Dulbecco's Minimal Essential Medium Research Articles

Prior Treatment with AICAR Causes the Selective Phosphorylation of mTOR Substrates in C2C12 Cells.

Metabolic stress in skeletal muscle cells causes sustained metabolic changes, but the mechanisms of the prolonged effects are not fully known. In this study, we tested C2C12 cells with the AMP-activated protein kinase (AMPK) stimulator AICAR and measured the changes in the metabolic pathways and signaling kinases. AICAR caused an acute increase in the phosphorylation of the AMPK target ULK1, the mTORC1 substrate S6K, and the mTORC2 target Akt. Intriguingly, prior exposure to AICAR only decreased glucose-6 phosphate dehydrogenase activity when it underwent three-hour recovery after exposure to AICAR in a bicarbonate buffer containing glucose (KHB) instead of Dulbecco's Minimum Essential Medium (DMEM). The phosphorylation of the mTORC1 target S6K was increased after recovery in DMEM but not KHB, although this appeared to be specific to S6K, as the phosphorylation of the mTORC1 target site on ULK1 was not altered when the cells recovered in DMEM. The phosphorylation of mTORC2 target sites was also heterogenous under these conditions, with Akt increasing at serine 473 while other targets (SGK1 and PKCα) were unaffected. The exposure of cells to rapamycin (an mTORC1 inhibitor) and PP242 (an inhibitor of both mTOR complexes) revealed the differential phosphorylation of mTORC2 substrates. Taken together, the data suggest that prior exposure to AICAR causes the selective phosphorylation of mTOR substrates, even after prolonged recovery in a nutrient-replete medium.

Comparison of media for a human peripheral blood mononuclear cell-based in vitro vaccine evaluation system.

Human peripheral blood mononuclear cell (PBMC)-based in vitro systems can be of great value in the development and assessment of vaccines but require the right medium for optimal performance of the different cell types present. Here, we compare three commonly used media for their capacity to support innate and adaptive immune responses evoked in PBMCs by Toll-like receptor (TLR) ligands and whole inactivated virus (WIV) influenza vaccine. Human PBMCs were cultured for different periods of time in Roswell Park Memorial Institute (RPMI), Dulbecco's minimal essential medium (DMEM), or Iscove's modified DMEM (IMDM) supplemented with 10% fetal calf serum. The viability of the cells was monitored and their responses to TLR ligands and WIV were assessed. With increasing days of incubation, the viability of PBMCs cultured in RPMI or IMDM was slightly higher than that of cells cultured in DMEM. Upon exposure of the PBMCs to TLR ligands and WIV, RPMI was superior to the other two media in terms of supporting the expression of genes related to innate immunity, such as the TLR adaptor protein gene MyD88 (myeloid differentiation factor 88), the interferon (IFN)-stimulated genes MxA (myxovirus resistance protein 1) and ISG56 (interferon-stimulated gene 56), and the leukocyte recruitment chemokine gene MCP1 (monocyte chemoattractant protein-1). RPMI also performed best with regard to the activation of antigen-presenting cells. As for adaptive immunity, when stimulated with WIV, PBMCs cultured in RPMI or IMDM contained higher numbers of IFNγ-producing T cells and secreted more immunoglobulin G than PBMCs cultured in DMEM. Taken together, among the different media assessed, RPMI was identified as the optimal medium for a human PBMC-based in vitro vaccine evaluation system.

Lovastatin Induces Apoptosis through Deprivation of Intracellular Geranylgeranyl Pyrophosphate and Induction of Puma Expression in C6 Glioma Cells

