| Project/Area Number |
12670985
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Hematology
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| Research Institution | Osaka University |
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Principal Investigator |
MACHII Takashi Osaka Univ., Graduate School of Medicine, Associate Prof., 医学系研究科, 助教授 (50124780)
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| Co-Investigator(Kenkyū-buntansha) |
KONAKURA Yuzuru Osaka Univ., Graduate School of Medicine, Professor, 医学系研究科, 教授 (20177489)
MIZUKI Masao Osaka Univ., Graduate School of Medicine, Assistant Prof., 医学系研究科, 助手 (80283761)
SHIBAYAMS Hirohiko Osaka University Hospital, Medical Staff, 医学部附属病院, 医員(臨床研究) (60346202)
池田 弘和 大阪大学, 医学系研究科, 助手 (10311755)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | PNH / PIG-A / GPI-anchor / CD59 / bone marorow failure / Aplastic anemia / clonal expansion / missense mutation / 造血幹細胞 / HMGA2 / PIG-A遺伝子 / レトロウイルスベクター |
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| Research Abstract |
A new sensitive method detecting PIG-A mutations was established, particularly, for those in small population of GPI-anchored protein (GPI-AP) deficient cells. We also established a sensitive CD59 assay. We conducted molecular studies for understanding the pathogenesis and pathophysiology of PNH, as described below. The methods were used in some of these experiments.
1. Small populations of CD59 deficient erythrocytes was detected in some patients with aplastic anemia (AA), or MDS, who have no significant abnormalities by the standard flow cytometry, and in some normal individuals.
2. We reanalyzed PIG-A gene in 9 patients with PNH, 6-9 years after initial examinations. In all of the patients, the previously dominant clone was still present and dominant in the present study, indicating one stem cell clone can sustain hematopoiesis for 6-9 years in patients with PNH.
3. Structural and functional analysis of the PIG-A missense mutations found in patients with PNH, showed that the genotype and phenotype were practically consistent with each other. Defective interaction of the PIG-A protein with PIG-H, GPI- 1, and/or PIG-P was recognized in 4 different missenses.
4. Cross culture experiments showed that the hematopoietic defect in PNH is not due to defective stroma, but is due to defective progenitor cell growth related to additional unknown factors.
5. To develop a gene therapy for PNH, a retroviral vector construct (MPIN) was made, containing the PIG-A complementary DNA and NGFR as a marker. Efficient and stable functional restoration of GPI-APs was seen in a variety of PIG-A-deficient hematopoietic cell types by MPIN transduction. MPIN was also transduced in BMMNC from a patient with PNH, and the cells restored surface expression of GPI-AP
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