【作者】 何军；

【导师】 赵晓昆；

【作者基本信息】 中南大学 ， 外科学， 2007， 博士

【摘要】 研究背景尿道下裂(Hypospadias)是指前尿道发育不全，导致异位的尿道口位于正常尿道口的近端至会阴部的途径上，部分病例可伴有阴茎下弯畸形(附录2，图1，图2)。它是人类泌尿生殖系常见畸形之一，在新生儿中发病率为250／100，000左右，仅次于先天性心脏病。而且近年来有上升的趋势。尿道下裂的发病机理很复杂，目前尚未完全清楚，常认为是多因素作用的结果。目前已证实的引起尿道下裂的病因包括：基因突变、内分泌失调、异常的细胞间信息传递、雄激素受体异常、表皮生长因子表达低下和环境因素等。Petiot等在FGFR2-ⅢB-／-(fibroblast growth factor receptor 2-exonⅢB)的小鼠模型中发现FGFR2-ⅢB的缺失可以引起尿道下裂；FGFR2-ⅢB-／-小鼠尿道下裂模型的尿道发生信号诱导区有SHH(Sonic Hedgehog)和FGF8(Fibroblast growth factor 8)基因的表达，但在尿道管形成中，尿道上皮祖细胞不能维持更新、尿道上皮细胞过早成熟或成熟终止，导致尿道上皮断裂。实验表明氟他胺和雄激素竞争雄激素受体(androgen receptor；AR)可导致FGFR2-ⅢB和FGF10(Fibroblast growth factor 10)在尿道上皮中的表达丧失，往往伴随尿道下裂。推测在外生殖器发育过程中，这些基因可能是雄激素受体调控的下游靶基因。另外，一些遗传疾病如：比尔一史蒂文生皮肤回旋综合征(Beare-Stevenson cutis gyrata syndrome)，手裂一足裂综合征(Split Hand／Split Foot syndrome)，肢体乳腺综合征(limb-mammary syndrome)缺指畸形—外胚层发育异常—唇裂腭裂综合征(ectrodactyly-ectodermal dysplasia-cleft lip／palate，EEC)和海—韦综合征(Hay-Wells syndrome)等常合并泌尿生殖器畸形，临床特征往往表现外生殖器的发育障碍：如尿道下裂和阴囊对裂。在这些遗传疾病患者中常发现有FGFR2错义突变。此外，部分研究者推测其他遗传学因素和环境因素也参与了负相调控FGFR2途径。以上研究均提示尿道下裂的发病机理和FGFR2有密切关系。目前，尿道下裂的研究大都和环境因素、基因突变、激素调控、受体的异常(雌，雄激素受体异常)的研究相联系，涉及尿道上皮的发生、发育和其相关的分子病理机制及相关基因研究甚少。尿道上皮发生、发育和FGFR2关系密切。但FGFR2缺陷是否导致人类尿道下裂及其相关分子病理机制尚不明确。本实验从调控尿道上皮发生、发育的FGFR2着手，进行探索性研究。通过检测FGFR2蛋白和FGFR2mRNA在尿道下裂患者包皮组织中的表达，初步探究FGFR2和尿道下裂的关系，为进一步研究FGFR2基因功能和揭示尿道下裂的病因奠定基础。第一章FGFR2蛋白在尿道下裂包皮组织中的表达【目的】检测FGFR2蛋白在尿道下裂组和正常对照组包皮组织中的表达情况，初步探究FGFR2蛋白表达和尿道下裂的关系。【方法】采用免疫组化实验和Western blotting(免疫印迹)方法；【结果】免疫组化结果：FGFR2蛋白定位于包皮组织真皮层纤维细胞；正常对照组中包皮组织FGFR2蛋白表达的阳性率(20／24)高于尿道下裂组中包皮组织FGFR2蛋白表达的阳性率(12／30)(P＜0.05)，尿道下裂患者轻度、中度、重度三组之间FGFR2蛋白表达比较(P＞0.05)，无统计学差异。Western blotting检测结果：正常对照组FGFR2蛋白相对定量值0.39±0.12，尿道下裂组FGFR2蛋白相对定量值0.19±0.09(P＜0.05)，尿道下裂组较正常对照组降低。尿道下裂轻度、中度、重度三组间FGFR2蛋白相对定量值分别为0.27±0.08、0.16±0.04、0.09±0.03；在尿道下裂轻度、中度、重度三组中，中度和重度之间比较无统计学差异(P＞0.05)。【结论】1，FGFR2蛋白定位表达在包皮组织真皮层基底部角质化细胞层和纤维细胞，阳性结果是细胞胞浆染色；2，正常对照组包皮组织FGFR2表达呈大部分强阳性或阳性，尿道下裂患者包皮组织FGFR2表达大部分呈阴性；3，尿道下裂患者包皮组织中FGFR2蛋白较正常对照组包皮组织中表达下调。提示进一步研究尿道下裂和FGFR2 mRNA表达情况，有助于明确FGFR2基因在尿道下裂发病机理中的重要地位。第二章FGFR2在尿道下裂包皮组织中的转录水平【目的】检测尿道下裂患者FGFR2 mRNA表达情况，初步探究尿道下裂和FGFR2基因功能的关系。【方法】采用RT-PCR和Real time PCR方法；【结果】正常对照组FGFR2mRNA表达量为1.30±0.069，尿道下裂患者组FGFR2mRNA的表达量为1.22±0.052，正常对照组FGFR2mRNA表达量高于尿道下裂组(P＜0.05)。在尿道下裂轻度、中度和重度三组中，其中轻度和中度之间FGFR2mRNA的表达量之间无统计学差异(P＞0.05)，重度尿道下裂患者FGFR2mRNA表达量低于其它两组。【结论】：尿道下裂患者中FGFR2mRNA的表达下调，提示FGFR2基因和尿道下裂关系密切，进一步研究FGFR2基因功能有可能揭示尿道下裂的发病机理。更多还原

【Abstract】 Hypospadias was anterior urethral hypoplasia, resulting in normal urethral meatus ectopic proximal to the perineum way, some patients with malformation of chordee of penis. It was the one common Congenital malformations in human urogenital system, the incidence in newborns was 250/100,000.Second only to congenital heart disease. The incidence of hypospadias has an upward trend in recent years. Little was known about The etiology of hypspasias was complicated and was not well elucidated。