【作者】 严辉；

【导师】 陈卫昌；

【作者基本信息】 苏州大学 ， 消化内科学， 2010， 博士

【摘要】 第一部分GKLF、IKLF在胃癌组织和胃癌细胞株中的表达及意义目的:研究胃癌组织和胃癌细胞株中GKLF、IKLF的表达及临床意义。方法:采用荧光实时定量PCR法检测胃癌组织和胃癌细胞株中GKLF mRNA、IKLF mRNA的表达,免疫组织化学法(IHC)检测胃癌组织及其远端正常胃组织中GKLF、IKLF蛋白的表达,Western blot和免疫细胞化学法(ICC)检测人正常胃上皮细胞株GES-1及胃癌细胞株MKN-28、SGC-7901、BGC-823、HGC-27中GKLF、IKLF蛋白的表达。结果:胃癌组织中GKLF mRNA的相对表达量明显低于远端正常胃组织、IKLF mRNA的相对表达量明显高于远端正常胃组织(P <0.05)、胃癌组织中GKLF mRNA、IKLF mRNA的表达水平与组织分化程度无关,GKLF蛋白总阳性率显著低于远端正常胃组织、IKLF蛋白总阳性率显著高于远端正常胃组织(P <0.05),GKLF、IKLF蛋白表达与胃癌患者的年龄、性别、分化程度、Lauren分型无关,与淋巴结是否有转移及pTNM分期有关(P <0.05);胃癌细胞株中GKLF mRNA和蛋白的相对表达量低于人正常胃上皮细胞株GES-1、IKLF mRNA和蛋白的相对表达量则高于细胞株GES-1(P <0.05)。结论:胃癌组织和胃癌细胞株中存在GKLF低表达、IKLF高表达,GKLF、IKLF在胃癌发生、发展中起一定的作用,检测GKLF、IKLF的表达有助于判断胃癌患者病情程度和预后。第二部分重组质粒pcDNA3.1-GKLF的转染及稳定细胞株的建立目的:建立高表达GKLF的稳定转染细胞株,以进一步观察其对人胃癌细胞株SGC-7901生物学行为的影响。方法:用脂质体转染法将重组质粒pcDNA3.1-GKLF转染人胃癌细胞株SGC-7901,通过G418筛选,建立稳定转染的SGC7901-pcDNA3.1-GKLF细胞株,用荧光实时定量PCR法、Western blot和ICC法检测GKLF mRNA和蛋白的表达。结果:建立了稳定转染的SGC7901-pcDNA3.1-GKLF细胞株,成功地表达目的基因。结论:成功建立稳定转染的SGC7901-pcDNA3.1-GKLF细胞株,为下一步探讨GKLF基因对胃癌生物学行为影响的体内外实验研究奠定基础。第三部分GKLF基因转染对人胃癌细胞株SGC-7901生物学行为影响的体外实验研究目的:体外观察外源性GKLF基因转染对人胃癌细胞株SGC-7901增殖活性和侵袭力的影响,探讨GKLF在胃癌中的作用机制。方法:应用MTT法(四甲基偶氮唑蓝比色法)、流式细胞仪、克隆形成实验和体外细胞侵袭实验分别研究GKLF基因转染前、后人胃癌细胞株SGC-7901增殖和侵袭活性的变化。RT-PCR法、Western blot法分别检测GKLF基因转染前、后人胃癌细胞株SGC-7901中细胞周期蛋白D1(Cyclin D1)、Ki-67、E-钙粘附分子(E-cadherin) mRNA和蛋白的表达。结果:与SGC-7901组和SGC7901-pcDNA3.1组相比,SGC7901-pcDNA3.1-GKLF组细胞生长速度减慢、细胞出现G0/G1期部分阻滞、克隆形成率低、细胞侵袭力明显减弱(P < 0.05)。SGC7901-pcDNA3.1-GKLF组细胞Cyclin D1、Ki-67、E-cadherin mRNA和蛋白的表达水平均低于SGC-7901组和SGC7901-pcDNA3.1组(P <0.05)。结论:GKLF基因可能通过下调Cyclin D1、Ki-67、E-cadherin的表达,而使人胃癌细胞株SGC-7901的增殖活性和侵袭力降低。第四部分GKLF基因转染对人胃癌细胞株SGC-7901生物学行为影响的体内实验研究目的:体内观察外源性GKLF基因转染对人胃癌裸鼠皮下移植瘤生长、转移的影响,并探讨GKLF在胃癌中的作用机制,从而为胃癌的基因治疗提供新的思路。方法:选用36只BALB/C-nu/nu裸鼠进行体内实验,分两批进行。每批裸鼠18只,各随机分为三组,每组分别注射等量的SGC7901-pcDNA3.1-GKLF、SGC7901-pcDNA3.1和SGC-7901细胞(2×107个/200μl/只)。第一批,将细胞注射于每只裸鼠右胁腹前肢皮下,接种6周后处死裸鼠,观察肿瘤生长状况、成瘤潜伏期,IHC法检测裸鼠皮下移植瘤组织内GKLF、Ki-67、E-cadherin、VEGF蛋白表达情况,测定皮下移植瘤组织中MVD值;同时用B超观察皮下移植瘤大小、血流信号及腹腔内转移情况。第二批,将细胞经尾静脉接种于裸鼠,接种6周后处死裸鼠,显微镜下观察有无肺转移。结果:与SGC-7901组和SGC7901-pcDNA3.1组相比,SGC7901-pcDNA3.1-GKLF组成瘤潜伏期延长、皮下移植瘤体积和瘤重较小(P <0.05),皮下移植瘤组织内GKLF蛋白表达升高、Ki-67、E-cadherin、VEGF蛋白表达均降低,微血管密度(MVD)值降低。B超显示:SGC7901-pcDNA3.1-GKLF组皮下移植瘤较小,血流信号I级。所有裸鼠均未见肺、腹腔转移。结论:GKLF基因可能通过下调Ki-67、E-cadherin、VEGF的表达,从而抑制肿瘤的生长,减少肿瘤中新生血管的生成,以GKLF为靶点的基因治疗有潜在的临床应用前景。更多还原

【Abstract】 Part 1 Expression and clinical significance of GKLF and IKLF in human gastric carcinoma tissues and cell linesObjective: To study the expression and clinical significance of GKLF and IKLF in human gastric carcinoma tissues and cell lines.Methods: Expressions of GKLF mRNA and IKLF mRNA in gastric carcinoma tissues and cell lines were detected by real-time fluorescence quantitative PCR.Expressions of GKLF protein and IKLF protein in gastric carcinoma tissues and adjacent normal gastric tissues were detected by immunohistochemistry(IHC) .Expressions of GKLF protein and IKLF protein in human normal gastric epithelium cell line (GES-1) and gastric carcinoma cell lines (MKN-28、SGC-7901、BGC-823、HGC-27)were