【作者】 戴毓欣；

【导师】 郎景和；

【作者基本信息】 中国协和医科大学 ， 临床医学， 2008， 博士

【摘要】 ●研究目的:1.应用基因表达谱芯片,筛选绝经后POP患者与正常绝经妇女主韧带组织中差异表达的基因2.分析差异表达基因,探讨POP发生的分子机制●材料和方法:1.在绝经状态、年龄、体重指数、产次等影响POP发生的重要因素匹配的前提下,选取3例POP与3例无POP对照的子宫主韧带组织标本。2.组织标本行HE染色,Masson’s三色染色验证组织来源,并观察主韧带组织形态结构。3.Trizol法提取总RNA,并对总RNA进行定量、质检。采用RNA扩增标记法合成生物素标记的cRNA,与Human Genome U133 Plus 2.0芯片杂交。4.用SAM软件筛选|Score(d)|≥2,FC值大于2或小于0.5的差异表达基因。用MAS2.0软件对差异表达基因进行基因功能分类注释(GO)和代谢通路分析(Pathway)。5.实时荧光定量PCR验证DKK1,SFRP1,FZD5,WNT16,COL1A1的差异表达。●实验结果:1.标本行HE染色、Masson’s三色染色观察,证实为子宫主韧带组织。POP组女性子宫主韧带组织中胶原纤维与平滑肌束失去正常的排列,形态紊乱,可见胶原纤维断裂,平滑肌束细碎。2.提取总RNA经定量和电泳检测,质量满足芯片杂交要求。杂交后芯片的各项检测参数均达到质控要求,子宫主韧带组织与基因表达谱芯片的杂交模型建立成功。3.筛选出179个在POP组和对照组之间差异表达的基因。其中包括20个功能未知的基因。107个基因在POP组中表达上调,72个基因在POP组中表达下调。差异基因涉及多种功能蛋白和代谢通路。其中Wnt信号转导通路变化最显著。4.实时荧光定量PCR证实DKK1,SFRP1,FZD5,WNT16,COL1A1在POP组中表达明显升高,与芯片结果一致。●结论1.POP患者子宫主韧带组织中胶原纤维与平滑肌束细碎,排列紊乱。POP中编码Ⅰ、Ⅲ、Ⅴ型胶原α1链蛋白的基因COL1A1、COL3A1、COL5A1的mRNA水平和MMP2的mRNA水平明显升高,提示POP中胶原合成增加,分解亦增强。说明POP中胶原的合成和分解活跃,可能以分解为主,引起韧带支持结构功能的改变,导致POP发病。2.人类全基因组表达谱芯片是一种有效的高通量筛选POP差异表达基因的研究方法。本实验以|Score(d)|≥2,FC≥2或FC≤0.5为标准筛选出179个在POP组和对照组之间差异表达的基因。其中包括20个功能未知的基因。107个基因在POP组中表达上调,72个基因在POP组中表达下调。3.POP的病理生理过程复杂,涉及多种功能蛋白和代谢通路。其中Wnt信号转导通路的拮抗剂DKK1、SFRP1介导的神经退行性变可能与POP的发病密切相关。4.实时定量荧光PCR验证目标基因DKK1,SFRP1,FZD5,WNT16,COL1A1在POP组的表达明显高于对照组,变化趋势与芯片一致,佐证了芯片结果的可信性。更多还原

【Abstract】 ●OBJECTIVES1.To identify the differentially expressed genes in cardinal ligament between patients with pelvic organ prolapse and those without POP by Human Genome Expression Chip.2.To further explore the possible molecular mechanism associated with pelvic organ prolapse(POP) based on the differential expression profile.●METHODS1.Cardinal ligament samples were obtained from three postmenopausal patients with POP-Q stageⅢand three age/BMI/parity matched postmenopausal subjects without POP.2.HE and Masson’s trichrome staining was used to verify tissue origin and evaluate histological characteristics.3.Total RNA from samples were used to synthesize biotin-labeled cRNA by One-Cycle cDNA Synthesis method and hybridized with Human Genome Chip.4.The differentially expressed genes were identified between POP and normal controls with the threshold:Fold Change(FC)＞2.0 or＜0.5.The data was further interrogated with Gene Ontology(GO) and Pathway Analysis.5.Five upregulated genes were confirmed by quantitative reverse transcription-polymerase chain reaction.●RESULTS1.Alterations in ligament architecture seen in our population included disarrangement and collapse of smooth muscle bundles and collagen fibers. 2.The quality of total RNA was ensured by quantification and electrophoresis.The hybridization module of Human Genome expression chip was successfully established.3.179 differentially expressed genes were screened between POP and normal cardinal ligament tissue,including 20 function-unknown genes.107 genes were upregulated in POP group,while 72 downregulated.This differential expression profile was related to multiple functional proteins and pathways by biological analysis.Among these,Wnt signaling pathway was the most predominant.4.Quantitative PCR validated the upregulated expression of five genes.●CONCLUSIONS1.Ligament architecture in POP undergoes great alteration with disarrangement and collapse of smooth muscle bundles and collagen fibers.The mRNA elevations of COL1A1、COL3A1、COL5A1、MMP2 indicate an increase in collagen synthesis and breakdown.Degradation is predominant in collagen turnover and lead to ligament tissue defect.2.Genome Expression microarray is an effective approach for the high-throughput analysis of gene expression profile and aids to further explore the underlying molecular mechanism in POP.179 differentially expressed genes were screened between POP and normal cardinal ligament tissue,including 20 function-unknown genes.107 genes were upregulated in POP group,while 72 downregulated.3.The pathophysiology of POP is complex covering multiple functional proteins and pathways.Among these,Wnt signaling pathway was the most predominant and its antagonist DKK1,SFRP1 may contribute a neurodegenerative role in POP development.4.Quantitative PCR validated the upregulated expression of five genes from microarray data.更多还原