基于组织连续切片的子宫主韧带内神经脉管数字化三维模型的构建

发布时间：2018-06-15 08:13

本文选题：连续切片 + 子宫主韧带　；参考：《南方医科大学》2014年硕士论文

【摘要】：经典的广泛性子宫切除术(Radical hysterectomy, RH)是早期宫颈癌患者的标准治疗术式,获得了较好的临床疗效,但由于在术中损伤了盆腔自主神经(pelvic autonomic nerve, PAN),在获得明显疗效的同时也带来了膀胱、肛门直肠、性功能障碍等多种术后并发症。PAN由腹下神经(Hypogastric nerve, HP),盆腔内脏神经(Pelvic splanchnic nerves, PSN)和盆丛(Plvic plexus, PP)组成。相关研究表明腹下神经走行于骶子宫韧带(Uterosacral ligament, USL)的外下方,盆腔内脏神经及盆丛走行于子宫主韧带(Cardinal ligament, CL)的下半部分,盆丛的分支走行于阴道旁复合体内。而在RH术中对上述韧带进行处理时损伤了PAN是导致上述并发症主要原因,其中对子宫主韧带的损伤是并发症产生的关键原因之一。1921年日本学者冈林首先提出在RH术中保留神经的想法,1961年小林隆在冈林的基础上进行改良,提出在切除子宫主韧带时保留支配膀胱的副交感神经,从一定程度上降低了膀胱功能障碍的发生率。1988年Sakamoto等对保留盆腔自主神经丛技术进行了第一次英文报道,即东京术式,他提出可将子宫主韧带分为血管部和神经部,通过触诊可识别血管和神经,并据此可保留子宫主韧带中的神经。1991年Yabuki正式命名该术式为保留神经的广泛性子宫切除术(nerve sparing radical hysterectomy, NSRH)。NSRH在经典RH术式的基础上保留了子宫主韧带神经部的盆腔内脏神经和部分下腹下神经丛,明显地降低上述并发症的发生。但由于只切除了子宫主韧带的血管部,即部分子宫主韧带,引起专家学者对疗效的质疑。因此,为了切除更多的子宫主韧带,并有效地保留PAN,有必要对子宫主韧带内神经脉管等结构进行详细的研究。虽然文献报道和本课题组前期研究已证实子宫主韧带主要由PAN、血管、淋巴管及各种结缔组织构成,也对神经分布进行了初步的定性定量研究,但目前对子宫主韧带内神经和脉管之间的立体结构缺乏详细的了解,国内外尚未见子宫主韧带内神经脉管结构重建的报道,而上述结构的解剖特点及关系恰恰为NSRH术中解剖的关键。因此,本课题选择计算机重建技术对子宫主韧带内神经脉管之间的立体结构进行数字化三维重建,为NSRH术式的改良提供立体解剖学依据。数字化三维重建技术是数字医学中的一个重要技术,它是指利用计算机技术将连续的二维图像变成三维图像,直观的立体图形更便于临床医师的观察研究。目前可利用的二维数据集主要有数字人数据集,CT或MRI数据集,组织连续切片数据集。数字人是通过对尸体标本的薄层铣切而获得数字化的二维人体数据集,继而通过计算机三维重建技术实现人体数据的三维可视化,其存在彩色、直观的优点,但采集难度大,重建困难较大地限制了其在临床中的广泛应用。近年来,随着影像学技术的发展,基于CT或MRI数据集的三维重建已逐渐在医学发挥其重要的作用,由于其来源广泛且具有个性化的特点,目前已较广泛地应用于临床并取得了较好的效果,显示了其在解剖研究中的优越性。但由于层距的限制,分辨率有限,同时受图像灰度的制约,目前尚未能完成毫米级以下的血管、神经、淋巴管的构建。基于组织连续切片的计算机三维重建技术即指通过对某一组织结构进行连续薄层切片并获得这一系列切片截面的二维图像,通过计算三维重建的方法而得到组织结构立体形态的一种方法。该方法对一些传统的影像学无法清晰、系统显示的结构如神经及毫米级以下的结构进行重建是一种很好的研究方法,目前已在小鼠肾小管、子宫内膜微血管、胎儿神经分布等方面得到了初步应用。本研究拟进一步在前期数字化三维重建研究的基础上,利用子宫主韧带组织连续切片染色数据集,结合图像识别、定位配准、三维重建等技术,构建子宫主韧带内神经及脉管的数字化三维模型,为NSRH手术提供子宫生韧带立体解剖结构依据。第一部分：基于组织连续切片子宫主韧带内神经脉管数字化三维重建定位材料的探讨 [目的] 比较经不同染色前后四种定位材料在玻璃切片上的粘附能力,探讨一种适合子宫主韧带组织连续切片三维重建的定位材料。 [方法] 获取因IB1期宫颈癌行广泛性子宫切除术的新鲜子宫主韧带标本1例(左侧),经固定、脱水、浸蜡后分别将1根普通缝线(3-0)、1根人发、1根可吸收缝线(8-0)及利用穿刺针将1条生物组织(利用穿刺针取自于肝脏组织,直径约0.6mm)作为定位杆标记与标本一起进行石蜡包埋,采用石蜡切片机对所取标本进行连续切片,切片厚度为5μm,共切取120张切片,用组织防脱载玻片进行贴片。将切片随机分成四组,各30张,分别进行常规HE染色、TH染色、VIP染色和D2-40染色,在显微镜下观察并记录各种材料染色前后定位点的数目,比较分析不同定位材料染色前后标记点的粘附情况。 [结果] 1、在人发、普通缝线、可吸收缝线与生物组织四种定位材料中,生物组织在染色后贴片率较其它三种材料高,经统计学检验差异有统计学意义(P0.01)。 2、在以上四种定位材料中,生物组织定位点脱失最少,四种染色前后贴片数量差异均无统计学意义(P=0.98)；非吸收性缝线、可吸收缝线与人发定位点染色前后均脱失严重,数量差异比较有统计学意义(P0.01)。 [结论] 利用穿刺针获取生物组织作为定位材料成功解决了定位标记点的脱失问题,其在HE染色和各种免疫组化染色中均具有很好的粘附效果。第二部分：子宫主韧带内神经脉管数字化三维模型的构建 [目的] 利用子宫主韧带组织连续切片数据集,构建子宫主韧带内血管、交感神经、副交感神经及淋巴管数字化三维模型,明确神经、血管及淋巴管在子宫主韧带中三维立体走行与分布情况。 [方法] 获取因IB1期宫颈癌行广泛性子宫切除术的新鲜子宫主韧带标本1例(左侧),将子宫主韧带用脱水盒固定方向。10%福尔马林固定8-12h后进行脱水,利用穿刺针穿取4条生物组织(直径约0.6mm)作为定位杆标记与标本一起进行石蜡包埋,冰冻30min后进行连续切片,切片厚度为5gm,连续4层为一组,每组间隔80μm。65℃烤箱中烘烤2-3h后进行分层染色。在第一层,切片用常规HE染色;在第二层,切片经TH染色,特异标记交感神经纤维；在第三层,切片经VIP染色,特异标记副交感神经纤维；在第四层,切片经D2-40染色,特异标记淋巴管。将4种染色后的组织切片置于显微镜下,对其中的各种显微组织成分观察。采用数字切片扫描系统对每张切片进行扫描。先用2×10倍下扫描地图,确定扫描范围,再转到10×10倍下对切片进行全自动扫描。利用Photoshop软件和Matlab软件的图像配准方法分别对4组组织切片图像进行自动配准。将配准后的4组切片数据集图片导入Photoshop软件中,应用画笔工具分别画出血管、交感神经、副交感神经及淋巴管的轮廓。利用ACDSEE软件将各组图片调至合适大小,分别导入Mimics软件中,经过自动定位图像、组织图片、内插值处理后,经三维重建分别构建出子宫主韧带、血管、交感神经、副交感神经、淋巴管的数字化三维模型,并对模型进行配准融合。利用Mimics软件的透明化、测量等功能对模型进行分析。 [结果] 基于子宫主韧带组织连续切片数据集,成功构建出了子宫主韧带内神经脉管数字化三维模型,通过对模型的分析与测量,我们发现： 1、子宫主韧带宽为26.77mm,长度为50.09mm,血管主要走行于上半部分,神经主要走行于下半部分,从宫颈侧至盆壁侧近、中、远段神经主干上缘与血管下缘的距离分别为8.59mm,7.18mm,4.87mm。 2、子宫主韧带内血管主要走行于子宫主韧带上半部分,从宫颈侧至盆壁侧近、中、远段血管上缘距离子宫主韧带上缘分别为11.99mm,9.36mm,4.89mm;血管下缘距离子宫主韧带下缘分别为14.01mm,13.74mm,12.70mm。 3、神经主要走行于子宫主韧带底部,在距离宫颈侧31.27mm处明显增多直至盆壁；交感神经主要走行于子宫主韧带下半部的底部,从宫颈侧向盆壁侧逐渐增多,从宫颈侧至盆壁侧近、中、远段交感神经主干距离韧带上缘分别为22.11mm,22.16mm,19.56mm;副交感神经相对较少,主要走行于子宫主韧带的底部,从宫颈侧至盆壁侧近、中、远段副交感神经距离韧带上缘分别为22.58mm,21.34mm,18.62mm。 4、淋巴管细小,在子宫主韧带中分布无明显规律,主要沿血管周围走行。 [小结] 本研究通过子宫主韧带连续切片的计算机辅助三维重建技术,构建了子宫主韧带内神经脉管数字化三维模型,精确地显示了子宫主韧带内的解剖结构及其之间的空间关系,形象地展示了子宫主韧带内神经、血管、淋巴管的分布与走行,真实地再现了子宫主韧带的三维立体结构。重建模型可以在空间位置上以任意轴线旋转任意角度,以便从不同位置对各结构的形态,空间位置及相互关系进行观察。同时,还可利用Mimics软件的测量工具对各个结构的距离进行测量,更加精确的显示子宫主韧带内结构的准确分布情况。
[Abstract]:The classic Radical hysterectomy (RH) is a standard treatment for early cervical cancer and has a good clinical effect. But because of the damage to the pelvic autonomic nerve (pelvic autonomic nerve, PAN) during the operation, the bladder, anorectal, sexual dysfunction and so on are also brought about. The postoperative complications of.PAN were composed of Hypogastric nerve (HP), pelvic visceral nerve (Pelvic splanchnic nerves, PSN) and pelvic plexus (Plvic plexus, PP). The lower part of ament, CL), the branches of the pelvic plexus walk in the paraplex compound. And the damage to PAN during the treatment of the ligaments during RH is the main cause of the complications, and the damage to the main ligament of the uterus is one of the key