Myocardin基因治疗双侧海绵体神经损伤大鼠勃起功能障碍的实验研究

发布时间：2018-05-17 23:25

本文选题：勃起功能障碍 + 海绵体神经损伤　；参考：《南方医科大学》2017年硕士论文

【摘要】：背景和目的前列腺癌根治术(radicalprostatectomy,RP)是局限性前列腺癌的一线治疗方法,但术后引起ED(RP-ED)的发病率高达30%-87%。同时,RP-ED的病理机制尚不清楚,且对5型磷酸二酯酶抑制剂(phosphodiesterase 5 inhibitor,PDE5i)治疗效果欠佳。伴随结构功能的不同,平滑肌细胞由收缩型转化为增殖型的过程称为表型转化。我们前期研究证实糖尿病大鼠阴茎海绵体平滑肌细胞存在表型转化,并且与糖尿病性大鼠的ED密切相关。Myocardin是平滑肌细胞表型调控过程中重要的调控因子,本课题组已证实,Myocardin基因治疗可改善糖尿病性大鼠阴茎勃起功能。本研究旨在建立双侧海绵体神经损伤(bilateral cavernous nerve injury,BCNI)大鼠,明确BCNI大鼠阴茎海绵体平滑肌细胞是否发生表型转化,初步探讨BCNI大鼠ED的病理机制,并试图通过Myocardin基因改善BCNI大鼠的勃起功能。方法1.双侧海绵体神经损伤大鼠阴茎海绵体平滑肌细胞表型特征建立双侧海绵体神经损伤大鼠及假手术组共36只,分别于3、5、7天对BCNI和NC组行勃起功能检测,计算出最大阴茎海绵体刺激压/平均动脉压比率(intraeavemous pressure/mean arterial pressure,CP/MAP),并取阴茎中段组织行 western blot 检测 a-sma、calponin、Myocardin、OPN 蛋白的表达。另外行HE染色和Masson染色观察大鼠阴茎组织组织形态学变化。2.Myocardin基因过表达对双侧海绵体损伤大鼠勃起功能及阴茎海绵体平滑肌细胞表型转化的影响30只SD大鼠分为 3 组,即 BCNI+Ad-Myocardin 组、BCNI+Ad-vector 组及NC+PBS组,构建Myocardin基因腺病毒,将纯化的Myocardin腺病毒导入BCNI大鼠阴茎海绵体内,注射21天后检测各组大鼠阴茎海绵体内压及颈动脉压,并取阴茎中段组织行 western blot 检测 a-sma、calponin、Myocardin、OPN 蛋白的表达。另外行HE染色、Masson染色和免疫组化和检测大鼠阴茎组织形态学变化以及平滑肌含量结果第一章:造模3天、5天、7天后采用测定阴茎海绵体内压及颈动脉压的方法评价大鼠勃起功能,在ICP及MAP测定中结果显示,BCNI大鼠ICP及AICP/MAP较NC组大鼠明显下降,达到统计学差异(P0.05);western blot检测发现第5天的BCNI大鼠阴茎海绵体组织中a-sma、Myocardin、calponin表达较NC大鼠显著下调,OPN的表达较NC组显著上调;在HE染色及Masson染色中BCNI大鼠及NC大鼠的平滑肌数量及形态无显著差异。第二章:各组大鼠注射21天后,检测阴茎海绵体内压及颈动脉压,结果显示:BCNI+Ad-Myocd大鼠ICP检测较BCNI+Ad-vector组的显著增高(P0.05),差异有统计学意义,但AICP/MAP仍低于NC组(P0.05)。HE染色可见,BCNI大鼠较NC大鼠阴茎组织平滑肌肌层变薄、细胞排列紊乱、内皮不连续,且BCNI+Ad-vector组更为严重;Masson染色可见平滑肌/纤维比率在BCNI大鼠中降低,且BCNI+Ad-vector组更为显著(P0.05)。免疫组化可见与BCNI+Ad-vector组对比,Myocardin及α-sma在NC组及BCNI+Ad-Myocd大鼠中表达显著增加(P0.05)。western blot检测,Myocardin治疗后的BCNI大鼠阴茎海绵体平滑肌组织a-sma、calponin、Myocardin蛋白表达较BCNI+Ad-vector组明显上调,OPN明显下调。结论1.BCNI大鼠发生勃起功能障碍,但早期BCNI大鼠阴茎海绵体平滑肌并无发生组织学变化。2.BCNI大鼠阴茎海绵体平滑肌细胞在造模后第5天发生表型转化。3.Myocardin基因治疗可以恢复BCNI大鼠部分勃起功能。4.Myocardin基因治疗可以维持BCNI大鼠阴茎海绵体平滑肌细胞的收缩表型。综合上述,我们得出:Myocardin基因通过维持阴茎海绵体平滑肌细胞的收缩表型,从而改善双侧海绵体神经损伤大鼠勃起功能。
[Abstract]:Background and objective radicalprostatectomy (RP) is a first-line treatment for localized prostate cancer, but the incidence of ED (RP-ED) is as high as 30%-87%. after operation. The pathological mechanism of RP-ED is not clear, and the treatment effect on type 5 phosphodiesterase inhibitor (phosphodiesterase 5 inhibitor, PDE5i) is not good. Different functions, the process of smooth muscle cells transformed from contractile type to proliferative type is called phenotypic transformation. Our previous study confirmed the phenotypic transformation of the cavernous smooth muscle cells in the penis of diabetic rats, and the close correlation with ED in diabetic rats was an important regulatory factor in the phenotype regulation of smooth muscle cells. The test group has confirmed that Myocardin gene therapy can improve the penile erectile function of diabetic rats. The aim of this study is to establish the bilateral cavernous nerve injury (BCNI) rat, to determine whether the phallic smooth muscle cells of the cavernous corpus cavernosum of the BCNI rats have phenotypic transformation, and to explore the pathological mechanism of ED in BCNI rats, and to try to find out the pathological mechanism of the ED in BCNI rats. Method 1. the erectile function of BCNI rats was improved by Myocardin gene. Methods the phenotypic characteristics of the cavernous smooth muscle cells in the cavernous corpus cavernosum of rats were established to establish bilateral cavernous nerve injury rats and 36 sham groups. The erectile function of the BCNI and NC groups was detected on 3,5,7 days, and the maximum penile corpus cavernosum stimulation