng/L, AUC=0.848, 95%CI 0.786?0.910. Sensitivity was 82.4%, specificity 74.5%, positive predictive value 62.1%, negative predictive value 87.3%, positive likelihood ratio 3.2, negative likelihood ratio 0.24. Conclusions The level of NT?proBNP>20 000 ng/L is mainly found in end?stage renal disease patients with HF. HF is a main factor for the increase of NT?proBNP in patients with renal failure in non?dialysis phase. High phosphorus viremia, anemia, and hypoalbuminemia are closely related to NT?proBNP. Therefore NT?proBNP predicting HF should take into account the effects of these confounding factors in these patients.

Objective To explore the expressions and correlation of COX-2, bcl-2 and Caspase-3 in diffuse large B-cell lymphoma(DLBCL). Methods The expression of COX-2, bcl-2 and Caspase-3 was studied in 29 cases of DLBCL and 10 cases of reactivited lymphoid tissue with immunohistochemistry of PowerVisionTM. Results The expression of COX-2 and bcl-2 are seperately 68.9 % and 72.4 %. The expression of COX-2 had correlation with Ann Arbor stage. The expression of COX-2 was positively correlated with that of bcl-2, the expression of bcl-2 was negatively correlated with that of Caspase-3, the expression of COX-2 was negatively correlated with that of Caspase-3. Conclusion COX-2, bcl-2 and Caspase-3 may be involved in the carcinogenesis of DLBCL, and their expression may be helpful in estimating the histology grade and prognosis of DLBCL.

Objective:Recent studies have shown that in contrast to decrease in distal gastric adenocarcinoma (DGA), incidence of adenocarcinoma of the esophagogastric junction (AEG) has increased noticeably in numerous counties. However, the reasons remain unclear. This study evaluated the possible differences in the expression of KLF4, SP1, and Cyclin D1 in AEG and DGA, and explored the potential carcinogenesis of AEG. Methods:Immunohistochemistry was performed on paraffin-embedded tissues to evaluate the pu-tative differences in the expressions of KLF4, SP1, and Cyclin D1 at protein level between AEG (n=58) and DGA (n=47). The patholog-ical significance of these markers between the two groups was also compared and analyzed. Results:The percentage of positive KLF4 expression was significantly lower in DGA than in AEG (P<0.05). Lower KLF4 expression was found both in well-or moderately dif-ferentiated cases and in poorly differentiated cases with DGA compared with their AEG counterparts (P<0.05). However, positive stain-ing for SP1 was significantly higher in DGA (P<0.05). No significant difference was found in the expression of Cyclin D1 between the two groups. Further analysis showed that in DGA, the positive expression of KLF4, SP1, and Cyclin D1 were significantly correlated with lymph node metastasis. In AEG, only Cyclin D1 expression was correlated with lymph node metastasis (P<0.05). No correlation was found among the expression of KLF4, SP1, and Cyclin D1 in AEG. In DGA, KLF4 was inversely correlated with SP1 and Cyclin D1 (r=-0.334 and r=-0.341, respectively, P<0.05), and SP1 was positively correlated with Cyclin D1 expression (r=0.340, P<0.05).Conclusion:Different expression patterns and clinicopathological significance of KLF4, SP1, and Cyclin D1 were observed between AEG and DGA, suggesting the putative difference in the carcinogenesis and progression of AEG and DGA.

Objective To investigate the effects of penehyclidine hydrochloride on activities of nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) during actue lung injury induced by blunt chest trauma-hemorrhagic shock and resuscitation (HSR) in rats.Methods Thirty male Sprague-Dawley rats,aged 8 weeks,weighing 250-300 g,were randomly assigned into 3 equal groups (n =10 each) using a random number table:sham operation group (group S),blunt chest trauma-HSR group (group THSR) and penehyclidine hydrochloride group (group PHCD).The model of actue lung injury induced by blunt chest trauma-HSR was induced by dropping a 300 g weight onto a precordium in anesthetized rats.Blood was withdrawn via the femoral artery 5 min later until MAP was decreased to 35-45 mmHg within 15 min and maintained at this level for 60 min,followed by resuscitation.In PHCD group,PHCD 2 mg/kg was injected intravenously at 60 min after hemorrhagic shock.At 6 h after the model was established,blood samples were obtained for measurement of concentrations of tumor necrosis factor-alpha (TNF-α) in serum.The lungs were then removed for determination of lung water content,myeloperoxidase (MPO) activaty (by colorimetric assay),NF-κB and AP-1 activaties (using electrophoretic mobility shift assay) in lung tissues,and for microscopic examination of pathologic changes (under light microscope).The left lung was lavaged,and lung permeability index (LPI) was calculated.Results Compared with S group,lung water content,LPI,serum TNF-α level and activites of MPO,NF-κB and AP-1 were significantly increased in THSR and PHCD groups.Compared with THSR group,lung water content,LPI,serum TNF-α concentrations and activites of MPO,NF-κB and AP-1 were significantly decreased in PHCD group.The pathological damage to lung tissues was significantly reduced in PHCD group as compared with THSR group.Conclusion PHCD can inhibit activities of NF-κB and AP-1 in lung tissues,thus mitigating acute lung injury induced by blunt chest trauma-HSR in rats.

Objective:To promote evidence-based practice in the pre-chemotherapy nursing assessment among adult cancer patients.Methods:The Joanna Briggs Institute Practical Application of Clinical Evidence System and Getting Research into Practice audit tools were used. The project was conducted in Shanghai Cancer Center of Fudan University from May to October 2018, 12 audit criteria were developed in the program including nursing training, patient medical and allergic history, medical diagnosis, lab data and so on.Results:A baseline audit of pre-chemotherapy nursing assessment among adult cancer patients was conducted, with a sample size of 68 patients and 36 nursing staff, during this stage, the compliance of audit 11 and 12 were 100%. After the implementation of systematic strategies, a follow-up audit involving similar sample as first audit was conducted using the same audit criteria. In the follow-up audit, except criterion 4 and 10, the compliance of the remaining 8 criteria had significantly improved, and χ2 value was 10.29-132.06, P<0.01. The result of history adverse reaction in the follow-up audit showed that among 68 patients, 3 had experienced chemotherapy infusion reactions in the past (The drugs were oxaliplatin, gemcitabine and paclitaxel), 39 had chemotherapy-related symptoms before admission (most of them were relieved at admission), of which the top five were loss of appetite, fatigue, nausea, neurotoxicity and vomiting. Conclusions:The aims of the project were fulfilled. We achieved increased compliance with evidence-based best practice recommended by JBI in most of audit criteria. Further audit will need to be carried out to improve the validity and quality of nursing assessment.