本文關(guān)鍵詞：TCTP在腫瘤耐藥細胞中的作用及機制研究，由筆耕文化傳播整理發(fā)布。

        化療是臨床腫瘤治療的必要手段之一，而長時間使用化學藥物后，腫瘤細胞的復發(fā)、轉(zhuǎn)移始終是制約最終療效的瓶頸問題。目前研究認為，腫瘤中少量存在的腫瘤干細胞是化療或放療后腫瘤復發(fā)的根源，腫瘤干細胞不僅具有自我更新和增殖能力，同時對化學藥物和射線等具有高度抗性。然而，關(guān)于腫瘤干細胞的來源始終存在較大爭議。有學者認為，腫瘤干細胞可能來源于正常干細胞的惡性轉(zhuǎn)化；但近期研究表明，在腫瘤化療過程中，DNA損傷可能一方面消除腫瘤細胞，同時也可能加劇基因組的不穩(wěn)定性，進而誘導普通腫瘤細胞演變成干細胞樣腫瘤細胞（stem-likecancer cells, SLCCs），其相關(guān)分子機制尚不明確�，F(xiàn)行的臨床腫瘤治療策略中，放療往往是在階段性化療后開展，那么，化療藥物誘導SLCCs的產(chǎn)生和累積可能對放療效果產(chǎn)生巨大影響。因此，闡明化療藥物誘導SLCCs產(chǎn)生的特點，進而圍繞DNA損傷修復和基因組穩(wěn)定性，尋找新的關(guān)鍵靶分子，對于深入探討化療過程中腫瘤細胞向SLCCs演變的分子機制，優(yōu)化臨床腫瘤治療策略，將具有重要參考意義。翻譯調(diào)控腫瘤蛋白（Translationally controlled tumor protein，TCTP）是一個在多種組織中，如肝癌、肺癌、胰腺癌、胃癌、黑色素瘤、宮頸癌、乳腺癌、骨肉瘤、神經(jīng)膠質(zhì)瘤、白血病等，廣泛表達的高度保守小分子蛋白質(zhì)。它不僅參與調(diào)控細胞周期、增殖和抗凋亡等多種重要生理過程，還與腫瘤的發(fā)生、發(fā)展密切相關(guān)。我們的前期研究證實，TCTP是維持細胞基因組穩(wěn)定的重要分子，在DNA損傷修復中發(fā)揮著關(guān)鍵作用。另有研究表明，TCTP參與了腫瘤細胞的惡性轉(zhuǎn)化調(diào)控，敲除TCTP可以有效促進乳腺癌細胞發(fā)生惡性逆轉(zhuǎn)，更可導致動物乳腺癌中“類腫瘤干細胞”的數(shù)量顯著減少，提示TCTP可能在SLCCs的生成及其生物學特性維持中具有重要作用。值得注意的是，既往研究還提示，TCTP的表達改變可能與腫瘤耐藥的產(chǎn)生相關(guān)，但其機制和意義尚未見報道�；赥CTP在維系基因組穩(wěn)定和促進細胞惡性轉(zhuǎn)化中的重要作用，本研究首先利用人膠質(zhì)瘤及乳腺癌臨床組織芯片和臨床腫瘤標本，明確TCTP蛋白表達在不同臨床分級腫瘤中的分布特點和意義；同時，結(jié)合現(xiàn)行的臨床腫瘤治療策略，選用不同組織來源（膠質(zhì)瘤和乳腺癌）、具有不同轉(zhuǎn)移潛能的五種腫瘤細胞系為對象，建立了九種耐藥細胞模型；在此基礎(chǔ)上，研究化療藥物誘導SCLLs產(chǎn)生的特點，并通過體外細胞試驗和在體裸鼠成瘤實驗，觀察了TCTP表達改變與耐藥細胞的干細胞樣生物學特性的相關(guān)性；利用siRNA轉(zhuǎn)染等技術(shù)，給予耐藥細胞復合輻射暴露，進一步探討了TCTP在維系耐藥細胞輻射抵抗中的作用機制。方法：1、利用膠質(zhì)瘤及乳腺癌臨床組織芯片，通過免疫組織化學方法，顯微鏡下，從每張免疫組織化學染色陽性的結(jié)果中隨機觀察5個不同視野。觀察在細胞漿和細胞核的陽性染色時才記分。評分標準如下：陽性細胞數(shù)為<10%，0分；10%-30%，1分；30%-50%，2分；50-70%，3分；>70%，4分。觀察TCTP在不同等級的臨床膠質(zhì)瘤及乳腺癌標本中的表達變化；同時，采集臨床膠質(zhì)瘤組織，利用Western-blot方法，觀察TCTP在不同等級的臨床膠質(zhì)瘤組織中的蛋白表達。2、選用人乳腺癌細胞MCF-7、MDA-MB-231和人膠質(zhì)瘤細胞系SHG44、U251和U87五種腫瘤細胞為對象，通過MTT實驗選擇合適的藥物劑量，對乳腺癌細胞進行三種方式（5-氟尿嘧啶：5-FU、鹽酸阿霉素:ADM及5-氟尿嘧啶+鹽酸阿霉素:5-FU+ADM）的長期化療藥物誘導篩選（MDA-MB-231連續(xù)給藥3次/代，MCF-7連續(xù)給藥2次/代，持續(xù)8個月），對膠質(zhì)瘤細胞給予TMZ誘導篩選（HG44、U251和U87連續(xù)給藥2次/代，持續(xù)6個月），建立腫瘤細胞耐藥模型。通過免疫印跡法實驗，觀察多藥耐藥蛋白ABCG2的表達變化。3、利用細胞劃痕、Transwell侵襲以及克隆形成等實驗，觀察篩選出的耐藥細胞模型中，其形態(tài)學、細胞增殖能力、遷移和侵襲能力的變化規(guī)律及特點。4、利用流式細胞技術(shù)，觀察乳腺癌耐藥細胞中的CD44+/CD24-干細胞亞群比例變化；采用免疫印跡法，檢測耐藥細胞的干細胞標記物Oct4和TCTP的蛋白表達變化。5、對裸鼠進行腫瘤細胞皮下注射實驗，在體觀察篩選出的耐藥細胞的成瘤性；通過免疫組織化學方法，觀察TCTP在不同細胞成瘤組織中的表達變化。6、利用TCTPsiRNA轉(zhuǎn)染技術(shù)，觀察降低TCTP表達后，乳腺癌耐藥細胞中OCT4的表達改變。7、耐藥細胞株接收0Gy，1Gy，2Gy，4Gy，8Gy X射線輻照后，通過克隆形成實驗評價其輻射抗性。8、通過siRNA轉(zhuǎn)染聯(lián)合輻射暴露，利用CCK-8試劑盒和臺盼藍染色實驗，觀察降低TCTP表達對乳腺癌耐藥細胞增殖能力和存活狀況的影響。9、通過siRNA轉(zhuǎn)染聯(lián)合輻射暴露后，利用凋亡檢測試劑盒，觀察降低TCTP表達對乳腺癌耐藥細胞凋亡的影響；應用western-blot方法，觀察TCTP表達改變對輻照后自噬標志物LC3B表達的影響。