超声造影评价小鼠Lewis肺癌血管生成及恩度的抗肿瘤血管生成治疗的疗效

发布时间：2018-03-05 02:00

本文选题：超声造影检查　切入点：Lewis肺癌　出处：《山东大学》2014年博士论文　论文类型：学位论文

【摘要】：第一部分超声造影定量分析评价小鼠Lewis肺癌肿瘤血管的生成研究背景及意义肿瘤血管生成即指在肿瘤原有微血管网的基础上通过内皮细胞的“芽生”方式形成新生血管的过程,它是肿瘤生长和转移的一个至关重要的因素。在肿瘤内及周围的血管生成被认为是恶性肿瘤细胞增殖的前提,这些新生血管不但能够提供肿瘤细胞增殖所需的营养,也为肿瘤细胞在体内的远处转移提供了通道。所以血管生成是恶性肿瘤的重要特征,它的检出对于肿瘤的诊断、治疗与预后相当重要。在过去的几年中,许多研究通过无创的影像学方法来评估肿瘤血管的生成,例如对比增强计算机断层扫描(contrast enhanced computed tomography, CECT)、动态对比增强磁共振成像(dynamic contrast enhanced magnetic resonance imaging, DCE-MRI),磁共振血管成像技术(magnetic resonance angiography, MRA）、正电子发射断层扫描(positron emission tomography,PET)和多普勒超声诊断技术等等。然而,以上这些方法在检测低速血流和微小血管上,具有明显的局限性。超声造影明显提高了对低速血流的检出能力,使评价组织内毛细血管和微血管的血流成为可能,并可提供关于肿瘤血管的详细信息。微血管密度即平均每平方毫米肿瘤面积内的血管数,是目前评价肿瘤血管生成的公认的参考标准。然而不同血管内皮细胞标记的微血管密度用于揭示不同肿瘤血管内皮细胞的分化程度以及结构和功能特点。在以往的实验研究中,较多使用CD31血管标志的微血管密度,用以评价组织的血管生成情况。由于CD31微血管密度在已分化和未分化的血管内皮细胞中均有表达；而另一常用的血管标记物CD34则只表达于已分化的内皮细胞中,故肿瘤血管的分化程度不同,其所代表的微血管密度亦有不同。小鼠Lewis肺癌(Lewis lung carcinoma, LLC)是将传代培养的Lewis肺癌细胞接种于C57/BL6小鼠的腋部皮下而成,已经多次反复应用于实验室的各项研究中,是较为成熟的动物瘤实验模型。虽然既往已有研究证实超声造影的定量分析参数与肿瘤的微血管密度存在一定的线性相关关系,到目前为止,还未有报道关于小鼠Lewis肺癌中不同血管标记的微血管密度同超声造影参数之间的相关性研究。研究目的本研究通过建立小鼠LLC的皮下移植瘤动物模型,应用增强脉冲序列(contrast pulse sequencing, CPS)的超声造影成像技术,结合时间-强度曲线,研究并比较超声造影的血流灌注参数与不同血管标记的微血管密度在LLC动物实验模型中的相关性,探讨超声造影在评价肿瘤血管生成方面的应用价值。研究方法建立25只小鼠LLC皮下移植瘤模型,待肿瘤大小约1.0cm左右行超声造影检查,应用超声造影定量分析软件进行脱机分析,获取峰值强度(peak intensity,PI)、达峰值时间(time to peak, TTP)和到达时间(arrival time,AT)等超声造影定量分析参数。超声检查结束后经颈椎脱位处死小鼠,完整切除肿瘤,免疫组织化学法分别检测肿瘤的CD31及CD34微血管密度的表达情况,并分别计算出微血管密度值(microvessel density,MVD)。结果 1.接种后7天出现肉眼可见的肿瘤,模型建立成功。肿瘤的平均直径约0.9cm,最大直径为1.3cm,大多呈椭圆形,成瘤率达100%,且无一例复种。 2.25例小鼠LLC的超声造影增强模式均表现为环状强化,即肿瘤组织周边部明显强化,而中央部呈现低强化或无强化。在时间-强度曲线上上述LLC肿瘤均呈现“快进、慢出”的表现,即上升支陡直,迅速达峰,下降支相对较缓。 3.在小鼠的LLC肿瘤中,存在两种代表血管内皮细胞不同分化程度的血管标记物：CD31和CD34. 4.超声造影的灌注参数峰值强度(PI)与CD31微血管密度呈正相关(r=0.428;P0.05)与CD34微血管密度无线性相关。 5.在小鼠的LLC肿瘤中,CD31微血管密度与CD34微血管密度呈负相关(r=-0.474;P0.05)。结论 1.本实验成功建立了小鼠Lewis肺癌的肿瘤模型,进一步证明了该模型是研究组织肿瘤的可靠实用的动物实验工具,并具有较好的可重复性。 2.在小鼠Lewis肺癌肿瘤模型中,存在两种不同分化程度的肿瘤血管：分别是代表未分化和已分化成熟血管内皮细胞的CD31微血管密度和只代表分化成熟的血管内皮细胞的CD34微血管密度。 3.超声定量分析参数与肿瘤微血管密度的相关性提示：超声造影(团注法)可以通过监测肿瘤的血流灌注参数来有效评价肿瘤血管生成；其中超声造影参数中的峰值强度可以在一定程度上评价肿瘤未分化的血管生成,指导临床应用抗血管治疗以及评估肿瘤的分级和预后上有着重要的临床实用价值。第二部分超声造影定量分析评价恩度对小鼠Lewis肺癌肿瘤的治疗疗效研究背景及意义抗肿瘤血管生成治疗是目前治疗肿瘤的研究热点和重点。由于抗肿瘤血管生成治疗的靶向作用于肿瘤内异常的新生血管或不成熟血管,其治疗会引起肿瘤内血容量、血流灌注速度及其它血流动力学指标的改变,监测这些血流动力学指标的改变成为临床上评价抗血管生成治疗的有效指标。既往多种影像学检查方法(如CT、 MRI及超声等)都曾来评价肿瘤血管的生成和抗肿瘤血管生成治疗的疗效。超声检查尤其是彩色多普勒超声由于具有实时、费用低、重复性好以及能直接评价肿瘤的血流灌注等优点,在评价肿瘤血管生成及抗血管生成治疗中起着重要的作用。但彩色多普勒超声对微血管的显示及评价微血管的血流灌注有一定的局限性,近年来超声造影更多的应用于临床,使对肿瘤内微小血管内血流的评价成为可能。在一定浓度范围内,超声造影的信号强度与血液内微泡浓度存在线性关系,故可利用时间-强度曲线软件对超声造影过程进行定量分析。