Lovastatin is widely used to decrease serum cholesterol levels and reduce the risk of cardiovascular diseases. Lovastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, an enzyme that converts HMG-CoA to mevalonate. Mevalonate is a precursor of various metabolic intermediates such as isopentenyl pyrophosphate, farnesyl pyrophosphate, geranylgeranyl pyrophosphate, and cholesterol. It has been reported that statins can induce apoptosis in various cell lines. In this study, we examined the mechanism of lovastatin-induced apoptosis in C6 and HCT116 cell lines. C6 is a rat glioma cell line and HCT116 is a human colon cancer cell line. Cells were maintained in Dulbecco's minimal essential medium containing 10% (v/v) fetal bovine serum. After treatment, the number of viable cells was estimated using a colorimetric assay based on tetrazolium salt. The percentage of dead cells was estimated using trypan blue cell viability assay or lactate dehydrogenase assay. The levels of Puma mRNA were estimated by reverse transcription quantitative polymerase chain reaction assay. The levels of PUMA were estimated by Western blot assay. Our data showed that lovastatin increased both mRNA and protein levels of Puma in C6 cells, which could be prevented in the presence of 100 μM geranylgeranyl pyrophosphate. Treatment with geranylgeranyl pyrophosphate, cycloheximide, and siPuma (siRNA against Puma) prevented the death of lovastatin-treated cells. Pifithrin-a, an inhibitor of tumor protein p53, neither prevented cell death nor decreased PUMA levels in lovastatin-treated cells. Pifithrin-a attenuated cell death and reduced PUMA levels in etoposide-treated C6 cells. Etoposide, an anticancer drug, has been known to induce p53-dependent apoptosis. To evaluate the role of p53 on the induction of Puma expression in lovastatin-treated cells, cytotoxic effects of lovastatin were examined in HCTm and HCTw cells. HCTm is a HCT116 cell line expressing inactive mutant p53 and HCTw is the wild-type. When cells were cultured in the presence of 25 μM lovastatin for 60 hours, the values of the percentage of cell death were 46% and 43% in HCTm and HCTw groups respectively: there was little difference in lovastatin-induced cell death between two groups. Etoposide-induced cell death was exacerbated in HCTw cells as compared to HCTm cells. When cells were cultured with 100 μM etoposide for 72 hours, the values of the percentage of cell death were 35% and 45% in HCTm and HCTw groups respectively. Pifithrin-a reduced etoposide-induced cell death in HCTw cells from 45% to 25%. Pifithrin-a did not attenuate etoposide-induced cell death in HCTm cells. Our data show that lovastatin depletes intracellular geranylgeranyl pyrophosphate to increase Puma expression in a p53-independent manner, resulting in apoptosis.

Preparation and In Vitro Biological Evaluation of Lawsone Loaded O‐Carboxymethyl Chitosan/Zinc Oxide Nanocomposite for Wound‐Healing Application

AbstractA biopolymer based nanocomposite with O‐Carboxymethyl chitosan (O‐Cmc), nano zinc oxide (ZnO) and Lawsone (Ln) with proving advantages in antibacterial wound healing application was prepared. The physiochemical characteristics of the nanocomposite (NCs) was analysed by Fourier Transmission Infrared spectroscopy (FT‐IR), X‐Ray Diffractometer (XRD), Scanning electron Microscopy (SEM), Transmission electron microscope (TEM) and Thermogravimetric analysis (TGA). The biological properties such as anti‐inflammatory, anti‐oxidant activities were investigated in vitro. The nanocomposite also possesses efficient drug encapsulation and releasing profile with greater stability in phosphate buffer saline (PBS) and Dulbecco's minimal essential medium (DMEM). The results of the antibacterial studies show its enhanced bactericidal nature. The results obtained from MTT assay, Ethidium bromide stained fluorescence image and in vitro wound scratch assay show greater cell viability with the closure of scratch wound obviously proves it′s prominent biocompatible, wound closure nature. These biological properties show that the prepared nanocomposite has a greater potential to act as an excellent biomaterial in the field of wound care applications.

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Investigation of the Anticancer and Proliferative Effect of Broccoli Extract on Du145 Prostate Cancer and MEF Healthy Fibroblast Cell Lines

In recent years, many researchers have focused on the health effects of plant-derived foods. In this context, foods with high content of flavonoids and phenolic substances have received a great deal of attention as potential agents for cancer prevention and treatment. Studies on the broccoli plant have revealed that broccoli has antioxidant and anti-carcinogenic properties. However, effective dose of broccoli needs to be determined for the cancer treatment. In this study, the effects of broccoli extracts on du145 prostate cancer and Mef fibroblast healthy cells were investigated at different doses for 48 hours. Du145 and Mef cells were grown with Dulbecco's Minimum Essential Medium (DMEM) and HAMS F 12 (1: 1) supplemented with 2% FBS. Broccoli extracts at ten different doses (0,19% - 100%) were added into cultures and incubated at 37°C for 24 and 48 h in 5% CO2. The viability of the cells was determined by the MTT method (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide). Probit analysis by SPSS software revealed that the broccoli extract at a dose of 4.070 % dose killed du145 cancer cells at 48 h. At the same time, MTT results showed that the viability of Mef cells was increased during 48 h of incubation. As a result, broccoli extract showed a significant level of anticancer activity in Du145 cells, while increasing the viability of Mef healthy cells. This result suggests that broccoli extract is a potential candidate for cancer treatment.

