【作者】 王洪；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 目的：检测1.Gal-3在人垂体腺瘤组织中表达情况。2.应用RNA干扰技术沉默肿瘤细胞中Gal-3。3.体外培养并建立人垂体腺瘤细胞系。4.为探讨Gal-3在垂体腺瘤侵袭机制中的可能作用；寻找提示垂体腺瘤侵袭性的可能的临床分子生物学标志；寻找侵袭性垂体腺瘤的可能治疗靶点。方法：1.免疫组化法检测垂体腺瘤组织中Gal-3的表达情况,明确何种亚型腺瘤中高表达,并在高表达的垂体腺瘤中比较侵袭性与非侵袭性腺瘤Gal-3表达的差异性。原位杂交技术检测Gal-3在高表达侵袭性或非侵袭性垂体腺瘤中的核酸情况。2.将RNA干扰技术与慢病毒载体相结合,建立一种慢病毒载体介导的能将siRNA表达盒稳定整合于细胞基因组的RNA干扰技术。构建抑制Gal-3蛋白表达的U6-siRNA的表达盒,将此表达盒克隆到载体pGCL-GFP中,选择MCF-7细胞为靶细胞模型进行感染,采用RT-PCR、Western blotting检测mRNA和蛋白的一致性,MTT和流式细胞仪检测其对细胞增殖、凋亡的干扰情况。3.建立体外培养人垂体催乳素腺瘤来源细胞-F培养体系的前提下,对垂体催乳素腺瘤来源细胞-F,进行光镜细胞形态学及扫描和透射电镜细胞超微结构观察。结果：1.Gal-3在垂体泌乳素腺瘤及ACTH腺瘤中均有表达,尤其是垂体泌乳素腺瘤高表达。2.在侵袭性垂体泌乳素腺瘤中Gal-3表达水平高于非侵袭性垂体腺瘤,且存在高的核分裂像。3.在侵袭性垂体泌乳素腺瘤中IHC检测阳性率越高其Gal-3-mRNA表达越强。4.成功构建并筛选了携带有人Gal-3基因的RNAi慢病毒载体及有效靶点。5.Gal-3的mRNA和蛋白表达一致降低,转染组mRNA表达量(0.028%)、阴性对照组为(0.617%)和空白对照组为(0.992%),转染组mRNA干扰效率达95%(P<0.05)；Gal-3蛋白表达明显下调、与阴性对照组和空白对照组相比差异明显(P<0.05),MTT显示：转染组为(0.40±3.69)%,阴性对照组(0.71±0.156)%；转染组和阴性对照组间差异均有统计学意义(P<0.05)。细胞凋亡百分率68.78%。6.在体外成功建立了垂体腺瘤细胞系的培养方法并完成了细胞系的体外培养。结论：1. Gal-3可作为一种新的术后判断侵袭性垂体泌乳素腺瘤和ACTH腺瘤的分子生物学标记。2.Gal-3结合高的核分裂像可以作为判断侵袭性垂体泌乳素腺瘤生物学行为的分子生物学标记3.应用显色原位杂交的方法能在侵袭性垂体泌乳素腺瘤病变原部位显示Gal-3的变化4.慢病毒载体的构建及靶点筛选,为研究Gal-3在肿瘤细胞中的作用提供实验平台5.慢病毒介导Gal-3-shRNA成功在体外敲减肿瘤细胞的目的蛋白,影响了肿瘤增殖凋亡。6.体外成功培养了具有独特形态的人垂体腺瘤细胞系更多还原

【Abstract】 1.To observe the expression of Galectin-3 in human pituitary adenomas2.Galectin-3 gene silencing was appliaed by RNAi3.To set up the cultured system of human pituitary adenoma lines4. hoping to evaluate its possible role in the mechanism of pituitary adenomas’invasion. To find a possible clinical marker of invasion. And to find a possible target of treatment of invasive adenomas.1 The expression of Galectin-3 in the human pituitary adenomas was detected by immunohistochemistry, and Galectin-3 is whose protein expression more strongly level was compared between patients of invasive and non-invasive adenoma;Galectin-3 is whose mRNA expression more strongly in invasion pituitary adenoma or noninvasion pituitary adenoma was detected by CISH.2 A high specific delivery system for siRNAs mediated by retrovirus vector was established. By this system, siRNAs can be integrated into the cell genome stably.We design and synthesized the shDNA, which can transcribe into shRNA and interfere with Galectin-3 gene. The shDNA duplex was ligated into the recombinant vector pGCL-GFP/U6 plasmid.the positive colonies of pGCL-GFP/U6 Gal-3shDNA-lwere selected by double restriction enzyme digestion with Hpa I and Xho I, Cell line was transfect with pGCL-GFP/U6 Gal-3shDNA-1 using pGCL-GFP/U6-s crambled shDNA as control. The expression level of Galectin-3 was detected by RT-PCR and Western Blot; then PI flow cytometric analysis were performed for apoptosis, MTT assay for cell proliferation.3 The prolactin adenoma derived cell-F cultured in vitro was in good condition and maintained the ability of proliferation and differentiation.1. The expression of Galectin-3 in the human pituitary prolactin adenomas and pituitary Adrenocorticotrophic hormone adenoma.2. In invasive adenomas, the Galectin-3 had a higher expression level than While in non-invasive adenomas, the difference was not so obvious.3. After when high the expression of Galectin-3-protein was detected by IHC method, CISH was used to detect the expression of Galectin-3-mRNA.4 A lentivirus RNAi vector containing shRNA targeting Galectin-3 gene was successfully constructed and be in efftct targeting Galectin-3 gene and will be used as method to develop Galectin-3 gene silencing therapeutics.5. The recombinant vector were successful constructed which was confirm by sequencing.The in vitro experiment indicated that the expression level of Galectin-3 were down regulated by western blot and RT-PCR analyses than control group (P<0.05); The interfering efficiency of Lentiviral was 95%, MTT assay showed that the proliferation of MCF-7 were suppress to(0.40±3.69)%than control group (0.71±0.156)%.FCM assay indicated that Lentiviral vector plasmid induce apoptosis of the cell about 68.78%.6 The cultured system of human prolactin pituitary adenoma derived cell-F was set up1. Galectin-3 might as a New molectin phyology marker of operation later invasion action of pituitary adenoma by judge2. Galectin-3 can judged phyology action of invasion prolactin pituitary adenoma3. Galectin-3 might have change in invasion pituitary adenoma was detected by chromogenic in situ hybridiza tion (CISH).4. A lentivirus RNAi vector containing shRNA targeting Galectin-3 gene was successfully constructed will be used as method to develop Galectin-3 gene silencing therapeutics.5. RNA interference can successful interfere with the expression of Galectin-3 in culture tumor cell and may affect the occurrence and development of the tumor.6. The cultured system of human prolactin pituitary adenoma derived cell-F have been set up.更多还原