causes of the complications. In.1921, the Japanese scholar, Kang Lin, first proposed to retain the nerve during RH. The idea that in 1961, Xiao Lin long was improved on the basis of the Kumin. It was proposed to retain the parasympathetic nerve that dominates the bladder during the removal of the main ligament of the uterus. To a certain extent, the incidence of bladder dysfunction was reduced to a certain extent by.1988 Sakamoto, the first English report on the preservation of the pelvic autonomic plexus technology, that is, the Tokyo operation. The main ligament of the uterus can be divided into the vascular and nerve parts, and the blood vessels and nerves can be identified by palpation. According to this, the.1991 year Yabuki of the main ligament of the uterus can be retained to formally name the operation for the extensive hysterectomy (nerve sparing radical hysterectomy, NSRH).NSRH to retain the uterus on the basis of the classical RH operation. The pelvic visceral nerve in the main ligament of the main ligament and the inferior lower abdominal nerve plexus obviously reduce the occurrence of the above complications. But because only the vascular part of the main ligament of the uterus is removed, that is, the main ligament of the uterus is a part of the main ligament of the uterus, which causes the experts and scholars to question the curative effect. Therefore, in order to excise more uterine main ligaments and retain the PAN effectively, it is necessary against the children. A preliminary study of the main ligaments of the uterus, including PAN, blood vessels, lymphatic vessels and various connective tissues, has also been preliminarily studied, although the preliminary studies of the main ligaments of the uterus have also been conducted preliminarily and quantitatively. There is no detailed understanding of the structure, and there is no report on the reconstruction of the vascular structure in the main ligament of the uterus at home and abroad. The anatomical features and relationships of the above structures are the key to the anatomy of the NSRH. Therefore, the computer reconstruction technique is selected to digitize the three-dimensional structure of the nerve vessels in the main ligament of the uterus. Provides the anatomical basis for the improved NSRH method.