Effect of Quinoa Plant on Metastasis and Ion Channels of Rat Brain Cancer Glioma Cell Lines

In this study, the effects of the quinoa plant on the rat brain cancer glioma cell line were examined. Rat C6 glioma cell lines were cultivated in Dulbecco's Minimum Essential Medium (DMEM) appendage with HAMS F 12 (1: 1) and 2% FBS. After proliferation, Quinoa plant was added into the cells and incubated at 37°C for 24 and 48 h in 5% CO2. The viability of the cells was identified by using the MTT method (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide). IC50 concentration was determined using the statistic software SPSS (Probit analysis). The effect of the quinoa plant on the invasiveness of the C6 cells was analyzed by the wound test and the changes in the ion concentrations in the cells were determined with ICP-MS. As a result of the MTT test, the IC50 value of the quinoa plant was determined as 50 ppb. Wound test showed that use of quinoa plant (50 ppb) inhibited metastasis in the glioma cells while the cell proliferation in the control group was continued. Furthermore, calcium, sodium and potassium ions, which are regulators of cell cycle, were found in higher concentrations in that the untreated control cells than quinoa treated cells. As a result of this study; ICP-MS analysis showed that higher levels of calcium, sodium, and potassium ions were found in the untreated cells, whereas the application of the quinoa plant decreased these values. This change in ion channels was thought to be associated with the invasion of glioma cells, and it was determined that quinoa had significant anticancer effects.

Gracilaria foliifera (Forssk.) Børgesen ethanolic extract triggers apoptosis via activation of p53 expression in HepG2 cells

Background: Hepatocellular carcinoma is one of the most common types of malignancy and causes significant morbidity and mortality worldwide. Gracilaria foliifera (Forssk.) Borgesen, a brown marine alga, is shown to have growth inhibitory potential against various cancer cell lines other than human hepatoma HepG2 cells. Objective: To investigate the cytotoxic potentials of G. foliifera in HepG2 cells. Materials and Methods: HepG2 cells were fed with culture medium supplemented with different concentrations of ethanolic extract of G. foliifera (20, 40, and 80 μg/mL). After 24 h of treatment, the cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Induction of early apoptosis was investigated by annexin V-fluorescein isothiocyanate immunofluorescence. Induction of late apoptosis and necroptosis was investigated by annexin V and propidium iodide (PI) staining. Nuclear chromatin condensation was evaluated by Hoechst staining. p53 protein expression was analyzed using Western blotting. Results: G. foliifera treatment in HepG2 cells caused a significant cytotoxic effect. Phosphatidylserine translocation confirms the induction of early apoptosis. Analysis of late apoptosis using annexin V/PI staining showed that the percentage of apoptotic cells was increased in a concentration-dependent manner. Hoechst nuclear staining further confirms the nuclear chromatin condensation. G. foliifera treatment also induced the tumor suppressor p53 protein expressions. Conclusion: The present study demonstrated that G. foliifera induced apoptosis in HepG2 cells through activation of p53. Abbreviations used: HCC: Hepatocellular carcinoma; DMEM: Dulbecco's minimum essential medium; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NCCS: The National Center for Cell Science; FBS: Fetal bovine serum; PI: Propidium iodide; PS: Phosphatidylserine.

Recovery of 3-Iodothyronamine and Derivatives in Biological Matrixes: Problems and Pitfalls.

