论文中英文摘要格式18693.docx

1、论文中英文摘要格式18693论文中英文摘要格式作者姓名：梁洁论文题目：朊蛋白促进胃癌增殖的分子机制作者简介：梁洁，女，1980年3月出生，2003年8月师从于第四军医大学樊代明教授攻读硕士学位，同年通过硕博直读选拔，面试攻读博士学位；于2008年6月获博士学位。中 文 摘 要【背景】朊蛋白是一种脂筏锚定蛋白质，分正常型PrPC和致病型PrPSc两种。尽管PrPC基因敲除小鼠的研究，提示PrPC缺失后小鼠会出现心律失常、昼夜节律改变等症状，但是PrPC的功能至今还不清楚。既往研究提示，PrPC编码基因的突变或多态性与朊病毒疾病相关，PrPC在神经系统发挥作用也与抗氧化、抑制细胞凋亡密切相关。此外

2、，PrPC在神经系统主要表达在突触及增殖较快的脑组织区域，在外周具有快速更新特征的胎盘等组织中富集，提示PrPC在细胞的增殖中发挥了一定的作用。本实验室在前期研究中最先发现PrPC在胃癌耐药细胞系中高表达，提示其与胃癌的多药耐药相关。Pammer等发现胃粘膜腺体颈处有PrPC弱阳性表达，且随着幽门螺旋杆菌的感染表达增加，而幽门螺旋杆菌感染是胃癌发生的重要原因之一，PrPC在胃癌中的表达、定位及其对胃癌增殖的作用和机制尚不清楚。本课题将对此进行探讨，明确PrPC对胃癌增殖的作用及机制。【目的】1、研究PrPC及其缺失突变体在胃癌中的表达情况；2、明确PrPC与胃癌细胞凋亡、增殖的相关性；3、研究

3、PrPC抑制胃癌细胞凋亡、促进胃癌细胞增殖的可能机制；4、探讨PrPC促进胃癌细胞增殖的信号转导通路及其在胃癌中高表达的可能原因。【方法】 1、利用免疫组织化学、免疫荧光技术观察PrPC在胃癌组织细胞中的表达、定位；2、通过DNA琼脂糖电泳及PAGE检测胃癌组织及细胞中PrPC编码基因的缺失突变情况；3、通过基因重组方法构建PrPC的正义全长、siRNA载体及各功能结构域的缺失突变体，分别转染胃癌细胞系构建筛选稳定转染细胞；4、利用电镜、流式细胞仪、免疫荧光显微镜等技术研究PrPC对胃癌细胞凋亡的影响；5、利用基因芯片筛选参与PrPC抑制胃癌细胞凋亡的相关基因，并通过Western Blot进

4、行验证；6、通过MTT、流式细胞仪检测细胞周期、细胞周期同步化阻滞、平板克隆和软琼脂克隆形成以及裸鼠体内成瘤等体内外实验研究PrPC对胃癌细胞增殖、生长的影响；7、用RT-PCR、Western Blot及双荧光素报告激酶实验检测各种转染有PrPC的胃癌细胞系对CyclinD的表达及转录激活作用；8、通过基因芯片筛选相关信号转导通路，通过Western Blot检测磷酸化Akt及总Akt的表达水平；9、通过缺氧孵箱或CoCl2诱导检测缺氧条件下PrPC的表达。【结果】1、PrPC及其缺失突变体(1-OPRD)在胃癌中高表达本研究利用胃癌、癌旁、正常胃粘膜组织标本和超英公司提供的胃癌、癌旁、正常

5、胃粘膜组织芯片，通过免疫组织化学和免疫荧光的方法，发现PrPC在胃癌组织中表达的评分(1.800.183)明显高于正常胃粘膜中表达的评分(0.740.075)(p0.05)。PrPC在胃癌中的表达与肿瘤分级和分期相关，但与患者的年龄和性别等因素无关，定位在胃癌细胞的细胞浆和/或细胞膜上。Western Blot检测发现其在各种胃癌细胞系中表达无明显差异，SGC7901表达相对较低，AGS表达相对较高。用特异性PCR及琼脂糖电泳检测发现，胃癌细胞系中存在着杂合或者纯合PrPC(1-OPRD)缺失突变体。MKN28和Kato为PrPC(1-OPRD)的纯合缺失突变体，SGC7901和BGC-823

