發(fā)布時間：2018-05-15 17:31

  本文選題：逼尿肌 + 粘連連接��； 參考：《大連醫(yī)科大學》2007年碩士論文

【摘要】： 目的:采用免疫組織化學方法及電鏡技術研究偶聯(lián)膀胱平滑肌細胞的粘連連接蛋白質構成,通過觀察粘連連接主要構成蛋白成分在下尿路梗阻并發(fā)膀胱過度活動患者中的變化,探討人逼尿肌粘連連接在膀胱不穩(wěn)定患者中的數(shù)量改變,進而分析粘連連接與膀胱過度活動之間的關系。揭示下尿路梗阻患者出現(xiàn)膀胱過度活動的肌源性發(fā)病機制,為臨床提供參考。 方法:隨機選取經(jīng)尿動力學檢查證實為逼尿肌不穩(wěn)定(detrusorinstability,DI)的BPH住院患者35例,為實驗組,年齡62-87歲,平均69.1歲;非梗阻性逼尿肌(取自膀胱癌患者)9例作為對照組,年齡57-80歲,平均66.3歲。均排除其它相關疾病。術前經(jīng)過IPSS評分。在擇期開放手術過程中獲得逼尿肌組織,并征得所有患者的書面同意(經(jīng)院倫理委員會同意)。用手術刀從膀胱前壁切取膀胱壁全層組織(約0.5×0.5×1.0cm大小),避免組織破壞和電烙造成假象。每個離體標本被立即分為兩份,一份約0.5cm×0.5cm大小,在1分鐘內被切割成約0.1cm×0.1cm的肌條存放入2%戊二醛中溶液中,用于電鏡觀察;另一份標本約0.5×0.5cm大小,存放入10%的福爾馬林中性溶液中保存。人膀胱上皮作為內部陽性對照,人正常闌尾組織、乳腺癌和子宮內膜癌組織作為已知的外部陽性對照。 標本固定好,將組織塊分別進行縱切與橫切,石蠟包埋,連續(xù)4μm切片,HE染色及用多克隆P-鈣粘蛋白抗體,單克隆α、β、和γ-連環(huán)蛋白抗體檢測,所有抗體都與粘連連接蛋白發(fā)生反應。 電鏡觀察實驗組和對照組逼尿肌細胞連接的超微結構。免疫組織化學方法測定各組標本中鈣粘蛋白、α-、β-、γ-連環(huán)蛋白的表達變化,每一逼尿肌至少評價切面上10個視野,計數(shù)可見粘連連接數(shù)量,并用計算機圖像分析系統(tǒng)對膀胱逼尿肌粘連連接構成蛋白的含量進行半定量分析,分別計算出每個樣本中鈣粘蛋白、α-、β-、γ-連環(huán)蛋白的平均含量。 分別進行以下比較: (1)實驗組與對照組平滑肌中粘連連接構成蛋白的含量; (2)穩(wěn)定膀胱逼尿肌與伴有膀胱過度活動逼尿肌中粘連連接的數(shù)量; (3)實驗組平滑肌粘連連接各構成蛋白量的變化; 結果: 1.經(jīng)免疫組織化學染色后光學顯微鏡觀察并經(jīng)計算機圖像分析系統(tǒng)進行半定量測定 (1)在非梗阻及伴有膀胱過度活動的逼尿肌中均檢測到鈣粘蛋白、及α、β、和γ-連環(huán)蛋白的特異性染色;并且檢測到平滑肌細胞間粘連連接的特異性染色。 (2)實驗組(DI)平滑肌切片中P-鈣粘蛋白和α、β、和γ-連環(huán)蛋白著色較淺并且不均勻,分布不規(guī)則。逼尿肌細胞間粘連連接明顯減少,著色淺,周圍見較多雜亂纖維。對照組中非梗阻逼尿肌切片著色較深并且分布均勻,見大量特異性濃染的P-鈣粘蛋白和α、β、和γ-連環(huán)蛋白,肌細胞排列規(guī)則,細胞間大量粘連連接,連接著色較深,肌細胞形態(tài)規(guī)則,連接集中。實驗組粘連連接蛋白的數(shù)量與對照組相比明顯減少(P0.01)。 (3)實驗組和對照組粘連連接的數(shù)量低于各組內膀胱上皮粘連連接的數(shù)量(P0.01)。 2.在電鏡下觀察 (1)實驗組(DI)肌纖維排列不整齊,肌細胞分布不均勻,肌膜周圍有高電子密度物質堆積,細胞間以胞突連接及縫隙連接(gapjunction,GP)為主;細胞間隙明顯增寬,可見大量膠原纖維;平滑肌細胞間連接明顯減少或消失,逼尿肌細胞出現(xiàn)肥大,線粒體腫脹、破裂; (2)對照組平滑肌纖維排列整齊,肌細胞分布均勻,肌細胞間隙細胞之間連接以粘連連接(adherensjunction,AJ)為主,細胞間的彈性纖維和膠原纖維含量較少,逼尿肌細胞內結構完整,排列規(guī)則。 結論: 1膀胱平滑肌細胞間粘連連接中含有P-鈣粘蛋白和α、β、和γ-連環(huán)蛋白; 2下尿路梗阻患者逼尿肌粘連連接構成蛋白的數(shù)量較正常減少; 3膀胱功能改變與膀胱逼尿肌粘連連接的數(shù)量變化有關。
[Abstract]:Objective: To study the protein composition of the adhesion and connection of the smooth muscle cells of the bladder by immunohistochemistry and electron microscopy, and to observe the changes of the main components of the adhesive connection in the patients with lower urinary tract obstruction and bladder overactivity. The relationship between adhesion and overactivity of bladder is analyzed. The myogenic mechanism of overactivity of bladder in patients with lower urinary tract obstruction is revealed, which provides reference for clinical practice.
Methods: 35 cases of BPH hospitalized patients with detrusorinstability (DI) were randomly selected as the experimental group. The experimental group was 62-87 years old and 69.1 years old. 9 cases of non obstructive detrusor (from the bladder cancer patients) were used as the control group, the age 57-80 years old and the average 66.3 years old. All the other related diseases were excluded. IPSS evaluation before operation was carried out. Detrusor tissue was obtained during elective open surgery, and the written consent of all patients (agreed by the Institute of ethics committee) was obtained. The whole bladder wall of the bladder wall was cut from the anterior wall of the bladder (about 0.5 x 0.5 x 1.0cm size) with a scalpel. The tissue damage and electrocautery were avoided. Each isolated specimen was immediately divided into two copies, one of which was about 0.5cm X. 0.5cm size, the muscle strips that were cut into about 0.1cm x 0.1cm within 1 minutes were stored in a solution of 2% glutaraldehyde and used for electron microscopy. The other specimen was about 0.5 * 0.5cm in size and stored in 10% formalin neutral solution. Human bladder epithelium was used as an internal positive control, human appendiceal, breast and endometrial cancer tissues were used as the human normal. A known external positive control.