Difficulties have been reported in quantitating 3-iodothyronamine (T1AM) in blood or serum, and tentatively attributed to problems in extraction or other pre-analytical steps. For this reason, even cell culture experiments have often be performed with unphysiological protein-free media. The aim of this study was to evaluate the recovery of exogenous T1AM added to a standard cell culture medium, namely Dulbecco's minimum essential medium (DMEM) supplemented with fetal bovine serum (FBS), and to other biological matrixes. Cell culture media (Krebs-Ringer buffer, DMEM, FBS, DMEM + FBS, used either in the absence or in the presence of NG108-15 cells) and other biological matrixes (rat brain and liver homogenates, human plasma, and blood) were spiked with T1AM and/or deuterated T1AM (d4-T1AM) and incubated for times ranging from 0 to 240 minutes. Samples were then extracted using a liquid/liquid method and analyzed using liquid chromatography coupled to mass spectrometry in order to assay T1AM and its metabolites, namely 3-iodothyroacetic acid (TA1), thyronamine, thyroacetic acid, N-acetyl-T1AM, and T1AM esters. In FBS-containing buffers, T1AM decreased exponentially over time, with a half-life of 6-17 minutes, depending on FBS content, and after 60 minutes, it averaged 0-10% of the baseline. T1AM metabolites were not detected, except for minimum amounts of TA1. Notably, d4-T1AM decreased over time at a much lower rate, reaching 50-70% of the baseline at 60 minutes. These effects were completely abolished by protein denaturation and partly reduced by semicarbazide. In the presence of cells, T1AM concentration decreased virtually to 0 within 60 minutes, but TA1 accumulated in the incubation medium, with quantitative recovery. Spontaneous decrease in T1AM concentration with isotopic difference was confirmed in rat organ homogenates and human blood. These results suggest binding and sequestration of T1AM and/or its aldehyde derivative by blood and tissue proteins, with significant isotope effects. These issues might account for the technical problems complicating the analytical assays of endogenous T1AM.

Viwithan, a Standardized Withania somnifera Root Extract Induces Apoptosis in Murine Melanoma Cells.

Background:Withania somnifera is an Indian medicinal herb known for the multipotential ability to cure various therapeutic ailments as described in the ayurvedic system of medicine.Objective:In the present study, we have evaluated the antiproliferative activity of a standardized W. somnifera root extract (Viwithan) against different human and murine cancer cell lines.Materials and Methods:The cytotoxicity of Viwithan was determined using thiazolyl blue tetrazolium blue assay and crystal violet staining. The apoptotic changes in B16F1 cells following treatment with Viwithan were observed by acridine orange/ethidium bromide (AO/EB) staining and DNA fragmentation assay. The binding affinity of withanolides in Viwithan with antiapoptotic proteins B-cell lymphoma 2, B-cell lymphoma-extra large, and myeloid cell leukemia 1 (MCL-1) were studied using in silico approach.Results:The half maximal inhibitory concentration (IC50) values of Viwithan against liver hepatocellular carcinoma, Henrietta Lacks cervical carcinoma cells, human colorectal carcinoma cell line, and Ehrlich ascites carcinoma cells were 1830, 968, 2715, and 633 μg/ml, respectively. Interestingly, Viwithan was highly effective against B16F1 cells with an IC50 value of 220 μg/ml after 24 h treatment. The morphological alterations of apoptotic cell death were clearly observed in the AO/EB-stained cells after treatment with Viwithan. Viwithan induced late apoptotic changes in treated B16F1 cells as evident by the ladder formation of fragmented DNA in a time-dependent manner. The findings of molecular docking showed that withanolides present in Viwithan have a more binding affinity with the antiapoptotic proteins, particularly MCL-1.Conclusion:We have reported for the first time that Viwithan with 5% withanolides has a potent cytotoxic effect, particularly against B16F1 murine melanoma cells among the different cancer cell lines tested.SUMMARY The present study reports for the first time that Viwithan, a standardized 5% Withania somnifera root extract, has potent cytotoxicity against B16F1 murine melanoma cellsWe have investigated the in vitro cytotoxicity of Viwithan in different human and murine cancer cells. Interestingly, we found that Viwithan was particularly very effective against B16F1 melanoma cells with a half maximal inhibitory concentration value of 220 μg/mlThe microscopic observations following acridine orange/ethidium bromide staining and DNA fragmentation assays clearly indicated that Viwithan might initiate late apoptosis in B16F1 cellsThe binding affinity of withanolides in Viwithan with antiapoptotic proteins of B-cell lymphoma 2 family was predicted using AutoDock tool. The results from in silico studies indicated a plausible synergistic effect of withanolides attributing to the Viwithan-induced apoptosis through suppression of intrinsic pathway for carcinogenesis. Abbreviations used: MTT: Thiazolyl blue tetrazolium blue; DMSO: Dimethyl sulfoxide; BSA: Bovine serum albumin; DMEM: Dulbecco's minimum essential medium; NCCS: National Centre for Cell Science; PBS: Phosphate-Buffered Saline; HepG2: Liver hepatocellular carcinoma; HeLa: Henrietta Lacks cervical carcinoma cells; HCT-116: Human colorectal carcinoma cell line; EAC: Ehrlich ascites carcinoma cells; IC50: Half maximal inhibitory concentration; AO/EB: Acridine orange/Ethidium bromide; BCL-2: B-cell lymphoma 2; BCL-XL: B-cell lymphoma-extra large; MCL-1: Myeloid cell leukemia 1; PDB: Protein Data Bank; ANOVA: Analysis of variance.

