【作者】 李乾；

【导师】 张桂英；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 研究背景胃癌是人类常见的恶性肿瘤之一,发病率和死亡率居我国各类恶性肿瘤的首位。临床早期胃癌大多无症状或症状不典型,早期诊断困难;临床就诊者大部分属于中、晚期,失去手术机会或术后易复发。因此,提高胃癌患者生活质量和无病生存期的化疗显得很重要。目前常用的化疗药物副作用大且疗效欠佳。因此寻找新的安全有效的非细胞毒化疗药物是目前胃癌等消化道肿瘤防治方面需要迫切解决的课题。细胞增殖和凋亡是细胞生物学行为的两个重要方面,肿瘤的发生即为细胞恶性增殖和凋亡失控所致。近年来,Fas和FasL在消化道肿瘤中尤其在胃癌前病变、胃癌中的表达及其与细胞凋亡之间的关系已引起广泛关注。Fas是细胞表面重要的死亡受体,与其配体FasL结合活化并传导凋亡信号是诱导细胞凋亡的重要途径,Fas与FasL结合后,通过胞浆内Fas相关死亡域蛋白（FADD）,再和门冬氨酸特异半胱氨酸蛋白酶（caspase-8）酶原发生相互作用,激活caspase-8酶原,进一步激活多种caspase联级反应,最终导致细胞凋亡。既往研究已证明胃癌前病变及胃癌组织表达FasL,不仅可以介导表达Fas的胃癌前病变及胃癌细胞凋亡,而且可能介导胃癌前病变及胃癌组织中表达Fas的肿瘤浸润淋巴细胞（TIL）发生凋亡,从而使胃癌细胞逃逸机体自身免疫清除,成为胃癌发生、发展的重要潜在生物学基础。深入研究胃癌及局部淋巴结组织Fas、FasL的表达,为进一步阐明Fas凋亡系统在胃癌发生、发展及免疫逃逸机制和防治方面提供实验依据。NSAIDS降低胃肠道肿瘤发生的危险性,已成为肿瘤防治领域的研究热点。国外多项大规模的流行病学研究、动物实验和临床研究均发现,长期规律服用非甾体抗炎药（NSAIDS）能使结肠癌发病的危险性降低。我们前期研究发现吲哚美辛（IN）和塞来昔布具有抗结肠肿瘤细胞增殖效应。然而传统的NSAIDS如IN,在抑制COX-2的同时抑制了COX-1,长期服用对胃肠道及肾等脏器产生损害,限制了其在临床上的广泛和长期应用。新型NSAIDS,选择性抑制COX-2,则既有抑瘤作用,又避免了胃肠道副作用。选择性COX-2抑制剂抑制胃癌细胞增殖,诱导细胞凋亡的研究,倍受肿瘤学专家们关注。国内外多个研究发现塞来昔布有抑制胃癌细胞增殖凋亡作用,对其作用的分子机理研究仅局限于对环氧化酶-2的活性抑制。一些研究表明COX-2抑制剂通过抑制环氧化酶-2的活性,减少前列腺素E2的释放,进而抑制细胞增殖,诱导细胞凋亡。但选择性COX-2抑制剂塞来昔布对细胞周期调控具有重要意义的细胞周期依赖性蛋白激酶抑制因子p16和p21表达的影响,及对细胞Fas、FasL凋亡途径中Fas、FasL蛋白表达和凋亡相关调控因子Bcl-2蛋白表达的影响,国内外尚未见相关文献报道。探讨选择性COX-2抑制剂塞来昔布对BGC-823胃癌细胞抑瘤作用,从细胞周期调控系统,Fas、FasL凋亡系统与凋亡信号转导通路调控等层面,“立体”和系统地研究胃肿瘤细胞增殖和凋亡调控网络,揭示选择性COX-2抑制剂塞来昔布抑制BGC-823胃癌细胞增殖和凋亡调控机理,将为包括塞来昔布在内的选择性COX-2抑制剂抗胃癌等肿瘤的临床应用提供实验依据,为发现新的分子靶标提供新的思路。第一章塞来昔布抑制BGC-823胃癌细胞增殖及对p16和p21表达的影响目的:研究塞来昔布对体外培养的BGC-823胃癌细胞增殖的影响及对细胞周期依赖性蛋白激酶抑制因子p16和p21表达的影响。方法:采用MTT比色法检测塞来昔布对BGC-823胃癌细胞增殖的抑制;通过流式细胞仪技术研究不同浓度的塞来昔布对BGC-823胃癌细胞周期的影响;通过RT-PCR技术检测塞来昔布对细胞周期依赖性蛋白激酶抑制因子p16 mRNA及p21 mRNA表达的影响。结果:①塞来昔布浓度为20,40,60,80,100,120μmol/L,24hr时抑制率（%）分别为:7.63±2.66,13.33±0.95,18.83±0.91,24.63±1.55,33.20±1.14,55.00±6.50;48hr时抑制率（%）分别为:10.72±3.20,25.16±2.70,37.22±1.60,42.42±2.41,46.79±4.57,91.64±4.14:72hr时抑制率（%）分别为:10.91±1.61,31.39±3.20,54.82±1.25,62.41±5.51,74.88±1.02,98.17±0.69。塞来昔布各浓度组均抑制胃癌细胞BGC-823的生长,并呈时间和浓度依赖性。②流式细胞仪检测结果显示:塞来昔布浓度为0,20,40,60,80,100μmol/L时,G₁期细胞数比例分别为0.603±0.016,0.643±0.006,0.671±0.005,0.708±0.008,0.750±0.007,0.787±0.006;S期细胞数比例分别为0.307±0.006,0.223±0.003,0.209±0.005,0.193±0.015,0.157±0.005,0.122±0.006。塞来昔布随浓度增加G₁期细胞数比例逐渐增加,S期细胞数比例逐渐减少。各浓度组间细胞数比例两两比较,具有显著性差异（P＜0.05）。③RT-PCR结果显示不同浓度塞来昔布作用于BGC-823胃癌细胞后,塞来昔布各浓度组p16 mRNA和p21 mRNA扩增产物均有表达。塞来昔布浓度为0,20,40,60,80,100μmol/L时,p16 mRNA表达的相对丰度分别为:0.223±0.006,0.296±0.012,0.381±0.008,0.438±0.017,0.494±0.002,0.663±0.068;p21 mRNA表达的相对丰度分别为:0.435±0.045,0.642±0.098,0.730±0.014,0.813±0.069,0.907±0.058,1.030±0.051,p16 mRNA和p21 mRNA表达的相对丰度随塞来昔布浓度增加而增加。各浓度组间相对丰度两两比较,具有显著性差异（P＜0.05）。结论:①塞来昔布抑制BGC-823胃癌细胞增殖,呈时间和浓度依赖性。②塞来昔布干预BGC-823胃癌细胞增殖,使G₁期细胞数增多,S期细胞数减少,阻滞胃癌细胞周期于G₁期。③塞来昔布通过上调细胞周期依赖性蛋白激酶抑制因子p21mRNA和p16 mRNA表达,抑制BGC-823胃癌细胞周期进展。第二章塞来昔布诱导BGC-823胃癌细胞凋亡及对Fas、FasL及Bd-2表达的影响目的:研究塞来昔布诱导BGC-823胃癌细胞凋亡及对凋亡相关蛋白Fas、FasL及Bcl-2表达的影响。