【作者】 易竞；

【导师】 江华；

【作者基本信息】 第二军医大学 ， 外科学， 2011， 博士

【摘要】 阴茎再造手术被广泛应用于治疗阴茎缺损、先天性性别畸形,以及女性易性病患者,具有重大的意义。当前,医学界在重塑阴茎外形、确保皮瓣存活与尿路通畅、植入支撑物获取硬度、甚至重建阴茎感觉功能等方面,均已形成比较成熟的临床治疗共识。但是,对于模拟阴茎在主观意识的控制下的勃起功能重建,其研究进展却相对滞后。为了克服依赖人工膨胀式假体可能出现的不良反应,并避免患者对纯外力依赖的勃起发动机制可能产生的心理阴影,帮助其实现主观自控的阴茎勃起发动过程,本实验采用保留神经支配、具备收缩功能的肌瓣移植来重建再造阴茎的勃起功能。长期以来,股薄肌因其解剖学特点及切除后对供区功能影响较小,已成为临床上常用的肌(皮)瓣,广泛应用于组织缺损修复与器官功能重建。1972年,Orticochea曾报道了1例利用股薄肌肌皮瓣再造阴茎的手术,术后通过股薄肌主动收缩,再造阴茎在假体支撑下较好地模拟了阴茎的勃起发动过程。然而,该手术操作复杂、疗程长,术后再造阴茎外观臃肿,影响了该术式的推广。当前,显微外科技术与皮瓣预制技术不断发展,手术操作大大简化,缩短了手术进程。而肌肉亚部化概念的提出与相应研究成果又为避免再造阴茎臃肿的外观带来希望,股薄肌的节段性应用已逐渐成为一种趋势。为了找到在改善术后外观与保留肌肉收缩功能之间的最佳平衡点,有必要进一步加深相关的解剖学与生理学研究。[研究目的]研究兔股薄肌肌内的神经支配与血管分布的解剖学特征,寻找能将其安全、简便地完全劈开的具体方式,构建应用节段性股薄肌肌皮瓣移植再造阴茎重建勃起功能的合适的动物模型,兼顾改善术后外观与保留肌肉收缩功能的手术要求。[研究方法]1、应用改良的Sihler’s肌内神经染色与乳胶血管灌注显色技术,研究兔股薄肌肌内神经支配与血管分布的解剖学特征。2、依照解剖学研究的结果,按照肌亚部的原则,将兔股薄肌近似二等分纵向完全劈开。应用HE染色观察术后肌束横断面的整体形态、Masson三色染色观察肌束内部发生结缔组织纤维化的程度、EnVision法检测肌细胞的增殖情况、TUNEL法检测肌细胞的凋亡情况、Western Blot检测肌内细胞增殖水平的动态变化；并运用多道生理信号采集处理系统同步检测劈开后肌束的等长收缩肌力与复合肌肉动作电位(CMAP)。3、构建应用节段性股薄肌肌皮瓣再造阴茎重建勃起功能的动物模型,并观测人为刺激肌束支配神经后再造阴茎的形态变化与CMAP。[实验结果]1、肌内神经染色与血管灌注显示：兔股薄肌的支配神经入肌后,各一级分支大体上相互平行由近端走向远端,基本上都有相应的血管分支伴行,不同来源的营养血管形成肌内丰富的吻合网。前部1/3-1/2肌肉的血供主要来自股动脉的分支,受股薄肌前侧的3支一级神经分支支配,后部1/2-2/3肌肉的血供主要依赖股深动脉的分支,其神经支配来自股薄肌支配神经后侧的2-3支一级分支。2、HE染色显示肌肉劈开后初期,肌束可发生较明显的水肿；Masson三色染色显示此时肌内纤维结缔组织增生活跃；增殖细胞核抗原(PCNA)的免疫组化检测与TUNEL法细胞凋亡检测显示肌纤维细胞与结缔组织细胞增殖、凋亡均较活跃,而凋亡更多发生于间质细胞内；肌束代偿性的肥大,两部分肌束湿重的总和增加。随术后时间延长,HE染色显示水肿逐渐消退；PCNA与TUNEL检测显示肌纤维与间质细胞的增殖均逐渐减弱,纤维结缔组织中凋亡阳性核的检出率更多,这与Masson三色染色所反映的肌内增生纤维结缔组织减少、比重下降之表现一致；肌束湿重的总和降低至接近术前水平。Western Blot检测显示肌细胞PCNA的表达水平也随术后时间延长逐步下降,并提示将兔股薄肌纵向劈开的手术干预对肌内细胞增殖的影响很小。3、在近似二等分纵向完全劈开兔股薄肌,应用前部肌束构建的相关动物模型中：通过电刺激支配神经直至肌束发生强直收缩,植入支撑假体的初步模型可模拟阴茎勃起时的位置变化；单纯肌束构型的初步模型可模拟阴茎勃起时的硬度变化；而有表面皮肤包被并植入银丝电极的完整模型能比较理想地模拟阴茎勃起发动时的形态变化。[结论]1、兔股薄肌肌内神经与血管的分布情况得以清晰、直观地显示,发现其肌内神经大体上相互平行由近端走向远端,各神经分支基本上都有相应的血管伴行,根据血供与神经支配的特点,肌肉的前、后两部分可以分为相对独立的亚单位结构。2、依据肌亚部原则,近似二等分纵向完全劈开兔股薄肌的手术操作不会明显影响肌束的长期存活；股薄肌分成两部分后,肌束各自的收缩功能也都能得到保留。3、按上述方式劈开兔股薄肌,选用前部肌束进行恰当构型、植入硅胶支撑假体以制备阴茎再造勃起功能重建的动物模型,通过刺激再造阴茎根部的银丝电极/支配神经可以引起肌束收缩,并使再造阴茎的形态发生改变,比较满意地模拟了正常阴茎的勃起过程。4、在按上述方法构建的再造阴茎勃起功能重建的动物模型中,能够观察到受动物自主控制的肌肉收缩与再造阴茎的阵发性持续抖动,充分体现了构建模型的手术方式的可行性,并佐证了兔股薄肌肌亚部划分的正确性。本实验是对股薄肌肌亚部的解剖学与生理学基础研究的补充,构建了比较理想的具备勃起功能的再造阴茎动物模型,达到了兼顾外观与功能的预期实验目的,为临床上进一步改良阴茎再造的手术方法提供了理论基础,也可为临床上应用节段性的股薄肌肌皮瓣移植整复面瘫、重建舌功能等手术提供相应的方法借鉴。更多还原

【Abstract】 Penis reconstruction surgery is widely used in the treatment of penile defects, congenital malformations of gender, and female to male sexually transmitted diseases, which is of great significance. Currently, doctors have formed a standardized clinical consensus in remodeling penis shape, ensuring flap survival and urinary tract unobstructed, implanting supportive structures for stiffness, and even the reconstruction of penile sensitivity, etc. However, the progress on simulating the situation from weakness to firmness when penis erectile happens under the patients’ subjective control has relatively lagged behind. In order to overcome the possible complications of artificial expansive implants, and to avoid the psychological shadow may happened in some patients for a erection initiating mechanism purely depending on an external force, we plan to use the innerved muscle transplantation to reconstruct the erectile function of penis.Gracilis has become a commonly used donor muscle for tissue defects and functional organ reconstruction, based on its anatomical characteristics and limited leg functional impact after its resection. In 1972, Orticochea reported a musculocutaneous gracilis transplanting surgery