結(jié)果：1、臨床組織芯片檢測結(jié)果顯示，在不同臨床分級的乳腺癌及膠質(zhì)瘤組織中，TCTP的表達隨著腫瘤惡性程度的增高而升高；Western-blot檢測結(jié)果顯示，惡性程度越高的臨床膠質(zhì)瘤組織中TCTP的蛋白表達量越高。2、腫瘤耐藥模型的建立：乳腺癌細胞系給予5-FU1μg/ml，ADM0.08-0.1μg/ml連續(xù)給藥8個月，膠質(zhì)瘤細胞系給予TMZ500μM-1000μM連續(xù)給藥6個月。同時，以ABCG2表達變化為主要耐藥標志物，研究顯示，同對照組相比，篩選出的五種化療抵抗細胞系中，ABCG2表達均顯著增高，提示本研究中的乳腺癌和膠質(zhì)瘤耐藥細胞模型建立成功。3、耐藥腫瘤細胞的生物學特性研究：①與對照組相比，六種乳腺癌耐藥細胞在形態(tài)上呈現(xiàn)出間質(zhì)細胞樣外觀，而三種膠質(zhì)瘤耐藥細胞形態(tài)上無明顯改變；②與對照組相比，耐藥細胞的劃痕傷口愈合能力均顯著增加，同時，Transwell小室穿膜細胞數(shù)量增多，提示耐藥細胞的遷移和侵襲能力明顯提高；③克隆形成實驗結(jié)果表明，與對照組相比，耐藥細胞的增殖能力顯著增強。4、干細胞樣腫瘤細胞（SLCCs）篩選分析表明，五種耐藥細胞株中的干細胞標記物OCT4表達均明顯升高，同時伴隨TCTP的表達升高；流式細胞術(shù)檢測顯示，與對照組相比，乳腺癌耐藥細胞群中的CD44+/CD24-細胞亞群比例顯著提升，提示干細胞樣乳腺癌細胞比例增加。5、裸鼠在體植瘤實驗結(jié)果表明，同對照組腫瘤細胞相比，耐藥細胞的皮下種植成瘤率明顯增高，并且腫瘤生長速度快；免疫組化染色發(fā)現(xiàn)，在耐藥腫瘤細胞的成瘤組織中，TCTP陽性顆粒的表達量明顯高于對照組。上述結(jié)果表明，TCTP的異常高表達與耐藥細胞的高增殖能力密切相關(guān)。6、利用siRNA質(zhì)粒轉(zhuǎn)染技術(shù)降低TCTP表達后可見，乳腺癌抗藥細胞的Oct4表達隨TCTP降低而降低，兩者變化呈正相關(guān)性。綜合4-6實驗結(jié)果分析表明，TCTP可能通過調(diào)控Oct4，在維持耐藥細胞的干細胞樣生物學特性中發(fā)揮關(guān)鍵作用。7、耐藥細胞的輻射抗性研究表明，不同劑量X射線照射后，與對照組相比，耐藥細胞的克隆形成率顯著增加，提示耐藥細胞輻射抗性明顯提高。8、乳腺癌耐藥細胞在siRNA轉(zhuǎn)染聯(lián)合輻射暴露后的實驗結(jié)果顯示，降低TCTP表達，乳腺癌耐藥細胞的增殖能力和存活能力均顯著降低。9、乳腺癌耐藥細胞在siRNA轉(zhuǎn)染聯(lián)合輻射暴露后的實驗結(jié)果顯示，降低TCTP表達，受照細胞的凋亡顯著增加，，自噬小體形成增多，同時，自噬標志物LC3B的表達明顯升高。結(jié)論：1、TCTP在膠質(zhì)瘤和乳腺癌組織的蛋白表達主要位于細胞質(zhì)和細胞核，并伴隨著腫瘤惡性程度的增加而增加，提示TCTP可能成為未來判斷腫瘤臨床預后或分級的新的標志物。2、腫瘤細胞持續(xù)使用化療藥物后，耐藥腫瘤細胞群中干細胞樣細胞亞群的比率顯著增加，篩選出的耐藥腫瘤細胞出現(xiàn)明顯的干細胞樣腫瘤細胞（SLCCs）生物學特性，包括OCT4表達增加、細胞增殖能力、遷移能力和克隆形成能力均顯著增加，TCTP主要通過維持上述SLCCs生物學特性，在耐藥細胞中發(fā)揮了重要作用。3、耐藥腫瘤細胞同時具有顯著的放療抗性，TCTP可能通過抗凋亡和抗自噬兩種方式參與化療耐藥細胞輻射抗性的調(diào)控。

    Chemotherapy is one of the necessary methods of clinical cancer treatment. Althoughmost malignancies initially respond to chemotherapeutic treatments, after an unpredictableperiod, developed chemoresistance and recurrence of tumor cells could always lead to thefailure of treatment. Multiple mechanisms which cancer cells may use to developresistance to the cancer treatment have been proposed. Notably, Cancer Stem Cells (CSCs),also named tumor-initiating cells and stem-like cancer cells (SLCCs) have been postulated recently as responsible for recurrent tumours after chemotherapy. CSCs not only have theability of self-renewal and proliferation, but also have developed highly resistant toionizing radiation and chemical drugs. Current studies showed that, during the process ofchemo-or radio-therapy, while DNA damage induced by ionizing radiation or chemicalsmay eliminate tumor cells, it