探討Oct3/4, Sox2, c-Myc, Klf4基因在癌症幹細胞中的表現量
一、計畫名稱
中文
探討Oct3/4, Sox2, c-Myc, Klf4基因在癌症幹細胞中的表現量
英文
Functional investigation on the role of Oct3/4, Sox2, c-Myc, Klf4 in cancer stem cell
二、計畫中文摘要(請於五百字內就本計畫要點作一概述，並依本計畫性質自訂關鍵詞。)
關鍵詞：stem cell, Oct3/4 , Sox2 , c-Myc , Klf4 , renew , iPS , CSC , reprogram
幹細胞(stem cell)研究是目前生物醫學領域最熱門的題目之一。所謂的幹細胞，是指一群尚未分化的細胞，這些細胞具有自我分裂增殖成另一些與自身相同之細胞的能力，因此幹細胞像癌細胞一樣在實驗室中能不斷培養繁殖。幹細胞在接受特定的生長激素刺激後，可以分化成特定功能的體細胞。在人體的骨髓、皮下、脂肪等多個組織都存在有幹細胞，以便進行造血、修補等功能。
哈佛大學於2006年所作的基因庫分析，發現Oct3/4, Sox2, c-Myc, Klf4等基因跟幹細胞的復原跟增生有關。像OCT3/4和SOX2就是轉錄因子，跟基因的表現與否有關，而MYC和KLF4則是和細胞的復原(renew)有關。同年，日本京都大學山中伸彌教授（Shinya Yamanaka)的團隊發現只需要將四個基因 (Oct3/4, Sox2, c-Myc, Klf4)植入已分化完全的小鼠纖維母細胞，即可以把纖維母細胞重新設定(reprogram)變回具備分化全能性的類胚胎幹細胞。他們將這種重新設定的細胞稱之為「誘導式多能性幹細胞」(induced pluripotent stem cells, iPS)。
研究者製造了許多已知致病基因的疾病的iPS細胞，包含肌肉萎縮症(Muscular Dystrophy)、高雪氏症(Gaucher disease)、漢丁頓舞蹈症(Huntington’s disease)、帕金森氏症(Parkinson’s disease)、青少年糖尿病(juvenile diabetes)等疾病的幹細胞細胞株以做為治療之用。
癌症幹細胞(cancer stem cell,CSC)是癌細胞(被發現於腫瘤與血癌(hematologic cancer)當中)，它最特別的地方在於它有類似於幹細胞的特徵，就我們從臨床試驗中抽樣出的癌組織來看。而這些癌症幹細胞與其它癌細胞比較起來更具有「致癌性(tumorigenic)」。癌症幹細胞最重要的特徵在於它有向幹細胞一般的功能，像是：復原(renew)、增生和分化至不同型式。
本實驗希望透過比較Oct3/4, Sox2, c-Myc, Klf4等基因在癌症幹細胞(cancer stem cell,CSC)上表現量，來確認Oct3/4, Sox2, c-Myc, Klf4等基因是否與癌症幹細胞(cancer stem cell,CSC)的復原、增生和分化能力有關，以期進一步運用於臨床醫療之中。
三、計畫英文摘要(請於五百字內就本計畫要點作一概述，並依本計畫性質自訂關鍵詞。)
Keywords：stem cell, Oct3/4 , Sox2 , c-Myc , Klf4 , renew , iPS , CSC , reprogram
The research of stem cell is one of the most popular field in the modern days . Stem cell is a group of un-differentiated cell , and these cells have ability to proliferate their self to more cell number .Therefore , stem cells are just like cancer cell that have the character to proliferate their-self in experiment . After accepting some special Growth Factor , the stem cell can differentiate into kinds of human adult cell that have normal physiologic function . Stem cell exist in normal human tissue , such as bone marrow , hypodermis , and fat . All of the differentiated stem cell have normal physiologic function .
