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.

Adenocarcinoma, Papillary ; blood supply ; metabolism ; pathology ; Adult ; Aged ; Carcinoma, Pancreatic Ductal ; blood supply ; metabolism ; pathology ; Chemokine CXCL12 ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lymph Nodes ; metabolism ; Lymphatic Metastasis ; Male ; Microvessels ; pathology ; Middle Aged ; Neoplasm Staging ; Neovascularization, Pathologic ; metabolism ; pathology ; Pancreas ; metabolism ; Pancreatic Neoplasms ; blood supply ; metabolism ; pathology ; Receptors, CXCR4 ; metabolism

Objective To explore the effect of sodium fluoride on DNA damage and apoptosis on ox peripheral blood lymphocyte cultured in vitro.Methods Using lymphoeytes separation medium lymphocytes were separated and different concentration of NaF(0,4,8,12,16 mg/L)were added into the cultual system of lymphocytes for 24 h.Cell viability was measured by MTT.nuclear changes stained by acridine orange-ethidium bromine staining (AO/EB)were observed under fluoroscope,DNA fragment was measured by agarose gel electrophoresis.DNA damage was detected by alkaline SCGE.The differences between each groups were compared.Results Cells were exposed to 4,8,16 and 24 mg/L NaF in 24 h,cell survival rates,respectively being(73.743±0.552)%,(69.184±0.724)%,(65.736±0.055)%and(63.651±0.287)%,decreased significantly compared to control group.There were distinctive cell apoptosis,evident DNA damage and visible dose-effect relation(R2=0.7456).Conclusions A certain concentration of sodium fluoride lcads to lymphocytes apoptosis and DNA damage.

Background Fibrin glue has been utilized to adhere the graft during the pterygium excision with conjunctival autografts.Several relevant clinical randomized controlled trial(RCT) and retrospective studies have been published abroad,but the samples for its effectiveness and safety issue of fibrin glue and suture are still underinvestigation.Objective Current study was to quantificationally assess the efficacy and safety of fibrin glue versus sutures in the application of pterygium excision with conjunctival autografts.Methods Based on established search strategy,a computerized literature search was conducted to identify all citations concerning the RCT for effectiveness and safety evaluation of fibrin glue and suture for the graft fixation during the pterygium excision with conjunctival autografts from MEDLINE ( from 2000 to October,2010 ),EMbase ( from 2000 to October,2010 ),Cochrane Central Register of Controlled Trials ( Issue 4,2010 ),CBMdisc ( 2000 to October 2010 ),CNKI ( 2000 to October 2010 ),and the relevant conference proceedings and references searched by hand was performed as supplement.The included literature was scored with Jadad table.The Cochrane Collaboration' s RevMan 5.0 software was used for the test of heterogeneity or test for overall effect.The effective indexes,such as operative duration,recurrence rate and complication,were evaluated by Meta analysis.Results Six RCTs involving 401 eyes of 377 participants were identified.These literatures were published with English in 2004-2010 from China,New Zealand,Sweden,Israel,Turkey and Malasia and the Jadad scores were 4-5.The quantitatively analysis revealed that fibrin glue appeared to short the operative time compared with suturing method (MD =14.23 ;95％ CI:- 16.18- 12.29;P=0.00) and drop the rate of recurrence ( RR =0.49,95％ CI:0.26 -0.95 ; P =0.03 ).No significant differences were found in the rate of postoperative graft dehiscence or absence (RR =3.41,95 ％ CI:0.85-13.68;P =0.08 ).Conclusions Fibrin glue shows the good effectiveness and easy application during the pterygium excision with conjunctival autografts.Long-term follow-up of multi-central RCTs with a larger number of cases are still needed to support this conclusion.