may aggravate the instability of genome and promote thetumor cells to stem-like cancer cells（SLCCs）. The molecular mechanism is still unclear.Translationally controlled tumor protein（TCTP）is a small molecular, which is highlyconservative in evolution and widely expressed in many tissues. It has been shown thatTCTP is involved in regulating a variety of important physiological processes, includingthe cell cycle, proliferation, anti-apoptosis and development of cancer. Our previous studyconfirmed that TCTP is one of the key molecules in the maintenance of genome stabilityand plays a critical role in DNA damage repair. Other studies have also shown that TCTPis involved in the regulation of tumor cell malignant transformation; knocking-down ofTCTP can effectively promote the malignant reversion of breast cancer cells and thenreduce the amount of the SLCCs. Interestingly, the change of TCTP expression in tumorcells has been suggested to associate with the chemoresistance, but its mechanisms hasnot yet been reported. The above studies implicates that TCTP may be involved in theprocess of SLCCs generation during chemotherapy.To learn the distribution characteristics and significance of TCTP in different clinicalclassification of tumor, we initiated our study by detecting the tissue and tissue chips ofglioma and breast cancer. Using5tumor cell lines, with different potential metastasis fromglioma and breast cancer, we set up9kinds of chemoresistant models through continualselection in6generation under different drug pressure. Then, we observed thecharacteristics of the generation of SCLLs which may be induced durng chemotherapy andfurther studied the correlation of the TCTP expression change and SCLLs biologicalcharacteristics.Results1. Clinical tissue chips detection shows that, the expression of TCTP increased withtumor malignant degree in different clinical classification of breast cancer and glioma tissues; Also, Western blot confirms that the amount of protein expression of TCTPincreased with tumor malignant degree.2. Through MTT analysis, we optimized the selection concentration of drugs,5-FU：1μg/ml，ADM：0.08-0.1μg/ml and TMZ:500μM-1000μM, respectively. As ABCG2expression changes the main resistance markers, compared with the control group,After continual selection through6generation, the expression of ABCG2, one of themarkers of chemoresistance, increased significantly in all9selected cell lines,which manifests that the chemoresistant cell