本文關(guān)鍵詞： 人舌鱗狀細(xì)胞癌 STAT3miR-21 順鉑 化療耐藥　出處：《天津醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：頭頸部惡性腫瘤是世界范圍內(nèi)常見的6大常見的惡性腫瘤之一,其中舌鱗狀細(xì)胞癌是頭頸部惡性腫瘤最常見的病理類型。晚期舌鱗狀細(xì)胞癌多數(shù)呈浸潤(rùn)性生長(zhǎng)并伴有頸部淋巴結(jié)轉(zhuǎn)移,手術(shù)很難完全切除已經(jīng)浸潤(rùn)周圍組織和發(fā)生遠(yuǎn)處轉(zhuǎn)移的腫瘤。眾所周知,順鉑(DDP)是被廣泛應(yīng)用于治療人舌鱗狀細(xì)胞癌的化療藥物,但DDP的不良反應(yīng)以及腫瘤對(duì)其耐藥性的產(chǎn)生嚴(yán)重影響了其治療效果,因此逆轉(zhuǎn)化療耐藥、提高舌鱗癌對(duì)其敏感性越來越受到關(guān)注。 STAT3在肝癌、乳腺癌、肺癌、結(jié)腸癌、頭頸部鱗狀細(xì)胞癌等上皮系統(tǒng)來源的惡性腫瘤中均有過表達(dá),并參與多種細(xì)胞增殖、凋亡、侵襲及轉(zhuǎn)移等相關(guān)基因轉(zhuǎn)錄調(diào)控。在多發(fā)性骨髓瘤及腦膠質(zhì)瘤中均發(fā)現(xiàn)STAT3能夠直接調(diào)控miR-21的轉(zhuǎn)錄。本課題選用TCA8113、TCA8113/DDP舌鱗癌細(xì)胞系,采用WP1066下調(diào)STAT3表達(dá),并選用臨床常用舌鱗癌化療藥物DDP,就WP1066聯(lián)合DDP影響人舌鱗癌細(xì)胞體內(nèi)外生長(zhǎng)能力的作用效果和機(jī)制進(jìn)行了以下體內(nèi)外實(shí)驗(yàn)研究。課題研究共分為三部分： 第一部分：首先運(yùn)用MTT法測(cè)定兩舌癌細(xì)胞WP1066、DDP的半數(shù)抑制濃度(IC50)；Western blot、實(shí)時(shí)定量PCR法檢測(cè)耐藥及敏感舌鱗癌細(xì)胞株STAT3、 miR-21表達(dá)差異；Western blot與免疫熒光法檢測(cè)檢測(cè)WP1066及DDP處理后舌鱗癌細(xì)胞STAT3/p-STAT3表達(dá)情況；運(yùn)用實(shí)時(shí)定量PCR法、原位雜交法檢測(cè)兩舌癌細(xì)胞WP1066、DDP處理后miR-21表達(dá)情況。運(yùn)用MTT法、平板克隆實(shí)驗(yàn)、Matrigel基質(zhì)生長(zhǎng)實(shí)驗(yàn)、Transwell體外侵襲實(shí)驗(yàn)、細(xì)胞劃痕實(shí)驗(yàn)、流式細(xì)胞術(shù)等研究WP1066、DDP抑制舌癌細(xì)胞體外牛長(zhǎng)的效果。 第二部分：rtPCR法、Western blot法檢測(cè)miR-21靶基因及蛋白的變化；CHIP實(shí)驗(yàn)、熒光素酶報(bào)告基因?qū)嶒?yàn)驗(yàn)證STAT3與miR-21調(diào)控關(guān)系及其對(duì)舌鱗癌耐藥的影響。 第三部分：建立裸鼠荷TCA8113/DDP舌鱗癌皮下動(dòng)物模型,瘤內(nèi)注射WP1066及DDP,動(dòng)態(tài)觀察腫瘤生長(zhǎng)情況,檢測(cè)WP1066抑制STAT3/pSTAT3表達(dá)后,miR-21表達(dá)情況,miR-21靶點(diǎn)蛋白及舌鱗癌生長(zhǎng)相關(guān)蛋白的變化情況,并且使用TUNEL法檢測(cè)細(xì)胞凋亡,對(duì)體外實(shí)驗(yàn)的結(jié)果進(jìn)一步驗(yàn)證。 結(jié)果： TCA8113、TCA8113/DDP細(xì)胞WP1066的IC50分別為3.1和3.5μmol/L。 DDP的ICso分別為0.9μM/L和10.9t.tM/L；順鉑耐藥細(xì)胞株STAT3、miR-21表達(dá)水平顯著高于順鉑敏感細(xì)胞株。WP1066能有效抑制pSTAT3表達(dá)；WP1066組、DDP組及WP1066+DDP組均出現(xiàn)腫瘤細(xì)胞增殖、克隆形成能力明顯下降,形成球形克隆、侵襲能力被抑制,遷移能力下降,凋亡率增加；聯(lián)合治療效果優(yōu)于單一處理組。WP1066處理后Bcl-2、mTOR、Ki-67、MMP2/9蛋白表達(dá)水平明顯下降,Caspase3表達(dá)水平上調(diào),DDP不能改變其表達(dá)。WP1066組與WP1066+DDP組miR-21表達(dá)水平低于對(duì)照組及DDP組。各組miR-21靶基因表達(dá)均無明顯變化,WP1066可抑制miR-21靶點(diǎn)蛋白表達(dá)；CHIP實(shí)驗(yàn)、熒光素酶報(bào)告基因?qū)嶒?yàn)證明miR-21是STAT3的調(diào)控靶點(diǎn)。腫瘤生長(zhǎng)曲線顯示W(wǎng)P1066及DDP治療組腫瘤生長(zhǎng)速度及體積相似,且均小于空白對(duì)照及DMSO組,WP1066+DDP組效果優(yōu)于單一治療；經(jīng)WP1066治療后腫瘤組織中STAT3/p-STAT3及miR-21表達(dá)水平均下降,DDP不能改變其表達(dá)；WP1066、 DDP治療組細(xì)胞凋亡數(shù)顯著均高于對(duì)照組,聯(lián)合治療后凋亡數(shù)明顯高于單一治療；Bcl-2、mTOR、Ki-67、MMP2/9表達(dá)下降,Caspase3、PTEN、PDCD4及TIMP3升高,DDP不能改變其表達(dá)。體內(nèi)外實(shí)驗(yàn)結(jié)果一致。 結(jié)論： 1. STAT3、miR-21過表達(dá)與人舌鱗癌順鉑耐藥相關(guān)。 2.WP1066可通過抑制STAT3磷酸化,轉(zhuǎn)錄抑制miR-21表達(dá),進(jìn)而減弱對(duì)miR-21靶點(diǎn)PTEN、PDCD4、TIMP3的轉(zhuǎn)錄后調(diào)控,分別在增殖、凋亡、侵襲及遷移方面抑制人舌鱗癌細(xì)胞的生長(zhǎng)。 3.WP1066與DDP聯(lián)合治療人舌鱗癌細(xì)胞及荷瘤裸鼠模型效果較單一處理(單用WP1066或DDP)效果明顯增強(qiáng)；STAT3作為人舌鱗癌中的新治療靶點(diǎn),其小分子抑制劑WP1066為逆轉(zhuǎn)化療耐藥及其治療提供了新的方向機(jī)可能性。
