不同超声微泡空化强度调控肿瘤血管毁损程度的研究

发布时间：2018-06-16 01:12

  本文选题：微泡 + 空化　； 参考：《第三军医大学》2014年硕士论文

【摘要】：背景： 血管生成是肿瘤生长、转移和侵袭的重要条件。已知直径超过1～2mm的实体肿瘤生长和转移离不开肿瘤血管生成。与正常血管相比，肿瘤新生血管发育不成熟，其结构上存在诸多缺陷：如血管内皮连接不紧密、基底膜不完整、缺少血管平滑肌层、周细胞连接疏松等。抗肿瘤血管生成靶向治疗主要利用肿瘤新生血管壁存在的特异性生物靶点，通过生物或者化学方法攻击和破坏肿瘤新生血管，以此达到治疗肿瘤的目的。然而，现有的靶向抗肿瘤新生血管的治疗方法，如血管生成抑制剂等，未取得突出的治疗效果，同时也会产生一些副作用影响到机体正常的生理过程（如伤口愈合、组织修复等），也无法避免长期使用产生的耐药性。 超声激励的血管腔内微泡空化可以物理破坏肿瘤微血管，阻断肿瘤血流灌注，其机制主要是空化效应产生的冲击波和微射流等机械效应对肿瘤血管壁造成的可逆性或不可逆性机械损伤。前期实验发现，采用峰值负压为4.8MPa的脉冲式聚焦超声激励微泡可以产生肿瘤血管毁损性损伤以及长达24h的微循环阻断，而当峰值负压降为2.6MPa时仅能使肿瘤血流阻断一小时。采用微泡超声空化治疗肿瘤具有安全、高效、操作简单、重复性好等优点，具有良好的发展前景。为了进一步了解超声空化强度与肿瘤微血管损伤的关系，我们采用三个水平峰值负压超声联合微泡治疗兔VX2肿瘤，并分析肿瘤超声空化治疗前后超声造影血流灌注情况及空化强度对微血管密度的影响。 目的： 1.通过观察不同水平峰值负压超声联合微泡空化对肿瘤血流灌注的影响，初步分析超声峰值负压对肿瘤微循环阻断程度的影响作用。 2.通过比较各水平超声峰值负压组与对照组肿瘤的微血管密度，探讨微泡超声空化治疗的超声峰值负压与微血管密度的量效关系。 材料方法： 1.实验材料 ⑴仪器设备：①DCT-700型新型数字化超声空化治疗仪，深圳市威尔德医疗电子有限公司生产。超声发射频率为1.0MHz，脉冲重复频率为100Hz，占空比为1.5%，峰值负压多档可调。②超声诊断仪，S2000型彩色多普勒超声诊断仪（西门子公司生产），配备有CPS超声造影成像模式，配有9L4高频线阵探头，发射频率为7～9MHz，中心频率8MHz。 ⑵实验动物：由第三军医大学附属新桥医院的实验动物中心提供，共46只雄性健康的新西兰大白兔，体质量在1.5～2.0kg之间。 ⑶实验试剂：①本实验中用于超声空化治疗与超声造影所用的微泡为第三军医大学附属新桥医院超声科所制的超声造影剂-脂氟显，，其中心气体为全氟丙烷，微泡浓度为（4～9）×109/mL，微泡直径为2～8μm，平均2μm。②小鼠抗人CD31单克隆抗体（即血小板内皮细胞黏附分子-1抗体），购自Abcam公司，保存于4°冰箱。 2.实验方法 ⑴不同水平峰值负压超声对兔VX2肌肉移植瘤空化治疗实验： 16只腿部肌肉种植VX2肿瘤荷瘤种兔，共30个肿瘤，随机分成3个不同水平超声峰值负压幅度处理组，分别为2184kPa组、1785kPa组、1019kPa组。各峰值负压组采用相应峰值负压参数进行超声辐照联合经静脉注射微泡（0.2ml）治疗肿瘤，并于治疗前、后进行超声造影检查，分别对超声造影峰值强度和曲线下面积值进行分析，即刻处死实验兔于治疗后，获取肿瘤组织标本进行HE（hematoxylin-eosin staining）染色，并观察肿瘤组织病理改变。 ⑵超声峰值负压与兔VX2肌肉移植瘤微血管密度量效关系研究实验： 30只腿部肌肉种植VX2肿瘤荷瘤兔随机分成4组：分别为上述三个水平峰值负压组（n=8）及对照组（n=6），对照组不予以任何处理。另三组给予超声激励微泡空化治疗肿瘤。治疗后即刻取出肿瘤组织，固定，包埋，切片，行免疫组化染色，对微血管密度（MVD）进行计数，并比较各组肿瘤微血管密度值。 结果： 1.不同水平峰值负压超声对兔VX2肌肉移植瘤空化治疗实验：峰值负压2184kPa组肿瘤治疗后造影血流灌注完全消失，其PI值由治疗前的（40.59.9）%降至治疗后的（11.67.8）%，AUC由（1299.1512.6）%s降至（280.1186.1）%s (P0.05)；1785kPa组治疗后造影肿瘤血流灌注明显下降，PI由（42.57.8）%降至（24.214.8）%，AUC由（1378.1494.6）%s降至（549.4463.4）%s (P0.05)；1019kPa组肿瘤治疗后造影与治疗前比较肿瘤血流灌注轻度增强，但治疗前后PI与AUC比较差异无统计学意义(P0.05)。病理检查各组肿瘤治疗后均可见部分肿瘤微血管断裂，红细胞溢出，肿瘤微血管出血，红细胞渗入组织间隙，肿瘤组织水肿，但2184kPa组肿瘤细胞较其它组水肿明显。 2.峰值负压为2184kPa、1785kPa、1019kPa的三组肿瘤MVD值分别为（157.15±124.37）、（141.12±60.45）、（311.64±73.93），均低于对照组肿瘤MVD值（619.73±282.78），差异有统计学意义(P0.05)，余各组两两比较差异无统计学意义(P＞0.05)。 结论： 1.超声峰值负压在1785kPa和2184kPa水平时超声空化可阻断VX2肿瘤微循环，且峰值负压越高，阻断效果越显著。峰值负压为1019kPa水平时不仅不能阻断肿瘤微循环，还可能造成肿瘤血流灌注轻度增强。 2.经静脉微泡联合超声空化可以减少肿瘤微血管数，可能与超声空化毁损性破坏肿瘤微血管有关；且峰值负压在2184kPa与1785kPa水平减少肿瘤微血管数要多于峰值负压在1019kPa水平。
[Abstract]:Background:
Angiogenesis is an important condition for tumor growth, metastasis and invasion. The growth and metastasis of solid tumors with known diameter of more than 1 to 2mm are inseparable from tumor angiogenesis. Compared with normal blood vessels, the neovascularization of the tumor is immature and there are many defects in its structure, such as the incompleteness of the vascular endothelium, incomplete basement membrane, and lack of smooth blood vessels. The targeting of tumor angiogenesis is mainly used to attack and destroy neovascularization through biological or chemical methods to achieve the purpose of treating tumor. However, the existing methods of targeting neovascularization, such as angiogenesis, have been used. As a inhibitor, there is no outstanding therapeutic effect, and some side effects will also affect the normal physiological processes of the body (such as wound healing, tissue repair, etc.), and the resistance to long-term use can not be avoided.
The intravascular microbubble cavitation stimulated by ultrasound can physically destroy the tumor microvessels and block the tumor blood flow. The mechanism is the reversible or irreversible mechanical damage caused by the mechanical effects such as the cavitation effect and the micro jets on the tumor vessel wall. In the previous experiment, the pulse type polymerization with the peak negative pressure of 4.8MPa was found. Jiao Chaosheng microbubbles can produce tumor vascular damage and 24h microcirculation blocking, but when the peak pressure drop is 2.6MPa, the tumor blood flow can only be blocked for an hour. Microbubble ultrasound cavitation is a safe, efficient, simple and reproducible, and has a good prospect. To understand the relationship between ultrasonic cavitation intensity and tumor microvascular injury, we used three horizontal peak negative pressure ultrasound combined with microbubbles in the treatment of rabbit VX2 tumor, and analyzed the effect of ultrasound contrast perfusion and cavitation intensity on microvascular density before and after ultrasound cavitation therapy.
