【作者】 林锐；

【导师】 郭剑明； 王杭；

【作者基本信息】 复旦大学 ， 泌尿外科学， 2010， 硕士

【摘要】 超声是临床上广泛应用的检查手段,具有无创伤性、应用面广、操作方便灵活、安全可靠而又价格低廉等优点,在泌尿外科,超声检查已在肾脏,输尿管,膀胱以及前列腺疾病的诊断中得到广泛应用,甚至已经成为首选检查手段,但是,常规二维超声有很多临床应用局限性,比如无法直观地反映脏器的立体形态,另一方面,目前关于肾脏肾盂发育形态学变化方面的研究虽然比较多,但往往需要处死动物解剖肾脏,或者使用有放射性危害的CT进行随访,这2种方式均对动物有较大损伤,而B超进行发育随访研究肾脏肾盂的形态学则有较多优势,可以针对动物个体肾脏发育情况进行连续跟踪随访,且随着B超三维重建技术的发展和成熟,将来也有望部分取代CTU等一些泌尿外科较为昂贵的检查项目。虽然三维超声已经问世,但由于各种原因,至今未得到临床应用,而关于二维超声三维重建的研究很少,本研究将以小鼠为研究对象,从技术手段的确立,高频显微超声三维重建随访肾输尿管畸形小鼠肾脏发育这两个方面,来研究B超三维重建是否能较为准确地对各种不同情况的肾脏进行检查,重建和测量,为进一步研究奠定基础。第一部分高频显微超声获取小鼠肾脏影像的技术手段的确立目的：通过实验建立使用Vevo 770 UBM超声扫描系统获取小鼠肾脏影像的操作流程,并熟练掌握操作过程中安全麻醉小鼠的方法。材料与方法：取正常C57BL/6新生小鼠5只,分别在生后第1天,第1,2,3及6-8周用Vevo 770 UBM系统高频显微超声对小鼠双侧肾脏进行扫描,重建获得的肾脏体积同文献报道的常用于测量小鼠肾脏体积的micro CT法所测得的体积用SPSS 15.0进行两组独立样本的成组t检验,了解高频显微超声三维重建测量小鼠肾脏体积的准确性,同时和经典的micro CT法及溢水法进行比较,了解超声三维重建的优点和缺点及应用前景。结果：5只小鼠从出生至8周龄检查结束,无一只因为麻醉意外,操作不当导致死亡,且每次试验结束后,小鼠均能被它的母亲接纳并继续抚养,也未出现其他相关合并症。预实验小鼠共10个肾脏,测得的体积见表1,平均体积为：183.18±30.90mm3,同文献中报道的micro CT法测得的肾脏体积用SPSS 15.0进行两组独立样本的成组t检验,p=0.817>0.05。结论：1.高频显微超声法和micro CT法三维重建检测小鼠肾脏体积结果无明显统计学差异,准确性较好。2.高频显微超声法具有安全性高,放射性小,可重复性高,无创性,准确性,便利性,成本低,速效性以及客观性等多种优点,也存在对技术人员要求较高,肾脏上下极处常显示不清,检测过程中存在的干扰因素较多,检测时间较长以及易引起技术人员疲劳等缺点,但总的来说,相较于micro CT法及溢水法,高频显微超声法的优势是明显地,同时我们也相信它的缺点和不足随着技术的进步能被逐步克服。第二部分高频显微超声三维重建随访肾输尿管畸形小鼠肾脏发育目的：利用第一部分建立的高频显微超声三维重建检测小鼠肾脏的技术手段,对肾输尿管畸形小鼠动物模型的肾脏发育过程进行随访,完善高频显微超声三维重建对积水肾脏和正常肾脏的检测技术,同时为将来进一步研究肾实质体积和功能的关系以及小鼠肾脏发育学打下基础。材料与方法：我们共选取4对ROBO2基因敲除的C57BL／6小鼠,分别交配产仔,建立正常和积水肾脏的动物模型,并使用第一部分建立的技术手段,对新生小鼠肾脏肾盂发育进行随访,将存活时间大于等于3周龄的小鼠入组,并用剪尾法测小鼠ROBO2基因型,了解基因型和表现性间的关系,并用SPSS 15.0软件对正常小鼠肾脏肾盂体积和积水小鼠肾脏肾盂体积进行2组独立样本的成组t检验,并描绘肾脏及肾盂发育箱图,了解小鼠肾脏肾盂的发育规律和特点,并通过三维重建立体图来展示正常肾脏的发育过程。结果：最后总共有3只积水小鼠和8只正常小鼠存活至3周龄以上,这些小鼠肾脏三维重建测得的数据列入表2中,剪尾法测小鼠基因型和小鼠最终的表现型和理论一致,正常小鼠和积水小鼠的肾脏体积在2周龄时无统计学差异,除此以外在其他周龄段都有较为显著的统计学差异,而正常小鼠和积水小鼠肾盂体积从出生一直到8周发育成熟每一个周龄段均有显著差异；而从肾脏肾盂发育箱图可以看出,可以看出正常小鼠肾脏体积随周龄的增加而增加,而肾盂的体积在2周龄和3周龄时出现波动,并不像肾脏体积一样呈现从始至终一致的增加趋势；积水小鼠肾脏体积和肾盂体积则均随周龄的增加而增加；通过肾盂占肾脏百分比的独立样本t检验,发现正常小鼠和积水小鼠在生后1天和1周龄时,肾盂占肾脏体积的百分比无显著统计学差异,而2周龄到6-8周龄时,正常和积水小鼠肾盂占肾脏体积的百分比均有显著统计学差异；通过正常小鼠和积水小鼠各自发育过程配对样本t检验,我们发现,正常小鼠肾脏肾盂发育过程中,肾脏体积在不同周龄段均有显著统计学差异,而肾盂体积则在2周龄到3周龄时无明显统计学差异,其余周龄段均有显著统计学差异；积水小鼠肾脏体积在2周龄到3周龄时无显著统计学差异,在其他各周龄段均有显著统计学差异,积水小鼠肾盂体积在2周龄和3周龄无显著统计学差异,在其他各周龄段均有显著统计学差异；同时通过三维立体图片及肾脏矢状面图片直观展示小鼠肾脏肾盂发育过程。结论：1.在三维重建过程中,积水肾脏的重建与正常肾脏的重建并无明显差别,除了超声表现和正常小鼠不同以外,三维重建的技术手段是和正常肾脏完全一样的,当然积水肾脏还会出现诸如侧壁回声失落等现象,导致误差的增大；2.关于2周龄时积水小鼠和正常小鼠肾脏体积无统计学差异的原因,一方面可能和样本量较少有关,另一方面,重度积水的小鼠基本上在2周龄前就已死亡,入组的积水小鼠一般为轻中度积水,因此肾脏体积较正常肾脏增大不明显；3.从小鼠肾脏肾盂发育箱图可以看出,2周龄时正常小鼠的肾盂体积大于3周龄时正常小鼠的肾盂体积,但差别较小,我们认为同样也是由于样本量较少以及不可避免的误差所造成的；4.不论正常小鼠还是积水小鼠肾盂的体积发育均在2周龄和3周龄时有一个减速和停滞的过程,而这个过程是否和积水小鼠肾脏体积在2周龄和3周龄时无明显统计学差异这一结果有关,还需增大积水小鼠的样本量,并进一步探讨尿路畸形小鼠在2到3周龄时积水程度一过性缓解的原因,方可得出结论。5.对积水小鼠来说,2周龄后肾盂积水的程度较正常小鼠明显加重,肾实质占肾脏总体积的百分比下降显著,且炎症反应的后果开始显现,表现为无功能的疤痕组织增生明显,因此,积水肾脏实质的体积会大于正常肾脏实质的体积,可功能却不如正常肾脏。更多还原

