光电成像系统在评价胚胎干细胞体外定向分化为心肌细胞中的应用

目的采用简易光电成像系统记录胚胎干细胞(ES细胞)体外定向分化心肌细胞的搏动频率,为药物诱导ES细胞体外定向分化心肌细胞提供量化评价指标。方法以简易光电成像系统记录心肌细胞搏动频率,并以维A酸和淫羊藿苷诱导ES细胞定向分化心肌细胞为例,收集分化过程中发育依赖性基因(α-肌球蛋白重链、心室肌球蛋白轻链及β-肾上腺素受体)和心肌特异性蛋白(α-辅肌动蛋白和肌钙蛋白T)表达情况,同步分析光电成像信号与分子生物学指标的一致性。结果光电成像系统可灵敏反映分化心肌细胞的搏动频率,在与维A酸和淫羊藿苷共培养体系中,搏动频率与心肌发育依赖性基因、蛋白表达和β-肾上腺素受体形成等一致,可体现心肌分化的不同时间段。结论简易光电成像系统能灵敏记录ES细胞定向分化心肌细胞的搏动频率,此搏动频率与细胞分化成熟程度一致,可望成为药物诱导分化心肌细胞初步评价的量化指标。     

Icariin promotes expression of PGC-1α, PPARα, and NRF-1 during cardiomyocyte differentiation of murine embryonic stem cells in vitro

AIM: To investigate the effect of icariin on the expression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1alpha), peroxisome proliferator-activated receptor alpha (PPARalpha), and nuclear respiratory factor 1 (NRF-1) on cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro. METHODS: The cardiomyocytes derived from murine ES cells were verified by immunocytochemistry using confocal laser scanning microscopy. Cardiac-specific sarcomeric proteins (ie alpha-actinin, troponin T) were evaluated when embryoid bodies (EB) were treated with icariin or retinoid acid. The expression of PGC-1alpha, PPARalpha, and NRF-1 were analyzed using both semiquantitative RT-PCR and Western blotting in cardiomyocyte differentiation. The phosphorylation of the p38 mitogen-activated protein kinase (MAPK) was studied in the differentiation process, and its specific inhibitor SB203580 was employed to confirm the function of the p38 MAPK on icariin-induced cardiac differentiation. RESULTS: The application of icariin significantly induced the cardiomyocyte differentiation of EB as indicated by the promoted expression of alpha-actinin and troponin T. The expression of PGC-1alpha, PPARalpha, and NRF-1 increased coincidently in early differentiation and the increase was dose-dependently upregulated by icariin treatment. The phosphorylation of the p38 MAPK peaked on d 6 and decreased after d 8, and the activation was further enhanced and prolonged when the EB were subjected to icariin, which was concurrent with the elevation of PGC-1alpha, PPARalpha, and NRF-1. Moreover, the inhibition of the p38 MAPK pathway by SB203580 efficiently abolished icariin-stimulated cardiomyocyte differentiation and resulted in the capture of the upregulation of PGC-1alpha, PPARalpha, and NRF-1. CONCLUSION: Taken together, icariin promoted the expression of PGC-1alpha, PPARalpha, and NRF-1 during cardiomyocyte differentiation of murine ES cells in vitro and the effect was partly responsible for the activation of the p38 MAPK.     

miR-149-3p在先天性心脏病胎鼠心脏组织中的表达及其对P19细胞分化的影响

目的 探讨miR-149-3p在先天性心脏病（CHD）胎鼠心脏组织中的表达及其对小鼠畸胎瘤P19细胞分化的影响。方法 建立CHD室间隔缺损胎鼠模型,于妊娠第19天,HE染色观察心脏组织病理学变化,实时荧光定量PCR(qPCR)检测心脏组织中miR-149-3p和热休克蛋白蛋白家族B成员6(HSPB6)mRNA表达水平。二甲基亚砜（DMSO）诱导P19细胞向心肌细胞分化,并收集分化第0、5、10天的细胞,qPCR检测诱导分化过程中心肌分化标志物GATA结合蛋白4(GATA4)、心肌肌钙蛋白T(cTnT)、心房利钠尿多肽（ANP）的mRNA表达水平以及HSPB6 mRNA和miR-149-3p表达水平。miR-149-3p过表达慢病毒和空载慢病毒感染P19细胞,DMSO诱导分化第10天,免疫荧光染色检测细胞中cTnI蛋白表达情况,qPCR检测心肌分化相关标志物mRNA表达水平。双荧光素酶报告基因实验验证miR-149-3p与HSPB6之间的靶向关系。结果 与正常组比较,CHD组胎鼠出现心脏室间隔缺损,心脏组织中miR-149-3p高表达,而HSPB6低表达（P<0.01）。P19细胞...     

