Sestrin2 mediates FOXM1 expression to block the EMT process in non-small cell lung cancer through the AMPK/YAP pathway.

Non-small cell lung cancer (NSCLC) is characterized by high incidence and mortality, severely threatening human health. The infinite growth and metastasis of NSCLC cells result in a poor prognosis. Therefore, our study was to investigate the mechanism of Sestrin2 on the epithelial-mesenchymal transition (EMT) process of NSCLC cells. Human embryonic lung fibroblasts, NSCLC cell lines, and nude mice were experimental subjects in this study. qRT-PCR and western blot were performed to evaluate the mRNA and protein expression of genes. CCK-8 and EdU assay were conducted to detect cell proliferation. The scratch test and Transwell assay were applied to examine cell migration and invasion. The bioinformatics analysis and Co-IP assay were employed to predict and consolidate the interaction between YAP and TEAD. We found the expression of Sestrin2 was declined but the expression of YAP was elevated in NSCLC cells. Sestrin2 sufficiency or YAP silencing could effectively impair cell growth and metastasis. Mechanistically, YAP interacted with TEAD to enhance FOXM1 expression. Additionally, the elevation of FOXM1 abolished the inhibitory influences of Sestrin2 sufficiency on NSCLC cell growth, invasion, and EMT process. Eventually, Sestrin2 elevation attenuated tumor growth in mice via modulation of the AMPK/YAP/FOXM1 axis, which was reversed by FOXM1 overexpression. Our consequences suggested Sestrin2 could inhibit the activation of YAP via prompting AMPK phosphorylation and then suppress FOXM1 expression through the interplay between YAP and TEAD to impair the capacities of NSCLC cell proliferation, migration, invasion, and EMT. This study provided a novel mechanism of Sestrin2 in NSCLC.

[Correlation of K-ras Gene Mutations with the Protein Expressions of TAK1 and MAP4K2 in Colorectal Cancer].

OBJECTIVE: To analyze the correlation of K-ras gene mutations with the protein expressions of transforming growth factor-ß activating kinase 1 (TAK1) protein and mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2) protein in colorectal cancer. METHODS: K-ras gene mutations were detected by DNA sequencing analysis, and the expressions of TAK1 protein and MAP4K2 protein were detected by immunohistochemical method in 76 cases of colorectal cancer tissues. RESULTS: In 76 cases of colorectal cancer tissues, the mutation rate of K-ras gene was 32.89% (25 cases), and K-ras gene mutations were correlated with the degrees of cell differentiation ( P<0.05). The positive rates of TAK1 protein and MAP4K2 protein were 48.68% and 46.05%, respectively. The protein expressions of TAK1 and MAP4K2 were positively correlated with the degrees of cell differentiation and lymph node metastases, respectively ( P<0.05). There was no correlation between K-ras gene mutation and either TAK1 protein or MAP4K2 protein expression ( P>0.05). In 25 cases of colorectal cancer with K-ras mutation, the expression of TAK1 protein was positively correlated with the expression of MAP4K2 protein ( P<0.05). CONCLUSION: K-ras gene mutation, TAK1 and MAP4K2 protein expressions were related to the degree of differentiation of colorectal cancer, but not to the depth of invasion. In colorectal cancer with K-ras gene mutation, the expression of TAK1 protein was positively correlated with the expression of MAP4K2 protein.

TRPV6 is a prognostic marker in early-stage cervical squamous cell carcinoma.

Transient receptor potential vanilloid 6 (TRPV6) has been shown to promote caner proliferation in several solid tumors, leading to unfavorable clinical outcomes. Our study aimed to elucidate the clinical significance of TRPV6 in patients with early-stage cervical squamous cell carcinoma (CSCC). The mRNA expression of TRPV6 was measured in 12 paired early-stage CSCC specimens and six cervical carcinoma cell lines using quantitative real-time PCR (qRT-PCR). Western blotting and immunohistochemistry (IHC) were employed to examine the protein expression level of TRPV6 in four paired specimens, 175 paraffin-embedded early-stage CSCC specimens, and 50 normal cervical tissues (NCTs), respectively. Statistical analyses were performed to evaluate the clinical significance of TRPV6 expression. The expressions of TRPV6 mRNA and protein were both significantly downregulated in early-stage CSCC tissues and cervical cancer cell lines. IHC analyses revealed that TRPV6 was downregulated in 136 (77.7 %) of 175 early-stage CSCC specimens. Moreover, TRPV6 expression in early-stage CSCC was significantly correlated with the tumor stage (P < 0.001), tumor growth type (P < 0.001), tumor size (P = 0.008), and differentiation grade (P = 0.003). The early-stage CSCC patients with a low TRPV6 expression level had a short progress-free survival (PFS) and overall survival (OS) duration. Univariate and multivariate analyses identified TRPV6 as an independent prognostic factor for early-stage CSCC patients' survival. We demonstrated that TRPV6 was downregulated in CSCC, which was correlated with unfavorable survival outcomes of early-stage CSCC patients. TRPV6 may be used as a novel prognostic marker for early-stage CSCC.