【Abstract】 B ultrasound is widely used in clinical examination, with the advantage of non-invasive, wide application, easy and flexible, secure and low cost,especially in the department of urology, which can help doctors in the diagnosis of kidney,urinary,bladder or prostate disease and even becomes the first selection of examination in urology.However,regular B ultrasound examination has much clinical limitation.For example,it can’t reflect the three-dimention shape of organs directly. Although three-dimention ultrasound examinations have advented,but for many reasons,they don’t applicate in clinic. And there is very little research of the three dimentional reconstruction of B ultrasound.This research will study from the establishment of technical process of three-dimentional reconstruction of high-frequency micro-ultrasound in mice to follow ing up the growth of kidney and ureter in mice for wondering w.hether the high-frequency micro-ultrasound can examinate,reconstruct and measure the normal or hydro kidney,laying the foundation of the further study.Part I The establishment of technical process for imaging the kidney of mice with high-frequency micro-ultrasoundObjective:Establish the technical process of imaging the kidney of mice with the Vevo 770 UBM ultrasound scanning system through the preliminary experiment and to be familiar with how to anesthesia the mice safely in the process of examination.Method:Five normal newborn C57BL/6 mice are used in our preliminary experiment. We scan the bilateral kidney of each mouse in the first day after the birthday,one-week old,two-week old,three week old and six-to-eight-week old with Vevo 770 UBM ultrasound system. And then we compare the volume of reconstructed kidney with the volume of mice’s kidney measured by micro CT in using of SPSS 15.0 for indepent t test for two indenpent samples.Our purpose is to realize the accuracy and compare the advantage and disadvantage of the high-frequency micro-ultrasound reconstructing method with classical micro CT method and overflow method,which could tell us the prospective application in clinic of our method.Results:None of the five mice is dead from anesthesia accidents or improper operation from its birthday to the end of examination in 8-week old. And each time after the experiment,the mouse can be accepted by its mother to get fed without any related complications.There are 10 reconstructed kidneys in five mice in the preliminary experiment.The volume of them is listed in Table 1.Its average volume is 183.18±30.90mm3.We compare the average that we reconstructed with the average,which is looked up in the paper using the method of micro CT,by SPSS 15.0 for indepent t test for two indenpent samples(p=0.817>0.05).Conclusion:1.There is no significant statistical difference showed in the method of high-frequency micro-ultrasound 3D reconstruction and micro CT 3D reconstruction.The accuracy of our method is good.2.The method of high-frequency micro-ultrasound shows the advantages of high safety and repeatability,low dosage of radiation,accuracy,non-invasion,convenience,low cost,rapid availability and impersonality.It also shows the disadvantages including high requirement of technical staff,unsharpness in the upper and lower pole of the kidney,interference in the examination process easy fatigue in technical staff and so