for penile reconstruction. Postoperatively, the reconstructive penis could move voluntarily when the initiative gracilis contraction happened. But the complicated surgical operation, rather long surgical duration, and the significantly overstaffed penis appearance had greatly affected the promotion of this surgical procedure. Recently, the progress on microsurgical techniques and flap prefabricating techniques makes it possible to simplify and shorten the surgical process. And the concept of muscle compartmentalization and its corresponding research achievements have brought hope to improve the overstaffed appearance of reconstructive penis at the same time. Yet, segmental flap application has also become a trend in musculocutaneous gracilis transplantation. In order to find the best balance between improving postoperative appearance and retaining muscle’s contractive function, it is necessary to promote the anatomic and physiologic studies on gracilis. [Objective]Study the intramuscular nerve branches’and blood vessels’distribution of rabbit gracilis muscle and find a specific safe, simple way to split it completely. Build a suitable animal model for the reconstruction of penile erectile function with segmental gracilis flap transplantation, which can meet the requirements of both improving postoperative reconstructive penis’s appearance and retaining muscle’s contractive function.[Methods]1. Using a modified Sihler’s intramuscular neural staining and red latex vascular perfusion technique to study the intramuscular nerve branches’and blood vessels’ distribution of rabbit gracilis muscle.2. According to the principles of muscle compartmentalization, vertically split the rabbit gracilis muscle into two approximately equal halves completely. Observe the overall postoperative situation of muscle bundles’cross-section with HE staining technique. Observe the connective tissue’s fibrosis occurred within the split muscle bundles with Masson’s three-color staining technique. Detect the proliferation of muscle cells with En Vision immunohistochemical technology. Detect the apoptosis of muscle cells with TUNEL. Detect the dynamic level of muscle cell’s proliferation with Western Blot. And using a multi-channel physiological signal acquisition and processing system to measure the isometric contraction forces and CMAP of muscle bundles synchronously.3. Build an animal model for the reconstruction of penile erectile function with segmental gracilis flap transplantation. Observe and record the morphologic changes and CMAP of the reconstructive penis when its nerve is electric stimulated.[Results]1. The intramuscular nerve primary branches of rabbit gracilis are generally parallel to each other and most of them are accompanied by a corresponding vessel branch. The blood vessels from different sources form an abundant microcirculation network through intramuscular vessel branches. The anterior 1/3-1/2 portion of the muscle is supplied by a branch of femoral artery and under the control of the anterior 3 primary nerve branches. While the posterior 1/2-2/3 portion of the muscle is supplied by a branch of profunda femoral artery and under the control of the anterior 2-3 primary nerve branches. 