【作者】 修建成；

【导师】 刘伊丽；

【作者基本信息】 第一军医大学 ， 内科学， 2004， 博士

【摘要】 在超声微泡技术应用过程中，微泡的空化经常发生。微泡空化一方面可以为声学造影诊断提供声学信号；另外一方面，微泡空化时产生的空化效应，也是超声微泡技术众多新用途的基础。在不同声强超声作用下，微泡可以发生稳定空化或者不稳定空化。微泡在较强声场作用下会发生破裂，发生不稳定空化，也称为惯性空化。微泡发生惯性空化破裂时可以释放出很大的能量，对附近细胞或组织产生空化效应，造成细胞或组织的损伤。 在超声微泡技术应用于不同用途时，需要我们控制损伤的程度。在诊断应用时，要尽量避免损伤的产生；在用于基因转染时，需要损伤即要保证外源大分子可以通过损伤的细胞膜进入细胞或通过受损的内皮屏障到达组织间隙，又要保证不会产生严重的后果；损伤导致细胞或组织坏死，则是治疗肿瘤时必须的。 目的： 通过观察白蛋白微泡(全氟显)在诊断和治疗剂量超声作用下对大鼠脊斜肌微血管的损伤情况，评估全氟显在诊断应用时的安全性；通过对微泡空化导致出血的微血管的透射电镜、溴化丙啶染色以及绿色荧光微球外溢情况观察，探讨微泡空化介导血管内基因传输的机理；在不同时间点，用透射电镜观察出血点，了解出血点微血管壁的结构变化情况；观察不同超声微泡条件对出血点产生的影响，初步探讨微泡空化介导血管内基因传输的优化条件，为体内基因转染实验提供超声微泡参数。 方法： 1、白蛋白微泡空化对大鼠微血管组织结构的影响 用Sequoia 512诊断超声机不同模式、最高机械指数和治疗超声机不同输出能量发射超声，经皮或直接照射大鼠脊斜肌内全氟显微泡，活体显微镜观察大鼠脊斜肌微血管的变化，光镜观察组织切片。2、微泡空化介导血管内基因传输的机理 用治疗超声直接照射大鼠脊斜肌内全氟显微泡，在脊斜肌上产生出血点，观察微血管澳化丙咤染色、绿色荧光微球外溢情况，透射电镜观察出血点处微血管壁结构;透射电镜观察不同时间点的出血点处微血管壁结构。3、影响微泡空化效应的超声微泡参数 观察不同能量超声、不同超声照射时间、不同微泡输入速度、不同超声脉冲间隔、不同输入途径以及不同种类微泡对微泡空化产生微血管损伤的影响。结果: 1、用诊断剂量超声，以2一D和PW模式最大机械指数(Ml=中心负压·频率一‘/z)，对大鼠脊斜肌经皮或直接发射超声，活体显微镜下未发现脊斜肌上有出血点，切片光镜观察未发现微血管和组织损伤。 2、治疗超声探头能量为0.5 w.。m一2时，超声照射后，活体显微镜下未发现红细胞外溢;活体显微镜下可见脊斜肌出现小出血点的超声能量闽值为0.75 w.cm.2，出血主要发生在毛细血管和微静脉，切片光镜观察发现红细胞外溢。改变触发间隔，微泡输入速度，微泡输入途径等对产生出血点的能量闽值影响不大。微循环观察发现，出血点部位微循环血流基本正常。探头能量为3w·cm一2时，经皮发射的超声作用于全氟显未导致大鼠脊斜肌出现出血点，切片光镜观察未发现毛细血管和明显肌肉组织损伤。 3、微泡空化介导外源大分子物质传输到组织间隙的外溢通道实质是血管内皮细胞的撕裂所致，撕裂发生在细胞核附近;这种外溢的通道可以是一过性产生，具有可修复性;红细胞可以通过小于自己直径的外溢通道。 4、超声能量越大、照射时间越长、微泡输入速度越快时，微泡空化在大鼠脊斜肌微血管造成的出血点越多，损伤越重;当超声脉冲间隔为5s时，微泡充满微循环，微泡空化造成的出血点最多;可以通过肺循环的微泡—全氟显，动或静脉输入对出血点产生数量影响不明显:结构相似的微泡，出血点产生数量相近。结论: 1、诊断剂量超声作用全氟显不会导致大鼠骨骼肌产生出血点。 2、超声作用全氟显导致游离体外大鼠骨骼肌的出现出血点的能量阐值为500至750 mvV.cm一，超声触发间隔、全氟显的输入速度以及输入途径对这一闽值影响不大。 3、微泡空化造成微血管内皮细胞损伤，在微血管内皮屏障上产生撕裂孔，红细胞和100nln荧光微球可以通过这些的孔洞外溢到组织间隙，这可能就是微泡空化介导血管内基因传输，增加基因转染的机理。内皮细胞的损伤具有可修复性。 4、影响微泡空化效应的超声微泡参数包括超声能量、超声照射时间、微循环内的微泡数目、微泡自身特性等等，增加超声能量、照射时间、增加微循环内的微泡数目可以增加红细胞的外溢，可能有利于提高基因传输的效率，但是可能带来更大副作用。用于基因传输的微泡最好能通过肺循环;不同结构的微泡基因传输的效率不同，与微泡的空化特性有关。更多还原

