Characteristics of microsomal phase II metabolic enzymes in mouse embryonic stem cell-derived liver tissue / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the characteristics of phase II metabolic enzymes in mouse embryonic stem (ES) cell-derived liver tissue.</p><p><b>METHODS</b>Mature hepatocytes were differentiated from embryonic stem cells in cultured mouse embryoid bodies (EB) at d18. Western blot was used to detect the expression of uridine 5'-diphosphate glucronosyl transferase (UGT1a1,UGT1a6) and microsomal glutathione S-transferases 1(mGST1) during the differentiation course.The derived liver tissue was incubated with UDPGA and 7-HFC,the formation of 7-HFC glucuronide was detected by HPLC to examine the total activities of UGT1a1 and UGT1a6. Furthermore, the microsomes were incubated with CDNB and GSH,and the mGST1 activity was measured by spectrometry.</p><p><b>RESULTS</b>An increase tendency of UGT1a1 expression was noticed during the differentiation course. UGT1a6 and mGST1 were not detected in the earlier stage until d18 of differentiation. The metabolic activity of mGST1 in the derived hepatocytes was 7.65 nmol/min/mg on d18.</p><p><b>CONCLUSION</b>The ES cell-derived liver tissue possesses partial metabolic function of phase II enzymes on d18 of differentiation,which might be used as a model for in vitro research on hepatic pathophysiology and phase II drug metabolism.</p>

Expression of Junctophilin 1 during cardiogenesis of mouse embryonic stem cells and rat embryos / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the expression of Junctophilin 1 (JP1) in cardiogenesis of mammalian.</p><p><b>METHODS</b>Cardiac differentiation of embryonic stem cells (ESCs) was generated by hanging drop method. Fetal heart was obtained from the rats aged d 14-20 of gestation. The expression of JP1 and JP2 during cardiogenesis of ESCs and rat embryos was analyzed by RT-PCR or Western blotting. Immunofluorescence staining was employed to reveal the distribution of JP1 and JP2 in embryoid body (EB), probing for merging of JP1 and JP2 and cardiac sarcomeric α-Actinin or Troponin-T. Percentage of JP1 and JP2-positive staining cells was analyzed quantitatively by FCS on d17.</p><p><b>RESULTS</b>JP1 mRNA was up-regulated at the early stage (d 5-11) and then decreased. The expression of JP1 protein was up-regulated at the early stage (d 7-9), then decreased gradually and disappeared after d 15. While JP2 gene and protein expression increased in a time-dependent manner during cardiogenesis of rat embryos. The results of immunofluorescence staining showed that there was a parallel co-localization of JP2 with Troponin-T or α-Actinin on d17, while JP1 failed to express in the sarcomeric positive area at the same time point. Furthermore, FCS analysis showed that about 16.59% of cells were JP2-positive, while no cells were stained positively for JP1 in d17 EBs.</p><p><b>CONCLUSION</b>JP1 gene is expressed during the whole process of cardiogenesis, while JP1 protein only appears on the early stage. The expression of JP1 in cardiogenesis of ESCs is consistent with that of rat embryos.</p>

Expression of metabotropic glutamate receptor 4 in cardiomyocytes differentiated from mouse embryonic stem cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the expression of metabotropic glutamate receptor 4 (mGluR4) in cardiomyocytes differentiated from mouse embryonic stem cells (ES cells).</p><p><b>METHODS</b>ES cells were differentiated into cardiomyocytes with hanging-drop cultures. Retinoic acid (RA) and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. The co-expression of cardiac sarcomeric protein (α-actinin or troponin-T) and mGluR4 were verified by immunocytochemistry and flow cytometry analysis. The mRNA and protein expressions of mGluR4 were verified by RT-PCR and Western blot analysis, respectively. Meanwhile, the expression of mGluR4 in prenatal mouse heart was also examined.</p><p><b>RESULTS</b>mGluR4 was expressed in both mouse ES cells and ES cell-derived cardiomyocytes. The level of mGluR4 protein expression decreased during the maturation of the cardiomyocytes. The co-expression rate of mGluR4 and Troponin T in the beating embryoid bodies (EBs) was only (3.00 ±1.00)%. On the other hand, mGluR4 gene and protein expressions showed remarkable down-regulation in the development of mouse fetal heart, which was not detected in mouse adult heart.</p><p><b>CONCLUSION</b>The expression of mGluR4 is down-regulated in the cardiomyocyte differentiation of ES cells. The trend of expression is consistent with that in the prenatal mouse heart development.</p>

Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope.</p><p><b>METHODS</b>Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10(-6)mol/L) and Isobavachin (IBA, 10(-7)mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes.</p><p><b>RESULTS</b>The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons.</p><p><b>CONCLUSION</b>Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.</p>

