In situ isolation of nuclei or nuclear proteins from adherent cells: a simple, effective method with less cytoplasmic contamination

BACKGROUND: Isolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100. RESULTS: First, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1-1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1-1% Triton X-100 for our method were determined via western blotting of eight extra-/ intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei. CONCLUSIONS: The in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

Induction of p450 enzymes by derivative of parthenolide / 中草药

Objective: In this study, primary cultured human hepatocytes were used to study the induction of P450 enzyme by the sesquiterpene lactone derivative ACT001, which provided reference for the clinical use of ACT001. Methods Three batches of frozen primary human hepatocytes were inoculated and cultured, and CYP1A2, CYP2B6 and CYP3A4 were induced by ACT001. Real-time fluorescence quantitative PCR was used to determine the mRNA expression level of P450 enzyme, and the activity of P450 enzyme was determined by LC-MS/MS method. Results The expression level of P450 enzyme mRNA and the activity of P450 enzyme showed that the P450 enzyme induction model was successfully established. Compared with the control group, the CYP1A2 mRNA expression level and enzyme activity of ACT001 1 μmol/L and 6 μmol/L group showed no significant changes. The mRNA expression level of CYP1A2 in ACT001 30 μmol/L group was significantly decreased, and the enzyme activity was decreased, but not as significantly as that of mRNA. With the increase of ACT001 concentration, the expression level of CYP2B6 mRNA was gradually increased. Compared with the control group, the expression level of CYP2B6 mRNA in the ACT001 group at 30 μmol/L was significantly increased, which was seven times higher than that in the control group, and the increase of enzyme activity was four times higher than that in the control group, which was 40% higher than that in the phenobarbital sodium induction multiple. Compared with the control group, the CYP3A4 mRNA expression level of cells in the ACT001 1 μmol/L group was significantly increased, which was four times higher than that of the control group, but did not reach 40% of the positive inducer rifampicin, and the CYP3A4 mRNA expression level was decreased gradually with the increase of ACT001 concentration. At the same time, there was no significant increase in CYP3A4 enzyme activity after ACT001 administration at different concentrations, which was less than two times of that in the control group. Conclusion The data indicated that ACT001 had no induction potential for CYP1A2 and CYP3A4, and had potential for CYP2B6 induction. In combination with CYP2B6 substrates, it should be avoided in clinical combination therapy to reduce adverse reactions caused by P450-mediated drug drug interaction.

Phagocytosis and nitric oxide production by peritoneal adherent cells in response to Candida albicans in aging: a collaboration to elucidate the pathogenesis of denture stomatitis

Abstract Elderly denture wearers are commonly affected by Candida-associated denture stomatitis (DS), an inflammatory process of the oral mucosa strongly associated with Candida spp and other microorganisms, as well as local and systemic factors. The impaired immune response against pathogens is among the inherent host factors that have been also associated with the pathogenesis of DS. Mononuclear phagocytes respond to the pathogens through phagocytosis followed by the production of several substances inside the phagosomes, among them are the reactive nitrogen species (RNS). A failure in these mechanisms may contribute to the DS development. Objective The aim of this study was to investigate the influence of aging on the internalization and the production of nitric oxide (NO) by peritoneal adherent cells (PAC), in response to Candida albicans (C. albicans). Material and methods PAC obtained from young and aged mice were challenged with dead or viable C. albicans by using predetermined proportions (cells:yeast) for 30 and 120 minutes. Phagocytosis was analyzed by acridine orange dye, and NO production by the Griess reaction. Results C. albicans phagocytosis by PAC from aged mice was similar to that of young mice, although the cells from older mice cells present more internalized fungi compared with matched control. In addition, a tendency towards impaired NO production by peritoneal mononuclear phagocytes from aged mice was observed. Conclusions PAC from aged mice may capture and store many fungi, which in turn may mean that these cells are effectively unable to eliminate fungi, probably due to impaired NO production. Therefore, considering the important role of C. albicans overgrowth in the pathogenesis of DS and the aspects observed in this study, aging may favor the onset and severity of local candidosis such as DS and its systemic forms.

