Analysis of the efficiency of the separation of normal hematopoietic stem cells of myelodysplastic syndrome for auto stem cell transplantation

骨髓增生异常综合征正常造血干细胞分离用于自体干细胞移植的效率分析

• 批准号: 11670991
• 负责人: MURATE Takashi
• 金额: $ 2.18万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670991/
• 关键词: HUMARA; hematopoiesis; clonality; PCR based HUMARA; methylation specific PCR; HUMARA-MSP; X chromosome inactivation; hematopoietic colonies; 造血クローン性; PCR based HUMARA法; 鉄芽球性貧血; p15メチル化; 造血幹細胞

The human androgen-receptor gene (HUMARA) has been used for analysis of X-chromosome inactivation (XCI) patter because of a polymorphic short tandem repeat (STR) near the 5'-promoter region correlated with XCI.We introduced a novel PCR based HUMARA method to analyze the clonality of the hematopoiesis of MDS patients named HUMARA methylation-specific PCR (HUMARA-MSP) assay, which analyzed methylation status of the HUMARA gene by bisulfite modification instead of a methylation-sensitive restriction enzyme. Although the original MSP method shows whether there is a methylated band, or not, our HUMARA-MSP method identifies the pattems of methylated and unmethylated bands. Because this method identifies either unmethylated or methylated alleles in each PCR tube and shows opposite band patterns dependent on methylation status, we can assess the XCI pattern independently twice. This method can avoid false results by incomplete enzyme digestion and incomplete bisulfite modification will not affect the results. Extremely small quantities of samples, such as hematopoietic colonies, were also available for HUMARA-MSP assay. The HUMAR-MSP assay may facilitate the analysis for pathogenesis of hematological disorders because of its simplicity, sensitivity and wide applicability.

人类雄激素受体基因（Humara）已用于分析X染色体失活（XCI）模式，因为在5'PROMOTER区域附近的多态性短串联重复（STR）与XCI相关。测定，通过硫酸硫酸盐修饰而不是甲基化敏感限制酶分析了Humara基因的甲基化状态。尽管原始的MSP方法显示了是否存在甲基化的带，但我们的Humara-MSP方法鉴定了甲基化和未甲基化带的烟。由于该方法在每个PCR管中识别未甲基化或甲基化的等位基因，并显示相反的带模式，依赖于甲基化状态，因此我们可以独立评估XCI模式两次。此方法可以通过不完整的酶消化避免错误结果，而不完整的亚硫酸盐修饰不会影响结果。 HUMARA-MSP分析也可以使用极少量的样品，例如造血菌落。 Humar-MSP分析可能会促进血液学疾病发病机理的分析，因为其简单，灵敏度和广泛的适用性。

项目成果

Aoki E,Uchida T,Murate T: "Methylation status of the P15 INK4B gene in hematopoietic progenitors and peripheral blood cell in myelodysplastic syndromes."Leukemia. 14. 586-593 (2000)

Aoki E、Uchida T、Murate T：“骨髓增生异常综合征中造血祖细胞和外周血细胞中 P15 INK4B 基因的甲基化状态。”白血病。

Uchida T,Ohashi H,Murate T: "Clonality analysis by methylation specific PCR for the human and rogen-receptor gene (HUMARA-MSP)."Leukemia. 14. 207-212 (2000)

Uchida T、Ohashi H、Murate T：“通过甲基化特异性 PCR 对人类和雄性受体基因 (HUMARA-MSP) 进行克隆分析。”白血病。

Suzuki H,Asano H,murate T: "Clonality analysis of refractory anemia with ring sideroblasts : simultaneous study of clonality and cytochemistry of bone marrow progenitors"Leukemia. 13. 130-134 (1999)

Suzuki H、Asano H、murate T：“环形铁粒幼细胞难治性贫血的克隆性分析：骨髓祖细胞克隆性和细胞化学的同步研究”白血病。

Uchida T, Kinoshita T, Murate T et al.: "CDK2 (MTS 1/p16INK4A) Gene alterations in adult T-cell Leukemia/Lymphoma"Leuk Lymphoma. 29. 27-35 (1998)

Uchida T、Kinoshita T、Murate T 等人：“成人 T 细胞白血病/淋巴瘤中的 CDK2 (MTS 1/p16INK4A) 基因改变”白血病淋巴瘤。

Kosugi H,Towatari M,Murate T: "Histone deacetylase inhibitos are the potent inducer lenhancer of differentiation in acute myeloid leukemia"Leukemia. 13. 1316-1324 (1999)

Kosugi H、Towatari M、Murate T：“组蛋白脱乙酰酶抑制剂是急性髓系白血病分化的有效诱导剂”白血病。