Korean J Hematol 1998; 33(2):
Published online June 30, 1998
© The Korean Society of Hematology
Retroviral vector를 이용하여 쥐 과립구 집락자극인자 유전자를 형질이입시킨 혈관평활근세포 이식 후 백혈구계의 변화
박국태, 김승택, 김영규, 오태근, 김동운
충북대학교 의과대학 내과학교실,
충북대학교 의과대학 신경외과학교실
Changes in White Blood Cells after Transplantation of Vascular Smooth Muscle Cells Retrovirally Transduced with Granulocyte Colony-Stimulating Factor Gene in Rats
Background: The granulocyte colony-stimulating factor(G-CSF) is crucial in neutrophil regulation. Since recombinant G-CSF became clinically available, it has been widely used in the treatment of neutropenia. Ex vivo therapy of recombinant G-CSF, however, requires large dose and frequent administration, which brings financial burden
on the patients. To overcome disadvantages of ex vivo therapy, we have tried to make an in vlvo G-CSF delivery system in rat using gene therapy technique.
Methods: We have tried to make an in vivo G-CSF delivery system using transduced vascular smooth muscle cells with G-CSF gene in a rat model. Retroviral vector plasmid containing rat G-CSF gene was made employing LXSN and LNFX
plasmid. Recombinant retrovirus was produced from PA317 packaging cells. Infection of the vascular smooth muscle cells with the virus and selection with G418 was done in
vitro. These transduced cells were transplanted to the balloon-injured carotid arteries of Fisher 344 rats, and complete blood count as well as differentials were measured in sequence.
Results: The virus titer was three times greater in case of LNFG than LGSN, whereas G-CSF production from infected vascular smooth muscle cell was lower in LNFG vector(0.1ng/10 6cells/day) than in LGSN vector(0.4ng/10 6cells/day). The increment of WBC count was observed until 25 days after transplantation, being 9,600±1,000/uL on seventh day after transplantation, which was significantly higher than that of controls, 7,300±540/uL.
The levels of neutrophil increased gradually after transplantation, reached to the peak after 1 week(3,250±1,099/uL in case of neutrophil count and 30±10% in case of differentials). The duration of increment, however, was relatively short, neutrophil count being decreased to the basal level within 4 weeks.
Conclusion: The effective increase of neutrophil count with low dose of G-CSF produced from vascular smooth muscle cells could make this gene therapy feasible in the clinical settings only if the problem of short duration of effect could be solved.
Keywords Gene therapy; Rat G-CSF gene; Retroviral vector; Vascular smooth muscle cell;
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Korean J Hematol 1998; 33(2): 231-242
Published online June 30, 1998
Copyright © The Korean Society of Hematology.
Retroviral vector를 이용하여 쥐 과립구 집락자극인자 유전자를 형질이입시킨 혈관평활근세포 이식 후 백혈구계의 변화
박국태, 김승택, 김영규, 오태근, 김동운
충북대학교 의과대학 내과학교실,
충북대학교 의과대학 신경외과학교실
Changes in White Blood Cells after Transplantation of Vascular Smooth Muscle Cells Retrovirally Transduced with Granulocyte Colony-Stimulating Factor Gene in Rats
Kuk Tae Park, Seung Taik Kim, Young Gyu Kim, Tae Geun Oh, Dong Woon Kim
Department of Internal Medicine, Neurosurgery, College of Medicine, Chungbuk National University
Abstract
Background: The granulocyte colony-stimulating factor(G-CSF) is crucial in neutrophil regulation. Since recombinant G-CSF became clinically available, it has been widely used in the treatment of neutropenia. Ex vivo therapy of recombinant G-CSF, however, requires large dose and frequent administration, which brings financial burden
on the patients. To overcome disadvantages of ex vivo therapy, we have tried to make an in vlvo G-CSF delivery system in rat using gene therapy technique.
Methods: We have tried to make an in vivo G-CSF delivery system using transduced vascular smooth muscle cells with G-CSF gene in a rat model. Retroviral vector plasmid containing rat G-CSF gene was made employing LXSN and LNFX
plasmid. Recombinant retrovirus was produced from PA317 packaging cells. Infection of the vascular smooth muscle cells with the virus and selection with G418 was done in
vitro. These transduced cells were transplanted to the balloon-injured carotid arteries of Fisher 344 rats, and complete blood count as well as differentials were measured in sequence.
Results: The virus titer was three times greater in case of LNFG than LGSN, whereas G-CSF production from infected vascular smooth muscle cell was lower in LNFG vector(0.1ng/10 6cells/day) than in LGSN vector(0.4ng/10 6cells/day). The increment of WBC count was observed until 25 days after transplantation, being 9,600±1,000/uL on seventh day after transplantation, which was significantly higher than that of controls, 7,300±540/uL.
The levels of neutrophil increased gradually after transplantation, reached to the peak after 1 week(3,250±1,099/uL in case of neutrophil count and 30±10% in case of differentials). The duration of increment, however, was relatively short, neutrophil count being decreased to the basal level within 4 weeks.
Conclusion: The effective increase of neutrophil count with low dose of G-CSF produced from vascular smooth muscle cells could make this gene therapy feasible in the clinical settings only if the problem of short duration of effect could be solved.
Keywords: Gene therapy, Rat G-CSF gene, Retroviral vector, Vascular smooth muscle cell,
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