Osteogenic Potential Differentiation of Human Amnion Mesenchymal Stem Cell with Chitosan-Carbonate Apatite Scaffold (In Vitro Study)

Background: Tissue engineering based approaches have received much attention. Incorporation of chitosan and carbonate apatite (CA) improve its capability. Human mesenchymal stem cells (hMSCs) is viable for xenogenic transplantation. The purpose of this study was to fabricate and evaluate the osteogenic potential differentiation of human amnion mesenchymal stem cell with carbonate apatite– chitosan scaffolds (CA-ChSs) for tissue engineering. Method: Human amniotic membrane was procured from using cesarean section. Soncini’s protocol was employed for the isolation procedure. The cells cultured on collagen-coated dishes using Dulbecco's minimal essential medium (DMEM)/F12 (1:1). A chitosan powder of medium molecular weight deacetylated chitin, poly(D(glucosamine) was used and mixed with CA. Immunocytochemistry and flowcytometry used for phenotypic characterization of hAMSC. Result: Amniotic membrane obtained using cesarean section under aseptic condition did not exhibit any growth of cell cultures which were not contaminated. Immunocytochemistry testing revealed that the target cells expressed strong mesenchymal stem cell marker CD 105. Characterization at passage 10 showed that CD44 was the most significant and abundant surface receptors. The number of viable cells in chitosan-carbonate apatite was 66.59%. Scanning electron microscope (SEM) observation revealed that CA-ChSs had three-dimensional structure with many pores and hAMSc could attached and proliferation among the porosity of the scaffold. The formation of calcium in the cell as an indicator of osteoblast cells was detected using Alizarin Red solution. Conclusion: hAMSc harvested from human amniotic membrane seeding in CA- ChSs had the capability for in vitro osteogenesis makes them be the one of the potential options for bone tissue engineering.

C-Rel is Required for the Induction of pTregs in the Eye but Not in the Gut Mucosa

ABSTRACTRegulatory T (Treg) cells play an integral role in maintaining immune homeostasis and preventing autoimmune diseases. Forkhead box P3 expression marks the commitment of progenitor cells to the Treg lineage. Although the essential function of the nuclear factor (NF)-κB family transcription factor c-Rel in the regulation of natural Treg cells has been firmly established, little is known about whether c-Rel is involved in the in vivo generation of peripheral Treg cells (pTregs), which develop from mature CD4+ conventional T cells outside of the thymus. We sought to answer this question through the induction of pTregs in the eye and gut mucosa using ovalbumin-specific T cell receptor transgenic mice that do or do not express c-Rel. Our results showed that Tregs can be induced in the eye in a c-Rel-dependent manner when immune-mediated inflammation occurs. However, c-Rel is dispensable for the induction of pTregs in the gut mucosa after oral antigen administration. Thus, c-Rel may play distinct roles in regulating the development of pTregs in different organs.Abbreviations ACAID: Anterior Chamber-Associated Immune Deviation; ATF: activating transcription factor; CREB: cAMP responsive element-binding protein; DMEM: Dulbecco minimum essential medium; HBSS: Hanks Balanced Salt Solution; NFAT: Nuclear Factor of Activated T cells; PBS: Phosphate-buffered saline; PE: Phycoerythrin; WT: wild type.

