發(fā)布時間：2018-08-28 17:14

【摘要】：食管癌是我國最常見的消化道腫瘤之一。雖然隨著外科、麻醉、圍手術(shù)期處理以及重癥監(jiān)護條件和技術(shù)取得較大的提高,但是單純手術(shù)治療效果仍不理想。其原因在于食管癌發(fā)生和發(fā)展的分子機制目前仍不清楚,導(dǎo)致食管癌療效欠佳。腫瘤細胞代謝異常是腫瘤細胞顯著區(qū)別于正常細胞的顯著特征之一。從腫瘤代謝的角度尋找食管癌的治療靶點很可能為食管癌的治療帶來新的希望。甲羥戊酸(MVA,Mevalonate)途徑除了終產(chǎn)物膽固醇外,還能產(chǎn)生許多中間代謝產(chǎn)物,如類異戊二烯,泛醌等。這些物質(zhì)在細胞重要的生理過程中發(fā)揮作用。甲羥戊酸代謝途徑在食管鱗癌發(fā)生中的功能目前仍不清楚。在本研究中,我們克隆了甲羥戊酸代謝限速酶3-羥基-3-甲基戊二酰輔酶A還原酶(3-Hydroxy-3-methylglutaryl coenzyme A reductase,HMGCR),并對其進行了功能和所涉機制的初步研究。在本研究中,我們收集食管鱗癌組織標(biāo)本,培養(yǎng)食管鱗癌細胞系,利用熒光定量PCR檢測食管鱗癌組織及其配對的正常組織中HMGCR的mRNA水平,利用蛋白印跡檢測食管鱗癌組織和食管鱗癌細胞中HMGCR的蛋白水平。在細胞水平上,我們通過脂質(zhì)體轉(zhuǎn)染食管鱗癌細胞,建立過表達HMGCR的食管鱗癌穩(wěn)定細胞株;設(shè)計RNA干擾序列,下調(diào)HMGCR在食管鱗癌細胞中的表達。我們利用MTT實驗、Boyden Chamber和軟瓊脂集落實驗分析HMGCR對食管鱗癌細胞生長、遷移和克隆形成的影響;利用GST-pull down和蛋白印跡檢測HMGCR對Ras-ERK信號通路的活化作用。最后,我們在食管鱗癌細胞中過表達或下調(diào)c-Myc的表達,通過熒光定量PCR和蛋白印跡檢測c-Myc的表達對HMGCR mRNA和蛋白水平的影響。我們的研究結(jié)果顯示,與正常食管粘膜組織相比,HMGCR在食管鱗癌組織中的mRNA水平和蛋白水平均呈現(xiàn)上調(diào)趨勢。HMGCR在食管鱗癌細胞株中的表達水平高于永生化的食管粘膜上皮細胞。在食管鱗癌細胞Eca109和正常食管粘膜細胞SHEE中過表達HMGCR促進食管鱗癌細胞生長、遷移和在軟瓊脂上克隆集落;在食管鱗癌細胞Eca109和Caes-17中下調(diào)HMGCR的表達抑制食管鱗癌細胞遷移和在軟瓊脂上克隆集落。在分子機制研究中,我們發(fā)現(xiàn)hmgcr過表達促進ras活化,上調(diào)erk磷酸化水平。在食管鱗癌細胞中,我們發(fā)現(xiàn)代謝的重要調(diào)控因子c-myc上調(diào)hmgcr的mrna水平和蛋白水平。綜上所述,我們的研究結(jié)果提供了充分的證據(jù),證明hmgcr在食管鱗癌發(fā)生發(fā)展過程中的重要功能。同時,我們的研究也提示hmgcr的抑制劑他汀用于食管鱗癌治療的可能性。本研究可以分為如下三個部分:第一部分hmgcr在食管鱗癌組織和食管鱗癌細胞中表達上調(diào)目的:研究hmgcr在食管鱗癌組織及配對的正常組織、食管鱗癌細胞株中的表達模式。方法:首先利用熒光定量pcr(real-timepcr)檢測hmgcr在40例食管鱗癌組織及配對的正常組織中的mrna水平;使用蛋白印跡法,檢測隨機挑選的6例食管鱗癌組織及配對的正常組織中hmgcr的蛋白水平,驗證熒光定量pc結(jié)果;最后,利用蛋白印跡檢測正常食管上皮細胞和食管鱗癌細胞中hmgcr的蛋白水平。結(jié)果:mrna水平的檢測結(jié)果顯示,食管鱗癌組織中hmgcr的mrna水平較配對的正常組織顯著升高;蛋白印跡實驗結(jié)果表明,食管鱗癌組織中hmgcr的蛋白水平較配對的正常組織顯著上調(diào),這一結(jié)果與熒光定量pcr的結(jié)果一致;最后,對多種食管鱗癌細胞和正常食管上皮細胞的蛋白印跡結(jié)果顯示,hmgcr在正常食管上皮細胞中的表達水平較低,在食管鱗癌細胞中的表達水平較高。結(jié)論:在食管鱗癌組織和細胞系中,hmgcr的mrna水平和蛋白水平均有較強的表達。與正常組織相比較,食管鱗癌組織中hmgcr的表達水平上升;與正常食管上皮細胞相比,hmgcr在鱗癌細胞中的表達水平升高。這些結(jié)果提示hmgcr在食管鱗癌發(fā)生發(fā)展過程中很可能起著非常重要的作用。第二部分hmgcr對食管鱗癌細胞生長、遷移和克隆集落的影響目的:研究hmgcr對食管鱗癌細胞惡性行為(如生長、遷移、克隆集落等)的影響,揭示hmgcr在食管鱗癌進展中的功能。方法:利用脂質(zhì)體轉(zhuǎn)染hmgcr的表達載體(myc-hmgcr,帶myc標(biāo)簽)至食管鱗癌細胞eca109和正常食管上皮細胞shee中;通過mtt法檢測hmgcr對食管鱗癌細胞eca109和正常食管上皮細胞shee生長的影響;通過軟瓊脂集落法研究hmgcr對食管鱗癌細胞克隆形成能力的影響;借助于細胞遷移模型揭示hmgcr對食管鱗癌細胞eca109和正常食管上皮細胞shee遷移的影響。同時,制備hmgcrrna干擾的病毒載體,在食管鱗癌細胞eca109、caes17中下調(diào)hmgcr的表達;利用軟瓊脂集落法揭示下調(diào)hmgcr的表達對食管鱗癌細胞克隆形成的影響;借助細胞遷移模型研究hmgcr表達下調(diào)對食管鱗癌細胞遷移能力的影響結(jié)果:通過脂質(zhì)體介導(dǎo)的穩(wěn)定轉(zhuǎn)染,在食管鱗癌細胞eca109和正常食管上皮細胞shee中建立了穩(wěn)定表達細胞株,過表達帶myc標(biāo)簽的hmgcr。mtt實驗結(jié)果表明,穩(wěn)定表達hmgcr促進食管鱗癌細胞eca109、正常食管上皮細胞shee的生長;細胞遷移實驗顯示,hmgcr在eca109和shee細胞中的表達增加食管鱗癌細胞的運動能力;軟瓊脂集落形成實驗顯示,hmgcr在eca109細胞中的表達促進其非錨定性生長。此外,我們構(gòu)建了hmgcrrna干擾的慢病毒載體,非常有效地干擾hmgcr在食管鱗癌細胞eca109和caes17中的表達。細胞遷移實驗表明,hmgcr表達下降削弱了食管鱗癌細胞eca109和caes17的運動能力;克隆集落形成實驗顯示,干擾hmgcr的表達使食管鱗癌細胞的非錨定性生長的能力受到抑制。