2. As the HE, Masson, En Vision IHC and TUNEL show:Early after the gracilis was split, the muscle bundles swelled obviously. The proliferation was active in both muscle cells and connective tissue cells. The activity of apoptosis was relatively higher in interstitial cells than it in muscle cells. The proportion of fibrous connective tissue increased greatly. The muscle bundles became compensatorily hypertrophy, which increased the muscle’s wet weight obviously. With the passage of time, the muscle bundles’swelling relieved gradually. The proliferation activity dropped in both muscle cells and connective tissue cells, while the positive apoptosis cores were more often found in connective tissue cells. The proportion of fibrous connective tissue decreased and the muscle’s wet weight reduced to near the preoperative level. The Western Blot showed an obvious trend that the PCNA expression level of muscle cells decreased gradually with postoperative time prolonged. It was found not only in the split muscle bundles, but also in the control side whole gracilis muscle. The Western Blot also implied that the surgical intervention of vertically splitting the rabbit gracilis muscle into two approximately equal halves would have little effect on the proliferation of muscle cells.3. After the rabbit gracilis muscle was vertically split into two approximately equal halves completely, the front muscle bundle was architected to build an animal model for the reconstruction of penile erectile function. When its dominant nerve was electric stimulated, especially when the muscle bundles occurred tetanic contraction, the 3 type of model were able to simulat the position changes, hardness changes and morphologic changes respectively, just like a normal erectile penis.[Conclusion]1. The intramuscular nerve branches’and blood vessels’distribution of rabbit gracilis muscle was displayed clearly and intuitively. Intramuscular nerve branches are generally parallel to each other and most of them are accompanied by a corresponding vessel branch. The anterior and posterior part of rabbit gracilis muscle seems to be regarded as two relatively independent sub-unit structures.2. The surgery according to the principles of muscle compartmentalization, which vertically split the rabbit gracilis muscle into two approximately equal halves completely, will not significantly affect the long-term survival of muscle bundles, nor shall it affect the contractive function of either muscle bundle respectively. 3. After the rabbit gracilis muscle was correctly split into two halves, the front muscle bundle was architected properly to build an animal model for reconstruction of penile erectile function, with a silicone stick implanted as a supporter. When its nerve was electric stimulated, the reconstructive penis could move satisfactorily, just simulating the way of a normal penis’s erection.4. We also observed that the reconstructive penis model was able to paroxysm dithering under the animal’s own control, which certified the feasibility of our surgical methods and supported the rabbit gracilis muscle compartmentalization.Our results have enriched the anatomic and physiologic study of gracilis compartmentalization. The reconstructive penis model has satisfactorily simulated the erectile function like that of a normal penis and has met the requirements of both improving postoperative reconstructive penis’s appearance and retaining muscle’s contractive function. It can be served as a useful reference for the further improvements of clinical penis reconstruction surgery. And it may also give some valuable advice on the application of segmental gracilis transplantation for the treatment of facial paralysis, the reconstruction of tongue function, and so on.更多还原