ABSTRACT
Objective To investigate the expression and role of β-catenin in small-for-size liver grafts during early stage of liver regeneration after liver transplantation in rats.Method The livers of male Sprague-Dawley rats were reduced to 30％ or 50％ of their original sizes and transplanted.The experiment was divided into 3 groups:small-for-size graft group (SSG),half-size graft group (HSG) and sham-operated group.Liver samples were harvested at various time points after transplantation (n =6 per time point) for Western blotting and immunohistochemistry.Six rats in each group were sacrificed at 3rd day after liver transplantation for estimating liver regeneration rate.Result Liver regeneration rate in SSG group was lower than that in HSG group.The expression of β-catenin was down-regulated in liver graft of both groups after being stored in cold Ringer solution for 2 h.The expression of β-catenin was significant up-regulated in HSG group from 5 min to 12 h after operation,while the down-regulated expression of β-catenin was persisted in SSG group at 5 min after operation,and mildly increased expression of β-catenin occurred at 2 h and 6 h,which was significantly lower than that in HSG group at the corresponding time points.The expression of active-β-catenin was low in each group before transplantation.Significant expression of active-β-catenin was found at 5 min in HSG group and persisted until 12 h after operation,mildly increased expression of active-β-catenin in SSG group was only found at 2 h,which was lower than that in HSG group at the same time points.Immunohistochemical staining revealed that β-catenin was mainly expressed on the hepatocyte membrane and in cytoplasm in the sham-operative group,many hepatocytes exhibited nuclear localization of β-catenin in HSG group from 5 min to 24 h,while only some hepatocytes exhibited nuclear localization of β-catenin in SSG group.The expression of Cyclin D1 in SSG group was significantly lower than that in HSG group,which was similar to the expression of C-Myc.Conclusion Attenuated activation of Wnt/β-catenin signaling and down-regulated expression of target genes during early regeneration of small-for-size liver grafts may be involved in the inhibition of liver regeneration of small for size liver grafts.
ABSTRACT
Poly (N-isopropylacrylamide) (PNIPAAm), a temperature-responsive polymer, can be potentially applied to replace enzymes or cell scrapers to recover attached cells. Taking full advantage of this unique function of PNIPAAm, cells can be protected from enzymatic hydrolysis and mechanical treatment, thereby to provide ideal seed cells with high quality for biomedical fields. In this review we describe the method to facilitate cell effective adhesion and rapid detachment on thermoresponsive two dimensional surfaces, including selecting special substrate, introducing hydrophilic group, adjusting reactant ratio, controlling polymer thickness/density, providing appropriate external force, so as to effectively improve adherent cell adaptability to thermoresponsive surfaces, depress the risk of bacterial contamination and reduce the effect of low-temperature treatment on the cells. The three dimensional cell culture systems involved in temperature-sensitive microcarriers, scaffolds and gels were briefly discussed. The application based on the platforms for cell culture was also presented.
Subject(s)
Acrylic Resins , Cell Adhesion , Cell Culture Techniques , TemperatureABSTRACT
To study the effect of sphingosine-1-phosphate (S1P) on the cardiomyogenic differentiation of human umbilical cord mesenchymal stem cells (UC-MSCs) and human adipose-derived mesenchymal stem cells (AD-MSCs), we seeded the cells in the culture plates and used cardiomyocyte culture medium (CMCM) combining with different concentration of S1P to induce UC-MSCs and AD-MSCs in vitro for 7, 14 and 28 days. Cardiomyogenic differentiations were identified through immunofluorescence staining, and the results were observed with fluorescence microscopy and confocal microscopy. The effects of S1P and CMCM on cell activity were evaluated by the methyl thiazolyl tetrazolium assay. The functional characteristic similar to cardiomyocytes was evaluated through detecting calcium transient. Our results showed that cardiomyogenic differentiation of UC-MSCs or AD-MSCs were enhanced with S1P concentration increasing, but cell activities declined. Results showed that the suitable differentiation time was 14 days, and the optimal concentration of S1P was 0.5 micromol/L. When working together with CMCM, S1P could promote the differentiation of UC-MSCs or AD-MSCs into functional cardiomyocytes, giving rise to specific electrophysiological properties (the calcium transient). Taken together, our results suggested that S1P could promote the differentiation of UC-MSCs or AD-MSCs into functional cardiomyocytes when being cultured in CMCM.
