OBJECTIVETo evaluate the killing effect of adenovirus(Ad)-mediated double suicide gene driven by kinase domain-containing receptor (KDR) promoter on gastric cancer MGC-803 cells.

METHODSThe 293 packaging cells were transfected by the plasmids pAdEasy-KDR-CDglyTK to generate infectious viruses. The gastric cancer MGC-803 cells were infected by the Ad followed by treatment with 5-FC and/or ganciclovir at different concentrations. The cell-killing effects were evaluated and the bystander effects analyzed after coculture of the cells without AdKDR-CDglyTK infection with the infected cells at different ratios. The cell cycle distribution was detected by flow cytometry and the pathological changes of the cells were observed by electron microscopy.

RESULTSThe infection rate of the resultant recombinant Ad in the cells increased gradually with increment of the multiplicity of infection (MOI) of the Ads. The killing effect of CD/TK fusion gene on the MGC-803 cells was much stronger than that of either of the single suicide gene (P<0.001), and considerable bystander effect was observed. The Ad infection caused MGC-803 cell growth arrest at G(1) phase with onset of apoptotic and necrotic morphologies of the cells as seen under electron microscope.

CONCLUSIONThe CD/TK fusion gene system driven by the KDR promoter possesses effective killing effect on the KDR-expressing gastric cancer MGC-803 cells.

Adenoviridae ; genetics ; Cell Line, Tumor ; Cytosine Deaminase ; genetics ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Genetic Vectors ; Humans ; Promoter Regions, Genetic ; Receptor Protein-Tyrosine Kinases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; Stomach Neoplasms ; pathology

OBJECTIVETo explore the clinical classification method of keloids and providing a thread for the treatment of keloids.

METHODSTo summarize the 600 cases of keloid patients we accepted and diagnosed from November 2004 to October 2012, and filling in keloid patients information sheet, recording the keloids form by photographs, analyzing the treatment, putting forward the classification method of keloids in clinic.

RESULTSAccording to the position and quantity that keloids grow, the keloid patients are divided into four major categories:one in single site, one in each site, more than one in single site and more than one in each site; According to the area and thickness of keloids, the keloid single lesion is divided into four subclasses: type of small area and thin, type of small area and thick, type of large areas and thin,type of large areas and thick; According to the number of lesions, keloid multiple lesions is divided into two subgenera: isolated multiple and dispersion multiple, different kinds of keloids suit different methods of treatment.

CONCLUSIONThe clinical classification method of keloids can be used to provide thought for the treatment of keloids, and have a good application value.

Humans ; Keloid ; classification ; pathology ; therapy

OBJECTIVETo investigate the selective killing of colorectal tumor cells by lentivirus-mediated double suicide gene under the regulation of KDR promoter.

METHODS293T packaging cells were transfected with the plasmid FGW-KDRP-CD/TK to obtain the infectious viruses. KDR-expressing LoVo cells and LS174T cells that did not produce KDR were transfected with the recombinant virus, and the transfection efficiency was evaluated by the fluorecence microscope. RT-PCR was employed to examine the expression of CDglyTK. After treatment of the cells with 5-FC and GCV, the killing effects on the two cell lines were evaluated.

RESULTSThe recombinant construct showed similar infection rate of the two cell lines. RT-PCR demonstrated that CDglyTK gene was expressed only in LoVo cells infected with FGW-KDRP-CD/TK but not in LS147T cells, and the sensitivity of the two cell lines to the prodrugs was significantly different (P<0.001). The killing effect of the double suicide gene was much stronger than that of single suicide gene administered (P<0.001).

CONCLUSIONThe double suicide gene driven by KDR promoter has specific killing effect on the KDR-expressing colorectal tumor cells.

Antimetabolites ; pharmacology ; Apoptosis ; drug effects ; Cell Line ; Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Cytosine Deaminase ; genetics ; metabolism ; Flow Cytometry ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Thymidine Kinase ; genetics ; metabolism ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; genetics

Using the isolated total RNA from osteosacoma cell line MG63, the cDNA encoding human OPG was amplified by RT-PCR. A recombinant adenoviral vector carrying cDNA of OPG was constructed and OPG expression in mouse myoblast C2C12 cells was confirmed by Western blot and ELISA. The secreted expression of OPG protein persisted more than 6 weeks in vitro, and the growth of C2C12 cells infected by recombinant adenoviral were in good state. Osteoclasts derived from mouse bone marrow cells infected with recombinant adenoviral made less number of TRAP positive cells and resorption pits formed on dentine slices.
Adenoviridae ; genetics ; Animals ; Blotting, Western ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; genetics ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Osteoclasts ; metabolism ; Osteoprotegerin ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the effect of adenovirus (Ad)-mediated fusion gene system driven by KDR promoter on the proliferation of human stomach adneocarcinoma SCG7901.