Objective:To explore the expression of MMP-3 and extracellular matrix metalloproteinase inducer(EMMPRIN)in benign and malignant breast lesions and its relevance.Methods:Using immunohistochemical method,we examined the expression of MMP-3 and EMMPRIN proteins in 58 breast cancer specimens,28 premalignant lesions,40 hyperplasia specimens,40 benign tumor specimens and 20 normal breast tissues.The expression of MMP-3 mRNA and EMMPRIN mRNA was examined using in situ hybridization in 20 breast cancer specimens,20 premalignant lesions,20 hyperplasia specimens,20 benign tumor specimens and 20 normal breast tissues.The values of integral optic density(IOD)of the expression was calculated by image analysis system.Results:The expression of MMP-3 and EMMPRIN proteins and mRNA was stronger in breast cancer and premalignant lesions than in the other lesions.The IODs of MMP-3 and EMMPRIN proteins and mRNA in breast cancer and premalignant lesion were significantly higher than those of hyperplasia,benign tumors and normal breast tissues(P

Objective: Study on the growth character of B16 melanoma cell which can express angiostatin. Methods: Angiostatin gene was constructed from human plasminogen cDNA by deletion mutation. A B16 melanoma cell clone named BAG28 which stably expresses angiostatin was established by introducing gene into it. Results: BAG28 in vitro had no changes in proliferation rate and the ability of clone formation in soft agar. Study in vitro showed that the tumor weight had reduced about 87% ( P

Objective: To clone Fd and light chain genes of monoclonal antibody HAb18 against human hepatoma and verify their accuracy and liability. Methods: Total RNA was extracted from hybridoma cell line secreting MAb HAb18, and Fd and light chain genes were amplified by RT-PCR. After PCR products were ligated into pMD18T vector, positive clones were screened and DNA sequences were tested and analysed by relative softwares. Then, light chain and Fd genes were sequential cloned into phage display vector pComb3. After recombinant vector was transformed into E.coli XL1-blue, recombinant vector was rescued by helper phage M13K07 and the specificity of phages to antigen was detected by indirect ELISA. Results: The size of amplified Fd and light chain genes was separately 665 bp and 668 bp. The results of sequence analysis showed that both VL and VH contained 2 characteristic cystines and CH1 was IgG1 classes and CL was ?. ELISA result identified that expressed Fab antibody could specially bind to corresponding antigen. Conclusion: Fd and light chain genes of MAb HAb18 were successfully cloned, which lay a good foundation for constructing a diversity of engineering antibody.

Objective To observe enhancing effect of nerve regeneration on peripheral nerve defect models bridged by silicone tube idled with valproic acid (VPA). Methods In present research we demon-strate the effect of VPA on peripheral nerve regeneration and recovery of motor function following sciatic nerve transaction in rats. An 8-mm sciatic nerve deficit was created in a rat mode land bridged by a 1-cm silicone tube.Then, 10 lad of 8% VPA were perfused into the silicone chamber in the VPA group. The same volume of normal saline was delivered in the control group. Results Each animal was observed sciatic nerve function index (SFI) at 2-week intervals and studied electrophysiology at 4-week intervals for 12 weeks. Histological and morphometrical analyses were performed at the end of the experiment, 12 weeks after operation. Using the digital image-analysis system, thickness of the myelin sheath was measured, and total numbers of regenerated axons were counted. There was a significant difference in SFI, electrophysiological index (motor-nerve conduct velocity, MCV), and morphometrical results (regenerated axon number and thickness of myelin sheath) in nerve regeneration between the VPA group and controls (P ＜ 0.05). Conclusion The results demonstrated that VPA is able to enhance sciatic nerve regeneration in rats, suggesting the potential clinical application of VPA for the treatment of peripheral nerve injury in humans.

Objective To analyze the characteristic and the clinical application of high -frequency stimulation ABR for the patients with unilateral sudden deafness .Methods 40 patients aged 17~45 with unilateral sudden deaf-ness(40 health ears ,subject group)and 20 normal volunteers (40 ears ,control group)were selected to receive high and low frequency stimulation ABRs and pure tone audiometry .Results There were no statistical differences be-tween subject group and control group in pure tone audiometry .The wave Ⅰlatency of high -frequency ABR was longer in the subject group than that in the control group(P<0 .001) .The abnormal rate of Ⅰ - Ⅴ interval between high and low frequency stimulation ABRs was 62 .5% (25/40) in the subject group ,and 5% (2/40) in the control group .There were statistical differences between the two groups (χ2 =29 .574 ,P<0 .001) .Conclusion The waveⅠlatency of high-frequency ABR was longer and the abnormal rate of Ⅰ - Ⅴ interval markedly increased in the health ear of patients with unilateral sudden deafness .This may suggest that the contralateral ear may have an ab-normal blood supply of the inner ear when single side sudden deafness happened .High frequency stimulation ABR can be used to detect the potential changes of contralateral ear before the hearing loss occurred .