【Abstract】 Cavitations occur regularly when ultrasound act on microbubble during the ultrasound microbubble technology application. On one hand, the cavitation supplies the acoustic signal for ultrasound diagnosis. On the other hand, the cavatition follow microbubble cavitation is the essential of many kinds of new application of ultrasound microbuble technology. Different kind of cavitations, stable or unstable cavitation, happens as different acoustic pressure act on the microbubble. At higher acoustic pressure, microbubble fragmentation, and unstable cavitation, also called inertial cavitation occurs. A lot of power is released when the microbubble rupture, which caused all kinds of effect on the nearby cell or tissue.We should control the extent of the damage induced by microbble cavitation freely when we use the technology of ultrasound microbubble on different purpose. In diagnosis use, the injure might as mild as possible. During gene transfection, the ruptures not only allow the macromolecular enter the cell through membrane, or pass through the endothelium barrier into tissue, but also do not induce serious subsequence. The injure cause cell or tissue regression is longed for in tumor therapy.Objective:To evaluate the safety of albumin microbubble used in diagnosis, we observed the state of injured microvessel caused by albumin microbubble destructed by diagnosis or therapy used ultrasound. To define the mechanism of intravascular gene transport by ultrasound microbubble, we observed the ruptured microvessel caused by ultrasound microbubble by electronic microscope, Propidium Iodide stain, and fluorescent polymer microspheres extraction. To investigated the change of microvessel structure by looking into the petechia at different time by electronic microscope. By observing theinfluence of different ultrasound microbubble condition on the extent of petechiae, intent to optimize the ultrasound microbuble induced intravascular gene transport condition primarily, and supply ultrasound microbuble parameter for in-vivo gene transfection.Method:1 The effect of albumin microbubble cavitation on the rat spinotrapezius microvessel structureAfter microbubble infusion, ultrasound was applied to the exteriorized spinotrapezius directly or to the intact rat spinotrapezius percutaneous by Sequoia 512 at the highest mechanical of different mode or ultrasound therapy device at different output. The changes of the structure of the spinotrapezius microvessel were observed by microscope under transillumination. The tissue slices were observed by microscope.2 The mechanism of microbubble cavitation induced intravascular gene transportSeveral frames of ultrasound were delivered to exteriorized spinotrapezius by a therapy ultrasound device directly. And petechias were induced by microbubble cavitation in the exteriorized spinotrapezius. The structure of the injured vessel structure was investigated by electronic microscope, Propidium Iodide stain, and fluorescent polymer microspheres extraction. The changes of microvessel structure were looked into at different time by electronic microscope.3 The influence of ultrasound or microbubble variables on microbubble cavatitionInfluence of ultrasound or microbubble variables on microvessel injure was observed by changing ultrasound output, ultrasound delivery time, microbubble infusion speed, pulsing interval, injection site, and different microbubble.Result:1 Diagnosis ultrasound delivery to spinotrapezius in-vivo or to intact rat spinotrapezius does not induced petechiae, no matter by the style of 2-D or PW in biggest mechanical index. No microvessel or tissue injure were found by microscope.2 No petechiae were caused by the therapy ultrasound device when the transducer output under 0.5 W cm-2. No microvessel or tissue injure were found by microscope. The therapy ultrasound device does induce petechiae in exteriorized spinotrapezius when the transducer output is above 0.75 W cm-2. The destructions of vessel were seen in capillaries and microvein. The threshold of tran更多还原