Objective To construct an expression vector harboring CYP2B1 suicide gene, and detect its expressions in tumor cell lines. Methods PCR amplification was performed using primers based on murine CYP2B1 gene sequence from gene bank and pc3/2B1 as template. PCR product was directly inserted an eukaryotic expression plasmid pcDNA3.0. The recombinants were analyzed and identified by restriction enzyme analysis, PCR and sequencing. Then the recombinant vector pcDNA3.0/CYP2B1 was transfected into three tumor cell lines by liposome-mediated method. The expressions of CYP2B1 gene in all the cell lines were detected by RT-PCR method. Results pCDNA3.0/CYP2B1 vector was successfully constructed, and could express CYP2B1 mRNA in the three tumor cell lines. Conclusion Eukaryotic expression vector pcDNA3.0/CYP2B1 containing CYP2B1 gene under the control of a CMV promoter is an novel effective expression vector for tumor gene therapy.

BACKGROUND: Collagen sponge is considered the most useful biomaterial owing to its excellent function and properties, easy processing, sterilization, and preservation and has been widely used in scientific research and clinical application. OBJECTIVE: To describe the properties of collagen sponge and to review the research progress of collagen sponge in clinical application in recent years. METHODS: A computer-based retrieval was performed by the first author to search for manuscripts published from January 2000 to August 2010 in Pubmeds and Elsevier databases and for manuscripts published from January 1993 to August 2010 in CNKI database using key words "collagen, collagen sponge, clinical application" in title and abstract items in English and Chinese language, respectively. RESULTS AND CONCLUSION: Collagen sponge, as a new biological material, has been widely applied in tissue engineering research in terms of hemostasis, wound healing, anti-infection, cartilage repair, and nerve repair. But at present, nearly all collagen is from animals and immunogenicity cannot be thoroughly eliminated. Researchers outside of China have synthesized recombinant human collagen using bioreactor and transgenic technology, but its efficacy and safety in clinical application needs further investigation and research.

Aim To investigate the protective effect of non-mitogenic human acidic fibroblast growth factor (nm-haFGF) on cerebral ischemia-reperfusion injury in mice. Methods Cerebral ischemia-reperfusion model was made by ligating bilateral carotid for 20 minutes in mice. These mice were randomly divided into model group( iv NS), two doses of nm-haFGF (iv 25、50 ?g?kg-1) groups, rhaFGF group(iv 50 ?g?kg-1) and sham- operated group. Step down test and Y-type electric maze were used to examine the effect of nm-haFGF on learning and memory of mice, then Even′s Blue(EB) level and NO level in brain of these mice were measured. Results The nm-haFGF significantly decreased numbers of errors of mice in 5 min in step down test and in Y-type electric maze test; EB and NO levels in brain of these mice were lower than those of model group respectively. Conclusion The nm-haFGF can protect cerebral ischemia-reperfusion injury in mice.

Objective To investigate the cytotoxic effects and mechanism of PNP-CD chimeric gene vector originated from PNP/MeP-dR system on HCC cells. Methods The fusion suicide gene PNP-CD obtained by site directed mutagenesis technique was subcloned into pcDNA3.0 to construct a eukaryotic expression vector containing a chimeric gene, pcDNA3.0/ PNP-CD. After being identified by recombinant enzyme, PCR and subsequent sequencing, it was transfected into HepG2 cells by liposome-mediation method. The G418-resistant cellular clone with stable transfection of pcDNA3.0/PNP-CD, HepG2/PNP-CD was established by selection. The expression of PNP-CD gene was also verified by RT-PCR and Western blotting. The curve of cellular growth was assayed by Trypan blue exclusion. The cellular sensitivity of HepG2/PNP-CD to its specific prodrugs and its bystander effects were also assayed by MTT method. Results The chimeric gene, PNP-CD, was inserted into pcDNA3.0 correctly, and the stable expression of pcDNA3.0/PNP-CD in HepG2 cells was confirmed.This cellular clone was highly sensitive to its corresponding prodrugs. It was indicated that its bystander effects with the synergetic treatment of its specific prodrugs were substantially higher than those caused by the same vector with the administration of only a single prodrug, MeP-dR. Conclusion The bi-functional fusion suicide gene vector, pcDNA3.0/PNP-CD, yields powerful cytotoxic effects on HCC cells in the presence of the synergetic treatment of its specific prodrugs, which would be a high-performance therapeutic vector in gene therapy for liver cancer.