Effects of hydrogen-rich medium on lipopolysaccharide-induced intestinal epithelial barrier dysfunction of human colon carcinoma cells

To investigate the effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction of human intestinal epithelial (Caco2) cells. Caco2 cells (passages 28-35) were purchased from the Cell Bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences in Shanghai, China, and they were cultured in Dulbecco minimum essential medium (DMEM) containing 20% fetal bovine serum. These cells were randomly divided into four groups: control group (group A), hydrogen-rich medium group (group B), LPS group (group C) and LPS + hydrogen-rich medium group (group D). Cells were cultured with normal medium in group A and group C or with hydrogen-rich medium in group B and group D. Meanwhile, 1 g/L LPS was simultaneously added into group C and group D, while an equivalent volume of normal saline was added into group A and group B instead. In vitro intestinal epithelial models were reproduced with monolayer filter-grown Caco2 and intestinal epithelium. The trans-epithelial electrical resistance (TEER) in models of each group was measured at different incubation times (0, 3, 6, 12, 24 and 48 hours). Cell viability and cytotoxicity were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release assay, respectively, after incubation for 24 hours. The expression levels of claudin-1 and occludin were respectively determined at 6, 12 and 24 hours of incubation by Western Blot assay. The morphological structure of claudin-1 and occludin was respectively observed after incubation for 24 hours with immunofluorescence staining. There was no statistical significance in variables between group A and group B. Compared with group A, it was shown that TEER was time-dependently decreased in groups C and D after 6 hours. Compared with group C, TEER in group D was increased after 6 hours. Compared with group A, the cell viability was significantly reduced in group C [(67.2±7.9)% vs. (100.0±0.0)%, P < 0.05] and cell injury was obvious [LDH release rate: (38.5±2.1)% vs. (1.2±0.3)%, P < 0.05]; the expression levels of claudin-1 and occludin at 6, 12, 24 hours were significantly down-regulated [claudin-1 (gray value): 0.351±0.079, 0.272±0.075, 0.190±0.049 vs. 0.518±0.030; occludin (gray value): 0.416±0.044, 0.290±0.062, 0.226±0.019 vs. 0.602±0.038, all P < 0.05], and the structure of claudin-1 and occludin were profoundly disrupted. Compared with group C, it was shown that the cell viability was significantly increased in group D [(88.8±7.4)% vs. (67.2±7.9)%, P < 0.05] and cell injury was significantly abated [LDH release rate: (16.4±4.3)% vs. (38.5±2.1)%, P < 0.05]; the expression levels of claudin-1 and occludin were significantly up-regulated at 24 hours [claudin-1 (gray value): 0.428±0.046 vs. 0.190±0.049, occludin (gray value): 0.466±0.071 vs. 0.226±0.019, both P < 0.05]; the disrupted structures of claudin-1 and occludin were partially recovered. Hydrogen-rich medium can effectively attenuate LPS-induced dysfunction of intestinal epithelial barrier in human Caco2 cells by ameliorating cell viability as well as regulating claudin-1 and occludin expression and structure.

Neuroprotective role of galectin-1 in central nervous system pathophysiology.

Neuroprotective role of galectin-1 in central nervous system pathophysiology.

Fuzheng Huayu recipe promotes the fenestration of capillarization in liver sinusoidal endothelial cells