方法:通过流式细胞仪技术观察不同浓度的塞来昔布对BGC-823胃癌细胞凋亡的影响;通过WesternBlotting技术检测塞来昔布对凋亡相关蛋白Fas、FasL和Bcl-2表达的影响。结果:①流式细胞仪检测发现:塞来昔布浓度在0,20,40,60,80,100μmol/L时,凋亡细胞比率（%）分别为:0.503±0.018,5.870±0.325,11.396±0.267,17.567±0.376,24.439±0.326,52.933±2.575;随塞来昔布浓度增加凋亡细胞比率增加。各浓度组间凋亡细胞比率两两比较,具有显著性差异（P＜0.05）。②WesternBlotting结果显示:塞来昔布浓度为0,20,40,60,80,100μmol/L时,作用BGC-823胃癌细胞48h后,Fas蛋白相对含量分别是0.1113±0.0037,0.1017±0.0065,0.1975±0.0100,0.3297±0.0053,0.4595±0.0045,0.5204±0.0148;FasL蛋白相对含量分别是0.8070±0.0268,0.7311±0.0780,0.6254±0.0501,0.5465±0.0180,0.4378±0.0081,0.3760±0.0034;Bcl-2蛋白相对含量分别是0.5448±0.0049,0.5213±0.0326,0.4256±0.0421,0.3934±0.0305,0.2659±0.0121,0.2294±0.0081。在0～100μmol/L浓度范围内Fas蛋白表达上调和FasL、Bcl-2蛋白表达下调,均呈浓度依赖性。各浓度组间蛋白相对含量两两比较,具有显著性差异（P＜0.05）。结论:①塞来昔布诱导BGC-823胃癌细胞凋亡,在一定范围内呈浓度依赖性。②塞来昔布在一定浓度范围内,Fas蛋白表达呈浓度依赖性上调,FasL、Bcl-2蛋白表达呈浓度依赖性下调。③塞来昔布诱导胃癌细胞凋亡可能与Fas蛋白上调,FasL、Bcl-2蛋白下调有关。第三章Fas、FasL在胃癌及局部淋巴结组织中表达意义目的:探讨胃癌及局部淋巴结组织中Fas、FasL蛋白的表达与胃癌发生发展及转移的关系。方法:通过免疫组化方法研究64例胃癌及局部淋巴结组织和20例正常胃组织Fas和FasL蛋白的表达,并分析其与年龄、性别、病理亚型和有无淋巴结转移的关系。结果:①正常胃组织Fas蛋白阳性表达率高,在胃癌组织中Fas蛋白阳性表达率低;正常胃组织FasL蛋白阳性表达率低,在胃癌组织中FasL蛋白阳性表达率高。二者间阳性表达均有显著性差异（P＜0.001）。②Fas蛋白在有侵犯组淋巴结组织中阳性表达率高,在无侵犯组淋巴结组织中阳性表达率低,二者间阳性表达有显著性差异（P=0.003）。FasL在两组淋巴结组织中阳性表达无差异（P=0.593）。③Fas蛋白在高中分化组胃癌组织中阳性表达率高,在低分化组胃癌组织中阳性表达低,二组间阳性表达有显著性差异（P=0.015）。反之,FasL蛋白在高中分化组胃癌组织中阳性表达率低,在低分化组胃癌组织中阳性表达率高,二组间阳性表达有显著性差异（P=0.021）。④胃癌组织中Fas、FasL蛋白阳性表达率与年龄、性别及是否有淋巴结转移无明显关系。⑤胃癌组织中Fas和FasL两者间阳性表达率,两两比较有显著性差异（P＜0.001）;两者表达无直线相关性（r_s=-0.425,P=0.575）。结论:①胃癌组织中Fas蛋白表达下调,FasL蛋白表达上调。②有侵犯淋巴结组织中Fas蛋白表达上调,无侵犯淋巴结组织中表达下调。③高分化胃癌组织中Fas蛋白阳性表达高于低分化胃癌组织;高分化胃癌组织中FasL蛋白阳性表达低于低分化胃癌组织。更多还原

【Abstract】 Background Gastric carcinoma is the most common human malignancies,morbidity and mortality in our country home of the first types of tumors.Clinically,most of the early gastric cancer has no symptoms or no typical symptoms,which has brought difficulty to early diagnosis;on the clinical the majority of doctors’ offices visiting of patients belong to advanced gastric cancer,which lose the opportunity to surgery or relapse easily after operation.As a result,improving the quality of life of patients with gastric cancer and disease-free survival of the chemotherapy appears to be very important.At present,common chemotherapeutics have more drug side effects,and the efficacy below the mark.Therefore,it is an urgent need to resolve the issue that the gastrointestinal cancer prevention and treatment need research and develop the new of safe and effective chemotherapy drugs.Cell proliferation and apoptosis in cell biology are two important aspects of behavior.The incidence of tumor cell proliferation and apoptosis is caused by out of control.In recent years,it has attracted great attention about the expression of Fas and FasL in the digestive tract