本文選題：HSP70　切入點：雷帕霉素　出處：《中南大學》2010年博士論文　論文類型：學位論文

【摘要】： 鼻咽癌(Nasopharyngeal carcinoma, NPC)是我國南方常見的惡性腫瘤之一,發(fā)病率為10-50／10萬人口,死亡率在全身惡性腫瘤中的比例,全國第八,兩廣居第三位。目前該病的治療仍然以放射治療為主。對放療不敏感、放療后復發(fā)以及中晚期的患者,可采取化學藥物治療。但傳統(tǒng)的放、化療存在特異性抗腫瘤能力低、殺傷指數(shù)過小、副作用大等缺點。 分子靶向治療作為腫瘤學研究的一個新興的熱點對于改善臨床鼻咽癌的療效具有重要意義。它針對可能導致細胞癌變的環(huán)節(jié),如細胞信號傳導通路、原癌基因和抑癌基因、細胞因子及受體、抗腫瘤血管形成、自殺基因等,從分子水平來逆轉(zhuǎn)這種惡性生物學行為,從而抑制腫瘤細胞生長,甚至使其完全消退的一種全新的生物治療模式。雷帕霉素作為一種新型的分子靶向治療藥物,在諸多實體瘤中取得了不同程度的療效,其中部分研究已經(jīng)進入了Ⅲ期臨床試驗,成果令人鼓舞,同時讓我們對雷帕霉素治療鼻咽癌的效果充滿了期待。如果雷帕霉素能夠成功用于鼻咽癌的分子靶向治療,不僅能造�；颊�,提高生存率,還能產(chǎn)生相當?shù)慕?jīng)濟效益和社會效益。 本研究以雷帕霉素為切入點,探討該藥物對不同生物學特性的鼻咽癌細胞所產(chǎn)生的生長抑制作用及其蛋白質(zhì)分子機制。并以此為基礎,進一步揭示沉默鼻咽癌中HSP70表達對雷帕霉素抑制作用的影響以及相關依據(jù)初探。整個研究內(nèi)容分三部分進行： 方法 通過MTS法確定mTOR抑制劑雷帕霉素作用三株鼻咽癌細胞產(chǎn)生最佳生長抑制率所需濃度和作用時間。 結(jié)果 1.雷帕霉素對不同生物學特性的鼻咽癌細胞均有抑制其生長的作用,該作用的發(fā)揮可能是通過靶向抑制mTOR激酶活性來實現(xiàn)的； 2.抑制率呈現(xiàn)劑量和時間依賴性,不同細胞株之間無顯著差別,基本穩(wěn)定在40%-50%之間,最高為53.9%; 3.對于三個細胞株而言,100nM雷帕霉素作用48小時均能讓藥物所致生長抑制率進入平臺期。 方法 1.采用Western Blotting法檢測不同濃度雷帕霉素作用下三株細胞內(nèi)PI3K/Akt/mTOR信號通路中關鍵性蛋白標記物(p-Akt Ser473、Akt、p-mTOR Ser2448、mTOR)表達情況； 2.采用Western Blotting法檢測100nM雷帕霉素作用不同時間后三株細胞內(nèi)PI3K/Akt/mTOR信號通路中關鍵性蛋白標記物(p-Akt Ser473、Akt、p-mTOR Ser2448、mTOR)表達情況。 結(jié)果 1.CNE-1和6-10B細胞p-mTOR Ser2448表達情況表現(xiàn)出一定的濃度、時間依賴性,而5-8F細胞中該指標不隨藥物濃度和作用時間改變而變化； 2.6-10B和5-8F細胞p-Akt Ser473隨藥物濃度增加而表達增高,而CNE-1則呈“上升-下降”趨勢； 3.三個細胞株p-Akt Ser473當100nM雷帕霉素作用不同時間后,在1hr時表達明顯增高,隨著藥物作用時間繼續(xù)延長,表達較lhr相比有下降,但是除6-10B外,均比0hr高； 4. mTOR、Akt、HSP70的表達保持相對穩(wěn)定,未呈現(xiàn)明顯時間、劑量依賴性改變。 方法 1.分別采用RT-PCR法和Western Blotting法初步驗證HSP70siRNA在鼻咽癌細胞株中使目標基因／蛋白產(chǎn)生滿意沉默效果所需作用時間； 2.采用MTS法進行Control siRNA組、HSP70 siRNA組、Control siRNA+100nM Rapa組和HSP70 siRNA+100nM Rapa組組間生長抑制率比較； 3.采用光學顯微鏡明視野觀察不同干預措施下的四組細胞形態(tài)學變化； 4.采用Western Blotting法檢測Control siRNA組、HSP70 siRNA組、Control siRNA+100nM Rapa組和HSP70 siRNA+100nM Rapa組細胞中PI3K/ Akt/mTOR信號通路中關鍵性蛋白標記物(p-Akt Ser473、Akt、p-mTOR Ser2448、mTOR)表達情況。 結(jié)果 1.RT-PCR法顯示siRNA作用48hrs,在基因水平可達到HSP70相對較高抑制率,而Western Blotting法顯示siRNA作用72hrs,可在蛋白質(zhì)水平達到HSP70相對較高抑制率； 2.三個細胞株中四個不同干預組互相比較,HSP70 siRNA+ 100nM Rapa組生長抑制率最高； 3.與Control siRNA組比較,Control siRNA+100nM Rapa組與第二部分的Rapa作用48hrs的情況相類似,能夠提升細胞中p-AktSer473表達,而HSP70 siRNA+100nM Rapa組中,p-Akt Ser473表達下降； 4.與單用siRNA組別比較,Control siRNA+100nM Rapa和HSP70 siRNA+100nM Rapa組中,CNE-1和6-10B細胞p-mTORSer2448表達下調(diào),而5-8F無明顯變化； 5.mTOR、Akt的表達組間比較無明顯變化。 結(jié)論 本文采用MTS法、Western Blotting法、脂質(zhì)體瞬時轉(zhuǎn)染技術并輔以逆轉(zhuǎn)錄-聚合酶鏈反應等實驗方法研究雷帕霉素對不同生物學特性的人鼻咽癌細胞株生長抑制作用,并通過檢測PI3K/Akt/mTOR信號通路中關鍵蛋白標記物揭示細胞生長抑制現(xiàn)象產(chǎn)生的相關機制,接著觀察雷帕霉素對HSP70表達下調(diào)的鼻咽癌細胞所產(chǎn)生的影響,最后初步探討HSP70 siRNA與雷帕霉素聯(lián)合抑制鼻咽癌細胞生長所涉及的相關機制,得出如下結(jié)論： 1.雷帕霉素在體外能夠一定程度抑制鼻咽癌細胞株的生長,不同細胞株之間抑制率無明顯差異； 2.雷帕霉素抑制p-mTOR Ser2448表達和mTOR的激酶活性是導致細胞生長抑制產(chǎn)生的原因； 3.雷帕霉素誘導p-Akt Ser473表達上調(diào)是導致藥物對鼻咽癌細胞株生長抑制率不高的原因之一； 4.雷帕霉素能夠引起部分細胞株p-mTOR Ser2448表達下調(diào),但是這并非反映生長抑制率情況的指標; 5. HSP70 siRNA和雷帕霉素聯(lián)合使用提高藥物對鼻咽癌細胞生長抑制作用； 6. HSP70 siRNA是通過沉默HSP70表達,調(diào)控Akt Ser473位點磷酸化水平使雷帕霉素對鼻咽癌細胞抑制率增高,為HSP70 siRNA與雷帕霉素聯(lián)合靶向治療鼻咽癌提供了有價值的實驗資料。