恩度(endostar)即重组人血管内皮抑素,是一种血管生成抑制药物,其可能作用机理是通过抑制血管内皮细胞的增殖、迁移达到抑制肿瘤新生血管的生成,阻断了肿瘤细胞的营养供给,从而达到抑制肿瘤的目的。临床主要应用于非小细胞肺癌的抗肿瘤血管生成治疗。由于血管生成抑制药物主要作用于肿瘤内的异常新生血管,治疗后不仅会引起组织内血管数目的变化,更重要的是会引起肿瘤血管的形态、以及功能的改变,上述改变往往继发了肿瘤内血流动力学的异常,因此监测此异常变化可以成为临床上评估抗血管生成治疗的一个可靠手段。研究目的本研究拟采用Lewis肺癌荷瘤小鼠模型应用腹腔内注射抗血管药物恩度治疗后与对照组的超声造影血流灌注参数进行对照性研究,并将超声造影参数与肿瘤的微血管密度进行相关性比较,旨在探讨超声造影对评价抗肿瘤血管生成药物疗效的作用。研究方法建立30只小鼠LLC皮下移植瘤模型,随机分为2组,各模型自建立次日起分别经腹腔注射生理盐水钠(normal saline, NS)、恩度(5mg/kg体重),每天给药,连续给药14天。用药后每天观察两组小鼠的一般情况,并每隔一天测定小鼠肿瘤的大小。末次给药24h后行超声造影检查,并应用超声造影定量分析软件进行脱机分析,获取峰值强度(PI)、达峰时间(TTP)和到达时间(AT)等参数。超声检查结束后颈椎脱位处死小鼠,并完整切除肿瘤,免疫组织化学检测肿瘤CD31的表达情况,并计算肿瘤的微血管密度(MVD)。 1.接种后7天出现肉眼可见的肿瘤,模型建立成功,肿瘤的平均直径约为0.9cm,最大直径1.3cm,大多呈椭圆形,成瘤率达100%,且无一例复种。 2.对小鼠一般情况的影响用药后生理盐水组小鼠随着移植瘤结节的逐渐长大,进食、饮水、活动均较前减少;恩度组小鼠进食、饮水较前无明显变化,活动正常,持续到实验结束。实验结束后,恩度药物干预组小鼠LLC皮下移植瘤体积(1762.72±53.08mm3)明显低于生理盐水对照组(2503.72±64.78mm3),两组之间有统计学差异(p0.05)。 3.对移植瘤微血管密度的影响以CD31作为血管内皮细胞的标志物,血管内皮细胞的胞浆被染成棕黄色。NS组及恩度组CD31MVD计数平均值分别为98.50±13.53、56.21±5.10。与NS组比较,恩度药物干预组MVD值减低且差异具有显著性(p0.05)。 4.超声造影软件定量分析实验结束后,恩度治疗组的峰值强度(PI)明显低于生理盐水对照组(p0.05),其峰值强度分别为50.74±5.82及88.56±25.99。 5.超声造影的定量分析参数与肿瘤微血管密度之间的关系实验结束后恩度组超声造影参数峰值强度(PI)与肿瘤的CD31微血管密度呈正相关(r=0.532,p0.05)。结论 1.重组人血管内皮抑素(恩度)能够有效抑制小鼠Lewis肺癌皮下移植瘤的生长。 2.超声造影定量分析(团注法)可以通过监测肿瘤的血流灌注参数的改变来评价恩度抗血管生成治疗的效果。 3.超声造影定量分析参数中的峰值强度可以有效反映恩度抗血管生成治疗引起的肿瘤血流灌注的改变。
[Abstract]:The first part of the quantitative analysis of contrast-enhanced ultrasound in the evaluation of tumor angiogenesis in Lewis lung cancer in mice
Research background and significance
Refers to the process of tumor angiogenesis and angiogenesis through endothelial cells "budding" based on the original tumor microvascular network, it is an essential factor for tumor growth and metastasis. In tumor angiogenesis in and around is considered to be the prerequisite for malignant tumor cell proliferation, these new blood vessels the proliferation of tumor cells can not only provide the necessary nutrition, but also provides a channel for the distant metastasis of tumor cells in vivo. Therefore angiogenesis is an important feature of malignant tumors, its detection for tumor diagnosis, treatment and prognosis is very important.
In the past few years, many studies by noninvasive imaging methods to evaluate tumor angiogenesis, such as contrast enhanced computed tomography (contrast enhanced computed tomography, CECT), dynamic contrast-enhanced magnetic resonance imaging (dynamic contrast enhanced magnetic resonance imaging, DCE-MRI), magnetic resonance angiography (magnetic resonance angiography, MRA), positron emission tomography (positron emission