Junchuan Qin

Transgene expression of human PON1 Q in mice protected the liver against CCl4-induced injury.

Oxidative stress, often in association with decreased antioxidant defenses, plays a pathogenetic role in both initiation and progression of liver injuries, leading to almost all clinical and experimental conditions of chronic liver diseases. Human paraoxonase 1 (hPON1) is a liver‐synthesized enzyme possessing antioxidant properties. Here, we investigate the effects of transgene‐expressed hPON1 Q on alleviating lipid peroxidation and preventing liver injury in a mouse model.

Comparative evaluation of the protective potentials of human paraoxonase 1 and 3 against CCl4-induced liver injury

We previously reported that electroporation mediated hPON1 or hPON3 gene delivery could protect against CCl(4)-induced liver injury. However, substantial evidence supported that the in vivo physiological functions of hPON1 and hPON3 were distinct. To compare the protective efficacies of hPON1 and hPON3 against liver injury, recombinant adenovirus AdPON1 and AdPON3, which were capable of expressing hPON1 and hPON3 respectively, were intravenously injected into mice before they were given CCl(4). Adenovirus mediated expression of hPON1 and hPON3 were demonstrated by elevated serum esterase activity, hepatic lactonase activity, and hPON1/hPON3 mRNA expression in liver. Serum transaminase assay, histological observation and TUNEL analysis revealed that the extent of liver injury and hepatocyte apoptosis in AdPON1 or AdPON3 treated mice was significantly ameliorated in comparison with control. Meanwhile, overexpression of hPON1 and hPON3 reduced the hepatic oxidative stress and strengthen the total antioxidant capabilities in liver through affecting the hepatic malondialdehyde (MDA), glutathione (GSH) and total antioxidant capability (T-AOC) levels, regardless of the exposure to CCl(4) or corn oil. Administration of AdPON1 or AdPON3 also suppressed inflammatory response by decreasing TNF-alpha and IL-1beta levels in CCl(4) mice. In this study, hPON1 exhibited a slightly higher efficacy than hPON3 in alleviating liver injury, but the difference between them were not significant.

Protective effects of transgene expressed human PON3 against CCl4-induced subacute liver injury in mice

Oxidative stress plays a crucial role in both initiation and progression of liver injury in almost all experimental and clinical liver diseases. Antioxidative therapy is therefore an effective means of preventing and attenuating oxidative stress related liver diseases. Human paraoxonase 3 (hPON3) is a lipid-associated enzyme with antioxidant activity. In the present study, hPON3 cDNA gene was cloned into pcDNA3.1 plasmid and electro-transferred into mouse skeletal muscle to maintain a higher serum PON3 activity. After gene delivery, serum PON3 activity was about 1.4 times higher than those of control and PON3 mRNA expression was also detected in mouse skeletal muscle. To investigate the role of hPON3 in protecting mice against liver injury, subacute liver injury model was induced by repeated CCl(4) administration and hPON3 gene was delivered into mouse skeletal muscle before progression or recovery phase, respectively, of liver injury. Afterwards, the mice were euthanized to evaluate liver marker enzymes, degrees of oxidative stress and liver histological architecture in order to reveal the effects of PON3 on subacute liver injury. In both damage phases, delivery of hPON3 gene significantly reduced serum aminotransferase level and improved liver histological architecture. Moreover, transgene expression of hPON3 attenuated oxidative stress by increasing hepatic glutathione content, superoxide dismutase (SOD) activity, total antioxidant capability (T-AOC), and reducing malondialdehyde (MDA) level.

Expression of a novel recombinant dual human stem cell factor in insect cells

Stem cell factor (SCF) is a hematopoietic cytokine that promotes the survival, proliferation, and differentiation of hematopoietic cells. A dual human stem cell factor (dhSCF) cDNA was constructed, which consisted of a full-length human stem cell factor cDNA plus a truncated hSCF cDNA (1-145aa), linked by a peptide (GGGGSGGGGSGG) coding region. The dhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the control of polyhedrin promoter. The Sf9 cells infected with the recombinant virus expressed rdhSCF up to 6000 U/10(6) cell in flask and 8300 U/10(6) cell in spinner flask. The rdhSCF was purified by two-step chromatography. The molecular mass of rdhSCF was examined by western blotting and HPLC analysis. The specific activity of rdhSCF was up to 3.1x10(6) U/mg, about 8.7 times as high as that of monomer rhSCF from Escherichia coli.

A novel recombinant dual human SCF expressed in and purified from silkworm, Bombyx mori, possesses higher bioactivity than recombinant monomeric human SCF.

A novel recombinant dual human stem cell factor (rdhSCF) gene was constructed which consisted of a full‐length hSCF cDNA plus a truncated hSCF cDNA (1–145 aa), linked by a peptide (GGGGSGGGGSGG) coding region. The rdhSCF gene was cloned into baculovirus transfer vector pAcSecG2T under the polyhedrin promoter control. Silkworm larvae infected with the recombinant virus expressed rdhSCF up to 15 800 units/mL in haemolymph. The specific activity of rdhSCF purified from the haemolymph was up to 3.0 × 106 units/mg, about 8.6 times as high as that of monomer rhSCF from Escherichia coli, and about 9.1 times as high as that of monomer rhSCF from insect cell. The binding affinity of rdhSCF to the cell surface receptor was higher than that of monomer rhSCF.

A novel thrombopoietin–stem‐cell factor fusion protein possesses enhanced potential in stimulating megakaryocyte proliferation and differentiation

TPO (thrombopoietin) and SCF (stem‐cell factor) are functionally related cytokines with overlapping but distinct haematopoietic effects. In the present study, a novel TPO–SCF fusion protein that combined the complementary biological effects of TPO and SCF into a single molecule was expressed in, and purified from, Sf9 [Spodoptera frugiperda (fall armyworm)] insect cells. The specific activity of rhTPO (recombinant human TPO)–SCF in megakaryoblastic Mo7e cell proliferation assays was 2.90±0.35×107 units/μmol, approx. 1.7 times as high as that of rhTPO. The specific activity of rhTPO–SCF in TF‐1 cells proliferation assays was 7.10±0.95×106 units/μmol, approx. 1.2 times as high as that of rhSCF (recombinant human SCF). In a megakaryocyte‐colony‐forming assay using human peripheral‐blood CD34+ cells, the SCF moiety of rhTPO–SCF worked in a synergistic way to augment the colony number and exhibited a higher potential to stimulate megakaryocyte colony growth. According to the results of EMSA (electrophoretic mobility‐shift assay) and semi‐quantitative RT (reverse transcriptase)–PCR, the synergistic effects of the SCF moiety were also reflected in increased STAT5 (signal transducer and activator of transcription 5) DNA binding and enhanced up‐regulation of p21 expression in Mo7e cells treated by rhTPO–SCF, suggesting that rhTPO–SCF could be more potent in promoting megakaryocyte proliferation and differentiation.