Digital 3D reconstruction is an important technology in digital medicine. It refers to the use of computer technology to transform the continuous two-dimensional images into three-dimensional images. The visual stereogram is more convenient for the clinicians to observe. The available two-dimensional data sets are mainly digital human data sets, CT or MRI data sets, and the number of continuous slices is organized. According to the data collection, the digital human is digitized by the thin layer milling of the corpse specimen, and then the three-dimensional visualization of the human body data is realized by the computer 3D reconstruction technology. It has the advantages of color and intuition, but it is difficult to collect, and the reconstruction is difficult to restrict its wide application in clinical. With the development of imaging technology, 3D reconstruction based on CT or MRI data sets has gradually played an important role in medicine. Because of its extensive and individualized characteristics, it has been widely used in clinical and achieved good results, showing its advantages in the study of anatomy, but because of the limit of layer distance and resolution. Limited, at the same time restricted by the gray level of the image, it has not yet completed the construction of the blood vessels, nerves, and lymphatics below the millimeter level. The three-dimensional reconstruction technique based on the continuous slice of tissue section refers to a two-dimensional image of a serial section of a certain tissue structure and a series of sections of the slice, by calculating the three-dimensional reconstruction. It is a very good method to reconstruct the stereoscopic structure of the tissue structure. It is a good method for the reconstruction of some traditional images, such as the structure of the nerve and below the millimeter level, which has been preliminarily obtained in the renal tubules of mice, the microvascular of the endometrium and the distribution of the fetal nerve. On the basis of the study of the early digital three-dimensional reconstruction, this study intends to construct the digital three-dimensional model of the internal nerve and pulse tube in the main ligament of the uterus by using the data set of continuous section of the main ligament tissue of the uterus, combining the image recognition, positioning and registration, and three-dimensional reconstruction, so as to provide the stereoscopic anatomical structure of the uterine ligaments for the NSRH operation. Basis.
The first part: To explore neural vascular three-dimensional reconstruction of digital positioning material of main ligament tissue in the uterus based on serial sections
[Objective]
The adhesion ability of four kinds of location materials on glass slices before and after different dyeing is compared, and a three-dimensional reconstruction material suitable for the continuous section of the main ligament tissue of the uterus is discussed.
[method]
1 specimens of fresh uterine ligamentous ligaments for IB1 cervical cancer were obtained. After fixed, dehydrated, 1 common sutures (3-0), 1 human hair, 1 absorbable sutures (8-0) and 1 biological tissues (from the liver tissue with a diameter of about 0.6mm) were used as the positioning rod markers. Paraffin embedding was carried out with paraffin paraffin, and the specimens were sectioned continuously with the paraffin slice machine. The thickness of the slice was 5 mu m, 120 slices were cut and the slides were cut by tissue. The slices were divided into four groups randomly, each of them was divided into four groups, each with conventional HE staining, TH staining, VIP staining and D2-40 staining. Under the microscope, observe and record each other. The number of anchor points before and after dyeing materials, comparative analysis of different positioning materials before and after dyeing adhesion marker points.
[results]
1, among the four kinds of location materials, such as human hair, common suture, absorbable suture and biological tissue, the rate of the patch was higher than that of the other three materials after dyeing, and the statistical difference was statistically significant (P0.01).
2, among the above four kinds of location materials, the location of biological tissue location was the least, and the number of the four kinds of dyed patches had no statistical significance (P=0.98). The absorbable suture, the absorbable suture and the localization point of the human hair were both serious, and the difference of quantity was significant (P0.01).
[Conclusion]
The use of needle puncture needle to obtain biological tissue as a location material has successfully solved the problem of loss of location marker. It has good adhesion effect in HE staining and all kinds of immunohistochemical staining.
The second part: the construction of the main ligament of the uterus of nerves and vessels in the digital 3D model
[Objective]
The three-dimensional model of the internal vascular, sympathetic, parasympathetic and lymphatic vessels of the main ligament of the uterus was constructed by using the data set of the main ligament of the uterus, and the three-dimensional walking and distribution of the nerve, blood vessel and lymphatic tube in the main ligament of the uterus were clearly defined.