A new modified watershed algorithm for image segmentation in cervical cancer cell adhesion / 军事医学

Objective To investigate an effective algorithm for image segmentation in cervical cancer cell adhesion , which enables accurate segmentation of the contour of adherent cells .Methods The images of target cells were extracted from the background area using level set methods , normalized with minimum values of transformation algorithms ,and multi-plied by the gradient image points in the region of interest ( ROI) to inhibit the undesired gradient information before the im-ages of adherent cells were segmented using labeled watershed algorithm .Results and Conclusion Compared to conven-tional watershed segmentation methods , this algorithm is not only effective in image segmentation of adherent cervical cancer cells with uneven staining and more accurate segmentation lines established around the contours of adherent cells , but of high clinical value .

Characteristics and response of mouse bone marrow derived novel low adherent mesenchymal stem cells acquired by quantification of extracellular matrix

PURPOSE: The aim of present study was to identify characteristic and response of mouse bone marrow (BM) derived low-adherent bone marrow mesenchymal stem cells (BMMSCs) obtained by quantification of extracellular matrix (ECM). MATERIALS AND METHODS: Non-adherent cells acquired by ECM coated dishes were termed low-adherent BMMSCs and these cells were analyzed by in vitro and in vivo methods, including colony forming unit fibroblast (CFU-f), bromodeoxyuridine (BrdU), multi-potential differentiation, flow cytometry and transplantation into nude mouse to measure the bone formation ability of these low-adherent BMMSCs. Titanium (Ti) discs with machined and anodized surfaces were prepared. Adherent and low-adherent BMMSCs were cultured on the Ti discs for testing their proliferation. RESULTS: The amount of CFU-f cells was significantly higher when non-adherent cells were cultured on ECM coated dishes, which was made by 7 days culturing of adherent BMMSCs. Low-adherent BMMSCs had proliferation and differentiation potential as adherent BMMSCs in vitro. The mean amount bone formation of adherent and low-adherent BMMSCs was also investigated in vivo. There was higher cell proliferation appearance in adherent and low-adherent BMMSCs seeded on anodized Ti discs than machined Ti discs by time. CONCLUSION: Low-adherent BMMSCs acquired by ECM from non-adherent cell populations maintained potential characteristic similar to those of the adherent BMMSCs and therefore could be used effectively as adherent BMMSCs in clinic.

Comparison of fracture site callus with iliac crest bone marrow as the source of plastic-adherent cells.

Background: Red marrow has been described as the main source of mesenchymal stem cells although its aspiration and isolation from bone marrow was reported to have significant donor site morbidity. Since secondary bone healing occurs through formation of callus as the result of proliferation and differentiation of mesenchymal stem cells, callus may become alternative source for mesenchymal stem cells. In this study, we compared the number of plastic-adherent cells from fracture site callus and bone marrow of iliac crest after two and four weeks of culture. Methods: Sixteen New Zealand rabbits were fracturized at the femoral shaft. Then, these rabbits were taken care. After two weeks of fracturization, 3 mL iliac crest bone marrow aspiration and callus extraction of eight rabbits were cultured (group I). The other eight rabbits were treated equally after four weeks of fracturization (group II). Simultaneously, the cultures were observed after one and two weeks. Four weeks later, they were harvested. Cells were counted using Neubauer hemocytometer. The average number of cells between the sources and groups were statistically analyzed using the unpaired t-test. Results: In group I, there were 2.6 ± 0.1 x 104 cells in the culture of iliac crest bone marrow aspirate and 2.5 ± 0.1 x 104 cells in culture of callus extract from fracture site (p = 0.34). In group II, there were 2.7 ± 0.1 x 104 cells and 2.1 ± 0.1 x 104 cells, respectively (p < 0.001). Conclusion: Fracture site callus at the second week post-fracturization may be potential as source of plastic-adherent cells compared with iliac crest bone marrow.

Immunopathological features developing in the mosquito midgut after feeding on anopheles gambiae mucin-1 / interleukin-12 cDNA immunized mice / Desarrollo de características inmunopatológicas en el intestino medio del mosquito después de alimentarse de ratones inmunizados con mucina-1 / interleucina-12 ADNc de anopheles gambiae