Subject(s)
Humans , Adipose Tissue , Cell Biology , Metabolism , Cell Differentiation , Cells, Cultured , Culture Media , Lysophospholipids , Pharmacology , Mesenchymal Stem Cells , Cell Biology , Myocytes, Cardiac , Cell Biology , Sphingosine , Pharmacology , Umbilical Cord , Cell BiologyABSTRACT
Objective To investigate the expressions of tyrosine kinase receptor (Trk)B and brain-derived neurotrophic factor (BDNF) protein in human gastric careinoma and compare them with those in epithelial cells of normal mucous, in order to evaluate their clinicopathological significance. Method The expressions of TrkB and BDNF protein in tumor tissues, matched with para-tumor mueosal tissues from 64 cases with gastric carcinoma and normal mucous of 20 cases were observed immunohistochemically and related to some of the clinicopathological parameters. Results The positive rates of TrkB and BDNF protein in tumor tissues were 60.9% and 59.4% respectively, but there was negative in matched para-tumor mueosal tissues and normal mucosal tissues. TrkB and BDNF protein expressions were related to invasive depth,lymph node metastasis and TNM stage of cancer, but not to sex, age and degrees of cancerous differentiation.The positive rates of TrkB and BDNF protein in cases with serosal infiltration, lymph node metastasis and TNM stage Ⅲ - Ⅳ were significantly higher than those in cases without serosal infiltration, lymph node metas-tasis, and TNM stage Ⅰ - Ⅱ (P< 0.01 ). In group with metastasis the positive rates of TrkB and BDNF protein were lower in metastatic foci (76.5%, 26/34, 70.6%, 24134) than those in primary tumor (85.3%, 29134,82.4%, 28/34 ), but statistically there was no significant difference between them (P > 0.05 ). Conclusions The expressions of TrkB and BDNF protein are closely relatedto tumorigenesis and progression of gastric carcinoma. The increased expression of TrkB and BDNF may promote the occurrance of local invasion and metastasis of gastric carcinoma.
ABSTRACT
BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are easily isolated and amplified, and facilitate the exogenous gene transfer and expression. In the human medicine, it is believed that BMSCs are ideal therapeutic cells and target cells in the gene therapy.OBJECTIVE: To investigate liposome-mediated cytosine deaminase (CD) gene transfecting rabbit BMSCs and its gene expression. DESIGN: A single sample observation. SETTING: Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University.MATERIALS: This study was performed at in the Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University from March 2006 to June 2007. New Zealand big-ear white rabbits of either gender, weighing 2.0-2.5 kg, with the age of 5 months old, were included in this study. METHODS: The CD gene was obtained from E.coli JM109 DNA by polymerase chain reaction (PCR). The fragment was cloned into pMD19-T vector. After restriction enzyme BamHI/XhoI digestion analysis and DNA sequence analysis, pIRES2-AcGFP1-CD eukaryotic expression plasmid was constructed. Meanwhile, BMSCs were harvested, cultured and identified. After enzyme digestion of eukaryotic expression plasmid, the rabbit BMSCs were transfected by Lipofectamine 2000-mediated method. Twenty-four hours after transfection, expression of green fluorescent protein was observed under an inverted fluorescent microscope. MAIN OUTCOME MEASURES: Construction of eukaryotic expression plasmid and identification of CD gene-transferred BMSCs. RESULTS: CD gene was cloned and connected to eukaryotic expression plasmid with green fluorescence. Twenty-four hours after transfecting rabbit BMSCs, it was found under an inverted microscope that under the excitation of 488 nm blue light, green fluorescence appeared in the pIRES2-AcGFP1-CD and pIRES2-AcGFP1 empty-plasmid transfected BMSCs, but not in the non-transfected ones. It indicates that CD gene successfully transferred BMSCs. CONCLUSION: BMSCs are ideal vectors in the CD gene therapy.