Establishment of optimized neuronal differentiation-promoting model derived from P19 embryonic carcinoma cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish an optimized primary drug screen model of neuronal differentiation using P19 embryonal carcinoma cells.</p><p><b>METHODS</b>The final concentration of retinoid acid (RA), days of suspension culture, manner of adherent culture, suitable cell density and adherent culture medium were tested, respectively. Two stages of neuronal differentiation were examined based on morphological changes and immunocytochemistry analysis of neuronal specific protein β-tubulin III.</p><p><b>RESULTS</b>On d 8 of differentiation culture, neuron-like cells were observed with final concentration of 1 μmol/L RA. Neuron-like network was formed on d 16 of neuronal differentiation. β-tubulin III was positively stained on both stages, indicating P19 cells were differentiated into neurons.</p><p><b>CONCLUSION</b>The model using RA to induce P19 embryonic carcinoma cells to differentiate into neuron-like cells has been successfully established, which may provide a rapid, phenotypic cell-based platform for primary screening of neurogenesis-promoting drugs.</p>

Construction of directional differentiation model from mouse embryonic stem cells to Leydig-like cells in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct a directional differentiation model from mouse embryonic stem cells into leydig-like cells in vitro.</p><p><b>METHODS</b>Mouse ES-D3 cells were transfected with plasmid containing steroidogenic factor 1 (SF-1) gene, then treated with RA and 8Br-cAMP, while the cells transfected with empty plasmid were used as the negative controls. The morphology of leydig-like cells differentiated from ES-D3 cells was observed with light microscopy. The expression levels of StAR, P450scc and 3β-HSD were detected by RT-PCR, Western Blot and fluorescence microscopy analysis in leydig-like cells derived from the ES cells.</p><p><b>RESULTS</b>ES-D3 cells were transfected with plasmid containing SF-1 gene successfully, and SF-1 was expressed 24 h after transfection. The SF-1-transfected ES-D3 cells were induced by RA and 8Br-cAMP to differentiate into leydig-like cells. The differentiated cells showed spindle shape with tentacles, which expressed the specific protein marker for leydig cells 3β-HSD1 and P450scc. Meanwhile, in these leydig-like cells, the expression of StAR increased compared with control group. 3β-HSD1, P450scc and StAR were not detected in negative control group.</p><p><b>CONCLUSION</b>When the ES-D3 cells are transfected with SF-1 plasmid and then treated with RA and 8Br-cAMP, the cells are able to differentiate into leydig-like cells, indicating that the model of directional differentiation of ES cells into leydig-like cells has been constructed successfully.</p>

Ginsenoside Rg1 antagonizes β-amyloid peptide-induced apoptosis in primarily cultured rat neurons via mitochondrial pathway / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To assess the neuroprotective effects of ginsenoside Rg1 against β-amyloid peptide (Aβ(25-35))-induced apoptosis in primarily cultured rat cortical neurons.</p><p><b>METHODS</b>Primarily cultured cortical neurons were obtained from embryonic (E18d) rat fetus and maintained in neurobasal medium for 7d. Primary neurons pretreated with 1 μmol/L, 10 μmol/L or 20 μmol/L Rg1 for 24 h were challenged with 10 μmol/L Aβ(25-35) for 72 h. Morphological changes of neurons were evaluated; mitochondrial membrane potential (ΔΨm) was measured; with JC-1 staining and the expression of neural apoptosis-related proteins was detected by Western blot analysis.</p><p><b>RESULTS</b>Exposure to Aβ(25-35) for 72 h caused serious neural cell insults. A pretreatment with Rg1 significantly reduced Aβ(25-35)induced cell death in a dose-dependent manner, with a maximal effect (-90%) obtained at 20 μmol/L. The JC-1 staining results demonstrated the loss of ΔΨm after Aβ(25-35) treatment, while Rg1 maintained the normal level of ΔΨm. A series of mitochondrion-mediated apoptotic events happened after Aβ(25-35) treatment, such as decrease of Bcl-2/Bax, release of cytochrome C and activation of caspase 9 and caspase 3, which were all blocked by Rg1 pretreatment. Both estrogen receptor (ER) antagonist ICI182, 780 and glucocorticoid receptor (GR) antagonist RU486 blocked the antiapoptotic effects of Rg1.</p><p><b>CONCLUSION</b>Ginsenoside Rg1 protects primary cultured rat cortical neurons from Aβ(25-35)-induced injury, which may be associated with mitochondrion-mediated antiapoptosis pathway.</p>