Objective:
1. by observing the effect of different levels of peak negative pressure ultrasound combined with microbubble cavitation on the blood perfusion of tumor, the effect of peak negative pressure on the blocking degree of tumor microcirculation was preliminarily analyzed.
2. the quantitative relationship between the peak negative pressure and the microvascular density of the microbubble ultrasound cavitation was investigated by comparing the microvascular density of the different levels of the ultrasonic peak negative pressure group and the control group.
Material methods:
1. experimental materials
(1) equipment: (1) a new type of DCT-700 digital ultrasonic cavitation treatment instrument, produced by Shenzhen Wilder Medical Electronics Co., Ltd., the ultrasonic emission frequency is 1.0MHz, the pulse repetition rate is 100Hz, the duty ratio is 1.5%, the peak negative pressure is multiple adjustable. 2. Ultrasonic diagnostic instrument, S2000 color color color ultrasonic diagnostic instrument (SIEMENS company production). The CPS ultrasound contrast imaging mode is equipped with 9L4 high frequency linear array probe. The transmitting frequency is 7 ~ 9MHz, and the central frequency is 8MHz.
(2) experimental animals: a total of 46 healthy New Zealand white rabbits were provided from the experimental animal center affiliated to the new bridge hospital affiliated to Third Military Medical University. The body mass of the rabbits was from 1.5 to 2.0kg.
(3) experimental reagents: (1) the microbubbles used in ultrasonic cavitation therapy and ultrasound contrast in this experiment are the ultrasound contrast agents made by the ultrasound department of the affiliated Xinqiao Hospital of Third Military Medical University. The central gas is perfluoropropane, the concentration of microbubbles is (4~9) * 109/mL, the diameter of microbubbles is 2~8 u m, and the average of 2 mu m. in mice against human CD31 monoclonal anti Body (that is, platelet endothelial cell adhesion molecule -1 antibody), purchased from Abcam company, is stored in the 4 degree refrigerator.
2. experimental method
(1) cavitation treatment of rabbit VX2 muscle xenograft with different level of peak negative pressure ultrasound:
16 leg muscles were planted with VX2 tumor bearing rabbits and 30 tumors were randomly divided into 3 different levels of ultrasonic peak negative pressure amplitude treatment group, group 2184kPa, group 1785kPa and group 1019kPa. The peak negative pressure groups were treated with corresponding peak negative pressure parameters for ultrasound irradiation combined with intravenous microbubbles (0.2ml) for the treatment of tumor before and after treatment. The peak intensity of contrast-enhanced ultrasound and the area under the curve were analyzed by ultrasound contrast examination. After the treatment, the rabbit tissue specimens were stained with HE (hematoxylin-eosin staining), and the pathological changes of the tumor were observed.
2. Study on the dose effect relationship between peak negative pressure and microvessel density of rabbit VX2 muscle transplantation tumor:
30 VX2 tumor bearing rabbits were randomly divided into 4 groups: the above three level negative pressure group (n=8) and the control group (n=6). The control group was not treated with any treatment. The other three groups were treated with ultrasonic stimulation microbubble cavitation for the treatment of tumor. Density (MVD) was counted, and tumor microvessel density values were compared.
Result锛

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