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BACKGROUND: The particular bystander effect of suicide gene can remarkably kill tumor cells. Meanwhile, it can be used together with radiotherapy as well as immune gene therapy, and overcome the defect of low gene transduction efficiency.Cytosine deaminase (CD) can generate a powerful bystander effect.OBJECTIVE: To observe the effect of a eukaryotic expression plasmid plRES2-AcGFP1-CD mediated by liposome transfected into bone marrow mesenchymal stem cells (BMSCs) and its gene expression.DESIGN, TIME AND SETTING: A cytologic experiment at genetic level was performed at Research and Development Center of Stem Cell and Tissue Engineering, Dalian University of Technology from May to December 2007.MATERIALS: A total of 6 C57BL mice of SPF degree and weighing 18-20 g were used for separation and culture of BMSCs.Escherichia coli DH5a was provided by Research and Development Center of Stem Cell and Tissue Engineering, Dalian University of Technology. Lipofectamine~(TM) 2000 was provided by Invitrogen, China.METHODS: The DNA plasmids were extracted from transformed Escherichia coli DH5a. plRES2-AcGFP1-CD plasmid was identified by BamHI/Xhol double digestion. The BMSCs derived from mouse femur and tibia were cultured and purified by adhesion method in vitro. Signal cell suspension prepared by BMSCs of the third generation was cultured by adding fluorescence-labeled CD44, CD45, CD90 and CD105 antibodies. BMSCs of the third generation were transfected by LipofectamineTM 2000 mediation.MAIN OUTCOME MEASURES: Identification of recombinant plasmids; the expressions of surface markers on BMSCs were detected by flow cytometry; the expressions of cytosine deaminase gene were observed at 36 and 48 hours after transfection under fluorescent inverted microscope.RESULTS: After agarose gel electrophoresis, a band appeared at 1.0-1.5 kb of the digested products of plRES2-AcGFP1-CD plasmids, and the band was accorded with the length of cytosine deaminase gene in the length. Flow cytometry demonstrated that the cells were negative for CD45 but positive for CD44, CD90 and CD105. Fluorescent inverted phase contrast microscope suggested that at 36 hours after plRES2-AcGFP1-CD gene transfection an expression of green fluorescent protein was found in the BMSCs; additionally, at 48 hours after transfection, the expression of green fluorescent protein remained and the intensity was remarkably increased.CONCLUSION: The liposome-mediated plRES2-AcGFP1-CD gene successfully expressed in BMSCs, and the expression peaked at 48 hours after transfection.
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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.
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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.
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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.
Assuntos
Humanos , Antígenos CD34 , Metabolismo , Reatores Biológicos , Técnicas de Cultura de Células , Métodos , Proliferação de Células , Células Cultivadas , Meios de Cultura , Citocinas , Farmacologia , Sangue Fetal , Biologia Celular , Células-Tronco Hematopoéticas , Biologia Celular , FisiologiaRESUMO
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.