6、为杂合缺失突变体，而胃癌细胞系AGS、MKN45 以及永生化胃粘膜上皮细胞系GES不存在PrPC(1-OPRD)的缺失突变。103例胃癌病人血液样品提取的基因组DNA，用特异性PCR检测出3例PrPC(1-OPRD)杂合缺失突变体(突变率2.9%)，205例正常人血液样品中只检测到1例突变(突变率0.5%)；56例胃镜下取样病理活检证实为胃癌组织的样品中检测到5例(突变率8.9%)；63例胃镜下取样病理活检证实为正常胃粘膜组织的样品中未检测到PrPC(1-OPRD)缺失突变体。说明PrPC(1-OPRD)缺失突变体在胃癌血液标本中的突变率明显高于正常血液样品中的突变率(p=0.047)，而Pr

7、PC(1-OPRD)缺失突变体在胃癌粘膜中的突变率也高于正常胃粘膜中的突变率(p=0.021)。2、PrPC通过上调Bcl-2、下调Bax，抑制细胞色素C和活性氧自由基的释放，抑制胃癌细胞凋亡将构建转染正义和siRNA PrPC的胃癌细胞系，通过血清剥夺诱导后利用电镜、流式细胞仪、免疫荧光等检测发现，转染PrPC的胃癌细胞具有抑制胃癌细胞凋亡及活性氧自由基释放的作用。用基因芯片筛选参与PrPC抑制胃癌细胞凋亡的相关分子，进一步通过Western Blot检测发现正义PrPC转染细胞后抗凋亡蛋白质Bcl-2呈高表达，而p53, Bax和细胞色素C呈低表达状态。用siRNA抑制PrPC表达之后可以

8、诱导胃癌细胞凋亡、增加活性氧自由基释放，可使p53, Bax和细胞色素C上调，而抗凋亡蛋白质Bcl-2下调。提示PrPC在胃癌中的高表达部分是通过依赖抑制活性氧自由基释放，促进Bcl-2表达，抑制细胞凋亡得以实现的。3、PrPC通过转录激活CyclinD、促进CyclinD表达，促进细胞周期G1/S期进展，从而促进胃癌细胞增殖及裸鼠体内成瘤将转染正义和siRNA PrPC的胃癌细胞系，进行MTT、平板克隆、软琼脂克隆形成、流式细胞仪检测及裸鼠体内成瘤等体内外实验，发现PrPC转染细胞的克隆形成、裸鼠体内成瘤能力增强，SGC7901/PrPC的增殖指数为(0.4490.017)，高于空载体对照细

9、胞(0.3730.023)和亲本细胞(0.3600.009) (p0.05)。而PrPC基因敲除细胞系AGS/PrPC(RNAi) 的增殖指数(0.4890.038)低于其空载细胞AGS/pSilencer (0.5370.049)和亲本细胞(0.5620.074) (p0.05)。用thymidine进行细胞周期同步化阻滞和流式细胞仪检测，提示PrPC促进胃癌细胞增殖主要是促进了细胞G1/S期进展。根据文献报导的PrPC功能结构域，利用重组PCR的方法分别构建N-端自由区域缺失的突变体SGC7901/PrPC(22-47)，脂筏定位序列缺失的突变体SGC7901/PrPC(24-90)，八肽

10、重复序列缺失的突变体SGC7901/ PrPC(51-90)，C-端球形区域缺失的突变体SGC7901/PrPC(90-230)，C-端GPI锚定序列缺失的突变体SGC7901/PrPC(230-253)，通过MTT和流式细胞仪检测其胃癌细胞SGC7901增殖的作用，发现缺失了八肽重复序列的缺失突变体SGC7901/PrPC(51-90)细胞的增殖与空载体对照细胞基本相似。说明PrPC八肽重复序列对胃癌细胞增殖和存活有重要的促进作用，将其缺失可以消除PrPC对胃癌细胞增殖存活的促进作用。用基因芯片筛选细胞周期进展相关分子，通过RT-PCR及Western Blot进行验证，提示胃癌细胞转染Pr

