本文選題：Rufy3 + FOXK1��； 參考：《南方醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:Rufy3蛋白參與細(xì)胞遷移、肌動(dòng)蛋白細(xì)胞骨架的運(yùn)動(dòng)、脂質(zhì)修飾、膜轉(zhuǎn)運(yùn)和細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)等多種細(xì)胞活動(dòng)過程的調(diào)控。有研究表明,p21活化激酶(PAK1)可以促進(jìn)Rufy3的表達(dá)且協(xié)同Rufy3促進(jìn)胃癌細(xì)胞的侵襲和遷移。另外,轉(zhuǎn)錄因子FOXK1參與胚胎發(fā)育、細(xì)胞代謝和分化、組織修復(fù)等活動(dòng)的調(diào)節(jié),且在多種腫瘤發(fā)生發(fā)展中扮演癌基因的角色。本課題旨在明確FOXK1和Rufy3在大腸癌增殖、侵襲轉(zhuǎn)移過程中發(fā)揮的作用及可能的作用機(jī)制。方法:1.免疫組織化學(xué)法檢測(cè)Rufy3和FOXK1在大腸癌組織和正常大腸黏膜組織中的表達(dá),分析Rufy3和FOXK1的表達(dá)量之間的關(guān)系、與大腸癌病人的病例特征、預(yù)后的關(guān)系;2.流式細(xì)胞周期技術(shù)、Western blot技術(shù)檢測(cè)Rufy3對(duì)大腸癌細(xì)胞周期的影響;3.構(gòu)建Rufy3穩(wěn)定表達(dá)株、EdU染色、裸鼠皮下移植瘤模型檢測(cè)Rufy3對(duì)大腸癌增殖的影響;4.Transwell侵襲實(shí)驗(yàn)、細(xì)胞劃痕、免疫熒光、羅丹明鬼筆環(huán)肽染色等實(shí)驗(yàn)檢測(cè)FOXK1和Rufy3對(duì)于大腸癌細(xì)胞上皮細(xì)胞間充質(zhì)轉(zhuǎn)化作用(EMT)、侵襲轉(zhuǎn)移功能的影響;5.運(yùn)用Smad2/3 siRNA和Smad4 siRNA、轉(zhuǎn)化生長因子-β1(TGF-β1)相關(guān)實(shí)驗(yàn)研究Rufy3作用于大腸癌細(xì)胞可能的TGF-β/Smad通路機(jī)制;6.構(gòu)建裸鼠原位肝轉(zhuǎn)移模型,免疫組化、qRT-PCR方法檢測(cè)E-cadherin、Vimentin的表達(dá)情況,研究在體內(nèi),FOXK1和Rufy3對(duì)大腸癌的作用。結(jié)果:1.Rufy3在大腸癌組織中的表達(dá)高于正常大腸粘膜組織,Rufy3高表達(dá)的大腸癌手術(shù)病人預(yù)后較低表達(dá)者差,Rufy3的表達(dá)與大腸癌的TNM分期、分化程度、AJCC分期、是否發(fā)生淋巴結(jié)轉(zhuǎn)移具有顯著的相關(guān)性;2.敲低Rufy3,處于G0/G1期的大腸癌細(xì)胞增多,G2/M細(xì)胞減少,相關(guān)周期蛋白表達(dá)發(fā)生改變;3.過表達(dá)Rufy3組的裸鼠皮下瘤較對(duì)照組生長快,Ki67表達(dá)量高;4.過表達(dá)Rufy3促進(jìn)Vimentin的表達(dá),抑制E-cadherin的表達(dá),加快劃痕愈合速度,增強(qiáng)大腸癌細(xì)胞穿透Transwell膜的能力,敲低Rufy3減弱TGF-β1對(duì)于大腸癌細(xì)胞的EMT作用;5.過表達(dá)Rufy3組的裸鼠較Vector組更多發(fā)生肝轉(zhuǎn)移,且E-cadherin表達(dá)降低;6.Rufy3和FOXK1在大腸癌細(xì)胞中存在相互作用,并在大腸癌組織中高表達(dá),表達(dá)量高者較低者平均生存時(shí)間短;7.Rufy3穩(wěn)定表達(dá)株,敲低FOXK1后,Vimentin表達(dá)下降,E-cadherin表達(dá)上升,劃痕愈合速度減慢,穿透 Transwell 膜的能力減弱;8.Rufy3 組較 Vector 組、Rufy3-FOXK1-siRNA組裸鼠形成的轉(zhuǎn)移結(jié)節(jié)多而大,Vimentin的表達(dá)量最高。結(jié)論:1.Rufy3在大腸癌組織中的表達(dá)高于正常大腸粘膜組織,Rufy3高表達(dá)負(fù)性影響大腸癌手術(shù)病人的預(yù)后;3.Rufy3過表達(dá)促進(jìn)大腸癌的增殖、侵襲轉(zhuǎn)移能力;4.Rufy3過表達(dá)在大腸癌細(xì)胞中可能通過TGF-β/Smad信號(hào)通路促進(jìn)EMT作用;5.Rufy3和FOXK1在大腸癌細(xì)胞內(nèi)存在相互作用,兩者在大腸癌組織中的表達(dá)呈正相關(guān),協(xié)同負(fù)性影響預(yù)后;6.Rufy3對(duì)大腸癌EMT、侵襲轉(zhuǎn)移能力的促進(jìn)作用能被FOXK1-siRNA 抑制。
[Abstract]:Aim to regulate the cellular activity of cell migration, actin cytoskeleton movement, lipid modification, membrane transport and cell signal transduction. Some studies have shown that p21 activated kinase PAK1) can promote the expression of Rufy3 and promote the invasion and migration of gastric cancer cells in combination with Rufy3. In addition, transcription factor FOXK1 is involved in the regulation of embryonic development, cell metabolism and differentiation, tissue repair, and plays the role of oncogene in the development of many kinds of tumors. The purpose of this study was to clarify the role of FOXK1 and Rufy3 in the proliferation, invasion and metastasis of colorectal cancer. Method 1: 1. Immunohistochemical method was used to detect the expression of Rufy3 and FOXK1 in colorectal carcinoma and normal colorectal mucosa. The relationship between the expression of Rufy3 and FOXK1 was analyzed. Flow cytometry and Western blot were used to detect the effect of Rufy3 on the cell cycle of colorectal cancer. The effect of Rufy3 on the proliferation of colorectal carcinoma in nude mice was detected by using Rufy3 stable expression strain Edu staining. 