HSP70调控雷帕霉素对鼻咽癌细胞生长抑制作用分子机制研究

发布时间：2018-03-12 10:37

本文选题：HSP70　切入点：雷帕霉素　出处：《中南大学》2010年博士论文　论文类型：学位论文

【摘要】： 鼻咽癌(Nasopharyngeal carcinoma, NPC)是我国南方常见的恶性肿瘤之一,发病率为10-50／10万人口,死亡率在全身恶性肿瘤中的比例,全国第八,两广居第三位。目前该病的治疗仍然以放射治疗为主。对放疗不敏感、放疗后复发以及中晚期的患者,可采取化学药物治疗。但传统的放、化疗存在特异性抗肿瘤能力低、杀伤指数过小、副作用大等缺点。分子靶向治疗作为肿瘤学研究的一个新兴的热点对于改善临床鼻咽癌的疗效具有重要意义。它针对可能导致细胞癌变的环节,如细胞信号传导通路、原癌基因和抑癌基因、细胞因子及受体、抗肿瘤血管形成、自杀基因等,从分子水平来逆转这种恶性生物学行为,从而抑制肿瘤细胞生长,甚至使其完全消退的一种全新的生物治疗模式。雷帕霉素作为一种新型的分子靶向治疗药物,在诸多实体瘤中取得了不同程度的疗效,其中部分研究已经进入了Ⅲ期临床试验,成果令人鼓舞,同时让我们对雷帕霉素治疗鼻咽癌的效果充满了期待。如果雷帕霉素能够成功用于鼻咽癌的分子靶向治疗,不仅能造福患者,提高生存率,还能产生相当的经济效益和社会效益。本研究以雷帕霉素为切入点,探讨该药物对不同生物学特性的鼻咽癌细胞所产生的生长抑制作用及其蛋白质分子机制。并以此为基础,进一步揭示沉默鼻咽癌中HSP70表达对雷帕霉素抑制作用的影响以及相关依据初探。整个研究内容分三部分进行：方法通过MTS法确定mTOR抑制剂雷帕霉素作用三株鼻咽癌细胞产生最佳生长抑制率所需浓度和作用时间。结果 1.雷帕霉素对不同生物学特性的鼻咽癌细胞均有抑制其生长的作用,该作用的发挥可能是通过靶向抑制mTOR激酶活性来实现的； 2.抑制率呈现剂量和时间依赖性,不同细胞株之间无显著差别,基本稳定在40%-50%之间,最高为53.9%; 3.对于三个细胞株而言,100nM雷帕霉素作用48小时均能让药物所致生长抑制率进入平台期。方法 1.采用Western Blotting法检测不同浓度雷帕霉素作用下三株细胞内PI3K/Akt/mTOR信号通路中关键性蛋白标记物(p-Akt Ser473、Akt、p-mTOR Ser2448、mTOR)表达情况； 2.采用Western Blotting法检测100nM雷帕霉素作用不同时间后三株细胞内PI3K/Akt/mTOR信号通路中关键性蛋白标记物(p-Akt Ser473、Akt、p-mTOR Ser2448、mTOR)表达情况。结果 1.CNE-1和6-10B细胞p-mTOR Ser2448表达情况表现出一定的浓度、时间依赖性,而5-8F细胞中该指标不随药物浓度和作用时间改变而变化； 2.6-10B和5-8F细胞p-Akt Ser473随药物浓度增加而表达增高,而CNE-1则呈“上升-下降”趋势； 3.三个细胞株p-Akt Ser473当100nM雷帕霉素作用不同时间后,在1hr时表达明显增高,随着药物作用时间继续延长,表达较lhr相比有下降,但是除6-10B外,均比0hr高； 4. mTOR、Akt、HSP70的表达保持相对稳定,未呈现明显时间、剂量依赖性改变。方法 1.分别采用RT-PCR法和Western Blotting法初步验证HSP70siRNA在鼻咽癌细胞株中使目标基因／蛋白产生满意沉默效果所需作用时间； 2.采用MTS法进行Control siRNA组、HSP70 siRNA组、Control siRNA+100nM Rapa组和HSP70 siRNA+100nM Rapa组组间生长抑制率比较； 3.采用光学显微镜明视野观察不同干预措施下的四组细胞形态学变化； 4.采用Western Blotting法检测Control siRNA组、HSP70 siRNA组、Control siRNA+100nM Rapa组和HSP70 siRNA+100nM Rapa组细胞中PI3K/ Akt/mTOR信号通路中关键性蛋白标记物(p-Akt Ser473、Akt、p-mTOR Ser2448、mTOR)表达情况。结果 1.RT-PCR法显示siRNA作用48hrs,在基因水平可达到HSP70相对较高抑制率,而Western Blotting法显示siRNA作用72hrs,可在蛋白质水平达到HSP70相对较高抑制率； 2.三个细胞株中四个不同干预组互相比较,HSP70 siRNA+ 100nM Rapa组生长抑制率最高； 3.与Control siRNA组比较,Control siRNA+100nM Rapa组与第二部分的Rapa作用48hrs的情况相类似,能够提升细胞中p-AktSer473表达,而HSP70 siRNA+100nM Rapa组中,p-Akt Ser473表达下降； 4.与单用siRNA组别比较,Control siRNA+100nM Rapa和HSP70 siRNA+100nM Rapa组中,CNE-1和6-10B细胞p-mTORSer2448表达下调,而5-8F无明显变化； 5.mTOR、Akt的表达组间比较无明显变化。结论本文采用MTS法、Western Blotting法、脂质体瞬时转染技术并辅以逆转录-聚合酶链反应等实验方法研究雷帕霉素对不同生物学特性的人鼻咽癌细胞株生长抑制作用,并通过检测PI3K/Akt/mTOR信号通路中关键蛋白标记物揭示细胞生长抑制现象产生的相关机制,接着观察雷帕霉素对HSP70表达下调的鼻咽癌细胞所产生的影响,最后初步探讨HSP70 siRNA与雷帕霉素联合抑制鼻咽癌细胞生长所涉及的相关机制,得出如下结论： 1.雷帕霉素在体外能够一定程度抑制鼻咽癌细胞株的生长,不同细胞株之间抑制率无明显差异； 2.雷帕霉素抑制p-mTOR Ser2448表达和mTOR的激酶活性是导致细胞生长抑制产生的原因； 3.雷帕霉素诱导p-Akt Ser473表达上调是导致药物对鼻咽癌细胞株生长抑制率不高的原因之一； 4.雷帕霉素能够引起部分细胞株p-mTOR Ser2448表达下调,但是这并非反映生长抑制率情况的指标; 5. HSP70 siRNA和雷帕霉素联合使用提高药物对鼻咽癌细胞生长抑制作用； 6. HSP70 siRNA是通过沉默HSP70表达,调控Akt Ser473位点磷酸化水平使雷帕霉素对鼻咽癌细胞抑制率增高,为HSP70 siRNA与雷帕霉素联合靶向治疗鼻咽癌提供了有价值的实验资料。
[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.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R739.63

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