[Effect of Notch Signaling Pathway on Differentiation of Mouse Embryonic Stem Cells into Hematopoietic Stem Cells or Hematopoietic Progenitor Cells by VEGF].

OBJECTIVE: To investigate the effect of Notch signaling pathways on differentiation of mouse embryonic stem cells(ESC) into haematopoietic stem cells or haematopoietic progenitors cells(HSC/HPC). METHODS: Mouse embryonic stem cells were proliferated in vitro to form embryoid bodies; the differentiation of embryoid bodies should be induced in vitro, the experiments were divided into BE, control, VEGF, DAPT and VEGF-DAPT groups; HSC/HPC ohenotype: CD117+D34+Sca1+ was detected by flow cytometry; the related gene expression was detected by RT-PCR. RESULTS: The number of VEGF-induced HSC/HPC in VEGF group was significantey higher than that in the control and EB group (P<0.05), suggesting that VEGF promotes ESC differentiation to HSC/HPC; the number of DAPT-induced HSC/HPC in DAPT group was significanty higher than that in the Control and EB groups(P<0.05), suggesting that DAPT promotes ESC differentiation to HSC/HPC; the number of VEGF+DAPT-induced HSC/HPC in VEGF-DAPT group was significantly higher than that in VEGF and DAPT groups(P<0.05), suggesting that DAPT and VEGF play a synergistic role to promote differentiation of ESC into HSC/HPC. CONCLUSION: Notch signal pathway inhibits differentiation of ESC into HSC / HPC by VEGF.

Hematopoietic stem cell-derived exosomes promote hematopoietic differentiation of mouse embryonic stem cells in vitro via inhibiting the miR126/Notch1 pathway.

Cell-derived exosomes (EXs) can modulate target cell differentiation via microRNAs (miRs) that they carried. Previous studies have shown that miR126 is highly expressed in hematopoietic stem cells (HSCs) and plays a role in hematopoiesis via modulating the Notch pathway that participates in progenitors' cell fate decisions. In this study we investigated whether HSC-derived EXs (HSC-EXs) could affect the differentiation of mouse embryonic stem cells (ESCs) into HSCs. We prepared HSC-EXscon, HSC-EXssc and HSC-EXsmiR126 from control HSCs and the HSCs transfected with scramble control or miR126 mimics, respectively. HSC-EXs were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis. We incubated the collected EXs with mouse ESCs over a 10-d differentiation induction period, during which HSC-EXs and a Notch pathway activator (Jagged1, 100 ng/mL) were added to the cultures every 3 d. After the 10-d differentiation period, the expression levels of miR126, SSEA1, CD117, Sca1, Notch1 and Hes1 in ESCs were assessed. The generated HSCs were validated by flow cytometry using antibodies against HSC markers (CD117, CD34 and Sca1). Our results revealed that: (1) transfection with miR126 mimics significantly increased miR126 levels in HSC-EXsmiR126. (2) HSC-EX co-culture promoted mouse ESCs differentiation into HSCs with the most prominent effect found in the HSC-EXsmiR126 co-culture. (3) HSC differentiation was verified by reduced SSEA1 expression and increased CD117 and Sca1 expression. (4) All the effects caused by HSC-EXs were accompanied by significant reduction of Notch1 and Hes1 expression, thus inhibition of the Notch1/Hes1 pathway, whereas activation of Notch by Jagged1 abolished the effects of HSC-EXsmiR126. In conclusion, HSC-EXs promote hematopoietic differentiation of mouse ESCs in vitro by inhibiting the miR126/Notch1 pathway.

[Effect of notch signaling pathway on VEGF promoting rat mesenchymal stem cell proliferation].

