【作者】 王海涛；

【导师】 金春顺；

【作者基本信息】 吉林大学 ， 耳鼻咽喉科学， 2009， 博士

【摘要】 目的:检测趋化因子受体(CXCR4)在喉癌组织中的表达,探讨CXCR4在喉癌发生及发展中的作用;应用RNA干涉技术沉默CXCR4基因,研究CXCR4基因沉默后对喉癌Hep-2细胞株增殖、凋亡、粘附和侵袭的影响。方法:1.应用免疫组织化学SP法检测CXCR4在喉癌组织、癌旁组织、对照组中的表达。2.应用分子生物学技术,模拟内源性miRNA,设计CXCR4基因的shRNAmir,用酶切鉴定、测序检测重组质粒。3.重组质粒用脂质体Lipofectimine 2000转染人喉癌Hep-2细胞。应用免疫组化SP,半定量RT-PCR检测转染后Hep-2细胞中CXCR4蛋白与mRNA的表达水平。4.四甲基偶氮唑盐(MTT)检测转染的重组质粒对喉癌Hep-2细胞体外增殖的影响。5.流式细胞术检测转染的重组质粒的喉癌Hep-2细胞体外凋亡情况。6.半定量RT-PCR检测转染的重组质粒对血管内皮生长因子VEGF与细胞间粘附分子E-CD的mRNA表达水平。7.体外粘附实验检测转染的重组质粒对Hep-2细胞体外与基质粘附能力的影响。8.Transwell小室体外侵袭实验检测转染的重组质粒对Hep-2细胞体外侵袭能力的影响。结果:1.CXCR4蛋白在喉癌组织中呈强阳性表达,蛋白表达水平显著高于对照组(P<0.01);在中低分化、淋巴结转移组蛋白表达水平均明显升高(P<0.05)。2 .构建的重组质粒( pCXCR4miR/GFP-262、pCXCR4miR/GFP-713、pCXCR4miR/GFP- 961)经酶切鉴定、测序比对后证明所构建的重组质粒成功。3.转染后免疫组化SP检测到72小时后3个重组质粒(pCXCR4miR/GFP-262、pCXCR4miR/GFP-713、pCXCR4miR/GFP-961)的CXCR4蛋白表达量显著下降;半定量RT-PCR显示3个重组质粒CXCR4的mRNA的表达量下降显著,尤以转染pCXCR4miR/GFP-713明显,选用pCXCR4miR/GFP-713用于后续实验。4.MTT比色法检测细胞的增殖,实验组(转染pCXCR4miR/GFP-713的Hep-2组)的细胞的增殖弱于空白对照组(Hep-2组)和阴性对照组(空质粒组),且差别具有显著性(P<0.01)。5.流式细胞仪检测显示实验组细胞凋亡率明显高于空白对照组和阴性对照组,差异有统计学意义(P<0.01)。6.体外粘附实验显示实验组细胞30、60、90、120分钟时的粘附抑制率分别为27.7%、28.9%、25.6%、31.1%,粘附抑制率均高于空白对照组和阴性对照组,差异有统计学意义(P<0.01)。7.半定量RT-PCR结果显示实验组VEGF与E-CD mRNA的相对表达水平分别为0.752±0.008, 0.815±0.010,与对照组比较,VEGFmRNA相对表达水平下降(P<0.05),E-CD mRNA相对表达水平上升(P<0.05)。8.Transwell小室体外侵袭实验显示细胞体外侵袭能力明显降低,实验组的细胞(30±4.119)与阴性对照组(69±6.437)及空白对照组(72±4.535)相比穿膜细胞数明显减少(P<0.01)。结论:CXCR4在喉癌组织中高表达并与喉癌的淋巴结转移、病理分化程度有关。RNA干涉沉默CXCR4基因可影响喉癌Hep-2细胞株增殖、凋亡、粘附和侵袭,CXCR4基因可以作为喉癌基因治疗的靶点,为喉癌的基因治疗提供一条新的途径。更多还原

