项目标书摘要：脐带血是研究造血干细胞最好的来源，因为脐带血具有容易捐赠、无危险、干细胞含量高及HLA相配较易等优点。脐带血干细胞已经被应用在抗癌和组织修护(如中风)等方面而有一定的成效。然而干细胞的分化(Differentiation)机制还未全盘了解，根据文献，研究人员发现基因甲基化扮演重要的脚色，尤其是CpG island的甲基化(methylation)与基因调控及细胞分化有密切关系。因此，本计划预计探讨的目标有(1)分别了解脐带血中造血干细胞和T(CD8+)细胞DNA甲基化的模式(2)了解有关造血干细胞的分化机制—如何以甲基化DNA来协助或主导调控及分化成T细胞(CD8+)(3)搜寻人类的基因体中未被注解的新」去甲基.」(demethylase),进而有助於了解造血干细胞分化过程中基因体甲基化和去甲基化的全貌，同时，有机会可利用反甲基化(reverse methylation)实验让各种细胞打开被抑制的基因。为了要达成这一个计划目标，敝人应用已知的技术加上两个小的自行组合的实验设计方法来应用於探讨发育(development)与甲基化、去甲基化的机转。一是 PCR选择性抑压杂交方法，而且另一个是应用酵母菌系统来搜寻人类新的去甲基.。本计划预估将找到相关基因群，其CpG island的甲基化可能分别地在造血干细胞和CD8+T细胞扮演一定的重要性。那些被甲基化的基因将提供我们有用的资料以了解关於干细胞的自我更新(self renew)与发育的基本机制，甚至有可能藉由阻断 DNA methyltransferase(DNMT)达到去甲基化而进一步了解甲基化的基因与发育关系。藉由本计划探讨细胞分化与发育等的控制机制，及人类的基因体甲基化和去甲基化的图谱﹔将有益於人类对器官再生、长寿和抗癌等的相关研究。
Application Abstract: Umbilical cord blood stem cells are the good source to obtain hematopoietic stem cells that are absence of risk for mothers and donors and also have advantage for less stringent criteria for HLA matching for donor-recipient selection.Hematopoietic stem cells have been studied for anti-cancer and tissue damage repaired.The mechanisms of hematopoietic stem cells differentiation are still unknown.Thus,this project aims to(1)understand the DNA methylation patterns in CpG islands of genes for hematopoietic stem cells and T(CD8+)cells respectively,(2)understand the differentiation that might relate to methylated DNA during hematopoietic stem cells developed into T cells(CD8+),(3)scan any possibility new global human demethylase from human genome and map the methylation and demethylation issues in hematopoietic stem cell differentiation.To achieve this project,the known technologies plus two my designed novel modified methods for examination of the methylation patterns will be applied.One is PCR selective suppression hybridization approach and the other is DNA demethylase screening in yeast system.Hundreds genes containing methylated CpG islands that might be discovered in hematopoietic stem cells and CD8+T cells respectively.Those methylated genes will provide us useful information to understand the basic mechanism about stem cells.More important,it is possible to reverse DNA methylation in replicating cells by blocking DNA methyltransferase(DNMT)during cell cycle.This project might be able to reveal the control mechanisms of cell development events,and also draw global methylation and demethylation patterns for Human genome.Therefore,organ duplication,longevity and anti-cancer will have lots of good news in the future.
