脐带间充质干细胞对尿道括约肌功能影响的实验研究

发布时间：2018-05-18 12:49

本文选题：压力性尿失禁 + 免疫组化　；参考：《暨南大学》2012年硕士论文

【摘要】：研究目的：本研究通过建立SUI大鼠模型，检测SD雌鼠尿流动力学变化，观察尿道及其周围组织学形态改变。应用免疫组化和图像分析方法，分析HUMSCs治疗前后desmin、MHC及PGP9.5三种因子在尿道中的表达情况，分析间充质干细胞治疗压力性尿失禁的效果和可能的机制，探讨两种不同方式干细胞移植后的效果，以指导临床。材料和方法：1.1选用SD（清洁级）雌鼠模拟人类妊娠、难产产伤、双侧卵巢去势后低雌激素建立SUI模型。测定实验动物的最大膀胱容量(MBC)、漏尿点压(LPP),喷嚏实验阳性率和组织学形态检验模型的成功与否。通过HE染色、Masson特殊染色、Mallory特殊染色、免疫组化方式对大鼠尿道括约肌的显微解剖进行研究。 2.2将建模成功好的20只大鼠分为三组：静脉注射组（A组，6只），尿道局部注射组(B组，8只)、对照组（不注射任何物质C组，6只)，D组正常组（6只）将获得MSCs分别以5*106个细胞/只通过尾静脉和尿道局部注射方式移植于SUI大鼠模型体内，移植4周及6周后分别用BL-410生物机能实验系统检测尿流动力学指标和喷嚏实验阳性率，以评估尿流动力学变化。 2.3注射细胞6周后处死所有大鼠，进行尿道及其周围组织取材，通过组织形态学和图像分析法观察尿道及其周围组织变化，定量评价desmin、MHC、PGP9.5含量和计算横纹肌占整个尿道阳性面积比及尿道括约肌占整个尿道的阳性面积比。结果：1.正常SD雌鼠尿道及其周围组织形态学观察尿道括约肌在整个尿道分布不均，在中下段开始增多，其中横纹肌呈环形分布。模拟人类妊娠、难产产伤、去势后低雌激素方式能建立SUI模型SD雌鼠:尿道括约肌肌层减少，排列松散，混乱，部分肌肉有断裂，形成空泡等现象，，肌相关蛋白desmin及MHC染色下降，甚至出现丢失现象。 2.LPP是反应尿控功能最重要的指标，其中LPP升高提示尿控功能得到部分恢复或完全恢复，本研究显示HUMSCs移植A、B两组LPP、MBC均增加，HUMSCs移植后4周LPP值A组、B组、C组、D组分别为：18.15±1.21mmHg，18.79±1.48mmHg,13.55±1.43mmHg，22.83±2.17mmHg，6周后LPP分别为18.86±1.14mmHg,19.40±0.85mmHg，11.82±0.91mmHg,24.36±3.26mmHg.组间两两比较有统计学差异(P0.01)，其中C组LPP最小，A组、B组及D组两两比较有统计学意义（P0.01），但是A与B组比较无统计学意义(P0.05)，HUMSCs治疗后4周与6周各组间LPP无统计学意义(P0.05)。 3．HUMSCs移植治疗后可以使尿道括约肌肌层得到明显的增厚，形态及功能得到明显的改善。图像分析显示治疗4周、6周后尿道横纹肌及括约肌阳性面积比较未治疗都有明显的增加，PGP9.5数目也较阳性对照组明显增多,A组、B组、C组、D组横纹肌阳性面积比(%)分别为(15.3±3.77)%,(15.2±4.79)%,(9.55±2.06)%,(16.49±2.4)%,；尿道括约肌阳性面积比(%)分别为(35.14±5.38)%,(36.88±3.93)%,(22.46±3.52)%,(37.9±1.80)%, PGP个数分别为7.75±1.26，8.14±1.68，5.2+2.05，10.25+1.89，上述三种因子的检测组间两两比较均有统计学差异(P0.01)，其中C组横纹肌阳性面积最小，与其他三组相比差异有统计学意义(P0.01)，但A组、B组及D组两两比较无统计学意义(P0.05)；结论:1.本部分研究通过妊娠、分娩阴道扩张及双侧卵巢切除去势的方法，成功建立了SUI的动物模型，为后续实验奠定了基础。 2.尿道括约肌损伤和神经纤维的减少导致SUI出现，提示尿道结构和功能的改变是引发SUI的直接原因。 3.HUMSCs移植到SUI模型SD雌鼠可以增加肌相关蛋白desmin、MHC的含量和神经轴突标记物PGP9.5数目。 4.局部注射治疗和静脉注射治疗的两种不同方式疗效无明显的区别。 5.HUMSCs移植到SUI模型SD雌鼠可修复薄弱的尿道括约肌，改善尿控功能。
[Abstract]:Objective : To investigate the changes of urinary flow dynamics of SD female rats and to observe the changes of histological morphology around the urethra and its surrounding tissues by establishing a rat model . The effects of mesenchymal stem cells in the treatment of stress urinary incontinence and possible mechanisms were analyzed . The effects of mesenchymal stem cells on stress urinary incontinence were analyzed , and the effect of two different methods of stem cell transplantation was discussed .