To explore the effect of Fuzheng Huayu (FZHY) recipe on the fenestration of capillarization in liver sinusoidal endothelial cells (LSECs). Ten Sprague Dawley (SD) rats were fed with 0.46 g/kg FZHY powder by intragastric administration. Two hours later, a second gavage were given to the rats. The serum from rat heart at 1 hour after second gavage was collected (FZHY group, n=10). Another ten SD rats was administrated with distilled water through the same process and served as the control (control group, n=10). The serum from both groups were separately diluted with Dulbecco minimum essential medium (DMEM) for 10% and served as the culture medium for LSECs. At the different conditions, the vWF and CD31 expressions were detected by immunocytochemistry and Western blot, while the changes of LSECs fenestrae structure were observed under scanning electron microscopy. 1) Immunocytochemistry and Western blot showed that the vWF and CD31 protein levels were lower in LSECs in the FZHY group than those in the control group. The gray levels of vWF and CD31 protein were 0.548±0.020 and 0.262±0.010 in the FZHY group, and 0.845±0.090 and 0.383±0.010 in the control group respectively, with statistical significant difference (t=5.18, 9.61, both P<0.05). 2) The results from scanning electron microscopy showed that the fenestration of LSECs was closed and almost lost in the control group, but many fenestra appeared in the LSECs in the FZHY group. FZHY recipe can suppress the expression of vWF and CD31, increase the fenestrae on the LSECs surface and reverse the capillarization of LSECs.

Establishment of C6 brain glioma models through stereotactic technique for laser interstitial thermotherapy research.

Objective:To establish C6 brain glioma models using stereotactic technique, and to study effects of laser interstitial thermotherapy (LITT) in rat models of glioma.Methods:C6 glioma cells were cultured in dulbecco's minimum essential medium (DMEM) cell culture medium. The in vitro C6 cell cultures were stereotaxically implanted into the right caudate nucleus of rat brain. Presence of tumor was confirmed with Factor VIII R, hematoxylin–eosin stain, staining of glial fibrillary acid protein, and S-100 immunohistochemistry. After magnetic resonance (MR) scanning and correction of tumor location, the models were divided into groups according to the treating time and laser power (2–10 W). Semiconductor laser optical fibers were inserted in tumors for LITT. Cortex's temperature conducted from the center target was measured using infrared thermograph, and deep-tissue temperature around the target was measured using a thermocouple.Results:Rat C6 gliomas were inoculated with optimized stereotactic technique. These gliomas resembled human glioma in terms of histopathological features. Such models are more reliable and reproducible, with 100% yield of intracranial tumor and no extracranial growth extension. The difference between cortex temperature conducted from center target and deep-tissue temperature around target was not statistically significant.Conclusion:The rat C6 brain glioma model established in the study was a perfect model to study LITT of glioma. Infrared thermograph technique measured temperature conveniently and effectively. The technique is noninvasive, and the obtained data could be further processed using software used in LITT research. To measure deep-tissue temperature, combining thermocouple with infrared thermograph technique would present better results.

Co-Culture of α TC-6 Cells and β TC-1 Cells: Morphology and Function

BackgroundIn vitro experiments using only β-cell lines instead of islets are limited because pancreatic islets are composed of four different types of endocrine cells. Several recent studies have focused on cellular interactions among these cell types, especially α- and β-cells. Because islet isolation needs time and experience, we tested a simple co-culture system with α- and β-cells. Their morphology and function were assessed by comparison to each single cell culture and pancreatic islets.Methodsα TC-6 cells and β TC-1 cells were maintained in Dulbecco's Minimal Essential Medium containing 5 mM glucose and 10% fetal bovine serum. Cells were mixed at a 1:1 ratio (5×105) in 6-well plates and cultured for 24, 48, and 72 hours. After culture, cells were used for insulin and glucagon immunoassays and tested for glucose-stimulated insulin secretion (GSIS).Resultsα TC-6 and β TC-1 cells became condensed by 24 hours and were more strongly compacted after 48 hours. β TC-1 cells showed both β-β and β-α cell contacts. GSIS increased with increasing glucose concentration in co-cultured cells, which showed lower secreted insulin levels than β TC-1 cells alone. The increase in the secreted insulin/insulin content ratio was significantly lower for co-cultured cells than for β-cells alone (P=0.04). Compared to islets, the α-/β-cell co-culture showed a higher ratio of GSIS to insulin content, but the difference was not statistically significant (P=0.09).Conclusionα TC-6 and β TC-1 cells in the co-culture system showed cell-to-cell contacts and a similar stimulated insulin secretion pattern to islets. The co-culture system may be used to better mimic pancreatic islets in in vitro assessments.