Harvard university found that octamer-binding transcription factor-3/4 (OCT3/4), SRY-related high-mobility-group (HMG)-box protein-2 (SOX2), MYC and Kruppel-like factor-4 (KLF4) have a connection with stem cells’ ability of renew and proliferation by gene bank analysis in 2006 . Oct3/4 and Sox2 are transcription factors that play an important role in gene expression .Moreover , c-Myc and Klf4 is crucial for cells’ renew . In the same year , the group of professor of Kyoto University , Shinya Yamanaka , had discovered that if they transferred Oct3/4, Sox2, c-Myc, Klf4 into mouse fibroblast , and then they could reprogram the mouse fibroblast into pluripotent fibroblast stem cells . It is called induced pluripotent stem cells, iPS .
The scientist had made kinds of iPS cell for disease associating to gene in clinical treatment , such as Muscular Dystrophy , Huntington’s disease , Parkinson’s disease and juvenile diabetes .
Cancer stem cells (CSCs) are cancer cells (found within tumor or hematologic cancer) that possess characteristics associated with normal stem cell, specifically the ability to give rise to all cell types found in a particular cancer sample. These cells are therefore tumorigenic (tumor-forming), perhaps in contrast to other non-tumorigenic cancer cells. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors.
In this paper , we want to compare and the cancer stem cell(CSC) with induced pluripotent stem cells (iPS) by detecting the expression of Oct3/4, Sox2, c-Myc and Klf4 . Then , we could identify that are the Oct3/4, Sox2, c-Myc and Klf4 play an important in renew , proliferation and differentiation in cancer stem cell(CSC). Finally , we could use these in clinical treatment.
四、計畫內容：
(一)研究計畫之背景及目標：請敘述本研究計畫之背景、目的、重要性以及國內外有關本計畫之研究情況。
Induced pluripotent stem (iPS) cells are human somatic cells that have been reprogrammed to a pluripotent state. There are several hurdles to be overcome before iPS cells can be considered as a potential patient-specific cell therapy, and it will be crucial to characterize the developmental potential of human iPS cell lines. As a research tool, iPS-cell technology provides opportunities to study normal development and to understand reprogramming. iPS cells can have an immediate impact as models for human diseases, including cancer .
Induced pluripotent stem (iPS) cells are the product of somatic cell reprogramming to an embryonic-like state. This occurs by the introduction of a defined and limited set of transcription factors and by culturing these cells under embryonic stem (ES)-cell conditions . The method was first described by Shinya Yamanaka and co lleagues using mouse fibroblasts, in which it was emonstrated that the retroviral-mediated ntroduction of four transcription factors octamer-binding transcription factor-3/4 (OCT3/4), SRY-related high-mobility-group (HMG)-box protein-2 (SOX2), MYC and Kruppel-like factor-4 (KLF4) — could induce pluripotency. The protocol has since been applied to other types of mouse cells2,3 and to human somatic cells4–7. Although the original gene set for reprogramming1 comprised Oct3/4, Sox2, Myc and Klf4, recent studies have shown that other combinations of factors can substitute for MYC and KLF4 and produce iPS cells. The reprogramming procedure is straightforward9, robust and has been independently replicated by multiple groups. However, the detailed mechanism for reprogramming is unknown and is a topic of intense scrutiny in several laboratories. iPS-cell technology is a novel method .
Cancer stem cells (CSCs) are cancer cells (found within tumor or hmmatological cancer) that possess characteristics associated with normal stem cell stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. These cells are therefore tumorigenic (tumor-forming), perhaps in contrast to other non-tumorigenic cancer cells. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastastic disease.
Existing cancer treatments were mostly developed on animal models, where therapies able to promote tumor shrinkage were deemed effective. However, animals could not provide a complete model of human disease. In particular, in mice, whose life spans do not exceed two years, tumor relapse is exceptionally difficult to study.
