ABSTRACT
Recombinant mutant human granulocyte colony stimulating factor (rmhG-CSF) was pegylated, purified and characterized. rhG-CSF was mutated in position 1,3,4,5,17, and cysteine was added in C-terminal. rmhG-CSF was pegylated by PEG-Mal 20000 and separated by ion-exchange chromatography, gel filtration chromatography. Analysis of SDS-PAGE showed thar the purity of the separated PEG-rmhG-CSF was greater than 95%. and in intro and in vivo bioactivity study showed that target modified PEG-rmhG-CSF kept full bioactivity which was better than traditional pegylation method, and longer half-life was proved in mice.
Subject(s)
Humans , Amino Acid Sequence , Base Sequence , Chromatography, Ion Exchange , Granulocyte Colony-Stimulating Factor , Chemistry , Genetics , Molecular Sequence Data , Mutant Proteins , Genetics , Polyethylene Glycols , Chemistry , Protein Sorting Signals , Recombinant ProteinsABSTRACT
Double antibody sandwich-type ELISA was used to detect rhG-CSF in serum to study the pharmacokinetics of rhG-CSF, PEG-rhG-CSF and rHSA-hG-CSF in mice and to confirm that PEGlyation and albumin fusion of rhG-CSF technology can prolong half-life of G-CSF. Pharmacokinetic parameters were calculated with 3P87 software. T1/2 s of rhG-CSF, PEG-rhG-CSF and rHSA-hG-CSF are 2. 1 , 14.2 and 10. 6 h, respectively. T1/2 s of PEG- rhG-CSF and rHSA-hG-CSF are 7, 5 times than T1/2 s of rhG-CSF, respectively. Tpeak s of PEG-rhG-CSF and rHSA-hG-CSF are 15, 13 times than Tpeak of rhG-CSF, respectively. The result of ELISA indicates that PEGlyation and albumin fusion of rhG-CSF technology can prolong half-life of G-CSF.
Subject(s)
Animals , Humans , Male , Mice , Area Under Curve , Enzyme-Linked Immunosorbent Assay , Methods , Granulocyte Colony-Stimulating Factor , Blood , Chemistry , Pharmacokinetics , Half-Life , Mice, Inbred ICR , Polyethylene Glycols , Chemistry , Recombinant Fusion Proteins , Blood , Chemistry , Pharmacokinetics , Recombinant Proteins , Serum Albumin , ChemistryABSTRACT
<p><b>OBJECTIVE</b>A mouse model of orthotopic bladder cancer simulating its human counterpart is of great importance in preclinical evaluation of new treatment modalities such as immunotxin therapy. The aim of the present study is to establish a novel nude mouse model with xenografted human bladder cancer.</p><p><b>METHODS</b>Single cell suspension of an established human bladder transitional cell carcinoma (TCC) cell line BIU-87 was instilled into nude mouse bladders which were pretreated with mild acid washing. The tumor growth in mouse bladder was assessed weekly by magnetic resonance imaging (MRI). At intervals following implantation and MRI tumor detection, the animals were sacrificed for necropsy, histological examination and immunocytochemical studies.</p><p><b>RESULTS</b>The overall tumor establishment was 92.9% (52/56 mice) at 7 - 36 days, while in the subgroup of animals sacrificed at 12 - 13 days, 40 out of 42 animals (95.2%) developed TCC, the majority of which was superficial. The tumor stages were assessed by gross and histopathology. Histological examination confirmed the presence of grade II - III TCC. Immunocytochemistry confirmed that the tumor model maintained the biological and immunological features of BIU-87 cells. The changes seen on MRI images well correlated with the extent of tumor invasion identified by histology. Carcinoma in situ could be detected histologically at 7 - 9 days post-inoculation and progressed into papillary or invasive tumors thereafter.</p><p><b>CONCLUSION</b>The orthotopic BIU-87 TCC model in nude mice is highly reproducible and is ideal for preclinical studies on experimental intravesical therapies.</p>