tumors,especially in precancerous lesions of gastric and gastric cancer, and its relationship with cell apoptosis.Fas as important death receptor of cell surface,binding FasL and activation of apoptosis signal transduction is an important way of induced apoptosis.FasL and Fas bind through intracytoplasmic Fas-associated death domain protein（FADD）,that interact to activate caspase-8 zymogen to further caspase activation together a variety of class reaction,eventually leading to apoptosis.Past research has proved the expression of FasL in gastric cancer and gastric precancerous lesions,which can mediate gastric cancer and gastric precancerous lesions cell with expression of Fas apoptosis and mediate TIL cells expression of Fas apoptosis in gastric precancerous lesions and gastric cancer tissue,which make the gastric cancer cells escape the body’s own immune clearance and become a potential biological basis of genesis and survival and development of gastric cancer.To depth study expression of Fas and FasL in gastric cancer and local lymph node tissues is of great significance to further clarify the Fas system in apoptosis of gastric cancer,development and immune escape mechanisms and provide experimental evidence for prevention and treatment.NSAIDS can reduce the gastrointestinal cancer risk,which has become another hot spot in recent years in the field of prevention and treatment tumor.Abroad,a number of large-scale epidemiological studies, animal experiments and clinical studies have found that long-term use of the law of non-steroidal anti-inflammatory drugs（NSAIDS） can reduce the risk of the incidence of colon cancer.Our preliminary studies found that indomethacin（IN） and celecoxib have anti-colon tumor cell proliferation effect.Traditional NSAIDs,such as IN,can inhibit COX-2 at the same time COX-1,and thus the gastrointestinal tract and kidneys organ damage because of long-term use will have to limit its wide range and long-term of clinical applications.New NSAIDs,selectively inhibiting COX-2,are both anti-tumor effect and avoiding the side effects.Oncology experts pay much attention to whether selective COX-2 inhibitors inhibit gastric cancer cell proliferation and induce apoptosis or not.Some study at home and abroad found that celecoxib has inhibited apoptosis and proliferation in gastric cancer cell,its role is limited to the molecular mechanism of celecoxib inhibition the activity of cyclooxygenase-2.Some studies show that COX-2 inhibitors can inhibit the activity of cyclooxygenase -2 and reduce the release of prostaglandin E2,thereby inhibiting cell proliferation and induced apoptosis.However, selective COX-2 inhibitors on gastric cancer cell proliferation and related regulatory genes p16 and p21 expression,and apoptosis-related