The efficacy of cancer treatments are, in the initial stages of testing, often measured by the amount of tumor mass they kill off. As CSCs would form a very small proportion of the tumor, this may not necessarily select for drugs that act specifically on the stem cells. The theory suggests that conventional chemotherapies kill differentiated or differentiating cells, which form the bulk of the tumor but are unable to generate new cells. A population of CSCs, which gave rise to it, could remain untouched and cause a relapse of the disease.
In this paper , we want to use kinds of method to compare and the cancer stem cell(CSC) with induced pluripotent stem cells (iPS) by detecting the expression of Oct3/4, Sox2, c-Myc and Klf4 , such as western , northern ,siRNA and so on . Then , we could identify that are the Oct3/4, Sox2, c-Myc and Klf4 play an important in renew , proliferation and differentiation in cancer stem cell(CSC). Finally , we could use these in clinical treatment.
Reference
http://en.wikipedia.org/wiki/Cancer_stem_cell
1. Takahashi, K. & Yamanaka, S. Induction of pluripotent
stem cells from mouse embryonic and adult fibroblast
cultures by defined factors. Cell 126, 663–676
(2006).
2. Aoi, T. et al. Generation of pluripotent stem cells from
adult mouse liver and stomach cells. Science 14 Feb
2008 (doi:10.1126/science.1154884)
3. Okita, K., Ichisaka, T. & Yamanaka, S. Generation of
germline-competent induced pluripotent stem cells.
Nature 448, 313–317 (2007).
4. Takahashi, K. et al. Induction of pluripotent stem cells
from adult human fibroblasts by defined factors.
Cell 131, 861–872 (2007).
5. Yu, J. et al. Induced pluripotent stem cells from adult
human somatic cells. Science 318, 1917–1920
(2007).
6. Park, I.-H. et al. Reprogramming of human somatic
cells to pluripotency with defined factors. Nature 451,
141–147 (2008).
7. Lowry, W. E. et al. Generation of human induced
pluripotent stem cells from dermal fibroblasts.
Proc. Natl Acad. Sci. USA 105, 2883–2888
(2008).
8. Blelloch, R. et al. Generation of induced pluripotent
stem cells in the absence of drug selection. Cell Stem
Cell 1, 245–247 (2007).
9. Takahashi, K., Okita, K., Nakagawa, M. &
Yamanaka, S. Induction of pluripotent stem cells from
fibroblast cultures. Nature Protoc. 2, 3081–3089
(2007).
10. Yamanaka, S. Strategies and new developments in the
generation of patient-specific pluripotent stem cells.
Cell Stem Cell 1, 39–49 (2007).
11. Cibelli, J. Development: is therapeutic cloning dead?
Science 318, 1879–1880 (2007).
12. Jaenisch, R. & Young, R. Stem cells, the molecular
circuitry of pluripotency and nuclear reprogramming.
Cell 132, 567–582 (2008).
13. Thomson, J. A. et al. Embryonic stem cell lines
derived from human blastocysts. Science 282,
1145–1147 (1998). And so on …
(二)研究方法及進行步驟：1.請詳述本計畫擬採用之研究方法與步驟，2.預計可能遭遇之困難及解決途徑。
Antibody Production
Oct3/4, Sox2, c-Myc and Klf4-specific peptide sequences will be selected following general principles for antigenicity. All candidate sequences have been subjected to stringent comparison by multiple-BLAST program in order to rule out possible cross-reactivity with other Oct3/4, Sox2, c-Myc and Klf4 members as well as reactivity towards other proteins. In many cases, specific peptide sequences with optimal antigenicity may not be available for certain proteins. In this case, we will subclone non-conserved portions of Oct3/4, Sox2, c-Myc and Klf4 and express it as bacteria derived recombinant proteins for use as antigen. Monoclonal and polyclonal antibodies will be produced following standard protocols. Further purification may be applied to polyclonal antibodies by affinity absorption with the original antigen.
Animal model
C57BL/6 mice were from the National Cancer Institute. B6.FVBTg ( EIIa-Cre)C5379Lmgd/J-transgenic mice and TNFR1-KO mice were from The Jackson Laboratory. All animal experiments followed protocols approved by the US National Cancer Institute Animal Care and Use Committee and guidelines of the US National Institutes of Health.