[Abstract]:Nasopharyngeal carcinoma (Nasopharyngeal carcinoma NPC) is one of the most common malignant tumor in southern China, the incidence rate was 10-50 per 100 thousand population, the proportion of mortality of malignant tumors in the human body of the eighth, Guangdong ranks third. The treatment of the disease remains to radiation therapy. Is not sensitive to radiotherapy, after radiotherapy and recurrence in patients later, chemotherapy should be considered. But the traditional chemotherapy, has specific anti-tumor ability low, killing index is too small, side effects and other shortcomings.
Molecular targeted therapy is a new hotspot in oncology research has important significance for improving the clinical curative effect of nasopharyngeal carcinoma. It is aimed at the links which may lead to cell carcinogenesis, such as cell signal transduction pathways, proto oncogenes and tumor suppressor genes, cytokine and receptor, anti-tumor angiogenesis, Dutch act to reverse the malignant genes. The biological behavior at the molecular level, thus inhibiting the growth of tumor cells, and even make a new biological treatment model of complete remission. Rapamycin as a new molecular targeted therapeutic drugs have achieved varying degrees of efficacy in many solid tumors, part of which has entered phase III clinical trial, the results were at the same time encouraging, let us on the effect of rapamycin treatment of nasopharyngeal carcinoma is full of expectations. If rapamycin can be successfully used in nasopharyngeal carcinoma molecular targeted treatment, not only can To benefit the patients and improve their survival rate can produce considerable economic and social benefits.
In this study, rapamycin as a starting point, to investigate the inhibitory effect and molecular mechanism of protein produced in nasopharyngeal carcinoma cells of the drug in different biological characteristics of growth. And on this basis, further reveal the inhibition effect on the basis of rapamycin and HSP70 expression in nasopharyngeal carcinoma. The research content is divided into three parts:
Method
The concentration and time of the optimal growth inhibition rate of three nasopharyngeal carcinoma cells with mTOR inhibitor, rapamycin, were determined by MTS.
