ABSTRACT
<p><b>OBJECTIVE</b>To investigate the pro-apoptotic effect of Her-2 targeted recombinant caspase-6 fusion protein on osteosarcoma SOSP-9607 cells.</p><p><b>METHODS</b>Recombinant immunocasp-6 was generated by sequential fusion of the genes of a signal peptide, a single-chain Her-2 antibody (e23sFv), a PEA translocation domain (PEA aa253-364) and an active caspase-6. The immunocasp-6 gene was cloned into pCMV plasmid to construct a kind of eukaryotic expression vector, i.e. pCMV-e23sfv-PE II-caspase-6 (abbr. pCMV-6) and transfected into SOSP-9607 cells. Murine xenograft models were randomly divided into two groups that received i.m. injections of liposome encapsulated pCMV-6 or pCMV alone. The tumor volume and weight of the nude mice and the tumor weight of the cured mice were observed and statistically analyzed. The morphological changes of the tumors were examined with HE staining, apoptotic morphology of the tumor was observed by TUNEL staining and the gene expression was analyzed by immunohistochemical staining.</p><p><b>RESULTS</b>The tumor growth of the mice in the treatment group was significantly slower than that of the control group (P = 0.001). The weight of the nude mice in the treatment group was significantly higher than that of the control group (P = 0.0002). The tumor weight of the mice in the treatment group was significantly lower than that of the control group (P = 0.0006). HE and TUNEL staining of the tumor of nude mice in the treatment groups showed typical characteristics of apoptosis, while normal structure was found in the control group. Furthermore, caspase-6 was not found in the tumor and muscle tissues in the control group, but only in the treatment group by immunohistochemistry.</p><p><b>CONCLUSION</b>Immunocasp-6 can selectively recognize and bind to and kill HER-2 positive osteosarcoma cells, therefore, to offer some foundation for the clinical treatment of osteosarcoma.</p>
Subject(s)
Animals , Humans , Mice , ADP Ribose Transferases , Genetics , Apoptosis , Bacterial Toxins , Genetics , Bone Neoplasms , Metabolism , Pathology , Caspase 6 , Genetics , Metabolism , Cell Line, Tumor , Exotoxins , Genetics , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Osteosarcoma , Metabolism , Pathology , Plasmids , Random Allocation , Receptor, ErbB-2 , Genetics , Recombinant Fusion Proteins , Genetics , Metabolism , Transfection , Tumor Burden , Virulence Factors , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the inhibitory effect of antisense oligonucleotides (ODN) on dopamine transporter (DAT) in rats and observe the response of the rats to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).</p><p><b>METHODS</b>The cannula was implanted in the substantia nigra compacta under a rat stereotaxic device, through which drugs were used. The rats with successful operation were divided randomly into four groups, and received injection of antisense, sense, missense oligonucleotides and saline respectively, in the substantia nigra compacta of each rat via the cannula, followed by MPTP (30 mg/kg) injection. Behavior of the rats was observed and immunohistochemistry was carried out to check the expression of DAT and apoptosis of dopamine cell.</p><p><b>RESULTS</b>The expression of DAT (positive unit, PU) in the substantia nigra compacta in rats was 6.65+/- 1.67 in the antisense ODN group, 12.41+/- 2.46 in saline group, 11.45+/- 1.17 in sense ODN group, and 10.35+/- 2.89 in missense ODN group. The expression of DAT was lower in the antisense ODN group than that of the other three groups (P< 0.01). The rotation of the rats induced by apomorphine was slower than that of the other three groups(P< 0.05). The apoptotic cells (21.4+/- 5.6) in the antisense ODN group (200x ) were less than that of the other three groups (61.6+/- 19.7, 56.5+/- 16.3, 52.2+/- 12.5 respectively), (P< 0.01).</p><p><b>CONCLUSION</b>The expression of DAT can be inhibited effectively by the antisense ODN, and the response of the rats to the MPTP was reduced upon DAT inhibition.</p>
Subject(s)
Animals , Male , Rats , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , Pharmacology , Apomorphine , Pharmacology , Dopamine Plasma Membrane Transport Proteins , Genetics , Physiology , Immunohistochemistry , Oligonucleotides, Antisense , Genetics , Rats, Sprague-DawleyABSTRACT
<p><b>OBJECTIVE</b>To investigate the clinical characteristics and analyze spastin gene mutation on a kindred with hereditary spastic paraplegia (HSP).</p><p><b>METHODS</b>All family members were studied through clinical examinations. The proband and another two patients in this kindred were subjected to electromyography (EMG) examinations. The proband was subjected to thoracic MRI examination too. Mutation analysis of spastin gene was screened by polymerase chain reaction combined with DNA sequencing in the proband and his father.</p><p><b>RESULTS</b>All patients in the kindred manifested as classical HSP. Thoracic MRI revealed atrophies of the spinal cord in the proband. No abnormal spastin gene mutation was detected in these two patients.</p><p><b>CONCLUSION</b>This kindred has typical clinical manifestations of HSP. The pathogenesis has no association with mutation of the exons of spastin gene.</p>
Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young Adult , Adenosine Triphosphatases , Genetics , Base Sequence , DNA Mutational Analysis , Electromyography , Exons , Genetics , Family Health , Magnetic Resonance Imaging , Mutation , Pedigree , Polymerase Chain Reaction , Spastic Paraplegia, Hereditary , Genetics , Pathology , SpastinABSTRACT
Objetive: To investigate whether apoptosis of SGC7901 cells can be induced by the expression of the recombinant gene of anti-HER2 ScFv/tBid. Methods: The recombinant anti-HER2 ScFv/tBid gene was cloned into vector pCMV and the recombinant plasmid was transfected into SGC7901 cells. The gene expression was detected by RT-PCR and immunofluorescent staining. Cell counting was carried out to show the effect of the gene transfection on cell growth. At the same time, significant apoptotic peak was detected by flow cytometry in recombinant anti-HER2 ScFv/tBid gene transfected cells. Results: The fusion protein of anti-HER2 ScFv/tBid was observed in the cytoplasm of transfected SGC7901 cells. The transfected cells displayed typical cell growth inhibition and apoptosis. Conclusion: Fusion protein of anti-HER2 ScFv/tBid can induce apoptosis of SGC7901.
ABSTRACT
<p><b>OBJECTIVE</b>To study the relationship of Chinese familial Parkinson disease with alpha-synuclein gene.</p><p><b>METHODS</b>Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and polymerase chain reaction-heteroduplex analysis(PCR-HA) were employed to detect the abnormal mobilization in the familial Parkinson disease and sporadic Parkinson disease patients, then it was verified by gene sequencing.</p><p><b>RESULTS</b>No mutation was found in alpha-synuclein gene exons 3 and 4 by PCR-SSCP together with PCR-HA. An inserted c and an inserted t were found in intron 4, position 23 and position 67 respectively.</p><p><b>CONCLUSION</b>(1) Exons 3 and 4 of alpha-synuclein gene are not the mutational hot spots of Chinese familial Parkinson disease. (2) Two polymorphisms were found in intron 4 of alpha-synuclein gene. They are 23 ins c and 67 ins t.</p>