Western blot
The western blot (alternatively, immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against many thousands of different proteins. Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments.
siRNA
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of 20-25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated.
Gene transfering
Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that apply to the direct manipulation of an organism's genes.Genetic engineering is different from traditional breeding, where the organism's genes are manipulated indirectly; genetic engineering uses the techniques of molecular cloning and transformation to alter the structure and characteristics of genes directly. Genetic engineering techniques have found some successes in numerous applications. Some examples are in improving crop technology, the manufacture of synthetic human insulin through the use of modified bacteria, the manufacture of erythropoietin in hamster ovary cells, and the production of new types of experimental mice such as the oncomouse (cancer mouse) for research.
RT-PCR
In molecular biology, reverse transcription polymerase chain reaction, abbreviated as RT-PCR, is a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule. The RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using polymerase chain reaction. This can either be a 1 or 2 step process. Reverse transcription PCR is not to be confused with real-time polymerase chain reaction (Q-PCR) which is also sometimes wrongly abbreviated as RT-PCR.
ELISA
Enzyme-Linked ImmunoSorbent Assay, also called ELISA, Enzyme ImmunoAssay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression. It takes its name from its similarity to the Southern blot technique, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot.Both techniques use electrophoresis and detection with a hybridization probe. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.
Flow cytometry
Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus.
(三)預期完成之工作項目及具體成果：1.請列述執行期限內預期完成之工作項目，2學術性及應用性價值。
本計劃之目的
1. 經由比較資料庫，為Oct3/4, Sox2, c-Myc and Klf4設計具有專一性的單株抗體，及以重組蛋白為抗原的多株抗體。
2. 用western 、RT-PCR和northern blot來偵測Oct3/4, Sox2, c-Myc and Klf4在protein、RNA和DNA的表現量
3. 用FLOW來偵測癌症幹細胞的表面標記和細胞狀況
4. 透過比較Oct3/4, Sox2, c-Myc, Klf4等基因在癌症幹細胞(cancer stem cell,CSC)上表現量，來確認Oct3/4, Sox2, c-Myc, Klf4等基因是否與癌症幹細胞(cancer stem cell,CSC)的復原、增生和分化能力有關?
學術性及應用性價值
1. 透過基因的分析釐清cancer stem cell和induced pluripotent stem cells在基因表現的差異
2. 藉此建立消滅cancer stem cell的機制
3. 建立anti-cancer藥物研發的基本知識
中文
探討Oct3/4, Sox2, c-Myc, Klf4基因在癌症幹細胞中的表現量
英文
Functional investigation on the role of Oct3/4, Sox2, c-Myc, Klf4 in cancer stem cell
二、計畫中文摘要(請於五百字內就本計畫要點作一概述，並依本計畫性質自訂關鍵詞。)
關鍵詞：stem cell, Oct3/4 , Sox2 , c-Myc , Klf4 , renew , iPS , CSC , reprogram
幹細胞(stem cell)研究是目前生物醫學領域最熱門的題目之一。所謂的幹細胞，是指一群尚未分化的細胞，這些細胞具有自我分裂增殖成另一些與自身相同之細胞的能力，因此幹細胞像癌細胞一樣在實驗室中能不斷培養繁殖。幹細胞在接受特定的生長激素刺激後，可以分化成特定功能的體細胞。在人體的骨髓、皮下、脂肪等多個組織都存在有幹細胞，以便進行造血、修補等功能。
哈佛大學於2006年所作的基因庫分析，發現Oct3/4, Sox2, c-Myc, Klf4等基因跟幹細胞的復原跟增生有關。像OCT3/4和SOX2就是轉錄因子，跟基因的表現與否有關，而MYC和KLF4則是和細胞的復原(renew)有關。同年，日本京都大學山中伸彌教授（Shinya Yamanaka)的團隊發現只需要將四個基因 (Oct3/4, Sox2, c-Myc, Klf4)植入已分化完全的小鼠纖維母細胞，即可以把纖維母細胞重新設定(reprogram)變回具備分化全能性的類胚胎幹細胞。他們將這種重新設定的細胞稱之為「誘導式多能性幹細胞」(induced pluripotent stem cells, iPS)。