6-Mercaptopurine (6-MP) induces cell cycle arrest and apoptosis of neural progenitor cells in the developing fetal rat brain

6-Mercaptopurine (6-MP), an analogue of hypoxanthine, is used in the therapy of acute lymphoblastic leukemia and causes fetal neurotoxicity. To clarify the mechanisms of 6-MP-induced fetal neurotoxicity leading to the cell cycle arrest and apoptosis of neural progenitor cells, pregnant rats were treated with 50 mg/kg 6-MP on embryonic day (E) 13, and the fetal telencephalons were examined at 12 to 72 h (h) after treatment. Flow-cytometric analysis confirmed an accumulation of cells at G2/M, S, and sub-G1 (apoptotic cells) phases from 24 to 72 h. The number of phosphorylated histone H3-positive cells (mitotic cells) decreased from 36 to 72 h, and the phosphorylated (active) form of p53 protein, which is a mediator of apoptosis and cell cycle arrest, increased from 24 to 48 h. An executor of p53-mediated cell cycle arrest, p21, showed intense overexpression at both the mRNA and protein levels from 24 to 72 h. Cdc25A protein, which is needed for the progression of S phase, decreased at 36 and 48 h. In addition, phosphorylated cdc2 protein, which is an inactive form of cdc2 necessary for G2/M progression, increased from 24 to 48 h. These results suggest that 6-MP induced G2/M arrest, delayed S-phase progression, and finally induced apoptosis of neural progenitor cells mediated by p53 in the fetal rat telencephalon.     

Induction of PUMA-α and down-regulation of PUMA-β expression is associated with benzo(a)pyrene-induced apoptosis in MCF-7 cells

Benzo(a)pyrene (BP) forms benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE)-DNA adducts in human breast adenocarcinoma MCF-7 cells, leading to p53 protein induction and phosphorylation. Although BP-induced apoptosis in rodent cells is known, it is still unclear in human cells. Here we have analyzed the effects of BP on p53 related apoptotic proteins, cell cycle and cell death in MCF-7 cells. PUMA-protein (p53 up-regulated modulator of apoptosis) levels were changed after BP exposure so that PUMA-α protein was statistically significantly increased whereas PUMA-β protein was statistically significantly decreased. PUMA-protein levels were also investigated in ZR-75-1 cells, where PUMA-α protein was statistically significantly increased. Cytochrome c, which is released from mitochondria during apoptosis to form the apoptosome, was increased in cytoplasmic fraction after BP exposure in MCF-7 cells. Increased apoptosis was also seen after 48 and 72 h BP exposure (2.5 and 5 μM). In addition, BP decreased dose dependently cell viability (2.5 and 5 μM) and increased ROS formation (1 and 10 μM). Our results suggest that PUMA-α protein is involved in BP-induced cell death most likely through a p53 dependent apoptotic pathway.     

Application of flow cytometry in the study of apoptosis in neonatal rat cardiomyocytes

Depending on the concentration, catecholamines activate various intracellular signaling pathways and can induce apoptosis in cardiac myocytes. Although 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1) has been previously used to study mitochondria in intact cardiomyocytes, there have been no reports on the detection of apoptosis in neonatal cardiomyocytes in combination with flow cytometry and confocal microscopy. In our study, neonatal rat cardiomyocytes were exposed to norepinephrine (NE) and isoproterenol (ISO) in concentrations of 1 and 10 microM for 48 h. NE concentrations of 1 and 10 microM decreased the number of viable cardiomyocytes by 18% (*p < 0.05) and 24% (**p = 0.01), respectively. ISO in a concentration of 1 microM increased the number of viable cardiomyocytes by 13% while 10 microM decreased the number of viable cardiomyocytes by 43% (***p < 0.001). Apoptotic cells were detected by flow cytometry and confocal microscopy. NE in concentrations of 1 and 10 microM increased the percentage of apoptotic cells by 12.2% and 34.3%, respectively, while ISO alone in a concentration of 10 microM increased the percentage of apoptotic cells by 11.3%. The results demonstrated that these two methods are reliable and suitable for the detection and study of apoptosis in cultures of neonatal cardiomyocytes.     