Acylglycerol kinase is over-expressed in early-stage cervical squamous cell cancer and predicts poor prognosis.

Acylglycerol kinase (AGK) had been shown to contribute to cancer progression and unfavorable clinical outcomes of patients. Our study aimed to investigate the expression pattern and clinical significance of AGK in patients with early-stage cervical squamous cell cancer (CSCC). The protein and messenger RNA (mRNA) expression of AGK was analyzed in six cervical cancer cell lines and four paired early-stage CSCC specimens and normal cervical tissues (NCT), using Western blotting and real-time PCR (RT-PCR). And we investigated the AGK protein expression in paraffin-embedded specimens from 140 patients with early-stage CSCC and 30 cases of NCT by immunohistochemistry (IHC). Statistical analyses were performed to evaluate the clinicopathological significance of AGK expression. The expressions of AGK protein and mRNA were significantly up-regulated in cervical cancer cell lines and cancer tissues. IHC analyses revealed that AGK was highly expressed in 93 (66.4 %) of 140 early-stage CSCC specimens, but in none of the NCT. Moreover, AGK expression in early-stage CSCC was significantly correlated with tumor stage (P < 0.001), tumor size (P < 0.001), and tumor type (P < 0.001). Early-stage CSCC patients with high AGK expression level had shorter progress-free survival (PFS) and overall survival (OS) time compared with patients with low AGK expression levels. Univariate and multivariate analyses identified AGK expression level as an independent prognostic factor for survival of early-stage CSCC patients. We showed that AGK was over-expressed in cervical cancer cell lines and clinical tissues, and over-expression of AGK was associated with poor survival outcomes of early-stage CSCC patients. AGK can be used as an independent prognostic marker for early-stage CSCC.

Generation of site-specific mutant mice using the CRISPR/Cas9 system.

The CRISPR/Cas9 system is a recently developed important technology for genome editing in cellular and animal models. Here we established a CRISPR/Cas9-based system of generating site-specific mutant mice using DNA double-strand breaks (DSBs) induced homologous recombination (HR)-dependent or independent repair mechanism. Through co-microinjection of Cas9 mRNA and single-guide RNA (sgRNA) targeting genomic DNA sequence corresponding to enzyme activity of lysine (K)-specific demethylase 2b (Kdm2b), both a frame-shifted Kdm2b null mutant and a Kdm2b enzyme activity disrupted mouse strain were obtained simultaneously. Moreover, sgRNA targeting flavin containing monooxygenases3 (Fmo3) gene and the corresponding single strand oligonucleotides (ssODN) donor template with point mutation were co-injected into the male pronucleus of one-cell mouse embryos stimulated HR-mediated repair mechanism. Genomic sequence analysis of F0 mice showed that frame-shifted Fmo3 knockout mouse and site-specific Fmo3 knock-in mouse with single base substitution were successfully generated, and these mutations could be stably transmitted to the next generation. Therefore, we successfully generated mouse strains containing site-specific mutations through HR-dependent and -independent DSB repair using the CRISPR/Cas9 system.

Optimization of seeding density of OP9 cells to improve hematopoietic differentiation efficiency.

