本文選題：壓力性尿失禁 + 恥尾肌　； 參考：《鄭州大學(xué)》2012年碩士論文

【摘要】：壓力性尿失禁(stress urinary incontinence,SUI)是女性常見疾病,根據(jù)大樣本流行病學(xué)調(diào)查顯示,約12.5%女性患有SUI,尤其中老年女性發(fā)病率較高約10%-27%,并常合并盆腔生殖器官的膨出或脫垂(Pelvic Organ Prolapse, POP),嚴(yán)重影響患者的生活質(zhì)量和身心健康。根據(jù)國(guó)際控尿協(xié)會(huì)(International Continence Society, ICS)的定義,女性SUI是指腹壓高于最大尿道壓時(shí),在無(wú)膀胱逼尿肌收縮狀態(tài)下,尿液不自主地由尿道外口溢出,多在咳嗽、打噴嚏、直立、勞累等腹壓增高時(shí)發(fā)生。壓力性尿失禁的發(fā)生機(jī)制現(xiàn)尚不明確。臨床研究表明SUI的發(fā)病與盆底組織的解剖缺陷和功能障礙有關(guān)。另外,被大多數(shù)專家學(xué)者認(rèn)同的關(guān)于SUI發(fā)病機(jī)制“完整理論”及“吊床假說”都強(qiáng)調(diào)了盆底支持組織在維持控尿過程及控尿系統(tǒng)結(jié)構(gòu)中的重要作用。其中,盆底支持組織神經(jīng)損傷可能是SUI主要發(fā)病機(jī)制之一。對(duì)于SUI神經(jīng)損傷機(jī)制的研究,國(guó)內(nèi)外研究學(xué)者多集中在盆底肌電圖和神經(jīng)傳導(dǎo)速度等電生理方面的研究,而本研究意在建立有效的SUI動(dòng)物模型,并在此基礎(chǔ)上,從分子生物學(xué)角度,通過觀察神經(jīng)纖維標(biāo)記物蛋白基因產(chǎn)物9.5(Protein gene product9.5,PGP9.5)和神經(jīng)肽類遞質(zhì)神經(jīng)肽Y(Neuropeptide Y,NPY)在SUI模型盆底肌肉及泌尿生殖結(jié)構(gòu)中的變化情況,以及盆底結(jié)構(gòu)功能障礙的微觀表現(xiàn),為SUI神經(jīng)損傷理論提供進(jìn)一步的證據(jù)。目的 1.研究模擬女性分娩損傷、難產(chǎn)、密產(chǎn)、絕經(jīng)后低雌激素水平等SUI發(fā)生危險(xiǎn)因素,根據(jù)檢測(cè)尿流動(dòng)力學(xué)指標(biāo)和噴嚏實(shí)驗(yàn),建立SUI動(dòng)物模型的可行性和穩(wěn)定性。 2.觀察SUI大鼠恥尾肌和泌尿生殖結(jié)構(gòu)組織形態(tài)學(xué)變化,并通過探尋蛋白基因產(chǎn)物9.5(PGP9.5)及神經(jīng)肽Y(NPY)的分布變化為SUI盆底失神經(jīng)支配學(xué)說尋找進(jìn)一步證據(jù)。為進(jìn)一步尋求保守治療SUI奠定基礎(chǔ)。 材料和方法 1材料 實(shí)驗(yàn)動(dòng)物鄭州大學(xué)實(shí)驗(yàn)動(dòng)物中心提供健康成年Sprague-Dawley雌鼠45只,重約180-220g,隨機(jī)分為兩組：實(shí)驗(yàn)組25只,對(duì)照組20只。Spraue-Dawley雄鼠5只，，重220-280g。 2方法： 2.1SUI模型建立妊娠SD大鼠分娩后通過兩次擴(kuò)張陰道模擬產(chǎn)傷及密產(chǎn)造成的損傷,并通過手術(shù)切除雙側(cè)卵巢模擬絕經(jīng),最終獲得SUI模型大鼠共15例(經(jīng)過尿動(dòng)力學(xué)監(jiān)測(cè)和噴嚏實(shí)驗(yàn)確定,見表1)。另有同等飼養(yǎng)條件下正常對(duì)照大鼠20例。 2.2采用硬膜外導(dǎo)管尿道置管方法,檢測(cè)尿動(dòng)力學(xué)指標(biāo)最大膀胱容量、腹壓漏尿點(diǎn)壓力,并根據(jù)噴嚏實(shí)驗(yàn),共同檢驗(yàn)?zāi)Ｐ褪欠窠⒊晒Α?2.3處死大鼠,取SUI模型大鼠及正常大鼠的恥尾肌、陰道前壁及尿道,通過HE染色,觀察恥尾肌及泌尿生殖道組織的病理形態(tài)學(xué)變化；并采用免疫組化的方法檢測(cè)標(biāo)本中PGP9.5.NPY表達(dá)情況。 3結(jié)果判定 HE染色觀察恥尾肌、陰道前壁及尿道形態(tài)肌肉含量,肌纖維形態(tài)。PGP9.5廣泛分布于中樞與周圍神經(jīng)系統(tǒng)神經(jīng)元、神經(jīng)纖維中,可特異性的標(biāo)記神經(jīng)纖維及小神經(jīng)束。免疫組織化學(xué)染色陽(yáng)性區(qū)域顯色為棕黃色至棕褐色。NPY免疫反應(yīng)性神經(jīng)纖維主要分布在血管平滑肌,免疫組織化學(xué)染色陽(yáng)性區(qū)域顯色為棕黃色至棕褐色。采用平均灰度值對(duì)各指標(biāo)陽(yáng)性表達(dá)染色區(qū)域的染色強(qiáng)度進(jìn)行分析比較。 4統(tǒng)計(jì)學(xué)方法 采用SPSS13.0統(tǒng)計(jì)學(xué)分析軟件進(jìn)行統(tǒng)計(jì)學(xué)處理,計(jì)量資料采用均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示；兩組組間比較采用配對(duì)樣本t檢驗(yàn)方法,計(jì)數(shù)資料比較采用x2檢驗(yàn)。統(tǒng)計(jì)檢驗(yàn)水準(zhǔn)均取α=0.05。 結(jié)果 1SUI模型建立情況 模擬人類妊娠分娩損傷、難產(chǎn)、密產(chǎn)以及絕經(jīng)后低雌激素水平可成功獲得穩(wěn)定的動(dòng)物模型。