压力性尿失禁大鼠模型的建立及耻尾肌和泌尿生殖道组织PGP9.5、NPY的表达及意义

发布时间：2018-05-01 10:09

本文选题：压力性尿失禁 + 耻尾肌　；参考：《郑州大学》2012年硕士论文

【摘要】：压力性尿失禁(stress urinary incontinence,SUI)是女性常见疾病,根据大样本流行病学调查显示,约12.5%女性患有SUI,尤其中老年女性发病率较高约10%-27%,并常合并盆腔生殖器官的膨出或脱垂(Pelvic Organ Prolapse, POP),严重影响患者的生活质量和身心健康。根据国际控尿协会(International Continence Society, ICS)的定义,女性SUI是指腹压高于最大尿道压时,在无膀胱逼尿肌收缩状态下,尿液不自主地由尿道外口溢出,多在咳嗽、打喷嚏、直立、劳累等腹压增高时发生。压力性尿失禁的发生机制现尚不明确。临床研究表明SUI的发病与盆底组织的解剖缺陷和功能障碍有关。另外,被大多数专家学者认同的关于SUI发病机制“完整理论”及“吊床假说”都强调了盆底支持组织在维持控尿过程及控尿系统结构中的重要作用。其中,盆底支持组织神经损伤可能是SUI主要发病机制之一。对于SUI神经损伤机制的研究,国内外研究学者多集中在盆底肌电图和神经传导速度等电生理方面的研究,而本研究意在建立有效的SUI动物模型,并在此基础上,从分子生物学角度,通过观察神经纤维标记物蛋白基因产物9.5(Protein gene product9.5,PGP9.5)和神经肽类递质神经肽Y(Neuropeptide Y,NPY)在SUI模型盆底肌肉及泌尿生殖结构中的变化情况,以及盆底结构功能障碍的微观表现,为SUI神经损伤理论提供进一步的证据。目的 1.研究模拟女性分娩损伤、难产、密产、绝经后低雌激素水平等SUI发生危险因素,根据检测尿流动力学指标和喷嚏实验,建立SUI动物模型的可行性和稳定性。 2.观察SUI大鼠耻尾肌和泌尿生殖结构组织形态学变化,并通过探寻蛋白基因产物9.5(PGP9.5)及神经肽Y(NPY)的分布变化为SUI盆底失神经支配学说寻找进一步证据。为进一步寻求保守治疗SUI奠定基础。材料和方法 1材料实验动物郑州大学实验动物中心提供健康成年Sprague-Dawley雌鼠45只,重约180-220g,随机分为两组：实验组25只,对照组20只。Spraue-Dawley雄鼠5只，，重220-280g。 2方法： 2.1SUI模型建立妊娠SD大鼠分娩后通过两次扩张阴道模拟产伤及密产造成的损伤,并通过手术切除双侧卵巢模拟绝经,最终获得SUI模型大鼠共15例(经过尿动力学监测和喷嚏实验确定,见表1)。另有同等饲养条件下正常对照大鼠20例。 2.2采用硬膜外导管尿道置管方法,检测尿动力学指标最大膀胱容量、腹压漏尿点压力,并根据喷嚏实验,共同检验模型是否建立成功。 2.3处死大鼠,取SUI模型大鼠及正常大鼠的耻尾肌、阴道前壁及尿道,通过HE染色,观察耻尾肌及泌尿生殖道组织的病理形态学变化；并采用免疫组化的方法检测标本中PGP9.5.NPY表达情况。 3结果判定 HE染色观察耻尾肌、阴道前壁及尿道形态肌肉含量,肌纤维形态。PGP9.5广泛分布于中枢与周围神经系统神经元、神经纤维中,可特异性的标记神经纤维及小神经束。免疫组织化学染色阳性区域显色为棕黄色至棕褐色。NPY免疫反应性神经纤维主要分布在血管平滑肌,免疫组织化学染色阳性区域显色为棕黄色至棕褐色。采用平均灰度值对各指标阳性表达染色区域的染色强度进行分析比较。 4统计学方法采用SPSS13.0统计学分析软件进行统计学处理,计量资料采用均数±标准差((?)