Effects of cigarette smoke components on myocardial differentiation of mouse embryonic stem cells

The heart is the first organ formed in the developing fetus, and abnormal development of the heart is a major cause of fetal death. The adverse effects of cigarette smoke on the heart have been well established, but it is not well understood how cigarette smoke components regulate signaling molecules and cardiac specific functions during the early differentiation stage of the embryonic heart. In this study, we identified changes in the size of mouse embryoid bodies (mEBs) in response to treatment with cigarette smoke extract (CSE) via regulation of HDAC2, p53, p21, and cyclin D1 protein expression, which are cardiac differentiation and cell‐cycle markers, respectively. In addition, exposure of mouse embryonic stem cells (mESCs) to cigarette smoke components inhibited myocardial differentiation and development through the expression of HDAC1, HDAC2, GATA4, NKX2‐5, TBX5, HAND1, and Troponin I. Long‐term exposure studies showed that CSE and nicotine may delay the development of mouse cardiomyocytes from mESCs and inhibit the contractibility, which is a fundamental function of the heart. Taken together, these findings suggest that cigarette smoke components, including nicotine, may affect abnormal myocardial differentiation and development.     

In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles

While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes.Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering.Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 μg/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested.Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis.     

Adrenomedullin upregulates M2-muscarinic receptors in cardiomyocytes from P19 cell line

1. The effects of AM on expression of muscarinic (M) receptors from P19-derived cardiomyocytes were examined. 2. RT-PCR experiments revealed expression of M(1)-M(4) receptor genes. Immuno-histochemistry indicated that M(2) expression is restricted to contractile cells. Carbachol inhibition of isoprenaline-induced increase in beating rate was prevented by atropine and methoctramine (pA(2): 8.1). Inhibition of [(3)H]-NMS binding by atropine (pK(i): -8.4+/-0.2) and methoctramine (pK(i): -8.3+/-0.2) suggests that M(2) is the functional expressed isoform. 3. [(3)H]-NMS binding and semiquantitative RT-PCR studies showed a dome shaped time course of M(2) expression with a maximum at 7 days of differentiation followed by a progressive decline. 4. AM concentration-dependently upregulated M(2) receptor mRNA during late differentiation stages in P19 cells but also in rat atrial cardiomyocytes. This effect was potentiated by factor H. AM (100 nM) plus factor H (50 nM) treatment of P19 cells for 24 h significantly increased [(3)H]-NMS-specific binding (B(max): 81+/-7 vs 31+/-6 fmol mg(-1) prot). The effect of AM on mRNA levels was prevented by AM receptor antagonist AM(22-52) (1 micro M) but not by CGRP antagonist, CGRP(8-37) (1 micro M). 5. The mRNA levels encoding CRLR receptor declined with culture duration, whereas those encoding L1/G10D receptor remained stable. 6. Our findings demonstrate that AM regulates M(2) receptors expression in cardiomyocytes probably through a mechanism involving L1/G10D receptors. The 'in vivo' significance of this phenomenon remains to be demonstrated.     

Identification of a small molecule 1,4-bis-[4-(3-phenoxy-propoxy)-but-2-ynyl]-piperazine as a novel inhibitor of the transcription factor p53

Aim:

To identify novel small compound inhibitor of p53 protein.

Methods:

Mouse embryonic fibroblasts (MEF) and mouse embryonic stem (ES) cells were tested. Cell proliferation rate was determined using a Cell Proliferation Kit. The mRNA and protein levels of p53-related genes were measured using real-time PCR and Western blotting, respectively. Global response in the p53 signaling network was analyzed using Illumina whole-genome expression BeadChips.