結(jié)論:hmgcr促進食管鱗癌細胞的生長、遷移、克隆形成能力,干擾hmgcr的表達抑制食管鱗癌細胞非錨定性生長和遷移。hmgcr對食管鱗癌的進展發(fā)揮促進作用。第三部分hmgcr在食管鱗癌細胞中激活ras-erk信號通路目的:研究hmgcr調(diào)控食管鱗癌細胞惡性行為(如生長、遷移和克隆集落)的分子機制。方法:利用gst-pulldown平臺,研究hmgcr的表達ras的活化作用及對erk磷酸化水平的影響;通過熒光定量pcr和蛋白印跡,檢測c-myc基因的表達對hmgcr的mrna水平和蛋白水平的影響。結(jié)果:GST-pull down分析顯示,過表達HMGCR促進Ras的活化,上調(diào)ERK的磷酸化水平。干擾HMGCR的表達抑制ERK的磷酸化。熒光定量PCR和蛋白印跡的實驗結(jié)果顯示,過表達c-Myc上調(diào)HMGCR的mRNA水平和蛋白水平,干擾cMyc的表達抑制HMGCR的mRNA水平和蛋白水平。結(jié)論:HMGCR在食管鱗癌細胞中激活Ras-ERK信號通路,代謝調(diào)控的關(guān)鍵分子c-Myc調(diào)控HMGCR的表達。
[Abstract]:Esophageal cancer is one of the most common gastrointestinal tumors in China. Although the surgical, anesthesia, perioperative management and intensive care conditions and techniques have been greatly improved, the effect of surgical treatment alone is still unsatisfactory. Abnormal metabolism of tumor cells is one of the significant characteristics that distinguish tumor cells from normal cells. Looking for therapeutic targets for esophageal cancer from the perspective of tumor metabolism may bring new hope for the treatment of esophageal cancer. Diene, ubiquinone and so on. These substances play an important role in the physiological process of cells. The function of mevalonate pathway in esophageal squamous cell carcinogenesis is still unclear. In this study, we cloned the mevalonate metabolic rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (3-Hydroxy-3-methylglutaryl coenzyme A reductase) In this study, we collected esophageal squamous cell carcinoma tissue samples, cultured esophageal squamous cell carcinoma cell lines, detected the mRNA level of HMGCR in esophageal squamous cell carcinoma tissues and their matched normal tissues by fluorescence quantitative PCR, and detected esophageal squamous cell carcinoma tissues and esophageal squamous cell carcinoma fine by Western blot. Protein level of HMGCR in esophageal squamous cell carcinoma cells. At the cellular level, we established a stable cell line of esophageal squamous cell carcinoma overexpressing HMGCR by liposome transfection. RNA interference sequence was designed to down-regulate the expression of HMGCR in esophageal squamous cell carcinoma cells. The effect of HMGCR on Ras-ERK signaling pathway was detected by GST-pull down and Western blotting. Finally, we overexpressed or down-regulated the expression of c-Myc in esophageal squamous cell carcinoma cells, and detected the effect of c-Myc expression on HMGCR mRNA and protein levels by fluorescence quantitative PCR and Western blotting. The results showed that HMGCR was up-regulated in both mRNA and protein levels in esophageal squamous cell carcinoma compared with normal esophageal mucosa. The expression level of HMGCR in esophageal squamous cell carcinoma cell lines was higher than that in immortalized esophageal epithelial cells. Promote the