±s)表示；两组组间比较采用配对样本t检验方法,计数资料比较采用x2检验。统计检验水准均取α=0.05。结果 1SUI模型建立情况模拟人类妊娠分娩损伤、难产、密产以及绝经后低雌激素水平可成功获得稳定的动物模型。模型组大鼠与对照组大鼠的最大膀胱容量标准差为(1.38±0.11vs.2.13±0.85)ml,模型组明显低于对照组,差异有统计学意义(t=24.139,P=-0.001)；漏尿点压力值标准差为(10.54±0.47vs.28.43±0.42)mmHg,模型组明显低于对照组,差异有统计学意义(t=88.604,P=0.001)。喷嚏实验检测共发现15只模型组大鼠喷嚏反射阳性,而对照组未见喷嚏反射阳性大鼠,差异有统计学意义(χ2=29.679,P=0.001)。 2SUI组耻尾肌及阴道前壁及尿道壁经HE染色后与各自对照组进行比较 (1)对照组耻尾肌肌纤维平行排列,其间有适当间隙,着色均匀,肌纤维膜完整；SUI组耻尾肌肌纤维明显萎缩,排列稀疏紊乱,着色不均匀,可见肌原纤维走行弯曲断裂及变性改变。 (2)对照组阴道前壁镜下可见上皮层和固有层,固有层主要为结缔组织,其中可见平滑肌组织、小神经组织、小血管和毛细血管等结构。固有层中平滑肌粗大、排列较整齐；SUI组阴道前壁固有层结缔组织排列稀疏,平滑肌比较细碎稀疏、排列紊乱,肌束间隙变宽,肌纤维出现断裂现象,神经组织含量减少。 (3)对照组尿道平滑肌为环形排列,与成纤维细胞、胶原纤维及其含有的血管神经等结构紧密相连,胶原为粉红色物质,呈束状均匀排列；SUI组尿道平滑肌细胞,胶原纤维排列稀疏变细,密度减低,呈灶性排列束间含少量血管神经等成分。 3经免疫组化和图像分析 PGP9.5和NPY在耻尾肌、阴道前壁及尿道壁中均有表达,切片中PGP9.5和NPY免疫组织化学染色阳性区域显色为棕黄色至棕褐色。 (1) SUI模型组与对照组相比较,SUI组耻尾肌中的PGP9.5低表达(112.01±6.83vs.131.71±5.05),差异有统计学意义(t=9.118,P=0.001)；SUI组耻尾肌中的NPY低表达(131.71±3.42vs.145.05±7.32),差异有统计学意义(t=8.292,P=0.001）。 (2)阴道前壁黏膜切片,可见阴道黏膜固有层内有神经纤维及小神经束分布,SUI组与对照组相比,SUI组中PGP9.5低表达(119.97±6.95vs.134.09±5.25),差异有统计学意义(t=7.349,P=0.001)；SUI组中NPY低表达（113.08±5.96vs.140.43±4.88),差异有统计学意义(t=18.321,P=0.001)。 (3)尿道粘膜上皮层及固有层均有神经纤维及小神经束分布,SUI组与对照组相比,SUI组中PGP9.5低表达(123.68±4.58vs.139.98±4.90),差异有统计学意义(t=6.990,P=0.001)；SUI组中NPY低表达(115.13±2.38vs.128.67±1.73),差异有统计学意义（t=5.781,P=0.001)。结论 1模拟分娩损伤、难产、密产以及绝经后低雌激素水平可成功获得稳定、有效的大鼠SUI模型。 2SUI组盆底组织功能和结构的改变以及PGP9.5和NPY低表达,提示盆底组织神经损伤可能参与了SUI的发病。
[Abstract]:According to the definition of International Society of Urology ( ICS ) , the pathogenesis of stress urinary incontinence is not clear .