METHODSThe KDR-expressing SCG7901 cells and HepG2 cells that did not express KDR were both transfected with AdEasy-KDR-CDglyTK followed by treatment with the prodrugs 5-FC and/or GCV at different concentrations. The killing effects of the transfection on the cells were evaluated.

RESULTSThe expression of green fluorescent protein (GFP) was observed in 95% of the infected SCG7901 and HepG2 cells with the multiple of infection (MOI) of the Ads of 100. Transfection of SCG7901 and HepG2 cells did not produce significant changes in the cell growth, and the infected cells exhibited different sensitivities to the two prodrug: SCG7901 cells infected with rAd were highly sensitive to the prodrugs, but the infected HepG2 cells were not (P<0.01). The killing effect of CDglyTK fusion gene on the target cells was much stronger than that of either the single suicide gene (P<0.01).

CONCLUSIONCDglyTK fusion gene system driven by KDR promoter selectively kills the KDR-CDglyTK SCG7901 cells and inhibits their proliferation.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Adenoviridae ; genetics ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Dose-Response Relationship, Drug ; Flucytosine ; pharmacology ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Neovascularization, Pathologic ; genetics ; metabolism ; pathology ; Prodrugs ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics

OBJECTIVETo express human osteoprotegerin (OPG) in E. Coli and analyze its bioactivity in vitro.

METHODSSynthetic oligonucleotides were used to amplify human OPG gene by RT-PCR from total RNA of human osteosarcoma cell line MG63. The OPG cDNA coding for 380 amino acid residues was inserted into prokaryotic expression vector pRSET-A, transformed into competent E. Coli BL21, and induced by 0.1 mmol/l IPTG. SDS-PAGE and Western blot were performed to identify OPG-6His fusion protein. After purified by affinity chromatography, 1,000 microg/L or 1,500 microg/L of OPG-6His were added into the mouse bone marrow cells culture medium. The number of tartrate-resistant acid phophatase (TRAP)-positive multinucleated cells and resorption pits were counted to assess the bioactivity of expression products.

RESULTSThe sequence of OPG mature peptide encoding cDNA obtained in this experiment was as same as reported. SDS-PAGE showed 24% of total bacterial protein was of OPG-6His fusion protein. Western blot assay demonstrated that the molecular weight of recombinant protein was about 46 KD and could react specifically with human anti-OPG antibody. The mouse bone marrow cells were induced by 1alpha, 25-dihydroxyvitaminD3 (10(-8) mol/L) and Dexamethasone (10(-7) mol/L) to form osteoclastic-like multinucleated cells. 1,500 microg/L of purified OPG-6His protein could decrease the number of resorption pits and TRAP-positive multinucleated cells in vitro (P < 0.05), but it didn't show the same effects when the concentration of OPG-6His fusion protein was of 1,000 microg/L.

CONCLUSIONSHuman OPG-6His fusion protein is expressed and purified in E. Coli. The expression products have moderate inhibitory effects on osteoclast differentiation and bone resorption in vitro only when excessive amount of proteins are added into the culture medium, indicating that prokaryotic expression of fuctionalal OPG protein awaits further investigation.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; genetics ; Glycoproteins ; biosynthesis ; genetics ; Humans ; Osteoclasts ; drug effects ; physiology ; Osteoprotegerin ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor ; Recombinant Fusion Proteins ; biosynthesis ; pharmacology

OBJECTIVETo evaluate the value of EBNA1-IgA and EA-IgG in serological diagnosis of nasopharyngeal carcinoma (NPC).

METHODSThe serum EBNA1-IgA and EA-IgG of 56 patients with NPC and 58 healthy adults were detected by ELISA. The sensitivity, specificity, positive predictive value, accuracy rate and odds ratio of the two tests used singly or in combination were compared with each other.

RESULTSThe sensitivity of EBNA1-IgA (91.07%) was higher than that of EA-IgG (87.50%), while the specificity of EA-IgG (87.93%) was higher than that of EBNA1-IgA (84.48%). The combination of EBNA1-IgA and EA-IgG could enhance the specificity (94.83%), positive predictive value (0.9375), likelihood ratio (15.5435) and odds ratio (75.0000) for serological diagnosis of NPC. Forty-five patients showed both positive EBNA1-IgA and positive EA-IgG. A positive EA-IgG was detected in 4 out of 5 patients with negative EBNA1-IgA and a positive EBNA1-IgA was founded in 6 out of 7 patients with negative EA-IgG.

CONCLUSIONAlthough relatively high sensitivity and specificity could be obtained by either EBNA1-IgA or EA-IgG test alone, the combination of these two tests with a complementary effect is able to enhance the reliability of serological diagnosis of NPC as most patients have positive ENBA1-IgA and EA-IgG concurrently.