[method]
1 specimens of the main ligaments of the main ligament of the uterus (left) for IB1 stage cervical cancer were obtained. The main ligament of the uterus was dehydrated after the.10% formalin was fixed in the fixed direction of the dehydrated box with the dehydration box for 8-12h, and 4 biological tissues (diameter about 0.6mm) were taken with the needle to be labeled with the specimen for paraffin embedding and frozen 30min After continuous slice, the slice thickness was 5gm, 4 layers were consecutively divided into one group, each group was bake 2-3H in 80 m.65 centigrade intervals for stratified dyeing. On the first floor, the slice was stained with conventional HE; on the second layer, the slice was stained with TH, and the sympathic nerve fibers were marked by TH, and the slices were stained by VIP, and the parasympathetic nerve fibers were specifically labeled. Fourth layers were stained by D2-40, specific markers of lymphatic vessels.
4 kinds of stained tissue sections were placed under the microscope to observe the various microstructure components of them. The scanning system of digital slice scanning was used to scan each slice. First, the scanning range was determined with 2 x 10 times, and then the slice was automatically scanned with 10 x 10 times. The images of the Photoshop software and the Matlab software were used. 4 groups of tissue slices were registered automatically by the registration method. The 4 sets of slice data set after registration were introduced into the Photoshop software. The contour of the vessels, sympathetic, parasympathetic and lymphatic vessels was painted by the brush tool.
ACDSEE software is used to adjust the pictures to the appropriate size and into the Mimics software. After automatically locating the images, organizing pictures and interpolating, the three-dimensional model of the main ligament, blood vessel, sympathetic nerve, parasympathetic nerve, and lymphatic tube of the uterus is constructed by three-dimensional reconstruction, and the model is registered and fused with Mimics. Transparent parts, measurement and other functions to analyze the model.
[results]
Based on the data set of the continuous section of the main ligament of the uterus, the digital three-dimensional model of the nerve pulse tube in the main ligament of the uterus was successfully constructed.
1, the uterine main toughening bandwidth is 26.77mm, the length is 50.09mm, the blood vessels mainly walk in the upper half, the nerve mainly walks in the lower half, from the cervical side to the pelvic wall, and the distance between the superior margin of the distal nerve trunk and the inferior border of the vessel is 8.59mm, 7.18mm, 4.87mm., respectively.
2, the blood vessels in the main ligament of the uterus mainly walk in the upper part of the main ligament of the uterus, from the cervix to the side of the pelvic wall, and the upper margin of the distal segment of the uterus is 11.99mm, 9.36mm, 4.89mm, respectively, and the lower margin of the inferior border of the uterus is 14.01mm, 13.74mm, 12.70mm., respectively.
3, the nerve mainly walked at the bottom of the main ligament of the uterus and increased to the wall of the cervix. The sympathetic nerve mainly walked at the bottom of the lower part of the main ligament of the uterus and increased gradually from the lateral pelvic side of the cervix from the cervix to the side of the pelvic wall, and the distal segment of the sympathetic trunk of the distal segment was 22.11mm, 22.16mm, and 19, respectively. .56mm, the parasympathetic nerve is relatively small, mainly walking at the bottom of the main ligament of the uterus, from the cervical side to the pelvic wall, and the distal parasympathetic nerve distance from the ligament is 22.58mm, 21.34mm, 18.62mm., respectively.
4, the lymphatic distribution is small, no obvious regularity in the main ligament of the uterus, mainly along the walk around the vessels.
[summary]
In this study, the digital three-dimensional model of the internal nerve pulse tube in the main ligament of the uterus was constructed by computer aided three-dimensional reconstruction of the main ligament of the uterus. The anatomical structure and the spatial relationship between the main ligaments of the uterus were accurately displayed, and the distribution and movement of the nerve, blood vessels and lymphatic vessels in the main toughened uterus of the uterus were displayed. The three-dimensional structure of the main ligament of the uterus is realistically reproduced. The reconstruction model can rotate any angle at any axis in space, so as to observe the shape, the space position and the relationship of each structure from different positions. At the same time, the distance of each structure can be measured by the measuring tool of Mimics software. Accurately display the distribution of the main ligament of the uterus internal structure accurately.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R713.4

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