Results:

Treatment of MEF cells with a small molecule 1,4-bis-[4-(3-phenoxy-propoxy)-but-2-ynyl]-piperazine (G5) at 10 μmol/L for 24 h markedly reduced the mRNA and protein levels of the p53 downstream genes MDM2 and p21. In G5-treated ES cells, a total of 372 differentially expressed genes were identified, and 18 among them were direct downstream genes of p53; 6 out of 9 p53-repressed genes were upregulated, and 5 out of 9 p53-activated genes were downregulated. In both MEF cells and ES cells, treatment of with G5 (10 μmol/L) up to 48 h neither affected the proliferation rate nor caused morphological alterations.

Conclusion:

G5 inhibits p53 activity and simultaneously preserves the normal growth and proliferation of cells, therefore is a new compound for studies of p53-mediated cell manipulation.     

蛇床子素通过p53信号通路诱导人乳腺癌细胞株MCF-7凋亡

目的 探讨蛇床子素对人乳腺癌细胞株MCF-7的作用及其机制。方法 对数生长期的MCF-7 细胞用0,25,50,100 mmol·L^-1的蛇床子素处理。细胞培养48 h后,利用MTT检测细胞增殖;流式检测细胞凋亡和膜电位。RT-PCR检测p53、p21、BCL-2、Bax和CytC mRNA水平;免疫印迹法检测p53、p21、BCL-2、Bax 和CytC 表达。结果 蛇床子素可以成浓度依赖性诱导MCF-7 细胞增殖抑制和凋亡,上调Bax、p53、p21和CytC表达,下调 Bcl-2的表达以及细胞膜电位。结论 蛇床子素可通过激活p53信号通路而诱导MCF-7细胞凋亡。     

Comparison of Matrigel and gelatin substrata for feeder-free culture of undifferentiated mouse embryonic stem cells for toxicity testing.

Murine embryonic stem (mES) cells have been used to evaluate cytotoxicity and developmental injury following exposure to embryotoxic agents. However, maintaining a homogeneous population of undifferentiated mES cells for this purpose has been complicated by the need for continuous co-culture with murine embryonic fibroblast (mEF) cells or limited passaging on plastic surfaces coated with gelatin. Here, we compare the synthetic basement membrane Matrigel with 0.1% gelatin substratum for feeder-free propagation of undifferentiated mES cells. Biomarkers of pluripotentiality, chromosome number, caspase-3 expression, and cardiomyocyte differentiation were monitored for mES cells cultured on Matrigel or 0.1% gelatin up to passage 7 (P7). Our results suggest that choice of substratum had no significant effect on population doubling time, cell viability, stage-specific embryonic antigen-1 (SSEA-1) expression, or early passage formation of beating cardiomyocytes (all P>or=0.09). In other comparisons, however, Matrigel supported significantly higher synthesis of alkaline phosphatase (7.7x10(-3)+/-0.8 vs 6.6x10(-3)+/-0.8 units/liter/cell, respectively, P=0.012), overall expression of activated caspase-3 following exposure to 5, 10, 50, 100 and 500 parts per billion (ppb) sodium arsenite (P<0.0001), and percent development to beating cardiomyocytes at P7 (P=0.01). Together, our findings suggest that Matrigel shows promise as a substrate for feeder-free propagation of undifferentiated mES cells for embryotoxicity endpoints.     

Quercetin-mediated cell cycle arrest and apoptosis involving activation of a caspase cascade through the mitochondrial pathway in human breast cancer MCF-7 cells