proteins Fas,FasL and Bcl-2 protein expression in small studies.We explore the selective COX-2 inhibitor celecoxib on gastric cancer cells BGC-823 anti-tumor effect from the cell cycle regulation and control system,Fas, FasL system and the apoptosis signaling pathway level,and so on, "Stereo" and systematically research stomach tumor cell proliferation and apoptosis regulation and control network,revealed a selective COX-2 inhibitor celecoxib inhibited gastric cancer cell proliferation and apoptosis regulation and control mechanism,provided the experimental basis for clinical applications a selective COX-2 inhibitor anti-gastric tumors including celecoxib,as well as to provide new ideas for detecting new molecular targets.ChapterⅠEffects of Celeeoxib on cell proliferation in human gastric cancer cell line BGC-823 and expression of p16 and p21Objectives:To explore the effects of Celecoxib on cell proliferation of human gastric cancer cell line BGC-823 and expression of cell cycle regulation -related factor p16 and p21.Methods:MTT assay were used to quantify the influence of Celecoxib on the proliferation of gastric cancer cell line BGC-823.Flow cytometry（FCM） were used to measure the influence of Celecoxib on cell cycle of difference concentration celecoxib on BGC-823 cell.The expressions of p16 and p21 mRNA with difference concentration celecoxib on BGC-823 cell were observed by RT-PCR analysis.Results:①Celecoxib concentration 20,40,60,80,100,120μmol/L respectively,cultivating for 24hr the inhibition rate（%） were 7.63±2.66,13.33±0.95,18.83±9.10,24.63±1.55,33.20±1.14,55.00±6.50; 48hr when the inhibition rate（%）,respectively:10.72±3.20,25.16±2.70,37.22±1.60,42.42±2.41,46.79±4.57,91.64±4.14;72hr when the inhibition rate（%）,respectively:10.91±1.61,31.39±3.20,54.82±1.25,62.41±5.51,74.88±1.02,98.17±0.69Difference concentration celecoxib inhibited the cell growth rates of gastric cancer cell line BGC-823 in a time -dependent and concentration -dependent manner.②FCM showed:Celecoxib concentration 20,40,60,80,100,120μmol/L respectively,the proportion of G1 phase cells were 0.603±0.016,0.643±0.006,0.671±0.005,0.708±0.008,0.750±0.007,0.787±0.006;S phase cell ratio were 0.307±0.006,0.223±0.003,0.209±0.005,0.193±0.015,0.157±0.005,0.122±0.006;Celecoxib increased the proportion of cells in G₁ phase,whereas decreased the proportion of cells in the S phase in a concentration-dependent manner from 0 to 100μmol/L in BGC-823 cells.The proportion of cells between various concentration celecoxib groups has significant differences,（P＜0.05）.