1 . To study the risk factors such as female labor injury , dystocia , dense labor and low estrogen level in postmenopausal women , and to establish the feasibility and stability of the animal model based on the detection of urinary flow dynamics and the experiments .

2 . To observe the morphological changes of the rat tail muscle and the urinary reproductive structure , and find further evidence through finding out the distribution of the protein gene product 9.5 ( PGP9.5 ) and the neuropeptides Y ( NPY ) .

Materials and Methods

1 Material

Forty - five healthy adult Sprague - Dawley rats weighing about 180 - 220g were randomly divided into two groups : experimental group ( n = 25 ) , control group ( n = 20 ) , sprague - Dawley male ( 5 , 220 - 280 g ) .

2 Method :

2 . The model was established to simulate the damage caused by two dilatation of vagina after giving birth to pregnant SD rats , and by surgical resection of bilateral ovaries to simulate the menopause , 15 of the model rats were finally obtained ( determined by the experiment of urodynamic monitoring and analysis , see Table 1 ) . There were 20 normal control rats in the same feeding condition .

2.2 The urethral catheterization method of epidural catheter was used to detect the maximum urinary bladder volume , abdominal pressure leakage and urine point pressure of the urinary dynamic index , and whether the model was successful was established according to the experiment .

2 . The pathological changes of the tail muscle , the anterior vaginal wall and the urethra of the rats and normal rats were sacrificed by HE staining .
The expression of PGP9.5 . NPY was detected by immunohistochemistry .

3 . Determination of results

The results showed that the color of NPY immunoreactive nerve fibers was brown - yellow to brown - brown . NPY immunoreactive nerve fibers were mainly distributed in the smooth muscle of vascular smooth muscle and the positive regions of immunohistochemical staining were brown - yellow to brown - brown .

4 Statistical Methods

SPSS 13.0 was used to analyze the statistical analysis software , and the mean 卤 standard deviation ( 卤 s ) was used to measure the data .
The paired t - test method was used for the comparison between the two groups , and x2 test was used to count the data . The statistical test level was taken as 伪 = 0.05 .

Results

Establishment of the Model 1sui Model

The standard deviation of the maximal urinary bladder capacity was ( 1.38 卤 0.11 vs . 2.13 卤 0.85 ) ml and the model group was significantly lower than that of the control group ( t = 24.139 , P = - 0.001 ) .
The standard deviation of urine leakage point pressure was ( 10.54 卤 0.47 vs . 28.43 卤 0.42 ) mmHg , and the model group was significantly lower than that of the control group ( t = 88.604 , P = 0.001 ) . A total of 15 model groups were found to be reflex - positive in 15 models , while the control group didn ' t see the reflex - positive rats . The difference was statistically significant ( 蠂2 = 29.679 , P = 0.001 ) .

After HE staining , the vaginal wall and anterior vaginal wall and anterior vaginal wall were compared with the control group .

( 1 ) In the control group , the myofibers were arrayed in parallel , with proper clearance , uniform color and intact muscle fiber membrane .
There were obvious atrophy , sparse irregularity and non - uniform staining of myofibers in the group of patients with the group , and it can be seen that the bending and degeneration of myofibrils were changed .

( 2 ) The epithelium and lamina propria were visible under the anterior vaginal wall of the control group . The lamina propria was mainly connective tissue , including smooth muscle tissue , small nerve tissue , small blood vessel and capillary vessel .
The connective tissue of the anterior vaginal wall was sparse , the smooth muscle was sparse , the arrangement was disturbed , the gap of the muscle bundle was widened , the muscle fiber appeared to break , and the content of nerve tissue was decreased .

( 3 ) The control group urethral smooth muscle was ring - shaped , which was closely linked with fibroblasts , collagen fibers and vascular nerves contained in the control group , and the collagen was pink substance , which was uniformly arranged in a bundle shape ;
The urethral smooth muscle cells and the collagen fibers were thinned , the density was reduced , and a small number of vascular nerves were included in the focal bundle .

3 Immunohistochemical and image analysis

The expression of PGP9.5 and NPY was expressed in both the anterior vaginal wall and the anterior vaginal wall . The positive regions of PGP9.5 and NPY were brown - yellow to brown - brown .

( 1 ) Compared with the control group , the low expression of PGP9.5 ( 112 . 01 卤 6 . 83 vs . 131 . 71 卤 5 . 05 ) was found to be statistically significant ( t = 9.118 , P = 0.001 ) .
The low expression of NPY was ( 131.71 卤 3.42vs . 145.05 卤 7.32 ) in the disgraceful tail muscle of the group , and the difference was statistically significant ( t = 8.292 , P = 0.001 ) .

( 2 ) In the anterior vaginal mucosa , the distribution of nerve fibers and small nerve bundles was found in the lamina propria of vagina , and the difference was statistically significant ( t = 7.349 , P = 0.001 ) compared with the control group .
There was a significant difference between NPY and NPY levels in the treatment group ( 13.08 卤 5.96 vs . 140.43 卤 4.88 ) ( t = 18.321 , P = 0.001 ) .

( 3 ) There were nerve fibers and small nerve bundles in the upper cortex and the lamina propria of the urethra mucosa , and the difference was statistically significant ( t = 6.990 , P = 0.001 ) compared with the control group .
There was a significant difference in NPY expression ( 115 . 13 卤 2 . 38 vs . 128 . 67 卤 1 . 73 ) in the group with stress ( t = 5.781 , P = 0.001 ) .

Conclusion

1 . Simulated labor injury , difficult labor , dense production and low estrogen level after menopause can be used to obtain stable and effective model of female stress in rats .

The changes of pelvic floor tissue function and structure and the low expression of PGP9.5 and NPY suggested that the pelvic floor tissue nerve injury might be involved in the pathogenesis .

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R-332;R694.5

【参考文献】