BACKGROUND: OP9 mouse stromal cell line has been widely used to induce differentiation of human embryonic stem cells (hESCs) into hematopoietic stem/progenitor cells (HSPCs). However, the whole co-culture procedure usually needs 14-18 days, including preparing OP9 cells at least 4 days. Therefore, the inefficient differentiation system is not appreciated. We aimed to optimize the culture conditions to improve differentiation efficiency. METHODS: In the experimental group, we set six different densities of OP9 cells and just cultured them for 24 h before co-culture, and in the control group, OP9 cells were cultured for 4 days to reach an overgrown state before co-culture. Then we compared the hematopoietic differentiation efficiency among them. RESULTS: OP9 cells were randomly assigned into two groups. In the experimental group, six different plated numbers of OP9 cells were cultured for 1 day before co-culture with hESCs. In contrast, in the control group, OP9 cells were cultured for 4 days at a total number of 3.1 × 104 cells/cm2 in a 6-well plate to reach an overgrown state before co-culture. Hematopoietic differentiation was evaluated with CD34 immunostaining, and compared between these two groups. We could not influence the differentiation efficiency of OP9 cells with a total number of 10.4 × 104 cells/cm2 in a 6-well plate which was cultured just for 1 day, followed by co-culture with hESCs. It reached the same differentiation efficiency 5 days earlier than the control group. CONCLUSION: The peak of CD34 + cells appeared 2 days earlier compared to the control group. A total number of 1.0 × 106 cells in a 6-well plate for OP9 cells was appropriate to have high differentiation efficiency.

Enhanced expression of RAGE/NADPH oxidase signaling pathway and increased level of oxidative stress in brains of rats with chronic fluorosis and the protective effects of blockers.

This investigation was designed to examine the potential involvement of RAGE/NADPH oxidase signaling in the damage to the brain caused by chronic fluorosis. Sprague-Dawley rats were divided randomly into 9 groups each containing 20 animals, Controls (C); rats receiving low (i.e., 10 ppm) (LF) or high does ( i.e., 50 ppm) (HF) of fluoride in their drinking water; and these same groups injected with FPS-ZM1, an inhibitor of RAGE, (CF, LFF and HFF, respectively) or administered EGb761, an active ingredient of Ginkgo biloba extract, intragastrically (CE, LFE, and HFE). Following 3 and 6 months of such treatment, the spatial learning and memory of the animals were assessed with the Morris water maze test; the levels of malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide dismutase (SOD) assayed by biochemical methods; and the levels of proteins related to the RAGE/NADPH pathway determined by Western blot and of the corresponding mRNAs by qPCR. After 6 months, the spatial learning and memory of the LF and HF groups had declined; their brain contents of MDA and H2O2 increased and SOD activity decreased; and the levels of the RAGE, gp91, P47, phospho-P47phox and P22 proteins and corresponding mRNAs in their brains were all elevated. Interestingly, all of these pathological changes caused by fluorosis could be attenuated by both FPS-ZM1 and EGb761. These findings indicate that the brain damage induced by fluorosis may be caused, at least in part, by enhanced RAGE/NADPH oxidase signaling and that FPS-ZM1 or EGb761 might be of clinical value in connection with the treatment of this condition.

Extract of Ginkgo biloba leaves attenuates neurotoxic damages in rats and SH-SY5Y cells exposed to a high level of fluoride.

BACKGROUND: Potential protection against the neurotoxic damages of high levels of fluoride on rats and SH-SY5Y cells by extract of Ginkgo biloba leaves, as well as underlying mechanisms, were examined. METHODS: The rats were divided randomly into 4 groups, i.e., control, treatment with the extract (100 mg/kg body weight, gavage once daily), treatment with fluoride (50 ppm F- in drinking water) and combined treatment with both; SH-SY5Y cells exposed to fluoride and fluoride in combination with the extract or 4-Amino-1,8-naphthalimide (4-ANI), an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1). Spatial learning and memory in the rats were assessed employing Morris water maze test; the contents of fluoride in brains and urine by fluoride ion-selective electrode; cytotoxicity of fluoride was by CCK-8 kit; the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) by appropriate kits; the level of 8-hydroxydeoxyguanosine (8-OHdG) was by ELISA; the content of ROS and frequency of apoptosis by flow cytometry; the expressions of phospho-histone H2A.X(Ser139), PARP-1, poly (ADP-ribose) (PAR) and Sirtuin-1 (SIRT1) by Western blotting or immunofluorescence. RESULTS: The rats with prolong treatment of fluoride exhibited dental fluorosis, the increased contents of fluoride in brains and urine and the declined ability of learning and memory. In the hippocampus of the rats and SH-SY5Y cells exposed to fluoride, the levels of ROS, MDA, apoptosis, 8-OHdG and the protein expressions of histone H2A.X(Ser139), PARP-1 and PAR were all elevated; the activities of SOD and GSH-Px and the protein expression of SIRT1 reduced. Interestingly, the treatment of Ginkgo biloba extract attenuated these neurotoxic effects on rats and SH-SY5Y cells exposed to fluoride and the treatment of 4-ANI produced a neuroprotective effect against fluoride exposure. CONCLUSION: Ginkgo biloba extract attenuated neurotoxic damages induced by fluoride exposure to rats and SH-SY5Y cells and the underlying mechanism might involve the inhibition of PARP-1 and the promotion of SIRT1.