ABSTRACT
BACKGROUND: Previous studies have demonstrated that neural stem cells play potential therapeutic effects on the repair of spinal cord injury. However, the time for acquiring the best allotransplantation effects remains unclear.OBJECTIVE: This study was designed to observe the repairing effects of allotransplantation of embryonic neural stem cells on the motor function of rat two posterior limbs after spinal cord injury and investigate the time effectiveness of the allotransplantation.DESIGN: A controlled observational experiment.SETTING: Laboratory of Molecular Biology, Dalian Medical University; Laboratory of Biomedicine, School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian, Liaoning Province, China.METHODS: This study was performed at the Laboratory of Molecular Biology, Dalian Medical University & Laboratory of Biomedicine, School of Environmental and Biological Science and Technology, Dalian University of Technology between July and August 2003. One albino rat of gestational 14-16 days was sacrificed for harvesting embryonic rat brain cells. Embryonic rat cerebral cortex and subcortical periventricular brain tissue were taken for in vitro culture of rat embryonic neural stem cells. An additional 30 adult Sprague Dawley rats were randomly divided into 3 groups with 10 rats in each group: control, early allotransplantation and delayed allotransplantation groups. All 30 rats were subjected to spinal cord transection injury, leading to rat paralysis of both lower extremities. Embryonic rat neural stem cells were transplanted into the rats in the early and delayed transplantation groups at 3 days and 3 weeks after injury, respectively. Following allotransplantation, motor function of rat two lower extremities was followed. At 4 weeks after allotransplantation of neural stem cells, rat spinal cord was harvested from transplanted region for immunohistochemistry in order to observe and compare the morphological change of rat spinal cord tissue among the 3 groups. The following protocol was performed in accordance with ethical guidelines stated in Guide for the use and care of laboratory animals, approved by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Commission on Life Scineces, National Research Council, China (1985).MAIN OUTCOME MEASURES: Motor functional recovery of rat two lower extremities after neural stem cell transplantation. Histomorphological change of rat spinal cord at 4 weeks after neural stem cell transplantation.RESULTS: Thirty rats were included in the final analysis. In the early and delayed transplantation groups, the motor function of rat two lower extremities was noticeably improved, in particular in the early transplantation group. In the two experimental groups, muscular strength of paralyzed rat two lower extremities began to recover 5 or 6 days after transplantation of neural stem cells. Two or three weeks later, all rats in the two experimental groups could crawl and four weeks later, two extremities could move actively (approximately approaching to score 3 prescribed as follows). In the control group, no recovery of paralyzed extremities was found. At 4 weeks after transplantation, in the early transplantation group, proliferative tissue could be visible in the spinal cord transplantation region. Through the use of microscope, a considerable number of new cells were found that presented with neuronal and glial cell-positive staining. In the control group, a cavity between two broken ends could be visible. Meanwhile, necrosis and vacuolar degeneration, and other symptoms in the stump of spinal cord were observed with a microscope. In the delayed transplantation group, the histomorphological change of spinal cord region was between the other two groups. No typical histomorphological change was found. A number of new cells were apparent with a microscope, but the number was less compared with the early transplantation group.CONCLUSION: Allotransplantation of embryonic neural stem cells promotes the recovery of rat motor function after spinal cord transection. Early transplantation acquires better therapeutic effects.
ABSTRACT
Rotating wall vessel (RWV) was used for the ex-vivo expansion of umbilical cord blood stem cells to meet the requirement of clinical application in the aspect of quantity and quality of the stem cells. The mononuclear cells (MNCs) from umbilical cord blood were cultured in T-flasks for 24 h, and then inoculated in RWV to culture for 200 h. The nucleated-cell numbers, pH and osmolality of the culture medium were determined every 24 h. The CD34+ cells content was measured and CFU-GM culture was carried out at 144 h and 197 h. Nucleated cells (NC) and CD34+ cells had a 435.5 +/- 87.6 fold expansion and a 32.7 +/- 15.6 fold expansion respectively in 197 h, and CFU-GM (colony-forming unit-granulocyte/macrophage) cells had a 21.7 +/- 4.9 fold expansion. In the whole course of culture, the pH and osmolality of the medium in the RWV were kept in the optimal hematopoietic stem cells' expansion conditions. pH was kept from 7.2 to 7.4, and the osmolality was kept from 290 mmol/kg to 310 mmol/kg. Owing to its structural particularity, the RWV could ensure cells to grow in the suspension state, could simulate the micro-environment of umbilical cord blood, and thus could make the hematopoietic stem cells expand largely in the RWV in short time.