【Abstract】 Laryngeal carcinoma , a malignant tumor of epithelial origin, represents 10% ENT of malignant tumor in china. surgery operation and radiotherapy are often adopted for the Laryngeal carcinoma in early stage.In the cases of middle and advanced stage ,surgery is used as the predominant therapy of comprehensive treatment.Since 1980s the treatment of the laryngeal carcinoma has made rapid progress. Operation,radiotherapy,chemotherapy and the application of the immunization therapy have greatly raised the survival rate of laryngeal carcinoma patients by five years.However,those therapies can cause high rate of disability and complications as well as many serious toxic and side effects.Recurrence and metastasis are still the major death cause of laryngeal carcinoma.In recent years, the tumor foundation research has obtained significantly progression and gene therapy displays good perspective to the patients with tumors. RNA interference(RNAi)is a newly discovered cellular pathway for the silencing of sequence-specific genes at the mRNA level by the introduction of the cognate double stranded RNA.It has rapidly developed into one of the most widely applied technologies with therapeutic potential in molecular and cellular research.The chemokines are soluble, small molecular weight (8?14 kDa) proteins, Chemokine receptors belong to the G protein-coupled receptor (GPCR) superfamily,Chemokines bingding with chemokine receptors can cause many biological events such as embryogenesis, wound healing, angiogenesis, leukocyte homeostasis and lymphoid organ development, inflammatory diseases, pro and anti-tumor responses.More than 50 different chemokines and 20 different chemo-kine receptors have been cloned. Chemokines usually bind to multiple receptors, and the same receptor may bind to more than one chemokine. However, there is one exception to this rule: the SDF-1, which binds exclusively to CXCR4 , has CXCR4 as its only receptor. This fact alone suggests that the SDF-1/CXCR4 axis plays a uniquely important biological role. Many studies disclosed that the biological axis of SDF-1/CXCR4 play important roles in proliferation,apoptosis ,adhesion ,angiogenesis,invasion and metastasis in tumer.So, from the gene level,employing the RNA interference technique to silence CXCR4 gene to block the biological axis of SDF-1/CXCR4 is recent study focus .We employed the RNA interference technique to silence gene expression of CXCR4 in laryngeal carcinoma cell line Hep-2 to study the effect of the proliferation apoptosis adhesion and invasion in vitro .To Study invasion and metastasis molecular mechanisms of laryngeal carcinoma. We suggest that CXCR4 is a novel target for gene therapy of laryngeal carcinoma.PART ONEExpression and significance of CXCR4 in human laryngeal carcinomaObjective: To observe the expression and significance of CXCR4 in human laryngeal carcinoma tissues .Methods: Immuohistochemistry was used to detect the expressions of CXCR4 in 65 samples of human laryngeal carcinoma. The relationship CXCR4 and the clinicpathological diameters was statistically analyzed.Results:①CXCR4 were positively expressed in laryngeal carcinoma tissues while they were negative in the control group. Their expressions were significantly higher than those of control group( P < 0. 01);②The expression of CXCR4 in laryngeal carcinoma declined with the rise of the different grade of laryngeal carcinoma( P < 0. 01),The expression of CXCR4 were significantly higher in the group with lymph node metastasis than those without lymph node metastasis( P < 0. 05).Conclusion:Chemokine Receptor CXCR4 are highly expressed in laryngeal carcinoma,and their expression were associated with differentiation grade of laryngeal carcinoma and neck lymph node metastasis.PART TWOThe construction of eukaryotic expression Plasmid of shRNAmir for CXCR4Objective: To construct the shRNAmir eukaryotic expression Plasmid specific for human CXCR4 gene and to observe its silencing effects on CXCR4 gene.Methods: Getting the number NM-003467 of CXCR4 in the database of GenBank to design single-stranded DNA oligos.using Invitrogen’s RNAi Designer, an online tool of Co. Invitrogen to design miR RNAi. Electing three sites 262, 713 and 961 because of high scores. Annealing was operated in the single-stranded oligos to generate a ds oligo. Cloning this ds oligo into pcDNA?6.2-GW/EmGFPmiR with T4 DNA Ligase ,Restriction Enzyme Digestion and sequencing were used to evaluate the recombinant,The expression Plasmids of (pCXCR4miR/GFP-262,pCXCR4miR/GFP-713and pCXCR4miR/GFP-961) and the control plasmid (pcDNA?1.2/V5-GW/lac),the negative control plasmid (EmGFPpcDNA6.2 ?GW/EmGFP ) were transfected by Lipofectimine 2000 into the Hep-2 cells, The expression of CXCR4 in transfected Hep-2 cells was evaluated by immuohistochemistry,Fluorescence microscopy and RT-PCR.Results:Compared with the control groups,Protein and mRNA expression were significantly decreased in Hep-2 cells transfected with expression Plasmid, pCXCR4miR/GFP- 713 expression Plasmid had the most eficient inhibitory efect, followed by pCXCR4miR/ GFP-262 and pCXCR4miR/GFP-961.Conclusion: The constructed CXCR4 shRNAmir Eukaryotic expression Plasmid can block the expression of CXCR4 in laryngeal carcinoma cells, and it can be used to study the effects of CXCR4 on the invasion, metastasis of laryngeal carcinoma cells.PART THREEThe effects of silencing CXCR4 gene by RNA interference on the proliferation,apoptosis, adhesion and invasion in transfected Hep-2 cells.Objective: To study the effects of expression Plasmid pCXCR4miR/GFP-713 on the proliferation,apoptosis, adhesion and invasion in transfected Hep-2 cells.Methods: By using MTT assay ,the proliferation capability of Hep-2 cells was tested.Apoptosis rate was evaluated by flow cytometric analysis.The abilities of invasion and adhesion were detected by Transwell chamber assay and cell ashedion assay.RT-PCR was used to detect mRNA of VEGF ,E-CD gene.Results:Compared with blank control group and Negative control group,Cell proliferation curve revealed that cell growth was significantly inhibited in experimental group (P<0.01) .FCM demonstrated that experimental group enhanced apoptosis of cells Compared with blank control group Negative control group, (P<0.01).The inhibitory rate on adhesion in experimental group was 27.7% at 30 min,28.9% at 60 min, 25.6% at 90 min and 31.1% at 120min, Compared with blank control group Negative control group, (P<0.01),The inhibition of invasion was observed by fewer penetrating cells by Transwell chamber assay(72±4.535. 69±6.437 and 30±4.119,respectively). (P<0.01) .RT-PCR revealed that The VEGF and E-CD mRNA expression were 0.752±0.008, 0.815±0.010 in experimental group respectively, compared with blank control group and Negative control group (P<0.05).Conclusion: By transfecting with the expression Plasmid pCXCR4miR/GFP-713,the cell proliferation, invasion ability of human Laryngeal carcinoma Hep-2 cells can be inhibited obviously,apoptosis was enhanced , the expression of VEGF mRNA can be down-expressed , cell-matrix adhesion can be enhanced but cell-cell adhesion be reduced significantly.更多还原