Multiple independent losses of the biosynthetic pathway for two tropane alkaloids in the Solanaceae family.

Hyoscyamine and scopolamine (HS), two valuable tropane alkaloids of significant medicinal importance, are found in multiple distantly related lineages within the Solanaceae family. Here we sequence the genomes of three representative species that produce HS from these lineages, and one species that does not produce HS. Our analysis reveals a shared biosynthetic pathway responsible for HS production in the three HS-producing species. We observe a high level of gene collinearity related to HS synthesis across the family in both types of species. By introducing gain-of-function and loss-of-function mutations at key sites, we confirm the reduced/lost or re-activated functions of critical genes involved in HS synthesis in both types of species, respectively. These findings indicate independent and repeated losses of the HS biosynthesis pathway since its origin in the ancestral lineage. Our results hold promise for potential future applications in the artificial engineering of HS biosynthesis in Solanaceae crops.

Three new vanilloid derivatives from the stems of Baccaurea ramiflora.

Three new (1-3) and four known (4-7) compounds were isolated from the stems of Baccaurea ramiflora. By analysis of 1D, 2D NMR, and MS data, the new compounds were identified as 4'-O-(6- O-vanilloyl)-beta-D-glucopyranosyl tachioside D (1), 6'-O-vanilloylpicraquassioside D (2), and 6'-O-vanilloylicariside B(5) (3). Compound 1 exhibited significant DPPH radical-scavenging activity, with an IC(50) value of 36.9 microM, while compound 4 revealed weak antioxidant activity against H (2)O(2)-induced impairment in PC12 cells.

[The killing effects of cytotoxic T lymphocytes stimulated by specific hAFP-derived peptides on hepatocellular cancer cells].

OBJECTIVES: To compare the in vitro killing effects of cytotoxic T lymphocytes (CTLs) induced by dendritic cells(DCs) that modified with 4 different specific hAFP-derived peptides. METHODS: DCs derived from normal human peripheral monocytes were activated by GM-CSF and IL-4. MTT assay was applied to analyse the proliferation activities of the DCs. To induce the specific CTLs, DCs modified with four immunodominant epitopes from alpha fetoprotein (AFP) were cocultured with MNC derived CD8+ lymphocytes. The acquired specific CTLs were cocultured with SMMC-7721 cells at different dilutions, and the killing effects were detected by flow-cytometry and Non-Radioactive Cytotoxity kit. RESULTS: The CTLs stimulated by DCs modified with the four immunodominant epitopes from alpha fetoprotein (AFP) had specific killing activities on the SMMC-7721 cells. And statistical differences of the killing effects existed between the hAFP(158-166) (FMNKFIYEI) group and the other three groups. CONCLUSION: CTLs induced by the DCs modified with the four immunodominant peptides had significant killing effects on the hepatocellular cancer cells in vitro. The hAFP(158-166) (FMNKFIYEI) peptide had a relatively higher efficiency in inducing DCs and stimulating specific CTLs.

Tarennanosides A-H, eight new lignan glucosides from Tarenna attenuata and their protective effect on H2O2-induced impairment in PC12 cells.

Eight new lignan glucosides, tarennanosides A-H (1-8, resp.), were isolated from the whole plant of Tarenna attenuata, together with three known compounds, fernandoside, (-)-lyoniresinol, and (-)-isolariciresinol. The planar structures of new compounds were elucidated mainly by analysis of physical and spectroscopic data, and the absolute configurations were determined by acid hydrolysis as well as CD spectroscopy. Compounds 1 and 2 exhibited potent antioxidant activities against H2O2-induced impairment in PC12 cells. Preliminary mechanism study by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method showed that these two compounds could act as radical scavengers.