growth, migration and cloning of esophageal squamous cell carcinoma cells on soft agar; down-regulate the expression of HMGCR in esophageal squamous cell carcinoma cells Eca109 and Caes-17, inhibit the migration of esophageal squamous cell carcinoma cells and clone colonies on soft agar. In the study of molecular mechanism, we found that overexpression of HMGCR promoted Ras activation and up-regulated ERK phosphorylation. In conclusion, our findings provide sufficient evidence to support the important role of HMGCR in the development of esophageal squamous cell carcinoma. Our findings also suggest that statin, an inhibitor of hmgcr, may be useful in the treatment of esophageal squamous cell carcinoma. This study can be divided into the following three parts: the first part is the up-regulation of HMGCR expression in esophageal squamous cell carcinoma and esophageal squamous cell carcinoma. The protein levels of HMGCR in 6 randomly selected esophageal squamous cell carcinoma tissues and matched normal tissues were detected by Western blot to verify the results of quantitative fluorescence PC. Finally, the protein levels of HMGCR in normal esophageal epithelial cells and esophageal squamous cell carcinoma cells were detected by Western blot. The results of Western blotting showed that the protein level of HMGCR in esophageal squamous cell carcinoma was significantly higher than that in matched normal tissues, which was consistent with the results of fluorescence quantitative pcr. Western blot analysis showed that the expression of HMGCR was lower in normal esophageal epithelial cells and higher in esophageal squamous cell carcinoma cells. Conclusion: The expression of HMGCR in esophageal squamous cell carcinoma tissue and cell line was higher than that in normal esophageal tissue. These results suggest that the expression of HMGCR may play an important role in the development of esophageal squamous cell carcinoma. Part two: The effects of HMGCR on the growth, migration and cloning of esophageal squamous cell carcinoma cells. The effect of Cr on malignant behavior of esophageal squamous cell carcinoma cells (e.g. growth, migration, colony cloning, etc.) and the function of HMGCR in the progression of esophageal squamous cell carcinoma were revealed. The effects of HMGCR on the growth of esophageal epithelial cells ECA 109 and normal esophageal epithelial cells shee; the effects of HMGCR on the clonal formation of esophageal squamous cell carcinoma cells were studied by soft agar colony assay; the effects of HMGCR on the migration of esophageal squamous cell carcinoma cells ECA 109 and normal esophageal epithelial cells shee were revealed by cell migration model. The expression of HMGCR was down-regulated in esophageal squamous cell carcinoma cells Eca109 and caes17; the