研究者製造了許多已知致病基因的疾病的iPS細胞，包含肌肉萎縮症(Muscular Dystrophy)、高雪氏症(Gaucher disease)、漢丁頓舞蹈症(Huntington’s disease)、帕金森氏症(Parkinson’s disease)、青少年糖尿病(juvenile diabetes)等疾病的幹細胞細胞株以做為治療之用。
癌症幹細胞(cancer stem cell,CSC)是癌細胞(被發現於腫瘤與血癌(hematologic cancer)當中)，它最特別的地方在於它有類似於幹細胞的特徵，就我們從臨床試驗中抽樣出的癌組織來看。而這些癌症幹細胞與其它癌細胞比較起來更具有「致癌性(tumorigenic)」。癌症幹細胞最重要的特徵在於它有向幹細胞一般的功能，像是：復原(renew)、增生和分化至不同型式。
本實驗希望透過比較Oct3/4, Sox2, c-Myc, Klf4等基因在癌症幹細胞(cancer stem cell,CSC)上表現量，來確認Oct3/4, Sox2, c-Myc, Klf4等基因是否與癌症幹細胞(cancer stem cell,CSC)的復原、增生和分化能力有關，以期進一步運用於臨床醫療之中。
三、計畫英文摘要(請於五百字內就本計畫要點作一概述，並依本計畫性質自訂關鍵詞。)
Keywords：stem cell, Oct3/4 , Sox2 , c-Myc , Klf4 , renew , iPS , CSC , reprogram
The research of stem cell is one of the most popular field in the modern days . Stem cell is a group of un-differentiated cell , and these cells have ability to proliferate their self to more cell number .Therefore , stem cells are just like cancer cell that have the character to proliferate their-self in experiment . After accepting some special Growth Factor , the stem cell can differentiate into kinds of human adult cell that have normal physiologic function . Stem cell exist in normal human tissue , such as bone marrow , hypodermis , and fat . All of the differentiated stem cell have normal physiologic function .
Harvard university found that octamer-binding transcription factor-3/4 (OCT3/4), SRY-related high-mobility-group (HMG)-box protein-2 (SOX2), MYC and Kruppel-like factor-4 (KLF4) have a connection with stem cells’ ability of renew and proliferation by gene bank analysis in 2006 . Oct3/4 and Sox2 are transcription factors that play an important role in gene expression .Moreover , c-Myc and Klf4 is crucial for cells’ renew . In the same year , the group of professor of Kyoto University , Shinya Yamanaka , had discovered that if they transferred Oct3/4, Sox2, c-Myc, Klf4 into mouse fibroblast , and then they could reprogram the mouse fibroblast into pluripotent fibroblast stem cells . It is called induced pluripotent stem cells, iPS .
The scientist had made kinds of iPS cell for disease associating to gene in clinical treatment , such as Muscular Dystrophy , Huntington’s disease , Parkinson’s disease and juvenile diabetes .
Cancer stem cells (CSCs) are cancer cells (found within tumor or hematologic cancer) that possess characteristics associated with normal stem cell, specifically the ability to give rise to all cell types found in a particular cancer sample. These cells are therefore tumorigenic (tumor-forming), perhaps in contrast to other non-tumorigenic cancer cells. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors.
In this paper , we want to compare and the cancer stem cell(CSC) with induced pluripotent stem cells (iPS) by detecting the expression of Oct3/4, Sox2, c-Myc and Klf4 . Then , we could identify that are the Oct3/4, Sox2, c-Myc and Klf4 play an important in renew , proliferation and differentiation in cancer stem cell(CSC). Finally , we could use these in clinical treatment.