BACKGROUND:It is still a research focus on constructing substitution of the human tissues and organs, or producing the alliance for grafting by engineering methods in tissue engineering. Among these researches, it is pivotal to choose appropriate materials. The prepared scaffolds should have suitable tensile strength and mechanical toughness to withstand the various outside forces without being damaged. So, it is very necessary to evaluate the biomechanical properties of candidated materials in tissue engineering, which can supply the references for selecting materials for tissue scaffolds and their designation.OBJECTIVE: To investigate the biomechanical properties of nine kinds of scaffold materials, in order to supply a biomechanical basis for the selection and design of scaffold materials for tissue engineering.DESIGN: A repetitive measurement study.SETTING: College of Bioengineering, Chongqing University.MATERIALS: The materials involved in this study were poly (DL-lactic-co - glycolic acid) (PLGA), sodium polymannuronate, gelatine, chitosan, collagen, acellular porcine dermis (APD), acellular vascular matrix (AVM),APD-PLGA, AVM-PLGA, modified gelatine and chitosan.METHODS: All the experiments related to this study were completed in the Biorheology laboratory of the College of Bioengineering, Chongqing University from April 2006 to March 2007. The nine materials above were prepared, gelatine and chitosan were modified. Stress-strain testing was performed at 10 mm per minute by a material testing machine (INSTRON 1011, USA). The Yang's modulus was calculated in the range of 0.005 to 0.02, the ultimate strain and stress were also obtained.MAIN OUTCOME MEASURES: The ultimate strain, ultimate stress and Yang's modulus of the nine materials were analyzed.polymannuronate ＞ AVM-PLGA ＞ collagen ＞ gelatine (P ＜ 0.05). The rate of burst straining of chitosan and PLGA were greater than those of other materials, 133% and 276% respectively (P ＜ 0.05). In addition, after being combined with ultimate stresses of APD and APD-PLGA were greater than that of other materials, i.e., their burst strengths were greater than those of other materials. The data also indicated that the burst strength of APD-PLGA was a little greater than that of APD (P ＞ 0.05). The burst strengths of gelatin, chitosan, and collagen were similar at the range of 7.67 to 9.51 MPa (P ＞ 0.05). The burst strengths of collagen and sodium polymannuronate were from 1.16 to 1.40 MPa, which were the least among all the materials. At the same time, being combined with PLGA, the burst strength of AVM-PLGA greatest, i.e., its rigidity was the greatest. The rigidity of APD was the least. After combined with PLGA, the rigidity of AVM and APD were increased (P ＜ 0.05), and corresponded with PLGA (P＞ 0.05). Except for gelatin, the order of rigidity in the materials was AVM-PLGA ＞ PLGA ＞ APD-PLGA ＞ AVM ＞ chitosan ＞ sodium polymannuronate ＞ collagen ＞ APD.CONCLUSION: AVM and APD have good biomechanical properties, which are very different from the water-soluble collagen. It is promising to improve the biomechanical properties of sodium polymannuronate, gelatin and chitosan by the complex of PLGA.

To investigate the killing effect of PNP/MeP-dR suicide gene system on hepatoma cells,pcDNA3. 0/PNP, an eukaryotic expression vector harboring E. coli PNP gene, was transfected into human hepatoma HepG2 cells by liposome-mediated method. A HepG2 cell line with stable PNP gene expression, HepG2/PNP, was established with presence of G418 selection. The cell growth curves were determined with trypan blue staining. The sensitivity of HepG2/PNP to MePdR and bystander effects were assayed by MTT and FCM methods. The enzymatic activity of the product of PNP gene was determined by HPLC method. The cytotoxic effects of MeP-dR on HepG2/PNP cells were obvious (IC50 =4.5μmol/L) and all HepG2/PNP cells were killed 4 days after the treatment with 100μmol/L MeP～dR. In mixed cultures containing increasing percentages of HepG2/PNP cells, total population killing was demonstrated when HepG2/PNP cells accounted for as few as 5% of all HepG2 cells 8 days after the treatment with 100μmol MeP-dR. Highpressure liquid chromatography (HPLC) demonstrated that the PNP enzyme could convert MePdR into 6-MP. PNP/MeP-dR suicide gene system had an advantage over traditional suicide gene systems for hepatoma gene therapy. Our e results suggest that high-level bystander effects of this system result in significant anti-tumor responses to hepatoma gene therapy, especially in vivo.

A scaffold fabricated with lysine/nerve growth factor (NGF)/poly (lactic acid coglycolic acid) copolymer (PLGA) and acellular pigskin was evaluated in vitro as a potential artificial nerve scaffold. Properties of the scaffold such as microstructure, mechanical property, degradation behavior in PBS and water, Schwann cell adhesion property, and release of NGF were investigated. Results showed PLGA had permeated into the porous structure of acellular pigskin; its breaking strength was 8.308 MPa, breaking extensibility was 38.98%, elastic modulus was 97.27 MPa. The porosities of the scaffold ranged from 68.3% to 81.2% with densities from 0.62 g/cm3 to 0.68 g/cm3. At 4 weeks of degradation in vitro, maximum mass loss ratio was 43.3%. The release of NGF could still be detected on the 30th day, and its accumulative release rate was 38%. Lysine added into the scaffold neutralized the acidoid preventing degradation of PLGA to maintain a solution pH value. Schwann cells had grown across the scaffold after co-cultivation for 15 days. These in vitro properties of the pigskin based composite might indicate its potentiality to be an artificial nerve scaffold.
Acellular Dermis ; Animals ; Biocompatible Materials ; Guided Tissue Regeneration ; Lactic Acid ; pharmacology ; Lysine ; pharmacology ; Nerve Growth Factors ; chemistry ; pharmacology ; Nerve Regeneration ; Polyglycolic Acid ; pharmacology ; Swine ; Tissue Engineering ; Tissue Scaffolds