The estimation of corrosion behavior of new TiNbTaZr alloys for biomedical applications

The corrosion behavior of two TiNbTaZr alloys (Ti25Nb8Ta10Zr and Ti21Nb15Ta6Zr) in Dulbecco's minimum essential medium (MEM) are evaluated and its protective ability is compared with that of Cp‐Ti, to ascertain their stability for biomedical application. All the samples were examined using electrochemical techniques: electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization curves. The electrochemical corrosion parameters obtained from the EIS and potentiodynamic polarization curves indicated a typical passive behavior for TiNbTaZr alloys. In addition, scanning electron microscopy was employed to observe the surface morphology after polarization test in MEM solution. Low anodic current density values were obtained from the polarization curves, indicating a typical passive behavior for TiNbTaZr alloys. EIS studies showed high impedance values for all samples, increasing with exposure time. The Ti21Nb15Ta6Zr alloy appears to possess superior corrosion resistance than the Ti25Nb8Ta10Zr alloy and Cp‐Ti in MEM solution.

Optimal Medium Formulation for the Long‐Term Expansion and Maintenance of Human Periodontal Ligament Stem Cells

Human periodontal ligament stem cells (hPDLSCs) are promising mesenchymal stem cells that are readily accessible. However, there is as yet no consensus as to the optimal culture medium for hPDLSCs. Thus, the purpose of the present study is to determine the optimal culture medium for long-term expansion of hPDLSCs. hPDLSCs were isolated from healthy third molars, and the most widely used medium formulations in previous studies were used: 1) an α minimum essential medium-based medium formulation (MBM); and 2) a Dulbecco's minimum essential medium-based medium formulation. Passage 5 (P5) and P8 were evaluated with the two media for cell proliferation, differentiation, and immunophenotype. hPDLSCs that were primarily cultured in MBM were far more proliferated than those grown in DBM. In general, application of the MBM for longer periods produced greater cell growth and osteogenic differentiation. Furthermore, MBM-precultured hPDLSCs exhibited a greater degree of cell proliferation and a greater production of mineralized tissue and alkaline phosphatase (ALP) activity in vitro, although the levels of both were dependent on the culture medium used. With respect to long-term expansion, the P5 hPDLSCs grew and produced the largest amount of mineralized nodules faster than the P8 hPDLSCs, but both passages exhibited a similar phenotype for stemness and ALP activity. The present study indicates that the inherent capacity of hPDLSCs could be maintained until a later passage, P8 in MBM, and MBM appears to be an optimal choice for manipulating the finest and most stable hPDLSCs.

Epithelial-mesenchymal transdifferentiation of retinal pigment epithelial cells induced by vitreous humor in vitro

Objective To observe the effect of epithelial-mesenchymal transdifferentiation (EMT) of human retinal pigment epithelial (RPE) cells induced by vitreous humor in vitro.Methods The third to fifth passage cultured RPE cells were divided into two groups of treatment by 10％ serum-containing Dulbecco minimum essential medium (DMEM)/F12 medium (group A),or the same medium supplemented with 25％ human vitreous (group B).The morphological changes were observed with a phase contrast microscrope.Cell migration,invasion and contractility were tested using a scratch wound assay,Transwell invasion assay and collagen gel contraction analysis.The expression levels of α-smooth muscle actin (SMA)and Snail1 were detected by reverse transcriptase polymerase chain reaction (RT-PCR).Results The RPE cells in group A were flat and gathered together.The RPE cells in group B grew as a fan-shaped projection at one edge and cone-shaped tail at the opposite edge,or spindle-shaped,and appeared to separate.In group A,filamentous actin distributed mainly at the margin of the cells with the distribution an oval shape.In group B,filamentous actin reorganized and formed fan-like flat pseudopodia at one edge of the cells.Compared to group A,the migration and invasion of the cells increased significantly (t=14.190,22.630;P＜0.05),but contractility decreased remarkably (t=6.221,P＜0.05) in group B.Compared to group A,the expression level of Snail1 mRNA increased significantly (t=3.218,P=0.032),but the expression level of α-SMA mRNA decreased (t=3.990,P=0.016).Conclusions Vitreous humor can induce the EMT of RPE cells.Increasing cell migration,cell invasion,and expression of Snail1 mRNA as well as up-regulated cells' contractility and expression of α-SMA mRNA may be the mechanism. Key words: Pigment epithelium of eye/growth & development; Cell, cultured; Cell transdifferentiation