The specimens were fixed, the tissue blocks were cut and cross cut, paraffin embedded, continuous 4 m slices, HE staining and polyclonal P- cadherin antibody, monoclonal alpha, beta, and gamma catenin antibody were detected, all the antibodies were reacted with adhesion connexin.
The ultrastructure of the detrusor cell connection in the experimental group and the control group was observed by electron microscopy. The changes in the expression of cadherin, alpha, beta, and gamma catenin were measured by immunohistochemical method. At least 10 fields of visual field were evaluated by each detrusor. The number of visible adhesion connections was counted, and the system of computer image analysis was used for the detrusor of the bladder. The content of protein was determined by semi quantitative analysis. The average content of cadherin, alpha -, beta - and gamma - Catenin in each sample was calculated.
The following comparisons are made, respectively.
(1) the adhesion between the experimental group and the control group was composed of protein.
(2) to stabilize the number of detrusor detrusor and bladder hyperactivity detrusor adhesion.
(3) the changes of smooth muscle adhesion and protein content in the experimental group.
Result:
1. after immunohistochemical staining, it was observed by optical microscope and semi quantitatively determined by computer image analysis system.
(1) the specific staining of cadherin, alpha, beta, and gamma catenin was detected in the detrusor with non obstruction and hyperactivity of bladder, and the specific staining of adhesion and connection between smooth muscle cells was detected.
(2) in the smooth muscle slices of the experimental group (DI), the P- cadherin and alpha, beta, and gamma catenin were coloured relatively shallow and uneven, and the distribution of the detrusor was obviously reduced. The coloring of the detrusor was shallow, and more disorderly fibers were found around it. The non obstructive detrusor slices in the control group were darker and evenly distributed, and a large number of specific concentrated P- calcium were found. Mucin and alpha, beta, and gamma catenin, the myocytes were arranged regularly, a large number of adhesions were attached to the cells, the joint coloring was deep, the morphology of the muscle cells was regular, and the connection concentration was concentrated. The number of adhesion connexins in the experimental group decreased significantly compared with the control group (P0.01).
(3) the number of adhesions in the experimental group and the control group was lower than that in each group (P0.01).
2. observation under electron microscope
(1) the muscle fibers in the experimental group (DI) had irregular arrangement of muscle fibers, uneven distribution of myocytes, high electron density materials around the musculi membrane, cell intercellular connections and gap junctions (GAPJUNCTION, GP), the intercellular space obviously widened, and a large number of collagen fibers were visible; the connection between the smooth muscle cells obviously decreased or disappeared, and the detrusor cells appeared hypertrophy. Swelling and rupture of the granules.
(2) the smooth muscle fibers in the control group were arranged neatly, the muscle cells were evenly distributed, the intercellular space cells were connected by adhesion connection (adherensjunction, AJ), and the content of the elastic fibers and collagen fibers between the cells was less. The structure of the detrusor cells was complete and arranged regularly.
Conclusion:
1 there are P- cadherin, alpha, beta and gamma catenin in bladder smooth muscle cell adhesion.
2 the number of detrusor adhesion junction proteins in patients with lower urinary tract obstruction is less than normal.
3 the change of bladder function is related to the quantity change of bladder detrusor adhesion.

【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R361

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