2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/648612摘要：第三部分：基因遺傳工程小鼠表現型，第四部分：血癌復發之分子機轉，分子治療標的。急性淋巴球性白血病（ALL）是兒童癌症中最常見的惡性腫瘤，目前使用預後危險因子為導向的化療法，可將完全緩解率（使疾病進入殘留癌細胞階段）提高至95％（Pui et at., 2006）。然而，根據我們TPOG（台灣兒童癌症研究群）初步研究數據顯示，在完全緩解後所發生的高復發機率及其治療抗藥性仍在影響著，即使是最成功的以危險因子為導向的化療方案的治療成績。有許多已經發現的可預測復發危險因子，雖可被用在初發病病童身上來評估復發的機率，但卻沒有一個是可完全信賴的（Pui et al., 1998）。最近的研究指出在臨床緩解期間以流式細胞儀來偵測其微量殘留癌細胞量，是獨立的、也是最重要的復發危險因子（Coustan-Smith et al., 2000）。根據我們的初步研究結果，已可利用流式細胞儀技術的方式，在病人臨床緩解階段時即可檢測微殘留癌細胞量來作為復發的預測性因子。微核醣核酸是一個非譯碼的小單股核醣核酸，他們在細胞的發育及生理過程（如造血）中扮演著重要的調節角色（Bartel et al., 2004）。最近的研究更指出微核醣核酸的失調已被發現與癌症的致癌基因及腫瘤抑制基因的調控有關（Chen, 2005）。我們的共同主持人已經發現5個新的微核醣核酸標幟，可用來預測非小細胞肺癌的存活率及復發（Yu et al., 2008）。在與基因體微陣列中心的合作下，我們的前驅性實驗發現了另外7個新的微核醣核酸標幟，可以用來預測兒童急性淋巴球性白血病的復發。我們的團隊也證明了擁有基因標靶疾病小鼠的繁殖與研究能力（Jou et at., 2002），並且也已建立超過30種基因剔除/基因替換並具生殖細胞傳遞能力的嵌和體小鼠（Lin SW）。基於上述的進展，我們將持續研究微核醣核酸在兒童急性淋巴球性白血病所扮演的角色：第一部分：（1）建立流式細胞儀檢測來確認並釐清微殘留癌細胞量的程度當作細胞層級可預測復發的因子。（2）利用q-RT-PCR比較此7個微核醣核酸在高復發風險（陽性微殘留癌細胞）以及低復發風險（陰性微殘留癌細胞）兩群病童檢體的表現模式以再鑑定此7微核醣核酸標幟的效度。（3）鑑定出更簡明的微核醣核酸標幟，來當作快速有效的（可在得到發病診斷的檢體後3個小時內完成）、創新的分子層級可預測復發的因子，以期降低目前以預後危險因子為導向的化療治療策略的復發機率。第二部分：藉由比較在陽性微殘留癌細胞的病童中，此7個微核醣核酸的表達方式，以找出2個最顯著有意義的微核醣核酸，來作為設計基因遺傳工程小鼠的標的，並據此建立2隻基因遺傳工程小鼠。第三部分：分析這兩隻微核醣核酸遺傳工程小鼠的表現型。第四部分：利用疾病小鼠模式進行進一步的分析：（1）闡明微核醣核酸在復發及治療抗藥性的分子機制中所扮演的角色與（2）進一步找出潛在的分子治療標的。<br> Abstract: Part II: genetically engineered mouse models; Part III: engineered mice phenotype, Part IV: molecular mechanism of relapse, molecular therapeutic targetThe acute lymphoblastic leukemia is the most common malignancy among the childhood cancers. The current prognostic risk factor-directed therapies have increased the complete remission rate (into the residual disease status) to greater than 95% (Pui et. at., 2006).However, our preliminary data of TPOG shows the high relapse rate after the complete remission and then therapeutic resistance continues to affect even the most successful risk-directed chemotherapy protocols.Many presenting characteristics (relapse predictive risk factor) can be used to estimate the relapse hazard in such patients, but none is completely reliable (Pui et al., 1998). Recent study revealed the detection of minimal residual disease (MRD) with flow cytometry during clinical remission appears to be independently the most important risk factor associated with relapse (Coustan-Smith et. Al., 2000).Our preliminary result demonstrates, by using flow cytometric techniques, we could detect the MRD as the relapse predictive factor in ALL patients in clinical remission.MicroRNAs are small non-coding single-stranded RNA. They play important regulation roles in cellular developmental and physiological processes such as hematopoiesis (Bartel et al., 2004). Recent study reported the dysregulation of miRNA has also been found to have links with cancers as oncogenes and tumor suppressors (Chen, 2005).Our co-P.I.s had already shown one novel 5-miRNA signature could predict survival and relapse in non-small –cell lung cancer (Yu et al., 2008). In collaboration with the Microarray Core on campus, our pilot study has identified another novel 7-miRNA signature which can predict the relapse in childhood ALL.Our team had also demonstrated the ability to generate and investigate the gene-targeted disease mice (Jou et. at., 2002) and set up more than 30 knock-out/knock-in chimeras with germline transmission in campus core facility (Lin SW).With the above progresses, we continue investigating the role of miRNAs in ALL relapse as the following:Part I: [1] To establish, check and clarify the MRD level by flow cytometry as the cellular relapse predictive factor. [2] By using the q-RT-PCR method, to compare the expression pattern of these 7 miRNAs between the high relapse risk (positive MRD) and low relapse risk (negative MRD) groups to revalidate our preliminary novel 7-miRNA signature and then, [3] to identify more concise miRNA signature, which could be available as soon as 3 hours after diagnostic sampling, as the novel miRNA-based molecular relapse predictive factor and modality for current risk-directed chemotherapy strategy to decrease the relapse rate.Part II: To identify the most significant 2 miRNAs among this novel 7-miRNA signature by comparison with their expression pattern in positive MRD patients as the targets to generate genetically engineered mouse models.Part III: To analyze the phenotypes of these two miRNA- engineered mice.Part IV: Use of the disease mice models for further analysis: [1] To elucidate the role of miRNA in the molecular mechanism of relapse/therapeutic resistance and [2] further identify potential molecular therapeutic target in ALL.兒童急性淋巴球性白血病微核醣核酸第一部分：微核醣核酸標幟流式細胞儀微量殘留癌細胞創新的疾病復發預測因子第二部分：基因遺傳工程小鼠模式childhood acute lymphoblastic leukemia (childhood ALL)microRNAPart I: miRNA signatureflow cytometryminimal residual diseasenovel relapse predictive factor