Dietary polyphenols have been correlated with a reduced risk of developing cancer. Quercetin (a natural polyphenolic compound) induced apoptosis in many human cancer cell lines, including breast cancer MCF-7 cells. However, the involvement of possible signaling pathways and the roles of quercetin in apoptosis are still undefined. The purpose of this study was to investigate the effects of quercetin on the induction of the apoptotic pathway in human breast cancer MCF-7 cells. When MCF-7 cells were treated with quercetin for 24 and 48 h and at various doses (10–175 μM), cell viability decreased significantly in time- and dose-dependent manners. Exposure of MCF-7 cells to 10–175 μM quercetin resulted in an approximate 90.25% decrease in viable cells. To explicate the mechanism underlying the antiproliferative effect of quercetin, cell cycle distribution and apoptosis in MCF-7 cells was investigated after exposure to 150 μM quercetin for 6–48 h. Quercetin caused a remarkable increase in the number of S phase (14.56% to 61.35%) and sub-G1 phase cells (0.1% to 8.32%) in a dose- and time-dependent manner. Quercetin caused S phase arrest by decreasing the protein expression of CDK2, cyclins A and B while increasing the p53 and p57 proteins. Following incubation with quercetin for 48 h, MCF-7 cells showed apoptotic cell death by the decreased levels of Bcl-2 protein and ΔΨ _m and increased activations of caspase-6, -8 and -9. Moreover, quercetin increased the AIF protein released from mitochondria to nuclei and the GADD153 protein translocation from endoplasmic reticulum to the nuclei. These data suggested that quercetin may induce apoptosis by direct activation of the caspase cascade through the mitochondrial pathway in MCF-7 cells.     

Effect of alisol B acetate, a plant triterpene, on apoptosis in vascular smooth muscle cells and lymphocytes.

Glucocorticoid-induced apoptosis is a well-recognized physiological regulator of T-cell number and function. Alisol B acetate, a triterpene from Alisma Plantago-aquatica, has a glucocorticoid-like structure, and may have a similar function like glucocorticoid-induced apoptosis in both vascular smooth muscle cell line (A7r5) and human acute lymphoblastic leukemia cell line (CEM cells). For exploring its mechanism, mitochondria membrane potential and apoptosis-related gene expression were discussed. Alisol B (10(-6)-10(-4) M) inhibited serum-stimulated DNA synthesis in a concentration-dependent manner (IC50) = 4.0 +/- 0.8 x 10(-6) M in A7r5 and 2.1 +/- 1.2 x 10(-6) M in CEM cells). The cell viability was reduced at 10(-4) M of alisol B. Similar results were seen in dexamethasone treatment (a synthetic glucocorticoid, 10(-6) M, 48 h). Apoptosis was induced after the cells were exposed to 10(-5)-10(-4) M alisol B or 10(-6) M dexamethasone for 48 h. The mitochondrial membrane potential (delta psi(m)) was significantly reduced after the alisol B treatment, indicating that the mitochondria might play a role in the alisol B induced cell apoptosis. Alisol B (10(-5)-10(-4) M) increased the levels of c-myc and bax mRNA and proteins, but not on the anti-apoptotic proto-oncogene, bcl-2, in A7r5 and CEM cells. In contrast, dexamethasone (10(-6) M) treatment only caused significant increase in c-myc mRNA levels. These results suggest that the increased ratio of Bax/Bcl-2 and the decreased mitochondrial membrane potential might be involved in the mechanisms of alisol B-induced cell apoptosis.     

First steps in establishing a developmental toxicity test method based on human embryonic stem cells.

The use of embryonic stem cells is currently the most promising approach to assess developmental toxicity in vitro. In addition, the possibility of using human embryonic stem (hES) cells will increase safety of consumers and patients as false classification of substances due to inter-species variations can be avoided. One validated test based on murine embryonic stem cells, the embryonic stem cell test (EST), consists of following endpoints: IC(50) values of fibroblasts and embryonic stem cells as well as the inhibition of differentiation of mES cells into cardiomyocytes. As a follow up of its successful validation study we established a cytotoxicity assay based on hES cells and human fibroblasts employing two developmental toxicants: 5-fluorouracil (5-FU) and all-trans retinoic acid (RA). The results were compared to historical data from the EST. For 5-FU, no significant differences were obtained between the different cell lines. However, for RA, both test systems produced higher IC(50) values for the fibroblasts than for the stem cells, which is a well-known effect of developmental toxicants. Moreover, the reliability and relevance of several marker genes as possible toxicological endpoints were tested. During early differentiation Oct-4, hTert and Dusp6 showed the most reliable results. Brachyury and GATA-4 were found to be best suited to monitor cardiac differentiation. The late cardiac marker gene TNNT2 demonstrated significant results until day 18. Therefore, these marker genes have the highest potential to serve as endpoints for a developmental toxicity test.