The mosquito midgut is the organ into which the blood meal passes and in which, within a peritrophic membrane secreted by the epithelium, the blood is retained during digestion and absorption. The mosquito midgut is lined with an actin filled microvilli that are exposed to the harsh environment of the gut lumen such as food particle abrasion, digestive hydrolases and attack by pathogens and parasites that are taken in by the blood meal. These microvilli are protected them these effects by the peritrophic matrix, the glycocalyx and the mucin proteins that line their epithelial surfaces. Immunization of BALB/c mice with AgMUC1/IL-12 cDNA has been shown to kill mosquitoes when fed on these mice. Mucin is one of the proteins produced in the mosquito midgut after a blood meal. The fine structure of the mosquito midgut epithelium interacting with immune factors such as antibodies or immune cells is of special significance for interpreting early events in the interaction between the mosquito midgut lining and the specific immune components present in the blood of AgMUC1/IL-12 cDNA immunized BALB/c mice. Following bright light microscopy, scanning electron and transmission electron microscopic observations of the features seen in mosquito midgut sections from An. gambiae mosquitoes fed on BALB/c mice immunized with AgMUC1/IL-12 cDNA, the most likely immune mechanisms responsible for mosquito killing could be cell mediated, most likely antibody dependent cellular cytotoxicity. Both necrotic and apoptotic processes that could be the cause of mosquito death were seen to take place in the cells lining the midgut epithelium.

El intestino medio es el órgano al cual pasa la sangre consumida por el mosquito y donde, mediante una membrana peritrófica secretada por el epitelio, esta sangre es mantenida durante la digestión y absorción. El intestino del mosquito está revestido por microvellosidades llenas de actina que son expuestas a las complejas condiciones en torno a la luz intestinal, tales como la abrasión producida por partículas de alimentos, hidrolasas digestivas y el ataque de patógenos y parásitos que son tomados en la sangre consumida. Estas microvellosidades se protegen de estos efectos mediante la matriz peritrófica, el glicocálix y las proteínas de mucina que revisten las superficies epiteliales. La inmunización con AgMUC1/IL-12 ADNc en ratones BALB/c ha demostrado ser útil para matar los mosquitos cuando se alimentan de estos ratones. La mucina es una de las proteínas producidas en el intestino medio del mosquito después de consumir sangre. La fina estructura del epitelio del intestino interactúa con factores inmunes tales como anticuerpos o células inmunes es de especial importancia para interpretar los eventos tempranos en la interacción entre el revestimiento del intestino medio y los componentes inmunológicos específicos presentes en la sangre de ratones BALB/c inmunizados con AgMUC1/IL-12 cDNA. Después de observar mediante microscopías de luz, electrónica de barrido y de transmisión las características de secciones del intestino medio del mosquito Anopheles gambiae alimentado de ratones BALB/c inmunizados con AgMUC1/IL-12 cDNA, mecanismos inmunes mediados por citotoxicidad celular dependiente de anticuerpos (ADCC) podrían ser los responsables de matar a los mosquitos. Los procesos necróticos y apoptóticos que pueden ser la causa de la muerte del mosquito tienen lugar en las células que recubren el epitelio del intestino medio.

Iliac crest and femoral bone marrow as the source of plastic-adherent cells.

Background: Many studies have used iliac crest as the source of mesenchymal stem cells. In cases of long bone shaft fracture, obtaining marrow from the fracture site offers more advantages. Nevertheless, due to the high number of fat cells in long bones, the yellow marrow of long bones is believed to contain lower number of mesenchymal stem cells than red marrow. Therefore the aim of this study is to compare the potency between red and yellow marrow as the donor site for the isolation of mesenchymal stem cell. Methods: Bone marrow of eight giant Flemish rabbits was aspirated from the iliac crest and femoral shaft. Mononuclear cells were isolated from both aspirates and expanded in low glucose DMEM. After eight weeks, the cells were harvested and counted using improved Neubauer hemocytometer. Comparison of the cell number between the two donor sites was then performed by t-test. Results: After 8 weeks, an average number of 2.93 ± 0.91 x 104 and 3.7 ± 2.50 x 104 cells were obtained from the iliac and femoral group respectively. No statistically significant difference was found between those two groups (p = 0.45). Conclusion: Plastic-adherent cells can be isolated and expanded from both iliac crest and femoral shaft.

