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.

由于人类雄激素受体基因（human androgen-receptor gene，HUMARA）5 '端启动子区存在一个与X染色体失活（X-chromosome inactivation，XCI）相关的多态性短串联重复序列（short tandem repeat，STR），因此该基因已被用于XCI的分析（HUMARA-MSP）测定，其通过亚硫酸氢盐修饰而不是甲基化敏感性限制酶分析HUMARA基因的甲基化状态。虽然原始MSP方法显示是否存在甲基化条带，但我们的HUMARA-MSP方法可识别甲基化和非甲基化条带的模式。因为该方法鉴定每个PCR管中的未甲基化或甲基化等位基因，并且显示依赖于甲基化状态的相反条带模式，所以我们可以独立地评估XCI模式两次。该方法避免了酶解不完全造成的假结果，亚硫酸氢盐修饰不完全也不会影响结果。极少量的样品，如造血集落，也可用于HUMARA-MSP测定。HUMAR-MSP法简便、灵敏、适用性广，可用于血液病的发病机制分析。

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。

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。

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。