【作者】 王振迪；

【导师】 曾甫清；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 第一部分大鼠精原细胞的分离与超微结构观察目的分离和纯化9日龄雄性SD大鼠睾丸内精原细胞,为深入研究精原细胞的生物学特性以及细胞形态的三维重建提供实验基础。方法选取3只9日龄大鼠,脱颈处死后取其睾丸,采取两步酶消化法制备细胞悬液;配置浓度为15%、25%、35%、45%的Percoll液,Percoll密度梯度离心法分离出精原细胞;细胞计数后固定,常规制作超薄切片,透射电镜下观察并对其拍照。结果经Percoll不连续密度梯度分层,分出5层细胞带。其中第Ⅲ带细胞光镜下观察体积大、圆形、胞质少、核大圆形,电镜下观察圆形或卵圆形,胞质中除见线粒体外其它细胞器不发达,偶可见高尔基体分布在细胞核周围,核浆比大,常染色体细密均匀,异染色质少,符合精原细胞的形态学特征。结论利用两步酶消化法、Percoll液密度梯度离心法可有效分离和纯化精原细胞。第二部分大鼠睾丸组织连续切片的三维重建目的在个人计算机(PC)Windows平台上利用大鼠睾丸组织的连续组织切片图像,重建其三维立体图像,探讨基于连续切片三维重建路线的可行性,为下一步基于细胞超薄连续切片的三维重建提供技术路线。方法我们选取大鼠睾丸进行连续切片,并在光学显微镜下放大40倍使用数码相机对其拍照,然后输入PC,Photoshop CS(Adobe)软件预处理后使用Amira 3.1(TGS)软件对得到的平面图像先后进行配准和三维重建。结果得到大鼠睾丸和附睾的三维图像,立体结构清晰,并能任意进行旋转、剖切等观察和操作。结论PC-Windows平台上可以利用生物组织的连续切片对其进行三维重建,且此操作在普通PC上即可实现,易于推广。第三部分精原细胞超薄连续切片的三维重建目的在个人计算机(PC)Windows平台上利用精原细胞超薄连续切片电镜图像,重建单个精原细胞的三维立体图像。方法我们选取大鼠睾丸分离纯化精原细胞后,固定制作成超薄连续切片,在透射电子显微镜下放大5800倍观察并对其拍照,然后将大小、分辨率统一的图像输入计算机。单个细胞经过编号以及肉眼对位后,使用Photoshop CS(Adobe)软件将其分割,最后使用Amira 3.1(TGS)软件对其进行进一步的配准和三维重建。结果共得到120层共7741张图像,经过肉眼对位后,所有的单个细胞中最多者有12个截面图像,通过后者能重建出部分的细胞超微结构,可以任意进行旋转、剖切等观察和操作。结论PC-Windows平台上可以实现细胞内外超微结构的三维重建,但配准方法尚需向计算机智能化发展,超薄连续切片数尚需进一步增加以充实细胞的三维立体信息。第四部分亲属活体肾移植术前供肾血管解剖的评估目的探讨PC-Windows平台上重建活体肾移植供体肾脏及其血管立体图像的方法,在供肾摘取术前和术中对手术医师提供指导。方法10名亲属活体肾移植供者行CT薄层扫描,图像以DICOM格式保存,在PC-Windows XP平台上读入TGS Amira 3.1软件后,通过图像裁剪、分割、三维重建等步骤进行处理。结果我们最终得到了供体肾脏及其血管解剖结构的立体图像和动画演示,其结果方便其它PC机查看,并可按要求输出。合成结果显示8名供体双侧肾脏均为单根动脉供应,2名供体存在副肾动脉,均符合术中所见。结论这项技术为活体肾移植中移植肾的术前评估和术中切取提供了便利,准确性高,花费时间少。相比CT工作站等昂贵的设备,这项技术所需设备价格低廉,更方便临床医生掌握。通过它,临床医生可以根据自己的实际需要灵活机动的对CT图像进行处理,更好的指导临床工作,因此具有广阔的应用前景。更多还原

【Abstract】 PartⅠSeparation and purification of spermatogonia in ratsObjective In order to study the mechanism of spermatogenesis and its influence factors, the spermatogonia should be separated and purified to provide technological groundwork and cell sources.Methods Continuous enzymatic digestion was used to prepare spermatogenic cell suspension of 9-day-old rats postnatal, and Percoll density gradient centrifugation combined with plating culture method was used to isolate and purify spermatogonia. Results Spermatogonia were mainly distributed in Percoll gradient between 25%-35%. These cells are spherical with large nuclei containing single or multiple nucleoli and small flakes of associated heterochromatin. The Golgi apparatus is distinct, and cytoplasmic organelles are concentrated in a perinuclear region. Conclusion Percoll density gradient centrifugation combined with continuous enzymatic digestion and plating culture method could effectively isolate and purify spermatogonia from 9-day-old rats postnatal. PartⅡComputer-assisted 3D reconstruction of rat testis from serial ultra-thin slice imagesObjective To obtain three-dimensional reconstruction of rat testis from serial sections on the PC-Windows platform.Methods Serial slice images of rat testis were obtained by a digital camera, which were inputted to the computer. After image preprocess in Adobe Photoshop CS, we aligned the serial images and reconstructed the 3D image in Amira 3.1(TGS).Results Internal and external structures of rat testis can be observed from any angles. Conclusion Within the help of Amira 3.1(TGS), 3D reconstructions of tissue can be performed in PC-Windows platform of a common PC, which is easy for popularizing.PartⅢThree-dimensional reconstruction of rat spermatogonia from ultra-thin serial slicesObjective To obtain three-dimensional reconstruction views of rat spermatogonia from ultra-thin serial sections on the PC-Windows platform.Methods Spermatogonia were isolated and purified from 9-day-old rats postnatal. Then serial ultra-thin slices were prepared and every single spermatogonium was observed and photographed by a Tecnai G2 12 TEM with magnification of 5800×, which were all inputted to a computer. After systemic numbering, we aligned the serial images by the naked eye and reconstructed the 3D image in Amira 3.1(TGS).Results A total of 7741 spermatogonia pictures in 120 serial sections were obtained. A spermatogonium which has 12 serial slices images was selected for 3D reconstruction. We reconstructed a part of cell stereostructure, furthermore internal and external structures of the model could be observed from any angles.Conclusion Within the help of Amira 3.1(TGS) and other softwares, 3D reconstructions of spermatogonia could be performed in PC-Windows platform of a common PC. Nevertheless, more convenient computer aided registration method should be raised, and the number of ultra-thin serial slices should be enriched for more abundant cell stereostructure information.PartⅣThree-dimensional reconstruction and virtual-reality technology facilitate living donor nephrectomyObjective To find a handy way to display the anatomy of the renal arterial and venous systems of potential donors on PC-Windows platform.Methods Dual-phase serial computed tomography (CT) images from ten living-related donors were loaded into TGS Amira 3.1 running on a PC with Windows XP. Image crop, segmentation and 3D reconstruction followed.Results We got the video clips as well as the 3D images of donor kidneys and blood vessels. According to the display which was confirmed by latter intraoperative findings, eight donors had single renal vessels, one had a left accessory renal artery and one had a right accessory renal artery.Conclusion This project offered a new approach to evaluate the renal vessel anatomy in living donor kidney transplantation, and it was favorable for accuracy and popularizing. Compared with expensive imaging workstations, it was much easier to be learned and manipulated by clinicians, and the clinicians can do their own interpretations according to the raw image data, it absolutely should be favorable for the accuracy of operation.更多还原