Adult ; Antigens, Viral ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epstein-Barr Virus Nuclear Antigens ; immunology ; Humans ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Nasopharyngeal Neoplasms ; diagnosis ; immunology ; Sensitivity and Specificity

OBJECTIVETo investigate the selectively killing effect of adenovirus (Ad) mediated double suicide gene under the regulation of KDR promoter on vascular endothelial cells and colorectal tumor cells.

METHODS293 packaging cells were transfected with the plasmids of pAdEasy- KDR- CDglyTK and pAdEasy- CMV- CDglyTK and the infectious viruses were generated. The KDR expressive cells of ECV304,SW620 and the KDR inexpressive cells of LS174T were infected by two Ads. The infection rate was observed and the expression of CDglyTK was detected by RT- PCR. After treatment with different concentrations of 5- FC and GCV,the killing effect and bystander effect on ECV304,SW620 and LS174T were examined.

RESULTSThe titers of these two purified Ads were 2.0 x 10(12 ) pfu/ml. There was no significant difference in infection rate between two recombinant Ads infecting various cells,and the infection rate increased in accordance with the enhancing titers of Ads. RT- PCR demonstrated that there existed the product of CDglyTK gene in all the cells infected by Ad- CMV- CDglyTK and the cells infected by Ad- KDR- CDglyTK except in the SL174T. The curative effect in this system on various cells was shown as follows: (1) All cells infected with Ad- CMV- CDglyTK and some cells of ECV304 and SW620 infected with Ad- KDR- CDglyTK were highly sensitive to the prodrugs,but there was no significant differences among them (P > 0.05); compared with ECV304 and SW620 cells,LS174T cells were not sensitive to the two prodrugs (P< 0.001). (2) The efficacy of double suicide gene was better than that of single suicide gene (P< 0.001). (3) The system had considerable bystander effect.

CONCLUSIONThe double suicide gene under the regulation of KDR promoter has specific killing effect on the KDR- expressing endothelial cells and colorectal tumor cells.

Adenoviridae ; genetics ; Cell Line, Tumor ; Endothelial Cells ; cytology ; Gene Expression Regulation ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; Humans ; Promoter Regions, Genetic ; Vascular Endothelial Growth Factor Receptor-2 ; genetics

OBJECTIVETo observe the injury on micro-skin induced by a self designed micro-skin machine.

METHODSMicro-skin was produced either with the machine or by hand. Cells at the edge of micro-skin were observed by transmission electron microscope. succinic dehydrogenase activity in supernatant of cultivated cells was analyzed, and the cell proliferation of micro-skin was assessed by (3)H-TdR. Twenty patients were enrolled in the study for the observation of the wound healing time between the two groups of micro-skin after being grafted.

RESULTSTransmission electron microscope examination revealed that the cellular injury at the edge of the micro-skin in machine-made group was mild compared with that in man-made group. (3)H-TdR rate was elevated but the activity of succinic dehydrogenase in the supernatant of cultured cells decreased in supernatant of cultured cells of machine produced micro-skin. Wound healing time was shortened in machine made group. (P < 0.05).

CONCLUSIONThe cellular injury at the edge of micro-skin in the machine made group was mild when compared with that in the man-made group with cell proliferation accelerated and wound healing time shortened.

Burns ; surgery ; Cell Division ; Epithelium ; pathology ; Humans ; Microscopy, Electron ; Skin ; ultrastructure ; Skin Transplantation ; methods ; Wound Healing

OBJECTIVETo establish an improved three-dimension (3D) and serum-free approach to differentiate human embryonic stem cells (hESCs) into endothelial cells, and detect the endothelial functions of the obtained cells.

METHODSWe cultured undifferentiated H9 human embryonic stem cell line in low-adhesion dishes to form embryonic bodies (EBs). After 12 days, EBs were harvested, re-suspended into rat tail collagen type I, and put into the incubator (37℃). After 30 minutes, EGM-2 culture medium was added to the solidified collagen, and the EBs were cultured for another 3 days to form embryonic body-sproutings (EB-sproutings). EB-sproutings were digested with 0.25% collagenase I and 0.56 U/ml Liberase Blendzyme for 20 minutes respectively, and the CD31(+) cells were sorted by FACS. The endothelial functions were tested by Dil-ac-LDL uptake assay and tube formation assay.

RESULTSThis approach raised the efficiency of endothelial differentiation to 18%, and also avoided the contamination with animal materials. The obtained hESC-derived endothelial cells (hESC-ECs) had the similar pattern of surface biomarkers as human umbilical vein endothelial cells (HUVECs), and their endothelial functions were confirmed by the uptake of Dil-ac-LDL and the tube formation on Matrigel.

CONCLUSIONSThe improved 3D approach can enhance the efficiency of differentiation from hESCs into endothelial cells. Furthermore, serum free differentiation system may be applied in future hESC-based therapies for various ischemic diseases.

Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Line ; Collagen Type I ; Culture Media ; Embryonic Stem Cells ; cytology ; Endothelial Cells ; cytology ; Humans