[Threshold Flux and Membrane Fouling Analysis of the Hybrid Pre-ozonation and CNTs Membrane Modification Process].

Polyvinylidene fluoride (PVDF) hollow fiber ultrafiltration membranes were modified with carbon nanotubes (CNT). Hybrid pre-ozonation and CNT modification were investigated by experimentally manipulating the ozonation process, threshold flux, and membrane fouling. The results showed that the threshold fluxes of the unmodified membrane and hybrid process were 45 L·(m2·h)-1 and 81 L·(m2·h)-1, respectively. Additionally, the fouling rate of the hybrid process was about 0.00137 kPa·min-1·L-1·m2·h, which was notably lower compared to other process. The results showed that the filtration volume under threshold flux was higher than that under critical flux with the same CNT loading mass and ozone dosage. This comparison indicated that membrane fouling was alleviated under threshold flux and that the corresponding operation period was extended. Through the carbon balance experiment, the fouling capacity and recoverability improved remarkably after CNT modification. Additionally, ozonation could enhance the recoverability of membranes. The hybrid process examined in this study could dramatically improve the permeability and extend the operation time of the ultrafiltration membrane.

[Insulino-mimetic effects of bis (alpha-furancarboxylato) oxovanadium (IV) in vitro].

To study insulino-mimetic effects of bis(alpha-furancarboxylato) oxovanadium (IV) (BFOV), a orally active antidiabetic vanadyl complex, on glucose uptake and lipogenesis in isolated rat adipocytes were determined by using 2-deoxy-D-[3H]-glucose and D-[3H]-glucose, respectively. Lipolysis was assayed by free fatty acids (FFA) released from isolated rat adipocytes treated with epinephrine. The results showed that BFOV, similar to insulin, concentration-dependently significantly enhanced the uptake of 2-deoxy-D-[3H]-glucose and the transformation from D-[3H]-glucose to lipid in isolated rat adipocytes, with the EC50 values of (0.31 +/- 0.08) mmol L(-1) and (0.49 +/- 0.12) mmol L(-1), respectively. Moreover, BFOV markedly inhibited FFA release from isolated rat adipocytes treated with epinephrine, and the IC50 value was (0.30 +/- 0.20) mmol L(-1). BFOV had insulino-mimetic effects such as enhancing glucose uptake and lipogenesis, as well as inhibiting lipolysis.

[Effect of Hybrid Process of Pre-ozonation and CNT Modification on Hollow Fiber Membrane Fouling Control].

Polyvinylidene fluoride (PVDF) hollow fiber ultrafiltration membranes were modified with carbon nanotube (CNT). Combined with the ozonation process, the effect of the hybrid pre-ozonation and CNT modification on fouling alleviation was investigated. The impacts of CNT loading mass and ozone dosage on the variation of flux and antifouling ability of the membrane modules were evaluated. Under a critical flux of 144 L·(m2·h)-1, CNT loading mass of 3 g·m-2, and ozone dosage(O3/DOC) of 0.22 mg·mg-1, the results revealed that the filtration volume of the hybrid process was promoted to 850 L·m-2, which was about 4.5 times higher than that of the original unmodified membrane. With a flux of 18 L·(m2·h)-1 and 15 day operation, the filtration volume was promoted to 3000 L·m-2, which was 10 times that of the unmodified membrane. The fouling membrane surface was observed using confocal laser scanning electron microscopy (CLSM). The results demonstrated that more living bacteria were present on the membrane surface of the unmodified membrane, which showed a rapid transmembrane pressure (TMP) increase. Both pre-ozonation and CNT modification decreased the total amount of microorganisms and the amount of the living bacteria as well, which mitigated the increase in TMP. After pre-ozonation, the presence of a CNT layer on the membrane surface further decreased the number of living bacteria. Although the CNT layer captured some dead bacteria, it had no obvious relationship with the increase in TMP.

Clinical application of oligo array-CGH for detecting balanced translocations in preimplantation genetic diagnosis.