detected respectively by Western blot and immunocytochemistry(ICC).Results: The expression level of GKLF mRNA in gastric carcinoma tissues was significantly lower than that in adjacent normal gastric tissues, the expression level of IKLF mRNA in gastric carcinoma tissues was significantly higher than that in adjacent normal gastric tissues(P <0.05).The levels of GKLF mRNA and IKLF mRNA in gastric carcinoma tissues were uncorrelated with differentiation.Positive expression rate of GKLF protein in the carcinoma tissues was significantly lower than that in adjacent normal gastric tissues .Positive expression rate of IKLF protein in the carcinoma tissues was significantly higher than that in adjacent normal gastric tissues(P <0.05).The expressions of GKLF protein and IKLF protein were uncorrelated with age,gender,differentiation and Lauren’s classification.They were correlated with lymph node involvement and clinical stage(P <0.05).The levels of GKLF mRNA and GKLF protein in four gastric carcinoma cell lines were lower than that in human normal gastric epithelium cell line (GES-1) . The IKLF mRNA and IKLF protein levels in four gastric carcinoma cell lines were higher than that in GES-1(P <0.05).Conclusion:The low expression of GKLF and high expression of IKLF were existed both in gastric carcinoma tissues and cell lines, GKLF and IKLF play roles in the carcinogenesis and progression of gastric carcinoma in a way.To examine the expression of GKLF and IKLF may be useful in judging the degree and prognosis of human gastric carcinoma.Part 2 The transfection of recombinant plasmid pcDNA3.1-GKLF and the establishment of stable transfected cell lineObjective:To establish stable transfected SGC7901-pcDNA3.1-GKLF cell line which expressed high level of GKLF.To observe the impact of biological behaviors of GKLF gene on human gastric carcinoma cell line SGC-7901.Methods: The recombinant plasmid of pcDNA3.1-GKLF was transfected into SGC-7901 cell by lipofectin-mediated method.After screening culture by G418,stable transfected SGC7901-pcDNA3.1-GKLF cell line was established,and the levels of GKLF mRNA and GKLF protein were identified by real-time fluorescence quantitative PCR、Western blot and immunocytochemistry(ICC).Results: The stable transfected SGC7901-pcDNA3.1-GKLF cell line was established.The GKLF gene was expressed successfully in the cell line.Conclusion: The establishment of stable transfected SGC7901-pcDNA3.1-GKLF cell line provide solid foundation for further experimental studies in vitro and in vivo of the impact of biological behaviors of GKLF gene on human gastric carcinoma.Part 3 Experimental study of the impact of biological behaviors on human gastric carcinoma cell line SGC-7901 by GKLF gene transfection in vitroObjective:To observe the effects of GKLF gene transfection on proliferation and invasion of human gastric carcinoma cell line SGC-7901 in vitro,to study the mechanism GKLF gene in human gastric carcinoma.Methods: Proliferation and invasion ability were measured before and after transfection by