2014-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/655943摘要：在台灣與全世界上，轉移性腦瘤發生率已超過原發性腦瘤，其中 50%以上的轉移性腦瘤是轉移自肺癌。另一方面，有 40%的肺癌病人會發生腦轉移並縮短病患的存活。然而，現今仍缺乏可靠的生物標記來預測肺癌病人是否會發生腦轉移。 在免疫缺陷鼠中，利用重複的原位注射乳癌細胞 MDA-MB-231已經成功地找出乳癌的肺、腦及骨頭轉移的生物標記。在此計畫中，我們將利用全基因體核糖核酸干擾策略找尋新穎的肺癌腦轉移生物標記。利用帶有小髮夾核糖核酸的慢病毒為載體，感染表現螢火蟲冷光酵素的 A549人類肺腺癌細胞株，並植入 NOD/SCID母鼠的左肺中。接著每周利用 IVIS追蹤腫瘤的生長及轉移。自腦轉移腫瘤取出的細胞會混和後，再一次注射到另外一批 NOD/SCID母鼠的左肺中，藉以挑選出更高轉移能力的細胞。這種活體篩選的方式會重複直到 75%的小鼠發生腦轉移。最後利用次世代定序鑑定篩選後的細胞中帶有那些小髮夾核糖核酸，並以體外與體內實驗進一步定性及確認其在腦轉移中所扮演的腳色。 因為，在腫瘤植入的小鼠模式中，小鼠的存活期會縮短，同時腦轉移的發生也需要較長的時間，或許在原位注射的模型中較難挑選出與腦轉移相關的基因。因此，我們也將同時發展將小鼠外頸動脈暫時阻斷後，從主頸動脈打入腫瘤的方式，以此方法處理的小鼠幾乎皆能產生腦轉移。我們將透過五次的活體篩選，來篩選出與腦轉移能力相關的基因。找出的基因將會利用體外腦組織切片培養模型以及活體腦部原位注射不同細胞數量的方式，驗證其腦轉移的能力。 透過這一系列研究，我們希望能找出新穎的生物標記已來評估肺癌病人腦轉移的風險。同時也能幫助發展新的治療策略，以降低肺癌病人的腦轉移發生。<br> Abstract: The incidence of metastasis brain tumors exceeds that of primary brain tumors in Taiwan and worldwide, of which about 50% are lung cancer origin. On the other hand, 40% of lung cancer patients will develop brain metastasis during tumor progression and significantly have shortened overall survival. However there is no reliable biomarker to predict brain metastasize in lung cancer yet. The repeated orthotopic implantation has been successfully used to identify the biomarkers for lung, brain, and bone metastasis of breast cancer by using breast cancer cell line MDA-MB-231 in immunodeficiency mice. In this proposal, we plan to use a genome-wide RNA interference approach to identify novel biomarkers for lung cancer metastasis, especially toward the brain. Lentivirus based A549 lung cancer cells harboring luciferase will be transfected with shRNA library and injected orthotopically into left lung lobes of female NOD/SCID mice. Tumorigenesis and metastasis of inoculated tumors will be tracked by IVIS weekly. The distal metastasis tumors and primary tumors in mice will be harvested. Cells from metastasis brain tumors will be pooled and orthotopically injected into left lung lobes of another mice to select out the higher brain metastatic clones. The in vivo selection will be repeated until 75% of mice develop brain metastasis. The shRNAs in those clones will be revealed by NGS and their roles in brain metastasis will be further characterized and validated in vitro and in vivo. Because of the long period required for development of brain metastasis and the short survival time of primary tumor-bearing mice, it might be difficult to identify the genes involved in brain-specific metastasis by orthotopic injection. Therefore, we will also develop an alternative approach to overcome the technology limits in which the shRNA library-bearing transfectants will be directly injected into the left common carotid arteries of NOD/SICD mice with temporally external carotid artery ligation. Since the possibility of develop brain metastasis is near 100% in this approach, five rounds of in vivo selection will be performed to discriminate the metastatic ability of injected cells. The candidate genes will be validated by an in vitro cultured brain slice system and by orthotopic brain injection of series diluted cells. Through the study we expect that several brain-specific metastasis related candidates will be identified and provide clues for development of new therapeutic strategies against brain metastasis of lung cancer in the future.