③RT-PCR results showed that there were expressions of p16 and p21 amplified product by different concentrations of celecoxib on BGC-823 cells, relative abundance of expressions of p16 and p21 amplified product increased with celecoxib concentration.Celecoxib concentration 20,40,60,80,100,120μmol/L respectively,p16 PCR products relative abundance,respectively:0.223±0.006,0.296±0.012,0.381±0.008, 0.438±0.017,0.494±0.002,0.663±0.068:p21 PCR products relative abundance,respectively:0.435±0.045,0.642±0.098,0.730±0.014, 0.813±0.069,0.907±0.058,1.030±0.051.The relative abundance of p16 and p21 between various concentration celecoxib groups has significant differences,（P＜0.05）. Conclusions:①Celecoxib inhibited BGC-823 gastric cancer cell proliferation,showing time and dose-dependent manner.②Celecoxib inhibited BGC-823 gastric cancer cell proliferation;G1 phase cells increased and S phase cells decrease in celecoxib on BGC-823gastric cancer cell,which block cell cycle in G1 phase.③Celecoxib inhibited cell cycle progress of BGC-823 gastric cancer cell,which may be associated with up-regulation of the expression of cell cycle dependent protein kinase p16 and p21,and alteration cell cycle progression.ChapterⅡEffects of Celecoxib on cell apoptosis in human gastric cancer cell line BGC-823 and expression of Fas,FasL and Bcl-2Objectives:To explore the effect of Celecoxib on cell apoptosis of human gastric cancer cell line BGC-823 and expression of Fas,FasL and Bcl-2.Methods:Flow cytometry was used to quantify the influence of Celecoxib on the apoptosis of gastric cancer cell line BGC-823.The expressions of Fas,FasL,bcl-2 proteins were observed by Western blot analysis.Results:①Flow cytometry found that the ratio of apoptotic cells increased with the concentration of celecoxib increased;Celecoxib concentration at 0,20,40,60,80100μmol / L,ratio of apoptotic cells（%） were:0.503±0.018,5.870±0.325,11.396±0.267,17.567±0.376, 24.439±0.326,52.933±2.575.The apoptotic rates of cells between various concentration celecoxib groups has significant differences,（P＜0.05）.②Western blotting analysis showed:Celecoxib concentration at 0,20,40,60,80,100μmol / L,cultivating for 48h on BGC-823 gastric cancer cells,Relative content of Fas protein were 0.1113±0.0037,0.1017±0.0065,0.1975±0.0100,0.3297±0.0053,0.4595±0.0045,0.5204±0.0148;Relative content of FasL protein were 0.8070±0.0268,0.7311±0.0780,0.6254±0.0501,0.5465±0.0180,0.4378±0.0081,0.3760±0.0034;Relative content of Bcl-2 protein were 0.5448±0.0049,0.5213±0.0326,0.4256±0.0421,0.3934±0.0305,0.2659±0.0121,0.2294±0.0081.The expression of Fas was up-regulated,whereas the expressions of FasL and bcl-2 proteins were down-regulated in a concentration-dependent manner from 0 to 100μmol/L in gastric cancer cell line BGC-823.The expressions of Fas,FasL and Bcl-2 proteins between various concentration celecoxib groups have significant differences（P＜0.05,respsctively）.Conclusions:①Celecoxib-induced gastric cancer BGC-823 cell apoptosis,to a certain range of dose-dependent manner.