models of breast cancer and glioma areestablished successfully.3. The study of biological characteristics of chemoresistant tumor cell lines shows that,①Compared with the control group, while interstitial cells emerge in two breastcancer resistant cells in morphology appearance, no obvious changes in threeTMZ-resistant glioma cells;②Compared with the control group, scratch woundhealing ability of five kinds of drug-resistant cells were significantly increased,meanwhile, Transwell Chambers in membrane the populations of cells through theTranswell Chambers membrane increased, which implicated that the migration andinvasion abilities of drug-resistant cell lines increased significantly;③Cloneformation assay shows that the proliferation ability of drug-resistant cells enhanced inthe culture medium containing drugs and3%FBS.4.Western blot analysis of SLCCs showed that, the expression of the stem cell markersOCT4in five chemoresistant cell lines were significantly increased, along with theup-regulation of TCTP; flow cytometry results show that,compared with the controlgroup, the percentage of CD44+/CD24-cells significantly increased inchemoresistant breast cancer cells. In vivo experiment in nude mice demonstrated that,compared with the control5. group, the growth rate of chemoresistant tumor cells which were subcutaneouslyinjected is much faster; immunohistochemical staining of tumor tissues show that thepositive staining particles of TCTP in groups of chemoresistant tumor cells weresignificantly higher than respective control groups. The above results suggested that TCTP expression is closely related to the proliferation ability of chemoresistant tumorcells.6. In chemoresistant breast cancer cells, down-regulation of TCTP by siRNA plasmidtransfection technology could also induce the reduction of Oct4expression. Thechange of TCTP and Oct4shows a positive correlation. Collectively, the results ofexperiments of four to six implicated that TCTP may play an important role inmaintaining the “stem” biological characteristics of SLCCs.7. We detected the radio-resistance of chemoresistant tumor cells by exposing the cellsto the different dose of X-rays. The clone formation analysis showed that thechemoresistant cells also have the stronger radio-resistant potentials. Interfering theexpression of TCTP by siRNA transfection would significantly reduced theproliferation and survival abilities of chemoresistant tumor cells when exposed to4GyX-rays.8. To