Transcription of cytoskeleton protein genes in differentiation of neurons from mouse embryonic stem cells induced by small molecules / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the transcription of cytoskeleton protein genes in differentiation of neurons from mouse embryonic stem (ES) cells induced by all-trans retinoic acid (RA), and to explore the possibility of setting up a method to screen small molecules with promoting or inhibiting effect.</p><p><b>METHODS</b>The hanging drop method was employed for embryonic body formation to mimic embryo development in vivo. Reverse transcriptase PCR (RT-PCR) was performed to investigate mRNA expression of the neuron-specific cytoskeleton proteins including Mtap2, Nefm and beta-tubulin III which were regarded as the inducing effect indexes of RA. Morphological evaluation and immunocytochemistry staining were conducted to identify the neural derivatives. Moreover, the inducing effects of six synthetic molecules were further evaluated.</p><p><b>RESULT</b>RA up-regulated the mRNA expression of Mtap2 and Nefm, especially Mtap2 increased by 1.27 times, which was consistent with the morphological alteration. However, there was no significant changes of beta-tubulin III expression. With addition of the six synthetic molecules, the transcription of Mtap2 was inhibited, while the Nefm mRNA expression was up-regulated in some degree, especially for molecule 1 and 3 that was increased by 1.4 and 1.2 times, which, however, was not parallel to the morphological changes.</p><p><b>CONCLUSION</b>The transcriptional levels of Mtap2 and Nefm are both up-regulated in the RA-induced differentiation of ES cells towards neurons. The up-regulation of Mtap2 is consistent with the morphological alteration, which might be the key landmark in the RA-induced differentiation of ES cells into neurons.</p>

Effect of concanavalin A on expression of MAPEG mRNA in Balb/c mouse brain / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the gene expression of MAPEG in the cortex of concanavalin A (Con A)-induced mouse immune inflammatory model and the effect of cyclosporine A (Cs A).</p><p><b>METHODS</b>Male Balb/c mouse immune inflammation model was developed by intravenous injection of Con A (20 mg/kg). Cs A (150 mg/kg) was intravenously infected prior to Con A administration. The MAPEG expressions were determined by RT-PCR.</p><p><b>RESULT</b>mGST1, mGST3, LTC(4)S, FLAP and mPGES-1 were detected by RT-PCR but not mGST2. Eight hours after Con A treatment, mGST1 level was up-regulated to 1.2 approximately 1.5 folds of control with or without Cs A treatment. mGST3ìLTC(4)S, FLAP and mPGES-1 mRNA levels were not influenced by Con A administration.</p><p><b>CONCLUSION</b>Immune mechanism may be not involved in mGST1 up-regulation in this model and Con A does not alter arachidonic acid metabolism in cortex.</p>

Directed differentiation of mouse embryonic stem cells into neuronal cells induced by icaritin in vitro / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To evaluate the inductive effects of icaritin (ICT) on the directed differentiation of mouse embryonic stem (ES) cells into neuronal cells in vitro.</p><p><b>METHODS</b>ES cells were cultured with embryoid body (EB) formation cultures, ICT in different concentrations was added in the cultural media and the cells were harvested in several differentiation phases. The expression spectrums of neuronal cell-specific genes and proteins were verified by semi-quantitative RT-PCR and immunocytochemistry analysis, respectively.</p><p><b>RESULTS</b>Differentiation of neurocyte phenotype from ES cells was promoted by ICT in a concentration-and time-dependent manner. ICT at 10(-7)mol/L significantly enhanced the differentiation toward neuronal cells, and up to 80 % of EBs outgrowth in d 8+8 incubation. The gene expressions of beta-tubulin III in neuron and GFAP in glial cells were detected in neuronal cell phenotype derived from EBs. Furthermore, nestin was detected in precursor cells, beta-tubulin III and GFAP were detected in the generated precursor neurocytes immunocytochemically.</p><p><b>CONCLUSION</b>Directed differentiation of neurons is facilitated by ICT in EB formation culture, which is associated with the expression of developmental-dependent gene and protein.</p>

Gene expressions of phase II enzymes in mouse embryonic stem cell-derived hepatocytes / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To elucidate gene expressions of phase II enzymes in the mouse embryonic stem (ES) cell-derived hepatocytes.</p><p><b>METHODS</b>Embryoid body (EB) formation cultures were applied in directed differentiation of ES to hepatic-like cells. The expressions of hepatic-specific genes, including AFP, ALB, Cyp7a1, were detected by RT-PCR during the differentiation course. Albumin was detected by immunocyto- chemistry. The gene expressions of mGST1 and UGTs family, including UGT1a1, UGT1a6, UGT1a9 and UGT2b5, were investigated using RT-PCR.</p><p><b>RESULTS</b>A notable gene expression of AFP and ALB was observed on d 8. On d 18, AFP gene failed to express, while ALB and Cyp7a1 genes were detected.Albumin-positive staining was detected in hepatic-like cells. Phase II enzyme genes expressed in variance during the differentiation course, UGT1a1 and UGT1a9 were expressed stably, UGT1a6 expression increased gradually, and UGT2b5 failed to express. Little mGST1 gene expression could been detected in the early course until d 18. In addition, all the enzymes gene expressions in the derived hepatocytes on d 18 were similar to those from mature mouse hepatocytes.</p><p><b>CONCLUSIONS</b>Mouse ES cell-derived mature hepatocytes express phase II enzyme UGTs and mGST1 genes similar to those in mature hepatocytes. The system may offer an alternative animal testing model related to phase enzymes in further research.</p>