11、PC后CyclinD mRNA和蛋白水平的表达增强。进一步构建CyclinD家族CyclinD1、CyclinD2和CyclinD3的启动子序列，通过双荧光素报告激酶实验，显示SGC7901/PrPC与 pGL3-CCND1, pGL3-CCND2 或 pGL3-CCND3 共转染的细胞激活活性分别为对照细胞的 4.94倍、3.36倍和 3.65倍，说明PrPC可以转录激活CyclinD的启动子，但是将八肽重复序列缺失后，PrPC对CylinD启动子的转录激活作用明显减低。以上实验说明，PrPC通过转录激活CyclinD，特别是CyclinD1的启动子，上调CyclinD1 mRNA和蛋白水平

12、的表达，促进细胞G1/S期进展，八肽重复序列在其中发挥了重要作用。4、PI3K/Akt信号转导通路介导PrPC对胃癌细胞增殖的促进作用根据文献报导和基因芯片的结果用磷酸化Western Blot检测相关信号通路分子，发现稳定转染PrPC的胃癌细胞磷酸化Akt水平明显高于对照空载体细胞和亲本细胞，而总Akt表达没有明显差异。PI3K/Akt特异性抑制剂LY294002可以通过抑制胃癌细胞G1/S期进展抑制其增殖，但在10M作用48小时的条件下，LY294002对亲本细胞生长的抑制作用不显著，故将此作为研究条件。用10M LY294002作用48小时后各细胞系克隆形成能力均下降，其在PrPC转染细

13、胞中最为显著。同样条件诱导后各细胞系中CyclinD1 mRNA、蛋白表达水平及启动子转录活性均降低，但是在PrPC转染细胞中的降低相对于对照细胞和亲本细胞更明显(p0.05)，说明PI3K/Akt信号通路介导了PrPC引起的CyclinD1转录激活、促进胃癌细胞周期G1/S期进展及胃癌细胞增殖作用。5、缺氧诱导胃癌细胞中PrPC表达增高利用缺氧孵箱及CoCl2诱导，通过RT-PCR和Western Blot检测胃癌细胞系中PrPC表达的变化，发现缺氧可以诱导PrPCmRNA和蛋白的表达水平增高，且呈时间依赖性，在8小时达到最高峰，至24小时逐渐恢复到常氧水平。对于缺氧诱导朊蛋白表达改变的机制

14、，利用含有和不含有热休克元件(Heat shock element, HSE)结构的朊蛋白启动子载体分别给予缺氧和常氧诱导后，用双荧光报告系统检测其转录活性。MKN28细胞用脂质体2000共转染pGL3-PRNP (-756+96)(不含HSE) 或PGL3-PRNP (-2253+289)(含有HSE) 和内参照pRL-TK混合体2g，孵育24h再给予缺氧处理后进行荧光报告激酶检测，发现缺氧诱导后朊蛋白启动子活性比常氧时启动子活性增强(p0.05)，且缺氧处理后含有HSE朊蛋白启动子pGL3-PRNP (-2253+289)的转录激活活性是不含HSE的朊蛋白启动子pGL3-PRNP (-75

15、6+96)及空载体pGL3对照的4.3倍。【结论】本研究揭示了PrPC分子的一种新功能，胃癌组织中的缺氧环境可能诱导PrPC高表达，PrPC的八肽重复序列介导了其转录激活CyclinD启动子，促进CyclinD表达增加从而促进细胞周期G1/S期进展，促进胃癌细胞增殖及裸鼠体内成瘤，其中PI3K/Akt信号转导通路发挥了重要作用。关键词：胃癌；增殖；朊蛋白 Cellular prion protein promotes proliferation of gastric cancerLiang Jie ABSTRACT【Background】Prion protein, a glycosylpho

16、sphatidylinositol (GPI)-anchored membrane protein with two forms (normal or cellular prion protein, PrPC and pathogenic or scrapie form of prion protein, PrPSc) was encoded by the gene PRNP. Although several different theories have been proposed like aberrant circadian rhythms, lectrophysiological a

17、bnormalities, high susceptibility to seizure in PRNP knockout mice, the exact physiological functions of PrPC remain unclear. Previous work suggested that approximately 10-15% of the human prion disease was inherited and more than 20 pathogenic mutations had been found. Another emerging function of

18、PrPC was its protective role for cell survival, as regards protection against oxidative stress or TNF-induced apoptosis. Meanwhile, PrPC was found to be expressed immediately adjacent to the proliferative region but not in mitotic cells in brain, as well as in placenta, where rapid cell renewing was