understand the mechanisms of TCTP involved in maintaining radio-resistance ofchemoresistant tumor cells, we detect the occurrence of apoptosis and LC3Bexpression, marker of autophagy, in cells exposed to X-rays with down-regulation ofTCTP. The expression of caspase-3and caspase-8are increase in somechemoresistant. Our results showed that TCTP participates in radio-resistance ofchemoresistant tumor cells through both anti-apoptosis and anti-autophagy functions.Conclusion1. The expression of TCTP in glioma and breast cancer tissues is mainly located incytoplasm and nucleus. The expression of TCTP increase significantly with the tumormalignant degree in both glioma and breast cancer tissue, which suggested that TCTPcould probably become a new promising marker in clinical prognosis or classificationof tumor.2. The ratio of SLCCs increased significantly when tumor cells experienced thecontinual treatment of chemotherapeutic drugs. The chemoresistant tumor cellsshowed SLCCs biological characteristics, including much higher abilities of cellproliferation, migration and colony-forming, and up-regulation of OCT4. Strikingly, TCTP plays an important role in chemoresistant tumor cells by maintaining theSLCCs biological characteristics, which may relate with the regulation of OCT4.3. Chemoresistant tumor cells also showed significant radio-resistance, TCTP may beinvolved in which through both anti-apoptosis and anti-autophagy functions.

TCTP在腫瘤耐藥細胞中的作用及機制研究

縮略語表5-7中文摘要7-12英文摘要12-16前言17-18文獻回顧18-29第一部分 TCTP 在人腦膠質(zhì)瘤和乳腺癌組織中的表達29-36    實驗一 TCTP 在人腦膠質(zhì)瘤和乳腺癌組織中的表達30-36        1 材料30-31        2 方法31-32        3 結(jié)果32-35        4 討論35-36第二部分 耐藥腫瘤細胞的干細胞樣生物學特性研究36-56    實驗二 耐藥腫瘤細胞模型的建立37-44        1 材料37-39        2 方法39-40        3 結(jié)果40-43        4 討論43-44    實驗三 耐藥腫瘤細胞的干細胞樣生物學特性研究44-56        1 材料44-45        2 方法45-47        3 結(jié)果47-55        4 討論55-56第三部分 TCTP 在維持耐藥腫瘤細胞干細胞樣特征中的作用研究56-71    實驗四 TCTP 在耐藥腫瘤細胞中的作用研究57-64        1 材料57-58        2 方法58-60        3 結(jié)果60-62        4 討論62-64    實驗五 TCTP 對腫瘤耐藥細胞成瘤性的在體實驗研究64-71        1 材料64-65        2 方法65        3 結(jié)果65-69        4 討論69-71第四部分 TCTP 在乳腺癌耐藥細胞輻射抵抗中的作用機制研究71-89    實驗六 TCTP 在乳腺癌耐藥細胞輻射抵抗中的作用研究72-81        1 材料72-73        2 方法73-74        3 結(jié)果74-79        4 討論79-81    實驗七 TCTP 在乳腺癌耐藥細胞輻射抵抗中的機制研究81-89        1 材料81-82        2 方法82-84        3 結(jié)果84-87        4 討論87-89小結(jié)89-91參考文獻91-100個人簡歷和研究成果100-102致謝102

  本文關(guān)鍵詞：TCTP在腫瘤耐藥細胞中的作用及機制研究，由筆耕文化傳播整理發(fā)布。