[Abstract]:Head and neck cancer is one of the most common in the world of the 6 common malignant tumors, including tongue squamous cell carcinoma is the most common pathological type of malignant tumor in head and neck. Most advanced tongue squamous cell carcinoma and infiltrative growth associated with cervical lymph node metastasis, surgical resection has been difficult to completely infiltrate the surrounding tissue and distant metastasis the tumor. As everyone knows, cisplatin (DDP) chemotherapy has been widely used in the treatment of squamous cell carcinoma of tongue, but the adverse reaction of DDP and tumor on the drug resistance seriously affect the treatment effect, thus reversing resistance to chemotherapy, improve the sensitivity of squamous cell carcinoma of the tongue has attracted more and more attention.
STAT3 in liver cancer, breast cancer, lung cancer, colon cancer, over expressed in head and neck squamous cell carcinoma and other malignant tumor derived from epithelial system, and participate in a variety of cell proliferation, apoptosis, invasion and metastasis related gene transcription. In multiple myeloma and glioma were found in STAT3 can directly regulate miR-21 the transcription. The project uses TCA8113 and TCA8113/DDP in tongue squamous cell carcinoma cell line, the WP1066 reduced the expression of STAT3, and the clinical commonly used chemotherapeutic drugs DDP tongue squamous cell carcinoma, the effect and mechanism of WP1066 combined with DDP of human tongue squamous cell carcinoma cells in vitro and in vivo growth ability were studied. The following in vivo research is divided into three parts:
The first part: firstly, the determination of two tongue cancer cell WP1066 by MTT method, DDP half inhibitory concentration (IC50); Western blot, real time quantitative PCR for the detection of resistant and sensitive tongue squamous cell carcinoma cell line STAT3, the difference of miR-21 expression in squamous cell carcinoma of the tongue; STAT3/p-STAT3 to detect the expression of WP1066 and DDP after treatment with Western blot and immunofluorescence detection method; application real time quantitative PCR method and in situ hybridization were used to detect two tongue cancer cell line WP1066, the expression of miR-21 after DDP treatment. Using MTT method, experimental plate cloning, Matrigel matrix growth experiment, Transwell in vitro invasion assay, cell scratch test of WP1066, flow cytometry, DDP inhibitory effect of tongue cancer cells in vitro bovine long.
The second part: rtPCR method, Western blot method to detect miR-21 target gene and protein changes; CHIP experiment, luciferase reporter gene experiment to verify the relationship between STAT3 and miR-21 and its impact on drug resistance of tongue squamous cell carcinoma.
The third part: the establishment of nude mice bearing subcutaneous TCA8113/DDP tongue squamous cell carcinoma animal model, intratumoral injection of WP1066 and DDP, the dynamic observation of tumor growth. The detection of WP1066 after inhibition of STAT3/pSTAT3 expression, miR-21 expression, changes in miR-21 target protein and growth associated protein in squamous cell carcinoma of tongue, and use the TUNEL method to detect apoptosis in vitro. The results of further validation.
Result錛，

本文編號(hào)：1486838