Array comparative genomic hybridization (array-CGH), which facilitates to detect unbalanced reciprocal translocation and allows screening aneuploidy for chromosomes, has been repeatedly verified to be valid for diagnosis of translocations in preimplantation human embryos. Currently, the main microarrays used for CGH are bacterial artificial chromosome (BAC)-based arrays. Compared with the BAC-based arrays, oligonucleotide (oligo)-based arrays have a relatively higher resolution and optimal coverage particularly in the subtelomeric regions. Herein, we described the clinical application of a newly designed oligo-based array by Agilent in preimplantation genetic diagnosis (PGD) and aneuploidy screening for balanced translocations. In the study, a total of 144 embryos from 9 couples carrying Robertsonian translocations and 5 carrying reciprocal translocations were biopsied on day 3 for array-CGH analysis. Overall, 135 (93.8%) embryos were successfully diagnosed to be free of either aneuploidies or unbalanced fragments. However, the remained 9 (6.2%) embryos failed to be amplified due to failed cell lysis, DNA damage or the absence of nuclei in the biopsied cells. Collectively, 23 embryos were identified as "euploid and balanced" and suitable to be transferred. Finally, 9 embryos of satisfactory quality were transferred to 6 women, among which 4 recipients exhibited positive hCG level. Fortunately, one recipient with positive hCG level has delivered one baby, and two pregnancies were continuing. Our study served as the first clinical application of oligo-based array CGH technology in PGD for both reciprocal and Robertsonian translocations concomitant with comprehensive aneuploidy screening.

Expression level of pluripotent genes in incomplete reprogramming.

OBJECTIVE: To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells (iPSCs), to explore the relationship between the expression of pluripotent genes and incomplete reprogramming. METHODS: Four genes (Oct4, Sox2, Klf4, C-Myc) were introduced into human foreskin fibroblasts (HFFs) by retroviruses. The HFFs were induced to reprogramming. Different forms of colonies were picked up, analyzed, and compared with iPSCs from different aspects, including the morphology of clones, alkaline phosphatase (AP) staining, immuno-fluorescence, and Q-PCR. RESULTS: In the reprogramming process, different colonies were emerged, some of them exhibited typical human embryonic stem cell morphology (eg., compact colonies, high nucleus-to-cytoplasm ratios, and prominent nucleoli). However, these colonies couldn't maintain these characters after passage. There was an intermediate state, named partially reprogramming. Through analysis and identification, AP staining results were weakly positive, compared with iPSC colonies. The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4. Q-PCR indicated that the expression of exogenous transcription factors was inappropriate, either at a high level or at a low level. Most of the endogenous pluripotency genes were expressed at a low level. CONCLUSIONS: It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed, and successful reprogramming may depend on a specific stoichiometric balance of Oct4, Sox2, Klf4 and c-Myc.

Cyclosporine A improves adhesion and invasion of mouse preimplantation embryos via upregulating integrin ß3 and matrix metalloproteinase-9.

Our previous study has demonstrated cyclosporin A (CsA) promotes the migration and invasiveness of human first-trimester trophoblast cells in vitro. Here, we further investigated the effect of CsA on the early implantation in vitro of mouse embryo. Female C57 mice were superovulated and mated, and then two-cell embryos were harvested from the oviducts and sequentially cultured in vitro in G1 and G2 media with 0, 0.1, 1.0 or 10 µM of CsA. Blastocyte formation, blastocyte cell number and apoptosis, embryo hatching were assessed in 4-6 dpc. The adhesion and stretching growth of hatched embryos in laminin coated dishes were evaluated from 5 dpc to 8 dpc, and the expressions of implantation serine proteinase 1 (ISP1), integrin (itg) ß3 and matrix metalloproteinase (MMP)-9 were determined by real time PCR and immunofluorescence, respectively. We showed there was no significant difference in blastocyst formation rates, hatching rates, number of whole embryonic cells, apoptotic cells, and distribution of inner cell masses (ICMs) and trophoblasts (TB) between the CsA- and control-treated groups. Expression of ISP1 mRNA was unaffected on 5 dpc. After hatching, adhesion rate of 7 dpc significantly increased in 0.1 and 1.0 µM of CsA treatment, and embryo area of 8 dpc stretch growing on laminin were increased in 1.0 µM of CsA. The mRNA and protein expression of itgß3 and MMP-9 on 7 dpc blastocyst were up-regulated. In conclusion, CsA in low dosage up-regulates itgß3 and MMP-9 expression, and enhances embryonic adhesion and invasion, which is beneficial to the embryo implantation.

[1,25(OH)(2)D(3) influences endothelial cell proliferation, apoptosis and endothelial nitric oxide synthase expression of aorta in apolipoprotein E-deficient mice].