四、計畫內容：
(一)研究計畫之背景及目標：請敘述本研究計畫之背景、目的、重要性以及國內外有關本計畫之研究情況。
Induced pluripotent stem (iPS) cells are human somatic cells that have been reprogrammed to a pluripotent state. There are several hurdles to be overcome before iPS cells can be considered as a potential patient-specific cell therapy, and it will be crucial to characterize the developmental potential of human iPS cell lines. As a research tool, iPS-cell technology provides opportunities to study normal development and to understand reprogramming. iPS cells can have an immediate impact as models for human diseases, including cancer .
Induced pluripotent stem (iPS) cells are the product of somatic cell reprogramming to an embryonic-like state. This occurs by the introduction of a defined and limited set of transcription factors and by culturing these cells under embryonic stem (ES)-cell conditions . The method was first described by Shinya Yamanaka and co lleagues using mouse fibroblasts, in which it was emonstrated that the retroviral-mediated ntroduction of four transcription factors octamer-binding transcription factor-3/4 (OCT3/4), SRY-related high-mobility-group (HMG)-box protein-2 (SOX2), MYC and Kruppel-like factor-4 (KLF4) — could induce pluripotency. The protocol has since been applied to other types of mouse cells2,3 and to human somatic cells4–7. Although the original gene set for reprogramming1 comprised Oct3/4, Sox2, Myc and Klf4, recent studies have shown that other combinations of factors can substitute for MYC and KLF4 and produce iPS cells. The reprogramming procedure is straightforward9, robust and has been independently replicated by multiple groups. However, the detailed mechanism for reprogramming is unknown and is a topic of intense scrutiny in several laboratories. iPS-cell technology is a novel method .
Cancer stem cells (CSCs) are cancer cells (found within tumor or hmmatological cancer) that possess characteristics associated with normal stem cell stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. These cells are therefore tumorigenic (tumor-forming), perhaps in contrast to other non-tumorigenic cancer cells. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastastic disease.
Existing cancer treatments were mostly developed on animal models, where therapies able to promote tumor shrinkage were deemed effective. However, animals could not provide a complete model of human disease. In particular, in mice, whose life spans do not exceed two years, tumor relapse is exceptionally difficult to study.
The efficacy of cancer treatments are, in the initial stages of testing, often measured by the amount of tumor mass they kill off. As CSCs would form a very small proportion of the tumor, this may not necessarily select for drugs that act specifically on the stem cells. The theory suggests that conventional chemotherapies kill differentiated or differentiating cells, which form the bulk of the tumor but are unable to generate new cells. A population of CSCs, which gave rise to it, could remain untouched and cause a relapse of the disease.
In this paper , we want to use kinds of method to compare and the cancer stem cell(CSC) with induced pluripotent stem cells (iPS) by detecting the expression of Oct3/4, Sox2, c-Myc and Klf4 , such as western , northern ,siRNA and so on . Then , we could identify that are the Oct3/4, Sox2, c-Myc and Klf4 play an important in renew , proliferation and differentiation in cancer stem cell(CSC). Finally , we could use these in clinical treatment.
Reference
http://en.wikipedia.org/wiki/Cancer_stem_cell
1. Takahashi, K. & Yamanaka, S. Induction of pluripotent
stem cells from mouse embryonic and adult fibroblast
cultures by defined factors. Cell 126, 663–676
(2006).
2. Aoi, T. et al. Generation of pluripotent stem cells from
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(二)研究方法及進行步驟：1.請詳述本計畫擬採用之研究方法與步驟，2.預計可能遭遇之困難及解決途徑。
Antibody Production
Oct3/4, Sox2, c-Myc and Klf4-specific peptide sequences will be selected following general principles for antigenicity. All candidate sequences have been subjected to stringent comparison by multiple-BLAST program in order to rule out possible cross-reactivity with other Oct3/4, Sox2, c-Myc and Klf4 members as well as reactivity towards other proteins. In many cases, specific peptide sequences with optimal antigenicity may not be available for certain proteins. In this case, we will subclone non-conserved portions of Oct3/4, Sox2, c-Myc and Klf4 and express it as bacteria derived recombinant proteins for use as antigen. Monoclonal and polyclonal antibodies will be produced following standard protocols. Further purification may be applied to polyclonal antibodies by affinity absorption with the original antigen.