Result
1. rapamycin can inhibit the growth of nasopharyngeal carcinoma cells with different biological characteristics, which may be achieved by targeting the inhibition of mTOR kinase activity.
2. inhibition rate showed dose and time dependence, there was no significant difference between different cell lines, basically stable between 40%-50%, and the highest was 53.9%.
3. for the three cell lines, the effect of 100nM rapamycin for 48 hours could allow the growth inhibition rate of the drug to enter the stage of the platform.
Method
1. Western Blotting method was used to detect the expression of p-Akt Ser473 (Akt, p-mTOR Ser2448, mTOR) in PI3K/Akt/mTOR signaling pathway of three cells under different concentrations of rapamycin.
2. Western Blotting method was used to detect the expression of p-Akt, Ser473, Akt, p-mTOR Ser2448 and mTOR in PI3K/Akt/mTOR signaling pathway of three 100nM cells after rapamycin treatment.
Result
The expression of p-mTOR Ser2448 in 1.CNE-1 and 6-10B cells showed a certain concentration and time dependence, while 5-8F cells did not change with the concentration and time of action.
The expression of p-Akt Ser473 in 2.6-10B and 5-8F cells increased with the increase of drug concentration, while CNE-1 showed a "rise - decline" trend.
3., three cell lines p-Akt Ser473. When 100nM rapamycin acted for different time, it increased significantly at 1hr. With the prolongation of drug action time, the expression decreased compared with LHR, but 6-10B was higher than 6-10B.
The expression of 4. mTOR, Akt, and HSP70 remained relatively stable, and did not show significant time and dose dependent change.
Method
1., the RT-PCR and Western Blotting methods were used to preliminarily verify the time needed for HSP70siRNA to produce satisfactory silencing effect of target gene / protein in nasopharyngeal carcinoma cell line.
2. the growth inhibition rates of Control siRNA group, HSP70 siRNA group, Control siRNA+100nM Rapa group and HSP70 siRNA+100nM Rapa group were compared by MTS method.
3. the morphological changes of four groups of cells under different intervention measures were observed by optical microscope.
4. Western Blotting method was used to detect the expression of key protein markers in the Control siRNA group, HSP70 siRNA group, Control siRNA+100nM Rapa group and HSP70 siRNA+100nM group.
Result
1.RT-PCR showed that siRNA acted on 48hrs and reached a relatively high inhibition rate at HSP70 level, while Western Blotting showed siRNA action 72hrs, which could achieve a relatively high inhibition rate of HSP70 at protein level.
2. of the three cell lines, four different intervention groups were compared with each other, and the growth inhibition rate of HSP70 siRNA+ 100nM Rapa group was the highest.
3. compared with Control siRNA group, the Control siRNA+100nM Rapa group was similar to the second part Rapa in the effect of 48hrs, which could enhance p-AktSer473 expression in cells, while HSP70 siRNA+100nM Rapa group decreased the expression of HSP70.
4., compared with single siRNA group, CNE-1 and 6-10B cell p-mTORSer2448 expression was downregulated in Control siRNA+100nM Rapa and HSP70 siRNA+100nM Rapa group, while 5-8F did not change significantly.
There was no significant change in the expression of 5.mTOR and Akt.
conclusion
This paper uses the MTS method, Western Blotting method, the growth inhibitory effects of liposome transfection technique combined with reverse transcriptase polymerase chain reaction experiment method to study the effect of rapamycin on the different biological characteristics of human nasopharyngeal carcinoma cell lines, and related mechanisms by detecting the PI3K/Akt/mTOR signal pathway key protein markers reveal cell growth inhibition phenomenon, then observe the effect from nasopharyngeal carcinoma cells down regulated by rapamycin on the expression of HSP70 and related mechanism of HSP70 siRNA finally combined with rapamycin inhibits the growth of nasopharyngeal carcinoma cells involved, draws the following conclusion:
1. in vitro, rapamycin can inhibit the growth of nasopharyngeal carcinoma cell lines to a certain extent, and there is no significant difference in the inhibition rate between different cell lines.
2. the inhibition of p-mTOR Ser2448 expression and mTOR kinase activity by rapamycin is the cause of cell growth inhibition.
3. the up-regulated expression of p-Akt Ser473 induced by rapamycin is one of the reasons for the low inhibitory rate of drug on the growth of nasopharyngeal carcinoma cell lines.
4. the expression of p-mTOR Ser2448 was down regulated by rapamycin, but it was not an indicator of the rate of growth inhibition.
Combined use of 5. HSP70 siRNA and rapamycin to improve the inhibition of the growth of nasopharyngeal carcinoma cells.
6. HSP70 siRNA, by silencing HSP70 expression, regulates the phosphorylation level of Akt Ser473 site, increases rapamycin inhibition rate on nasopharyngeal carcinoma cells, and provides valuable experimental data for HSP70 siRNA combined with rapamycin in the treatment of nasopharyngeal carcinoma.

【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R739.63

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