Materials and Methods : 1.1 Female rats with SD ( clean grade ) were selected to simulate human pregnancy , which was difficult to produce and produce low estrogen after castration . The results showed that the maximal bladder capacity ( MBC ) , urine leakage point pressure ( lpp ) , the positive rate and the histological morphology of the experimental animals were successful or not . The microscopic anatomy of the urethral sphincter in rats was studied by HE staining , special staining of the Mallory , and immunohistochemical method .

2.2 All 20 rats with successful modeling were divided into three groups : IV group ( group A , 6 rats ) , local injection group ( group B , 8 rats ) , control group ( no substance C group , 6 rats ) , normal group D group ( 6 rats ) .

2 . After 6 weeks of injection , all the rats were sacrificed and the urethra and its surrounding tissues were taken , and the changes of the urethra and its surrounding tissues were observed by histomorphology and image analysis . The contents of Desin , MHC , PGP9.5 were quantitatively evaluated .

Results : 1 . The morphology of the urethra and its surrounding tissues in normal SD female rats showed that the urethral sphincter was not evenly distributed throughout the urethra , and the middle and lower segments began to grow .

There was significant difference between group A , group B , group C and D in group A , group B , group C , and D group ( P 0.01 ) , but there was no statistical difference between group A and group B ( P0.05 ) , but there was no statistical difference between group A and group B ( P0.05 ) .

3 . After the transplantation of HUMSCs , the thickness , shape and function of the urethral sphincter muscle were obviously improved . The results showed that the positive area of the striated muscle and sphincter was increased significantly after 4 weeks and 6 weeks . The positive area ratio of the striated muscle in group A , group B , group C and D was ( 15.3 卤 3.77 ) % , ( 15.2 卤 4.79 ) % , ( 9.55 卤 2.06 ) % , ( 16.49 卤 2.4 ) % , respectively .
The positive area ratio ( % ) of the urethral sphincter was ( 35.14 卤 5.38 ) % , ( 36.88 卤 3.93 ) % , ( 22.46 卤 3.52 ) % , ( 37.9 卤 1.80 ) % , respectively ( P < 0.01 ) .

Conclusion : 1 . In this part , this part studies the method of pregnancy , vaginal dilation and bilateral oophorectomy and lays a foundation for the follow - up experiment .

2 . The urethral sphincter injury and the decrease in nerve fibers lead to the occurrence of the occurrence of the disease , suggesting a change in the structure and function of the urethra is a direct cause of the cause of the disease .

3 . The transplantation of HUMSCs into the model SD female mice could increase the content of myoassociated proteins , and the content of MHC and the number of nerve axon markers PGP9.5 .

4 . There was no significant difference between the two different methods of local injection therapy and IV therapy .

5 . The transplantation of HUMSCs into the model SD female mice could repair the weak urethral sphincter and improve the urinary control function .
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】