本文關(guān)鍵詞： ENO1 基因沉默 藥物敏感性 成瘤性 侵襲轉(zhuǎn)移　出處：《蘭州大學》2014年碩士論文　論文類型：學位論文

【摘要】：腫瘤細胞增殖所需能量主要是通過有氧糖酵解途徑代謝產(chǎn)生,糖酵解是細胞惡變過程中最為基礎的代謝改變之一,是腫瘤細胞代謝的一個重要特征。Q-烯醇化酶(ENO1)是一種重要的糖酵解酶,在有氧糖酵解過程中起到關(guān)鍵作用。此外,ENO1的功能具有多樣性,它既可催化糖酵解過程中2-磷酸甘油酸(PGA)向磷酸烯醇式丙酮酸(PEP)的轉(zhuǎn)化,又可以編碼c-myc啟動子結(jié)合蛋白(MBP-1),下調(diào)原癌基因c-myc的活性,發(fā)揮抗腫瘤作用。本研究的目的是利用慢病毒介導的RNA干擾技術(shù),構(gòu)建ENO1基因穩(wěn)定沉默的Hela和SiHa細胞系,探討ENO1對人宮頸癌細胞生長,藥物敏感性及侵襲轉(zhuǎn)移能力的影響。 本研究采用慢病毒介導的RNA干擾技術(shù),建立ENO1基因穩(wěn)定沉默的Hela和SiHa細胞系,并通過western-blot方法驗證ENO1蛋白的表達水平。實驗分為ENO1沉默組(pLKO.1shENO1)、陰性對照組(scr)和正常細胞組。采用MTT的方法來觀察宮頸癌細胞Hela和SiHa在沉默ENO1基因后對化療藥物敏感性的變化。細胞克隆形成實驗觀察宮頸癌細胞成瘤能力的變化,同時采用細胞劃痕實驗、Transwell小室、免疫細胞化學技術(shù)檢測細胞侵襲轉(zhuǎn)移能力的變化及可能的機制。 本研究結(jié)果表明,成功建立ENO1基因沉默的宮頸癌細胞系,通過MTT的方法檢測三組細胞48h對不同濃度順鉑和紫杉醇的敏感性,結(jié)果顯示不同濃度藥物對ENO1基因沉默組細胞的抑制率要高于對照組,結(jié)果具有統(tǒng)計學意義�？寺⌒纬蓪嶒灲Y(jié)果表明,ENO1基因沉默后細胞的肉眼可見克隆數(shù)目顯著低于正常細胞和空載體組細胞,Hela pLKO.1shENO1組及SiHa pLKO.1shENO1組克隆形成率分別是5.17%和6.33%,明顯低于對照組細胞,P0.05,差異具有統(tǒng)計學意義。細胞劃痕實驗檢測細胞遷移能力的變化,發(fā)現(xiàn)ENO1沉默組細胞遷移速率降低。采用無基質(zhì)膠小室進行細胞遷移能力的檢測,ENO1沉默組細胞遷移至下室的細胞明顯減少,光鏡下每組細胞隨機選取5個視野,取平均數(shù)后進行統(tǒng)計分析,差異具有統(tǒng)計學意義。上室鋪基質(zhì)膠后進行細胞侵襲能力的檢測,同樣發(fā)現(xiàn),ENO1沉默組細胞穿過基質(zhì)膠的數(shù)量明顯減少,差異具有統(tǒng)計學意義。采用免疫細胞化學技術(shù)檢測SiHa細胞ENO1基因沉默后,侵襲轉(zhuǎn)移相關(guān)因子VEGF, EGFR, MMP9,COX2,Notch2的表達情況,DAB染色后,ENO1沉默組細胞表達均較空載體組明顯下降。 本研究結(jié)論：沉默ENO1基因能夠增強宮頸癌細胞Hela和SiHa對化療藥物紫杉醇和順鉑的藥物敏感性,減低成瘤能力,抑制細胞的侵襲轉(zhuǎn)移�？傊�,ENO1與宮頸癌細胞生長及侵襲轉(zhuǎn)移密切相關(guān)。
[Abstract]:The energy needed for the proliferation of tumor cells is mainly produced by aerobic glycolysis. Glycolysis is one of the most basic metabolic changes in the process of cell malignancy. Q- enolase ENO1) is an important glycolytic enzyme that plays a key role in aerobic glycolysis. It can not only catalyze the transformation of 2-phosphoglyceric acid PGA to PEP (phosphoenolpyruvate) during glycolysis, but also encode c-myc promoter binding protein MBP-1, which can down-regulate the activity of proto-oncogene c-myc. The aim of this study was to construct Hela and SiHa cell lines with stable silencing of ENO1 gene by lentivirus mediated RNA interference, and to investigate the effects of ENO1 on the growth, drug sensitivity and invasion and metastasis of human cervical cancer cells. In this study, ENO1 gene stable silencing Hela and SiHa cell lines were established by lentivirus-mediated RNA interference technique. The expression level of ENO1 protein was verified by western-blot. The experiment was divided into two groups: ENO1 silencing group (pLK0.1 shENO1) and normal cell group. MTT method was used to observe the sensitivity of Hela and SiHa to chemotherapeutic drugs after silencing ENO1 gene. To observe the change of tumorigenic ability of cervical cancer cells by cell clone formation assay. At the same time, cell scratch test was used to detect the change of cell invasion and metastasis ability and its possible mechanism. The results showed that the cervical cancer cell line silenced by ENO1 gene was successfully established. The sensitivity of three groups of cells to different concentrations of cisplatin and paclitaxel was detected by MTT method. The results showed that the inhibition rate of different concentrations of drugs on ENO1 gene silencing group was higher than that of control group. The results showed that the number of clones in Hela pLKO.1shENO1 group and SiHa pLKO.1shENO1 group was significantly lower than that in normal cells and empty vector group. The clone formation rates of Hela pLKO.1shENO1 and SiHa pLKO.1shENO1 were 5.17% and 6.33, respectively. It was significantly lower than that of control group (P 0.05), the difference was statistically significant. Cell scratch assay was used to detect the change of cell migration ability. It was found that the cell migration rate of the ENO1 silencing group was decreased. The cell migration ability of the ENO1 silencing group was measured by using the non-matrix colloidal chamber. The number of cells migrating to the lower chamber in the ENO1 silencing group was significantly reduced, and 5 visual fields were randomly selected for each group under light microscope. After taking the average, the difference was statistically significant. The number of cells passing through the matrix glue in ENO1 silencing group was significantly decreased. The expression of VEGF, EGFR, MMP9 COX2Notch2 in SiHa cells after ENO1 gene silencing was detected by immunocytochemistry. Conclusion: silencing ENO1 gene can enhance the sensitivity of Hela and SiHa to the chemotherapeutic drugs paclitaxel and cisplatin, and reduce the tumorigenesis. ENO1 is closely related to the growth, invasion and metastasis of cervical cancer cells.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R737.33

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