②At a certain celecoxib concentration scope, Fas protein increased and FasL,Bcl-2 protein reduced were dose-dependent manner.③Celecoxib induced cell apoptosis in gastric cancer cell line BGC-823,which may be associated with up-regulation of the expressions of Fas protein and down-regulated of the expressions of FasL and bcl-2 protein. ChapterⅢExpression significance of Fas and FasL in gastric cancer tissues and local lymph nodes tissuesObjective:To investigate the relationship between the expression of Fas and FasL protein with genesis,development and transfer of gastric cancer.Methods:FasL and Fas expression of 64 cases gastric cancer and 20 cases normal gastric tissue were studied by immunohistochemistry, and analysis the relationship between their age,gender,pathological subtype and lymph node metastasis with FasL and Fas expression.Results:①Expression of Fas protein in normal gastric tissue is high, expression of Fas protein in gastric cancer tissue is low.Expression of FasL protein in normal gastric tissue is low;expression of FasL protein in gastric cancer tissue is high.The positive expressions between the two groups have significant differences,（P<0.001）.②Expression of Fas in infringement lymph node tissue is high;Expression of Fas in non-infringement lymph node tissues is low.Fas positive expressions between the two groups have significant differences,（P=0.003）.FasL positive expression between the two groups lymph tissues have no difference,（P=0.593）.③Expression of Fas protein in highly and moderately differentiated gastric cancer tissue is high;Expression of Fas protein in poorly differentiated gastric cancer tissue is low.Expression of FasL protein in highly and moderately differentiated gastric cancer tissue is low;Expression of FasL protein in poorly differentiated gastric cancer tissue is high.The positive expressions between the two groups have significant differences,（P=0.015 and P=0.021,respectively）.④There is no significant relationship between age,sex and whether or not lymph node metastases with expression of Fas and FasL in gastric cancer.⑤There was a significant difference between Fas and FasL positive expression rate in gastric cancer（P＜0.001）;both the expression of non-linear correlation（rs=-0.425,P=0.575）.Conclusions:①Fas expression in gastric cancer down-regulation,FasL expression in gastric cancer up-regulation.②Fas expression in infringement lymph node tissue up-regulation,in non-infringement lymph node tissues down-regulation.③Fas expressions in the well-differentiated gastric cancer were higher than it in poorly differentiated gastric cancer;FasL expressions in the well-differentiated gastric cancer tissues were lower than it in poorly differentiated gastric cancer.更多还原