19、 common. As far as we know, limitless replicative potential is one of the characteristics of tumor cells. All above data pointed to the fact that PrPC is involved in the proliferation of certain types of cells.Our previous studies demonstrated that PRNP was upregulated in multidrug-resistant (MDR) g

20、astric cancer cell lines, and forced expression of PrPC affected drug accumulation. Pammer et al found that the expression of PrPC was negative or weak in the neck region of gastric mucosa, but upregulated in gastric mucosa of the patients infected with Helicobacter pylori (Hp). Since Hp infection w

21、as thought to play an important role in carcinogenesis of gastric mucosa, it is interesting to investigate the relationship between PrPC and gastric cancer.Hence, the expression, localization of PrPC and its role in the proliferation of gastric cancer were investigated in the present work.【Objective

22、s】 (1) To examine the expression and localization of PrPC in gastric cancer; (2) To explore the relationship between PrPC and cell proliferation of gastric cancer; (3) To study on the possible mechanisms underlying inhibiting apoptosis and promoting proliferation of PrPC on gastric cancer cells; (4)

23、 To investigate the possible mechanisms of overexpressed PrPC in gastric cancer and related signal pathways.【Methods】 (1) The subcellular localization and expression of PrPC in gastric cancer tissues or gastric cancer cell lines were determined by immunohistochemistry and immunofluorescence; (2) The

24、 mutation of PRNP gene in gastric cancer samples or gastric cancer cell lines were investigated by DNA agarose electrophoresis or PAGE; (3) Full-length, siRNA and functional region deletion mutants of PrPC were constructed and transfected into gastric cancer cell lines. Then stable transfectants wer

25、e screened; (4) The effects of PrPC on apoptosis of gastric cancer cells were investigated by FACS, electron microscope and immunofluorescence assay; (5) Downstream molecules involved in inhibiting apoptosis of PrPC was screened by gene array and then proved by Western Blot; (6) The effects of PrPC

26、on proliferation of gastric cancer cells in vivo and vitro were studied by MTT, FACS, thymidine synchronization, plate colony formation assay, soft agar colony formation assay and tumor growth in nude mice; (7) Expression and activation of CyclinD at mRNA, protein and transcriptional levels were eva

27、luated by RT-PCR, Western Blot and dual luciferase reporter assay; (8) Possibly involved signal pathway molecules were screened by microarray. The expression of total and phosphate-Akt was examined by Western Blot; (9) Expression of PrPC in hypoxia condition was determined by Western Blot and RT-PCR

28、.【Results】1. PrPC and PrPC(1-OPRD) were overexpressed in gastric cancer.To determine the expression and localization of PrPC in gastric cancer, we compared the expression of PrPC in patients with gastric carcinoma, two slides of tissue array provided by the ChaoYing Company with immunohistochemistry

29、 or immunofluorescence. It was found the expression of PrPC was significantly higher in gastric cancer tissues (1.800.183) than that in normal gastric mucosa (0.740.075) (p0.05), which was related with tumor grade and stage, but not with age or sex. The expression of PrPC was widely located in gastr

30、ic cancer cell lines, mainly in the cytoplasm and/or plasma membranes, which was relatively low in SGC7901 and high in AGS.To study on possible mutation of PRNP gene in gastric cancer samples and cell lines, DNA sequencing and gel electrophoresis were used. It was found one octapeptide-repeat deleti

31、on (1-OPRD) variant occurred with high frequency in gastric cancer cell lines or tissues. Human gastric cancer cell lines MKN28 and Kato were homozygotes for 1-OPRD and those in cell lines SGC7901 and BGC-823 were heterozygous. However, gastric cancer cell lines AGS, MKN45 and human gastric mucosa e

32、pithelial cell line GES contained normal octapeptide form of N-terminal PrPC in their genome. Three cases for 1-OPRD in gastric cancer samples and one case in normal samples were found by PCR from DNA extracted from human blood. All were heterozygous for 1-OPRD. Five cases of heterozygous for 1-OPRD were also detected in gastric cancer tissue samples while none observed in tissue extracted DNA in normal samples. The occurrence frequency of 1-OPRD variant was significantly higher in gastric cancer blood samples (2.9%; p=0.047) and tissue samples (8.9%; p=0