This study was purposed to investigate the effect of Notch signaling pathway on VEGF promoting the proliferation of rat mesenchymal stem cells (MSC). Rat MSC were cultured in vitro, and the cells in logarithmic growth phase were used for experiments. The inhibitor DAPT was used to block Notch signaling pathway, and the effect of the pathway on VEGF promoting proliferation of MSC was observed. The experiment was divided into 4 groups: control, VEGF, DAPT and VEGF+DAPT. The CCK-8 was used to assay the cells proliferation of each group, while RT-PCR was used to detect the changes of related genes (Notch1, Notch2, Flk-1, Hes-1) at mRNA levels. The results indicated that the cells survival rate MSC in DAPT group and VEGF+DAPT group was low in each time point (24 h, 48 h, 72 h), the cell number decreased, and the cells became rounded. The survival rate of MSC in VEGF group was the highest; the difference of cell survival rate was statistically significant between the groups (P < 0.01); Compared with the control group, the mRNA expression level of Notch1, Notch2 and Flk-1 in VEGF group was raised, while the expression level of Notch1 and Notch2 in DAPT group and VEGF+DAPT group come down, with statistically significant differences (P < 0.05); whereas the mRNA expression level of Hes-1 in VEGF group was down-regulated, but that in DAPT group and VEGF+DAPT group was up-regulated, and the difference was statistically significant (P < 0.05). Flk-1 mRNA level in DAPT group and VEGF+DAPT group was slightly lower, but the difference was not statistically significant (P > 0.05). It is concluded that Notch signaling pathway plays an important role in promoting the proliferation of rat MSC, treated with VEGF, however, the DAPT can weaken this effect.

Studies on interaction between Vitamin B12 and human serum albumin.

The binding reaction between Vitamin B12 (B12, cyanocobalamin) and human serum albumin (HSA) was investigated by fluorescence quenching, UV-vis absorption and circular dichroism (CD) spectroscopy. Under simulative physiological conditions, fluorescence quenching data revealed that the quenching constants (Ksv) are 3.99 x 10(4), 4.33 x 10(4), 4.76 x 10(4) and 5.16 x 10(4) M(-1) at 292, 298, 304 and 310 K, respectively. The number of binding sites, n is almost constant around 1.0. On the basis of the results of fluorescence quenching the mechanism of the interaction of B12 with HSA has been found to be a dynamic quenching procedure. Thermodynamic parameters DeltaHTheta= -13.38 kJ mol(-1), DeltaSTheta=66.73 J mol(-1) K(-1) were calculated based on the binding constant. These suggested that the binding reaction was enthalpy and entropy driven, and the electrostatic interaction played major role in stabilizing the reversible complex. The binding distance r=5.5 nm between HSA and B12 was obtained according to Förster theory of energy transfer. The effect of B12 on the conformation of HSA was analyzed by synchronous fluorescence and CD spectroscopy. Synchronous spectra indicated that the polarity around the tryptophan (Trp214) residues of HSA was decreased and its hydrophobicity was increased; however, the alpha-helix content of the protein was predominant in the secondary structure but the CD spectra indicated that B12 induced minor conformational changes of HSA.

Effective treatment of manganese-induced occupational Parkinsonism with p-aminosalicylic acid: a case of 17-year follow-up study.

OBJECTIVE: Chronic manganese (Mn) intoxication induces syndromes resembling Parkinson disease. The clinical intervention has largely been unsuccessful. We report a 17-year follow-up study of effective treatment of occupational Mn parkinsonism with sodium para-aminosalicylic acid (PAS). METHODS: The patient, female and aged 50 at the time of treatment, was exposed to airborne Mn for 21 years (1963-1984). The patient had palpitations, hand tremor, lower limb myalgia, hypermyotonia, and a distinct festinating gait. She received 6 g PAS per day through an intravenous drip infusion for 4 days and rested for 3 days as one therapeutic course. Fifteen such courses were carried out between March and June 1987. RESULTS: At the end of PAS treatment, her symptoms were significantly alleviated, and handwriting recovered to normal. Recent follow-up examination at age 67 years (in 2004) showed a general normal presentation in clinical, neurologic, brain magnetic resonance imaging, and handwriting examinations with a minor yet passable gait. CONCLUSIONS: This case study suggests that PAS appears to be an effective drug for treatment of severe chronic Mn poisoning with a promising prognosis.