pressure / average was calculated. Intraeavemous pressure/mean arterial pressure (CP/MAP) and the expression of a-SMA, calponin, Myocardin, OPN protein in the middle segment of the penis, calponin, Myocardin, OPN protein. The effect of the erectile function and the phenotypic transformation of the penile cavernous smooth muscle cells (cavernous smooth muscle cells) in 30 SD rats were divided into 3 groups, namely, group BCNI+Ad-Myocardin, group BCNI+Ad-vector and group NC+PBS, to construct Myocardin gene adenovirus, and the purified Myocardin adenovirus was introduced into the cavernous cavernous cavernous body of BCNI rats, and the internal pressure of the cavernous corpus cavernosum in each group was detected after 21 days of injection. Western blot was used to detect the expression of a-SMA, calponin, Myocardin, OPN protein in the middle section of the penis. In addition, HE staining, Masson staining and immunohistochemistry and detection of the morphological changes of the penis and the results of smooth muscle content in the first chapter: 3 days, 5 days, and 7 days, the internal pressure and neck movement of the penis corpus cavernosum were measured. The method of pulse pressure was used to evaluate the erectile function of rats. The results of ICP and MAP showed that ICP and AICP/MAP in BCNI rats were significantly lower than those in the NC group (P0.05), and Western blot detected the a-SMA in the tissue of the cavernosum of the penis of the fifth days of the BCNI rats. There was no significant difference in the number and shape of smooth muscle of BCNI rats and NC rats in HE staining and Masson staining. Second: after 21 days of injection, the internal pressure of cavernosum and carotid pressure in the penis were detected. The results showed that the ICP detection in BCNI+Ad-Myocd rats was significantly higher than that in the BCNI+Ad-vector group (P0.05), but the difference was statistically significant, but AICP was statistically significant. /MAP was still lower than group NC (P0.05).HE staining. Compared with NC rats, the smooth muscle layer of the penis tissue was thinner, the cell arrangement was disorderly, the endothelial discontinuity was discontinuous, and the BCNI+Ad-vector group was more serious; Masson staining showed that the ratio of smooth muscle / fiber was decreased in BCNI rats, and BCNI+ Ad-vector group was more significant (P0.05). Immunohistochemical staining can be seen and observed. In group tor, the expression of Myocardin and alpha -sma in NC and BCNI+Ad-Myocd rats increased significantly (P0.05).Western blot detection. The smooth muscle tissue of the cavernous corpus cavernosum was a-SMA, calponin, and obviously down regulated in the BCNI rats after Myocardin treatment. Conclusion erectile dysfunction occurred in the rats. But there is no histological change in the smooth muscle of the cavernous corpus cavernosum in the early BCNI rats. The phallic smooth muscle cells of the cavernous corpus cavernosum in the.2.BCNI rat fifth days after the model transformation, the.3.Myocardin gene therapy can restore the BCNI rat's partial erectile function.4.Myocardin gene therapy can maintain the contraction of the smooth muscle cells of the corpus cavernosum of the BCNI rat In general, we conclude that the Myocardin gene can improve the erectile function of the bilateral cavernous nerve injury by maintaining the contractile phenotype of the cavernous smooth muscle cells of the penis.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R698.1

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