4. Transwell invasion assay, cell scratch, immunofluorescence. The effects of FOXK1 and Rufy3 on epithelial mesenchymal transformation and invasion and metastasis of colorectal cancer cells were detected by rhodamine penicyclopeptide staining. Smad2/3 siRNA and Smad4 siRNAs were used to study the mechanism of TGF- 尾 / Smad pathway induced by Rufy3 in colorectal cancer cells. An in situ liver metastasis model was established in nude mice. The expression of E-cadherinus vimentin was detected by immunohistochemical qRT-PCR. The effect of FOXK1 and Rufy3 on colorectal carcinoma was studied in vivo. Results 1. The expression of Rufy3 in colorectal carcinoma tissues was higher than that in normal colorectal mucosa tissues. The prognosis of patients with lower expression of Rufy3 was lower than that of patients with lower expression of Rufy3. The expression of Rufy3 in colorectal carcinoma was lower than that in patients with colorectal cancer. The expression of Rufy3 in colorectal carcinoma was different from that in TNM stage. There is a significant correlation between lymph node metastasis and lymph node metastasis. With low Rufy3 knockout, the number of G2 / M cells in G0/G1 phase increased and the expression of cyclin was changed. The expression of Ki67 in subcutaneous tumor of nude mice with overexpression of Rufy3 was higher than that in control group. Overexpression of Rufy3 promoted the expression of Vimentin, inhibited the expression of E-cadherin, accelerated the healing rate of scratches, enhanced the ability of colorectal cancer cells to penetrate Transwell membrane, and knocked down Rufy3 to attenuate the EMT effect of TGF- 尾 1 on colorectal cancer cells. In nude mice with overexpression of Rufy3, liver metastasis was more than that in Vector, and the expression of E-cadherin decreased. 6. There was interaction between Rufy3 and FOXK1 in colorectal cancer cells, and high expression was found in colorectal carcinoma. The average survival time of those with higher expression was shorter than that of Vector, and the stable expression of Rufy3 was higher than that of Vector. The expression of Vimentin increased, the rate of scratch healing slowed down, and the ability to penetrate the Transwell membrane decreased. The expression of Vimentin in group Rufy3 was higher than that in group Vector (Rufy3-FOXK1-siRNA), but the expression of Vimentin was the highest. Conclusion: 1. The high expression of Rufy3 in colorectal carcinoma is higher than that in normal colorectal mucosa. The high expression of Rufy3 negatively affects the prognosis of colorectal cancer patients. 3. The overexpression of Rufy3 promotes the proliferation of colorectal cancer. 4. The overexpression of Rufy3 in colorectal cancer cells may promote the interaction between EMT and FOXK1 through TGF- 尾 / Smad signaling pathway. The expression of Rufy3 and FOXK1 in colorectal cancer cells is positively correlated. 6. Rufy3 can promote the invasion and metastasis of colorectal carcinoma and the ability of invasion and metastasis can be inhibited by FOXK1-siRNA.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.34

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