Protective effect of calycosin-7-O-ß-D-glucopyranoside against oxidative stress of BRL-3A cells induced by thioacetamide.

BACKGROUND: Calycosin-7-O-ß-D-glucopyranoside (CG) is a natural isoflavone found in traditional Chinese medicines Astragali Radix (AR). OBJECTIVE: Calycosin-7-O-ß-D-glucopyranoside, an isoflavone isolated from AR, has been found to have potent antioxidantive effects. This study was designed to investigate whether CG prevents oxidative stress induced by thioacetamide (TAA). MATERIALS AND METHODS: BRL-3A cells were pretreated with different concentrations of CG (10, 20, 40 mg/mL) for 12 h and then exposed to 0.18 mol/L TAA for 2 h. The cell viability were examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium assay, total antioxidant capacity, malondialdehyde (MDA) and the activity of antioxidant enzymes, including catalase, glutathione peroxidase and superoxide dismutase were determined by microplate method. Reactive oxygen species (ROS) generation was quantified by the 2',7'-dichlorofluorescin-diacetate method. Protein and mRNA expression of CYP2E1 were determined by western blotting and real-time PCR. RESULTS: The cell oxidative stress was significantly increased after 2 h of TAA exposure. Pretreatment of BRL-3A cells with CG significantly increased the activities of antioxidant enzymes, scavenged ROS and reduced MDA production. CG decreased the expression of CYP2E1, and ultimately decreased TAA-induced BRL-3A cells oxidative stress. CONCLUSIONS: Calycosin-7-O-ß-D-glucopyranoside has a protective effect against TAA-induced oxidative stress in BRL-3A cells, and that the underlying mechanism involves in scavenging of ROS and the modulating expression of CYP2E1.

Transfection of mouse bone marrow mesenchymal stem cells with Lipofectamine-mediated cytosine deaminase genes / 中国组织工程研究

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.

Construction of pIRES2-AcGFP1-CD eukaryotic expression plasmid and its expression in bone marrow mesenchymal stem cells / 中国组织工程研究

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.

Effects of leptin supplementation in in vitro maturation medium on meiotic maturation of oocytes and preimplantation development of parthenogenetic and cloned embryos in pigs.

The objective of the present study was to investigate the effects of leptin addition in in vitro maturation (IVM) medium on meiotic maturation of oocytes and preimplantation development of parthenogenetic and cloned embryos in pigs. In experiment 1, oocytes were matured in North Carolina State University 23 (NCSU-23) medium supplemented with various concentrations of leptin: 0, 1, 10 and 100 ng/ml. IVM medium added with 10 or 100 ng/ml leptin significantly increased the rate of oocytes reaching metaphase II compared to the control (76.8% and 73.8% versus 61.7%). In experiment 2, the influence of the timing of leptin addition in IVM medium on meiotic maturation of porcine oocytes was assessed, and maximum maturation rate of oocytes developing to metaphase II was achieved when supplemented during the first half (0-22 h), the latter half (22-44 h) or the entire maturation period (0-44 h) compared to the control (80.5%, 84.7% and 78.1% versus 70.4%). In experiment 3, leptin strikingly increased the blastocyst rate of parthenogenetic embryos at the concentration of 10 ng/ml (37.5% versus 21.7%) and this increase was independent of the addition timing (0-44, 0-22, 22-44 h) compared to the control (32.5%, 34.6% and 31.5% versus 16.2%). Moreover, total cell number per blastocyst of parthenogenetic embryos was obviously increased in the 10 and 100 ng/ml leptin treatments as compared with the control (36, 38 versus 28). In experiment 4, 10 ng/ml leptin treatment significantly increased the rate of cleavage (72% versus 56%) of cloned embryos. Meanwhile, the rate of blastocyst formation was also improved although no significant difference was found (12.8% versus 7.1%). Collectively, our results indicate that leptin supplementation in IVM medium may be beneficial not only for developmental potential of oocytes but for subsequent developmental competence of embryos produced by parthenogenetic activation and the cleavage of embryos derived by somatic cell nuclear transfer (SCNT).

Studies on tissue culture and rapid propagation technique of Iphigenia indica / 中草药

Object To establish a tissue culture and rapid propagation system for the medicinal plant Iphigenia indica Kunth. Methods Clusters of seedlings and protocorms and induction were studied on MS media with different parts of the plant, such as corms, stems, leaves and root tips as explants. Results The proper media for protocorm inducing, propagation and rooting were found in this paper, rapid propagation system of I. indica was established and the plant could put in large scale production. Conclusion Sprout can be successfully induced from corm and stem on MS media. MS+6-BA 2 mg/L+NAA 0.5 mg/L can induce both sprout and protocorm, and fit for large scale production. 1/2 MS+IBA 0.5 mg/L+NAA 0.2 mg/L is good for rooting.