multiple factors were involved in The genesis of hypospadias. It has been demonstrated that the etiology of hypospadias include: gene mutations, endocrine disturbance, intracellular sigal transmation , androgen receptor dysfunction and low expression of epidermal growth factor and environmental factors etc’.Petiot et al.’s reports had discovered that FGFR2-ⅢB absence can provoke hypospadias with animal model.In the FGFR2-ⅢB---/- rat model of hypospadias, urethral signaling regions, as indicated by SHH and FGF8 expression;however,cell proliferation arrests prematurely and maturation of the urethral epithelium is disrupted. FGFR2-ⅢB-/- mutants fail to maintain the progenitor cell population required for uroepithelial renewal during tubular morphogenesis. They show that flutamide antagonism of the androgen receptor (AR) leads to loss of FGFR2-ⅢB and FGF10 expression in the urethra, and an associated hypospadias phenotype, suggesting that these genes are downstream targets of AR during external genital development. In addition, Malformations of the urogenital tract also occur in Beare-Stevenson cutis gyrata syndrome,Split Hand/Split Foot syndrome, limb-mammary syndrome, ectrodactyly-ectodermal dysplasia-cleft lip/palate (EEC) and Hay-Wells syndrome,which was characterized by external genital defects such as hypospadias and bifid scrotum, And Mis-sense mutations in FGFR2 was found in these hereditary disease. Besides that ,by either genetic or environmental factors, may therefore involve negative regulation of the FGFR2 pathway. The studies above indicated that the etiology of hypospadias has intimate corelation with FGFR2 pathway.Currently, environmental factors, genetic mutation, hormone regulation, the abnormal receptor (ER,AR abnormal) most associated with the study of the hypospadias, involving epithelial the urethra, and its development and its associated molecular pathogenesis-related genes and little of this research. The between the development and growth of urethral epithelium and FGFR2 was dosed. But it is not well understood that whether FGFR2 defection lead to human hypospadias and the related molecule molecule mechanism.The expression of FGFR2 protein and mRNA was deteced in the preputial tissue of hypospadias patients and normal control in our exprement to explore the correlation between FGFR2 expression and hypospadias and to laid foundation for futher research of FGFR2 gene function in the pathogenesie of hypospadias.Chapter 1: The expression of tibroblast growth factor receptor 2 in patients’ preputial tissue with hypospadias[Objective] To Study the expression of fibroblast growth factor receptor 2 in patients of hypospadias group and the normal control group in preputial tissue for explore the relationships between fibroblast growth factor receptor 2 proteinum and hypospadias.[Methods] Immunohistochemistry and Western blotting technique was used to detect the expression of fibroblast growth factor receptor 2 in both groups.