Neural cell induction with human placenta derived adherent cells / 基础医学与临床

Objective The expression of hematopoietic stem cell and neural markers on placenta adherent cells and their association were investigated, the potential of neural differentiation of placenta adherent cells may demonstrate the relationship between placenta adherent cells and hematogenesis and neurogenesis. Methods Isolated adherent cells in human placenta tissue were stained immunocytochemically with Nestin, MAP_2, MBP, CD133, CD34 antibody to detect protein production and induced neural differentiation with β-mercaptoethanol and retinoic acid. Results Isolated placenta adherent cells confirmed to be heterogenous expressed CD133, Nestin, MAP_2 weakly and CD34 none. While induced cells showed neural like morphology, strong positive for Nestin, MAP_2 and weak positive for MBP and GFAP in immunocytochemical staining. Conclusion Human placenta adherent ceils can be induced into neural like cells in vitro with histological characteristics of mesenchymal stem cell(MSC) ,it provides a new source for clinical application of hMSCs.

Measurement of annexin V uptake and lactadherin labeling for the quantification of apoptosis in adherent Tca8113 and ACC-2 cells

Phosphatidylserine (PS) exposure occurs during the cell death program and fluorescein-labeled lactadherin permits the detection of PS exposure earlier than annexin V in suspended cell lines. Adherent cell lines were studied for this apoptosis-associated phenomenon to determine if PS probing methods are reliable because specific membrane damage may occur during harvesting. Apoptosis was induced in the human tongue squamous carcinoma cell line (Tca8113) and the adenoid cystic carcinoma cell line (ACC-2) by arsenic trioxide. Cells were harvested with a modified procedure and labeled with lactadherin and/or annexin V. PS exposure was localized by confocal microscopy and apoptosis was quantified by flow cytometry. The detachment procedure without trypsinization did not induce cell damage. In competition binding experiments, phospholipid vesicles competed for more than 95 and 90 percent of lactadherin but only about 75 and 70 percent of annexin V binding to Tca8113 and ACC-2 cells. These data indicate that PS exposure occurs in three stages during the cell death program and that fluorescein-labeled lactadherin permitted the detection of early PS exposure. A similar pattern of PS exposure has been observed in two malignant cell lines with different adherence, suggesting that this pattern of PS exposure is common in adherent cells. Both lactadherin and annexin V could be used in adherent Tca8113 and ACC-2 cell lines when an appropriate harvesting procedure was used. Lactadherin is more sensitive than annexin V for the detection of PS exposure as the physical structure of PS in these blebs and condensed apoptotic cell surface may be more conducive to binding lactadherin than annexin V.

Neural cell induction with human placenta derived adherent cells / 基础医学与临床

Objective The expression of hematopoietic stem cell and neural markers on placenta adherent cells and their association were investigated,the potential of neural differentiation of placenta adherent cells may demonstrate the relationship between placenta adherent cells and hematogenesis and neurogenesis.Methods Isolated adherent cells in human placenta tissue were stained immunocytochemically with Nestin,MAP2,MBP,CD133,CD34 antibody to detect protein production and induced neural differentiation with ?-mercaptoethanol and retinoic acid.ResultsIsolated placenta adherent cells confirmed to be heterogenous expressed CD133,Nestin,MAP2 weakly and CD34 none.While induced cells showed neural like morphology,strong positive for Nestin、MAP2 and weak positive for MBP and GFAP in immunocytochemical staining.Conclusion Human placenta adherent cells can be induced into neural like cells in vitro with histological characteristics of mesenchymal stem cell(MSC),it provides a new source for clinical application of hMSCs.

Fetal Membrane Derived Adherent Cells: a Novel Source for Mesenchymal Stem Cells / 中国生物工程杂志

Objective: To establish a method for isolation and purification of fetal membrane derived adherent cells (FMDACs) , and investigate their biological characteristics. Method: FMDACs were isolated with trypsin inducing and cultured in vitro. FMDACs were induced to differentiate into osteoblasts and adipocytes. FACS and immunocytochemistry technique were used to examine the cell surface antigen. The genetic stability was verified by karyotype analysis. Results: FMDACs were successfully isolated and expanded in vitro. They had strong proliferative ability. FMDACs were positive for CD44 and CD29, but negative for CD34, CD14 and CD45. FMDACs were differentiated into osteoblasts and adipocytes after inducement. The karyotype was stable in the sixth-passaged FMDACs and the tumorigenicity was not found. Conclusion; FMDACs have the possibility of multipotent stem cells, which have strong capacities of self-renewal and multidirectional differentiation. The genetic background of FMDACs is stable. FMDACs may be used as a kind of novel seed cells for tissue engineering.