effect of down-regulated expression of HMGCR on the formation of esophageal squamous cell carcinoma cell clones was revealed by soft agar colony assay; the effect of down-regulated expression of HMGCR on the migration ability of esophageal squamous cell carcinoma cells was studied by cell migration model; the results showed that stable transfection mediated by liposome could induce fine esophageal squamous cell carcinoma cells. A stable expression cell line was established in Eca109 cells and normal esophageal epithelial cells shee. over-expression of myc-tagged hmgcr. MTT assay showed that stable expression of HMGCR promoted the growth of esophageal squamous cell carcinoma cell Eca109 and normal esophageal epithelial cells shee. cell migration assay showed that the expression of HMGCR in Eca109 and shee cells increased the fineness of esophageal squamous cell carcinoma. In addition, we constructed a lentiviral vector interfered by hmgcrrna, which was very effective in interfering with the expression of HMGCR in esophageal squamous cell carcinoma cells ECA 109 and caes17. Cell migration assay showed that the decrease of HMGCR expression weakened the expression of caes17 and ECA 109 cells. The motility of esophageal squamous cell carcinoma cells Eca109 and caes17 was inhibited by interfering with the expression of hmgcr. conclusion: HMGCR can promote the growth, migration and clonal formation of esophageal squamous cell carcinoma cells, and interfere with the expression of HMGCR can inhibit the Non-anchoring growth and Non-anchoring growth of esophageal squamous cell carcinoma cells. The third part is the activation of ras-erk signaling pathway by HMGCR in esophageal squamous cell carcinoma cells. Objective: To study the molecular mechanism of HMGCR in regulating malignant behavior of esophageal squamous cell carcinoma cells (such as growth, migration and clonal colony). Results: GST-pull down analysis showed that over-expression of HMGCR promoted the activation of Ras and up-regulated the phosphorylation level of ERK. Interference of HMGCR expression inhibited the phosphorylation of ERK. The results of Western blotting showed that overexpression of c-Myc up-regulated the mRNA and protein levels of HMGCR, interfered with the expression of cMyc and inhibited the mRNA and protein levels of HMGCR. Conclusion: HMGCR activates Ras-ERK signaling pathway in esophageal squamous cell carcinoma cells, and c-Myc, the key molecule of metabolic regulation, regulated the expression of HMGCR.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.1

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10 肖斌;施瑞華;杜琰萍;朱宏;凌亭生;林艷;;載體介導(dǎo)shRNA特異性抑制食管鱗癌細胞Eca-109綠色熒光蛋白基因的表達[A];中華醫(yī)學(xué)會第七次全國消化病學(xué)術(shù)會議論文匯編（下冊）[C];2007年

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