[Results] The expression of fibroblast growth factor receptor 2 in preputial tissue of normal and hypospadias was significantly (P＜0.05,). The expression of fibroblast growth factor receptor 2 in patients with hypospadias was weakened. Between the mild, moderate, severe three groups, the expression of fibroblast growth factor receptor 2 was no statistical significance (P＞0.05) .The FGFR2 protein located in the preputial tissue endochylema of the keratinocyte and fibroblast cell.The amount of protein of Western bloting resulted that the hypospadias patients preputial tissue fibroblast growth factor receptor 2 protein concentrations lower than the normal control group. Correspondence quantitation of FGFR2 protein was 0.39±0.12 in normal control group. Correspondence quantitation of FGFR2 protein was 0.19±0.09 in hypospadias group. Correspondence quantitation of FGFR2 protein was 0.27±0.08 in mild group, 0.16±0.04 in moderate group,0.09±0.03 in severe group . Between Hypospadias group and the normal control group was statistically significant(P＜0.05). In the three groups, no comparison between moderate and severe groups. (P＞0.05).[Conclusion]1, Positive result was coloration in the preputial tissue epidermis fibroblast cell and keratinize cell of basilar part in the preputial tissue.2, Immunohistochemistry : hadro- masculine and masculine in bulk normal preputial tissue, Negative in bulk hypospadias.3,The expression of FGFR2 of preputial tissue in hypospadias was down regulation than normal.The degression in hypospadias patients’s preputial tissue of the expression of fibroblast growth factor receptor 2 suggested that the relationship of fibroblast growth factor receptor 2 and hypospadias was closely. Further research of FGFR2 gene function in hypospadias may reveal the pathogenesis of hypospadias.Chapter 2 The expression of transcriptional level of Fibroblast growth factor receptor 2 in preputial tissue with hypospadias[Objective] To detect the expression of fibroblast growth factor receptor 2 mRNA in hypospadias patients’s preputial tissue. Exploration the relationships of hypospadias and fibroblast growth factor receptor 2 gene function.[Methods] We used RT-PCR and Real time PCR technique to detect the expression of fibroblast growth factor receptor 2 mRNA in both groups.[Results] The expression of hypospadias patients’s fibroblast growth factor receptor 2 mRNA was1.22±0.052,the normal wasl.30±0.069. Between the two groups was significant(P＜0.05). Mild and moderate of hypospadias in those groups ,the expression of fibroblast growth factor receptor 2 mRNA was no significant(P＞0.05). Based on the statistics, The expression of fibroblast growth factor receptor 2 mRNA in severe levels lower than the other two groups.[Conclusion] The degression of fibroblast growth factor receptor 2 in hypospadias patients.It cued that the relationship of fibroblast growth factor receptor 2 gene and hypospadias was closely.The further study of the fibroblast growth factor receptor 2 gene maybe explore the pathogenesy of hypospadias.更多还原