Animal model
C57BL/6 mice were from the National Cancer Institute. B6.FVBTg ( EIIa-Cre)C5379Lmgd/J-transgenic mice and TNFR1-KO mice were from The Jackson Laboratory. All animal experiments followed protocols approved by the US National Cancer Institute Animal Care and Use Committee and guidelines of the US National Institutes of Health.
Western blot
The western blot (alternatively, immunoblot) is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. There are now many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against many thousands of different proteins. Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments.
siRNA
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of 20-25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated.
Gene transfering
Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that apply to the direct manipulation of an organism's genes.Genetic engineering is different from traditional breeding, where the organism's genes are manipulated indirectly; genetic engineering uses the techniques of molecular cloning and transformation to alter the structure and characteristics of genes directly. Genetic engineering techniques have found some successes in numerous applications. Some examples are in improving crop technology, the manufacture of synthetic human insulin through the use of modified bacteria, the manufacture of erythropoietin in hamster ovary cells, and the production of new types of experimental mice such as the oncomouse (cancer mouse) for research.
RT-PCR
In molecular biology, reverse transcription polymerase chain reaction, abbreviated as RT-PCR, is a laboratory technique for amplifying a defined piece of a ribonucleic acid (RNA) molecule. The RNA strand is first reverse transcribed into its DNA complement or complementary DNA, followed by amplification of the resulting DNA using polymerase chain reaction. This can either be a 1 or 2 step process. Reverse transcription PCR is not to be confused with real-time polymerase chain reaction (Q-PCR) which is also sometimes wrongly abbreviated as RT-PCR.
ELISA
Enzyme-Linked ImmunoSorbent Assay, also called ELISA, Enzyme ImmunoAssay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries. In simple terms, in ELISA an unknown amount of antigen is affixed to a surface, and then a specific antibody is washed over the surface so that it can bind to the antigen. This antibody is linked to an enzyme, and in the final step a substance is added that the enzyme can convert to some detectable signal. Thus in the case of fluorescence ELISA, when light of the appropriate wavelength is shone upon the sample, any antigen/antibody complexes will fluoresce so that the amount of antigen in the sample can be inferred through the magnitude of the fluorescence.
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression. It takes its name from its similarity to the Southern blot technique, named for biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analyzed in the northern blot.Both techniques use electrophoresis and detection with a hybridization probe. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University.
Flow cytometry
Flow cytometry is a technique for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical and/or electronic detection apparatus.
(三)預期完成之工作項目及具體成果：1.請列述執行期限內預期完成之工作項目，2學術性及應用性價值。
本計劃之目的
1. 經由比較資料庫，為Oct3/4, Sox2, c-Myc and Klf4設計具有專一性的單株抗體，及以重組蛋白為抗原的多株抗體。
2. 用western 、RT-PCR和northern blot來偵測Oct3/4, Sox2, c-Myc and Klf4在protein、RNA和DNA的表現量
3. 用FLOW來偵測癌症幹細胞的表面標記和細胞狀況
4. 透過比較Oct3/4, Sox2, c-Myc, Klf4等基因在癌症幹細胞(cancer stem cell,CSC)上表現量，來確認Oct3/4, Sox2, c-Myc, Klf4等基因是否與癌症幹細胞(cancer stem cell,CSC)的復原、增生和分化能力有關?
學術性及應用性價值
1. 透過基因的分析釐清cancer stem cell和induced pluripotent stem cells在基因表現的差異
2. 藉此建立消滅cancer stem cell的機制
3. 建立anti-cancer藥物研發的基本知識
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