MTT assay、flow cytometry、colony formation assay and cell invasion assay. The mRNA and protein levels of Cyclin D1、Ki-67、E-cadherin were detected respectively by reverse transcription-polymerase chain reaction(RT-PCR) and Western blot . Results: The proliferative speed of SGC7901-pcDNA3.1-GKLF group was markedly slower than that of SGC-7901 and SGC7901-pcDNA3.1 groups(P <0.05).GKLF gene transfection caused part of the G0/G1 arrest,decreased clone formation rate and the invasion ability(P <0.05).The mRNA and protein levels of Cyclin D1、Ki-67、E-cadherin of SGC7901-pcDNA3.1-GKLF group were lower than that of SGC-7901 and SGC7901-pcDNA3.1 groups ( P <0.05).Conclusion: Transfection with GKLF gene might suppress proliferation and invasion ability of human gastric carcinoma cell line SGC-7901 by downregulation the expressions of Cyclin D1、Ki-67、E-cadherin.Part 4 Experimental study of the impact of biological behaviors on human gastric carcinoma cell line SGC-7901 by GKLF gene transfection in vivoObjective:To observe the effects of GKLF gene transfection on growth and metastasis of human gastric carcinoma cell line SGC-7901 xenograft in nude mice,to explore the potential mechanism of GKLF gene in gastric carcinoma and to bring a new method of gene therapy for human gastric carcinoma.Methods: We carried out an in vivo study with 36 BALB/C-nu/nu nude mice which divided into two parts.Every 18 nude mice were randomly divided into 3 groups.SGC7901-pcDNA3.1-GKLF、SGC7901-pcDNA3.1、SGC-7901 were injected into 3 groups nude mice .Every group nude mice injected with the same cell and volume(2×107 cells/200μl/mouse) .In part one, cells were injected into right costal abdomen subcutis of nude mice.The mice were killed 6 weeks later after injection and the changes of weight、volume、latency、state were observed. The expressions of GKLF、Ki-67、E-cadherin、VEGF and CD34 in xenograft tissues were stained by immunohistochemistry(IHC).The microvessel density (MVD) of xenograft tissues were evaluated.The size of tumour、blood stream signal and abdominal cavity were examined for tumour metastasis by ultrasonic.In part two,cells were injected into the tail vein of nude mice.The mice were killed 6 weeks later after injection and lung were examined for tumour metastasis by microscope. Results:Compared with SGC7901-pcDNA3.1、SGC-7901 groups,for SGC7901-pcDNA3.1-GKLF group,the stage of latency lengthened and growth of xenograft were inhibited,and the volume and weight of xenograft tissues were small(P <0.05).GKLF protein of xenograft tissues in SGC7901-pcDNA3.1-GKLF group increased.The expressions of Ki-67、E-cadherin、VEGF and MVD value decreased.The size of xenograft tissues were small, blood stream signal were in stage I.No metastasis sites of lung and abdominal cavity were seen in all nude mice.Conclusion: GKLF gene might inhibit the growth of tumour and reduce the formation of new blood vessel by downregulating the expressions of Ki-67、E-cadherin、VEGF.So GKLF gene has potential application value in clinical of being target of gene therapy.更多还原