on.But overall,compared to micro CT and overflow method,the advantage of high-frequency micro-ultrasound method is obviously.And we also believe that we will conquer the disadvantage with the progress of technique.Part II Follow up the development of kidneys of mice by using high-frequency micro-ultrasound reconstructionObjective:We follow up the development of kidneys in newborn mouse model of CAKUT(congenital anomalies of kidney and urinary tract) using the technical process we establish in the Part I.Our purpose is to consummate the technique in the scanning of hydro-kidney and lay the foundation of further study to the relationship between function and volume of renal parenchyma simultaneously.Method:We have selected four pairs of C57BL/6 mice which are knocked out the ROBO 2 gene and let them generate newborn mice seperately to establish the normal and hydro mouse model. And follow up the development of kidneys of newborn mice using the technical process in the Part I.We enroll the mice which are older than two-week old and cut their tail to sequence their type of ROBO 2 gene to study the relationship between genotype with phenotype.Simultaneously we use SPSS 15.0 software to compare the normal kidneys and pelvis with them of hydro-mice by independent t test for two independent samples and depict the box map of development of kidneys and pelvis in two groups to realize the rules and characteristics of their development. We will show the 3D pictures of kidneys and pelvis in the development of mice.Results:Finally,a total of three hydro-mice and eight normal mice survived for more than 3-week old.The data of their volume of kidney and pelvis is listed in the Table 2. The genotype and phenotype of the mice are consistent with that in theory.There are significant statistical differences in volume of kidney between the normal mice and hydro-mice except that when they are two-week old.Otherwise,the volume of pelvis is significant statistically difference in them on each time of the examination. We also show the 3D pictures and saggital image of the developing process of the normal kidney and pelvis of mice in this part.Coclusion:1.In the process of 3D reconstruction,there is no obvious differences in the technique between normal kidney and hydro-kidney,except the image display in the B-ultrasonography.2. About the reason of differences in the volume of kidneys bitween normal mice and hydro-mice,we think it could be relative with less amount of the samples,otherwise,all of the severe hydro-mice are dead before the time of two-week-old,so the enrolled mice’s diseases are mild or moderate.3.From the box map of the development of mice’s kidneys,we can realize that the average volume of pelvis in two-week-old normal mice is larger than that of three-week-old normal mice.But the difference is not obvious.We think it is also caused by the less amount of samples and the inevitable error. And no matter they are normal mice or hydro-mice,their volume of pelvis’ development are a little slower in two to three weeks age.更多还原