逼尿肌不稳定粘连连接超微结构变化的免疫组化监测和电镜观察

发布时间：2018-05-15 17:31

本文选题：逼尿肌 + 粘连连接　；参考：《大连医科大学》2007年硕士论文

【摘要】： 目的:采用免疫组织化学方法及电镜技术研究偶联膀胱平滑肌细胞的粘连连接蛋白质构成,通过观察粘连连接主要构成蛋白成分在下尿路梗阻并发膀胱过度活动患者中的变化,探讨人逼尿肌粘连连接在膀胱不稳定患者中的数量改变,进而分析粘连连接与膀胱过度活动之间的关系。揭示下尿路梗阻患者出现膀胱过度活动的肌源性发病机制,为临床提供参考。方法:随机选取经尿动力学检查证实为逼尿肌不稳定(detrusorinstability,DI)的BPH住院患者35例,为实验组,年龄62-87岁,平均69.1岁;非梗阻性逼尿肌(取自膀胱癌患者)9例作为对照组,年龄57-80岁,平均66.3岁。均排除其它相关疾病。术前经过IPSS评分。在择期开放手术过程中获得逼尿肌组织,并征得所有患者的书面同意(经院伦理委员会同意)。用手术刀从膀胱前壁切取膀胱壁全层组织(约0.5×0.5×1.0cm大小),避免组织破坏和电烙造成假象。每个离体标本被立即分为两份,一份约0.5cm×0.5cm大小,在1分钟内被切割成约0.1cm×0.1cm的肌条存放入2%戊二醛中溶液中,用于电镜观察;另一份标本约0.5×0.5cm大小,存放入10%的福尔马林中性溶液中保存。人膀胱上皮作为内部阳性对照,人正常阑尾组织、乳腺癌和子宫内膜癌组织作为已知的外部阳性对照。标本固定好,将组织块分别进行纵切与横切,石蜡包埋,连续4μm切片,HE染色及用多克隆P-钙粘蛋白抗体,单克隆α、β、和γ-连环蛋白抗体检测,所有抗体都与粘连连接蛋白发生反应。电镜观察实验组和对照组逼尿肌细胞连接的超微结构。免疫组织化学方法测定各组标本中钙粘蛋白、α-、β-、γ-连环蛋白的表达变化,每一逼尿肌至少评价切面上10个视野,计数可见粘连连接数量,并用计算机图像分析系统对膀胱逼尿肌粘连连接构成蛋白的含量进行半定量分析,分别计算出每个样本中钙粘蛋白、α-、β-、γ-连环蛋白的平均含量。分别进行以下比较: (1)实验组与对照组平滑肌中粘连连接构成蛋白的含量; (2)稳定膀胱逼尿肌与伴有膀胱过度活动逼尿肌中粘连连接的数量; (3)实验组平滑肌粘连连接各构成蛋白量的变化; 结果: 1.经免疫组织化学染色后光学显微镜观察并经计算机图像分析系统进行半定量测定 (1)在非梗阻及伴有膀胱过度活动的逼尿肌中均检测到钙粘蛋白、及α、β、和γ-连环蛋白的特异性染色;并且检测到平滑肌细胞间粘连连接的特异性染色。 (2)实验组(DI)平滑肌切片中P-钙粘蛋白和α、β、和γ-连环蛋白着色较浅并且不均匀,分布不规则。逼尿肌细胞间粘连连接明显减少,着色浅,周围见较多杂乱纤维。对照组中非梗阻逼尿肌切片着色较深并且分布均匀,见大量特异性浓染的P-钙粘蛋白和α、β、和γ-连环蛋白,肌细胞排列规则,细胞间大量粘连连接,连接着色较深,肌细胞形态规则,连接集中。实验组粘连连接蛋白的数量与对照组相比明显减少(P0.01)。 (3)实验组和对照组粘连连接的数量低于各组内膀胱上皮粘连连接的数量(P0.01)。 2.在电镜下观察 (1)实验组(DI)肌纤维排列不整齐,肌细胞分布不均匀,肌膜周围有高电子密度物质堆积,细胞间以胞突连接及缝隙连接(gapjunction,GP)为主;细胞间隙明显增宽,可见大量胶原纤维;平滑肌细胞间连接明显减少或消失,逼尿肌细胞出现肥大,线粒体肿胀、破裂; (2)对照组平滑肌纤维排列整齐,肌细胞分布均匀,肌细胞间隙细胞之间连接以粘连连接(adherensjunction,AJ)为主,细胞间的弹性纤维和胶原纤维含量较少,逼尿肌细胞内结构完整,排列规则。结论: 1膀胱平滑肌细胞间粘连连接中含有P-钙粘蛋白和α、β、和γ-连环蛋白; 2下尿路梗阻患者逼尿肌粘连连接构成蛋白的数量较正常减少; 3膀胱功能改变与膀胱逼尿肌粘连连接的数量变化有关。
[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.

【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R361

【参考文献】