OBJECTIVE: To study possible influences of 1,25(OH)(2)D(3) on endothelial cell proliferation, apoptosis and endothelial nitric oxide synthase (eNOS) expression of aorta in apolipoprotein E-deficient (apoE(-/-)) mice and to explore the relationship between vitamin D and atherosclerosis. METHOD: Endothelial cell of aorta in apoE(-/-) mice were isolated and cultured, and the influence of 1,25(OH)(2)D(3) on endothelial cell proliferation were observed by MTT, apoptosis of cells were quantitated by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling, Bcl-2 mRNA, fas mRNA and eNOS mRNA was detected by reverse transcription-polymerase chain reaction. RESULT: Endothelial cell proliferation rate of aorta did not significantly change in the two control groups (0.162 ± 0.031 vs. 0.158 ± 0.006, P > 0.05). Compared with control groups, 1,25(OH)(2)D(3) stimulated endothelial cell proliferation of aorta (P < 0.05), but endothelial cell proliferation rate did not significantly change in different 1,25(OH)(2)D(3) concentration groups [1,25(OH)(2)D(3) concentration: 10(-4)mol/L, 10(-5) mol/L, 10(-6) mol/L, 10(-7) mol/L, 10(-8) mol/L, endothelial cell proliferation rate: 0.189 ± 0.013 vs. 0.285 ± 0.011 vs. 0.296 ± 0.026 vs. 0.284 ± 0.017 vs. 0.233 ± 0.010, P > 0.05]. 1,25(OH)(2)D(3) research concentration as chosen as 10(-6) mol/L. In 1,25(OH)(2)D(3) 10(-6) mol/L group, the expression of Bcl-2, eNOS mRNA was significantly increased (0.78 ± 0.16 vs. 0.46 ± 0.21 vs. 0.42 ± 0.17, 0.56 ± 0.16 vs. 0.39 ± 0.13 vs. 0.35 ± 0.11, 0.46 ± 0.2 vs. 10.42 ± 0.17 vs. 0.78 ± 0.16, 0.79 ± 0.21 vs. 0.81 ± 0.20 vs. 0.43 ± 0.12), apoptotic index, Fas mRNA was significantly decreased (15.14 ± 3.19 vs. 18.94 ± 4.22 vs. 19.27 ± 4.58, 0.43 ± 0.12 vs.0.79 ± 0.21 vs. 0.81 ± 0.20)(P < 0.05). The quantity of eNOS gene expression was inversely associated with apoptosis index and Fas mRNA, was positively associated with Bcl-2 mRNA (r = -0.676, -0.758, 0.762, P < 0.01). CONCLUSION: 1,25(OH)(2)D(3) stimulated endothelial cell proliferation, inhibited apoptosis and increased eNOS expression of aorta in apoE(-/-) mice. These results may deepen understanding of the pathogenesis of atherosclerosis.

[EphrinB2 gene transfection promotes the differentiation of bone marrow mesenchymal stem cells into vascular endothelial cells].

OBJECTIVE: To study the effects of ephrinB2 gene transfection on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into vascular endothelial cells. METHODS: Wistar rat BMSCs were isolated by density gradient centrifugation and purified on the basis of their adhesion ability. The BMSCs were transfected with a lenti-virus vector encoding a constitutively active form of human ephrinB2 gene, and the cell markers including CD105, CD73, CD44, von Willebrand factor (VWF) and vascular growth factor receptor 2 (KDR) were detected using flow cytometry. The potential of ephrinB2-BMSCs for differentiation into osteoblasts and adipoblasts in vitro were tested, and the differentiation of the cells into endothelial-like cells was induced by culture in the presence of 2% fetal bovine serum and 50 ng/ml vascular endothelial growth factor. RESULTS: EphrinB2-BMSCs were positive for the markers CD105, CD73 and CD44, and negative for the typical endothelial markers like VWF and KDR, and retained high potentials for differentiation into osteoblasts and adipoblasts in vitro after cultivation in respective media. After induced differentiation, ephrinB2-BMSCs expressed VWF and KDR and showed greater ability of differentiation into vascular endothelial cells and formation of capillary structures on matrix gel than the BMSCs without transfection. CONCLUSIONS: EphrinB2 gene transfection efficiently promotes the differentiation of BMSCs into vascular endothelial cells. These genetically engineered cells provide valuable sources for new therapies of coronary heart disease.