Subject(s)
Humans , Antigens, CD34 , Metabolism , Bioreactors , Cell Culture Techniques , Methods , Cell Proliferation , Cells, Cultured , Culture Media , Cytokines , Pharmacology , Fetal Blood , Cell Biology , Hematopoietic Stem Cells , Cell Biology , PhysiologyABSTRACT
When the size of a neurosphere cultured in vitro reaches a certain critical value, a necrotic core will appear inside the neurosphere because of the limitation of oxygen or other nutrients transport from medium to the cells in the neurasphere. Large necrotic core will greatly reduce the expansion of NSCs. The cellular automaton (CA) model is applied in this article to model the growth of NSCs in sphere state. The appearance and enlargement of the necrotic core in a neurosphere is calculated by coupling the CA model with the nutrient diffusion analysis in bioreactors. The calculation results indicate that the culture conditions, such as seeding density, the concentration of nutrients in medium and the mass transfer coefficient between a neurosphere and medium, have some effects on the appearance of the necrotic core. However, the necrotic core mainly depends on the inner diffusion. It will certainly appear if the size of the neurosphere is large enough even the outside mass transfer is in a good condition in bioreactors. Additionally, the appearance of the necrotic core resulting from the shortage of oxygen is earlier than that caused by the limitation of glucose. And the growth of the necrotic core is very fast after its appearance, and the whole neurosphere may become necrotic. The model developed with cellular automaton and mass transfer is a good qualitative representation of NSCs growth in bioreactors.
Subject(s)
Bioreactors , Cell Culture Techniques , Methods , Cell Differentiation , Cell Proliferation , Cells, Cultured , Computer Simulation , Models, Biological , Neurons , Cell Biology , Spheroids, Cellular , Stem Cells , Cell Biology , Tissue Engineering , MethodsABSTRACT
The nanoparticle-modified surfaces were built up by alternating deposition of oppositely charged Al2O3 and SiO2 nanoparticles (from 10 nm to 500 nm) solutions. The properties of these nanoparticle-modified surfaces and the controls were investigated by Atomic Force Microscope for topography analysis. Pseudomonas Fluorescence (PF) cell adhesion was evaluated by microscopic determination of the numbers of cells that adhered to the produced slides exposed to PF cell suspensions on static and dynamic condition. The results show that adhesion of PF to both surfaces readily increases with the time of exposition but the adhered numbers of PF on produced surfaces are considerably higher than that on controls in static condition. Cell morphologies on these nanoparticle-modified surfaces studied by inverted microscope show that the adhered PF on the produced surfaces are more in presence of clusters, which contributes more to the total adhering numbers in the late of cell adhesion assays. Meanwhile on controls the cells rarely attained confluence and had a single shape. The significant statistical correlation observed between nanoparticle-modified surfaces and control adds a new concept to the studies of substratum topography influence on cell behavior. The results suggest that nanoparticle-modified surfaces may enhance the interactions between PF cell and slides.
Subject(s)
Bacterial Adhesion , Fluorescence , Nanoparticles , Pseudomonas , Cell Biology , Silicon Dioxide , Surface PropertiesABSTRACT
Purpose:To study the expression of survivin a nd its relationship with the expression of bcl-2,p53,PCNA in the dysplasia of gastric mucous epithelium and gastric carcinoma.Methods:The expression of survivin,bcl-2,p53,PCNA was detected in 60 cases of gastric carcinoma, 30 cases of dysplasia of gastric mucous epithelim and 8 cases of normal gastric mucous tissue by using immunohistochemical SP method. Results:The positive rates of survivin were 10%,13%,80% and 82%, respectively, in mild, moderate and svere dysplasia and gastric carcinoma. The positive rates of survivin in mild and moderate dy splasia were significantly lower than in severe dysplasia and carcinoma (P0 05). There was a relationship between survivin gene expression with the invasive depth of gastric carcinoma. lymph node metastases and survival stage (P