模型組大鼠與對(duì)照組大鼠的最大膀胱容量標(biāo)準(zhǔn)差為(1.38±0.11vs.2.13±0.85)ml,模型組明顯低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=24.139,P=-0.001)；漏尿點(diǎn)壓力值標(biāo)準(zhǔn)差為(10.54±0.47vs.28.43±0.42)mmHg,模型組明顯低于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(t=88.604,P=0.001)。噴嚏實(shí)驗(yàn)檢測(cè)共發(fā)現(xiàn)15只模型組大鼠噴嚏反射陽(yáng)性,而對(duì)照組未見噴嚏反射陽(yáng)性大鼠,差異有統(tǒng)計(jì)學(xué)意義(χ2=29.679,P=0.001)。 2SUI組恥尾肌及陰道前壁及尿道壁經(jīng)HE染色后與各自對(duì)照組進(jìn)行比較 (1)對(duì)照組恥尾肌肌纖維平行排列,其間有適當(dāng)間隙,著色均勻,肌纖維膜完整；SUI組恥尾肌肌纖維明顯萎縮,排列稀疏紊亂,著色不均勻,可見肌原纖維走行彎曲斷裂及變性改變。 (2)對(duì)照組陰道前壁鏡下可見上皮層和固有層,固有層主要為結(jié)締組織,其中可見平滑肌組織、小神經(jīng)組織、小血管和毛細(xì)血管等結(jié)構(gòu)。固有層中平滑肌粗大、排列較整齊；SUI組陰道前壁固有層結(jié)締組織排列稀疏,平滑肌比較細(xì)碎稀疏、排列紊亂,肌束間隙變寬,肌纖維出現(xiàn)斷裂現(xiàn)象,神經(jīng)組織含量減少。 (3)對(duì)照組尿道平滑肌為環(huán)形排列,與成纖維細(xì)胞、膠原纖維及其含有的血管神經(jīng)等結(jié)構(gòu)緊密相連,膠原為粉紅色物質(zhì),呈束狀均勻排列；SUI組尿道平滑肌細(xì)胞,膠原纖維排列稀疏變細(xì),密度減低,呈灶性排列束間含少量血管神經(jīng)等成分。 3經(jīng)免疫組化和圖像分析 PGP9.5和NPY在恥尾肌、陰道前壁及尿道壁中均有表達(dá),切片中PGP9.5和NPY免疫組織化學(xué)染色陽(yáng)性區(qū)域顯色為棕黃色至棕褐色。 (1) SUI模型組與對(duì)照組相比較,SUI組恥尾肌中的PGP9.5低表達(dá)(112.01±6.83vs.131.71±5.05),差異有統(tǒng)計(jì)學(xué)意義(t=9.118,P=0.001)；SUI組恥尾肌中的NPY低表達(dá)(131.71±3.42vs.145.05±7.32),差異有統(tǒng)計(jì)學(xué)意義(t=8.292,P=0.001）。 (2)陰道前壁黏膜切片,可見陰道黏膜固有層內(nèi)有神經(jīng)纖維及小神經(jīng)束分布,SUI組與對(duì)照組相比,SUI組中PGP9.5低表達(dá)(119.97±6.95vs.134.09±5.25),差異有統(tǒng)計(jì)學(xué)意義(t=7.349,P=0.001)；SUI組中NPY低表達(dá)（113.08±5.96vs.140.43±4.88),差異有統(tǒng)計(jì)學(xué)意義(t=18.321,P=0.001)。 (3)尿道粘膜上皮層及固有層均有神經(jīng)纖維及小神經(jīng)束分布,SUI組與對(duì)照組相比,SUI組中PGP9.5低表達(dá)(123.68±4.58vs.139.98±4.90),差異有統(tǒng)計(jì)學(xué)意義(t=6.990,P=0.001)；SUI組中NPY低表達(dá)(115.13±2.38vs.128.67±1.73),差異有統(tǒng)計(jì)學(xué)意義（t=5.781,P=0.001)。 結(jié)論 1模擬分娩損傷、難產(chǎn)、密產(chǎn)以及絕經(jīng)后低雌激素水平可成功獲得穩(wěn)定、有效的大鼠SUI模型。 2SUI組盆底組織功能和結(jié)構(gòu)的改變以及PGP9.5和NPY低表達(dá),提示盆底組織神經(jīng)損傷可能參與了SUI的發(fā)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 鹵 standard deviation ( 鹵 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 偽 = 0.05 .

Results

Establishment of the Model 1sui Model

The standard deviation of the maximal urinary bladder capacity was ( 1.38 鹵 0.11 vs . 2.13 鹵 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 鹵 0.47 vs . 28.43 鹵 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 鹵 6 . 83 vs . 131 . 71 鹵 5 . 05 ) was found to be statistically significant ( t = 9.118 , P = 0.001 ) .
The low expression of NPY was ( 131.71 鹵 3.42vs . 145.05 鹵 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 鹵 5.96 vs . 140.43 鹵 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 鹵 2 . 38 vs . 128 . 67 鹵 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 .

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R-332;R694.5

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