tomography, PET) and Doppler ultrasound technology and so on. However, these methods to measure low flow and small blood vessels in the inspection, has obvious limitations. CEUS can improve the detection capability of low flow, so that capillary tissue and microvascular evaluation the blood becomes possible, and can provide detailed information about tumor angiogenesis. MVD is the average per square millimeter of tumor area The number of blood vessels, is currently the evaluation of tumor angiogenesis as a reference standard. However, the microvessel density of different vascular endothelial cell markers to reveal the degree of differentiation and the structural and functional characteristics of different tumor endothelial cells. In previous experimental studies, more microvascular density by CD31 vascular markers, with vascular production evaluation organization. Because the CD31 microvessel density in differentiated and undifferentiated vascular endothelial cells expressed; and another common vascular marker CD34 is only expressed in the differentiated endothelial cells, the tumor vascular differentiation, microvascular density is also different.
Lewis Lewis lung cancer (lung carcinoma, LLC) is a subcutaneous injection of subcultured Lewis lung cancer cells into the axilla of C57/BL6 mice. It has been repeatedly applied to various studies in the laboratory, and is a relatively mature animal tumor experimental model.
Although there is a linear relationship between the microvessel density in previous studies have confirmed the quantitative parameters of contrast-enhanced ultrasound and tumor, so far, there is no reports of related research on different vascular markers in mice with Lewis lung cancer in microvessel density with contrast-enhanced ultrasound parameters. The purpose of the study
This study by subcutaneous transplantation tumor animal model of mouse LLC, application of enhanced pulse sequence (contrast pulse sequencing, CPS) of the ultrasound contrast imaging technology, combined with time intensity curve, the correlation study of microvessel density and blood perfusion parameters comparison of contrast-enhanced ultrasound with different vascular markers in LLC animal models, to explore the application value of contrast-enhanced ultrasound in the evaluation of tumor angiogenesis.
research method
The establishment of 25 mice transplanted LLC tumor model, the tumor size is about 1.0cm underwent contrast-enhanced ultrasound, contrast-enhanced ultrasound quantitative analysis software for off-line analysis, to obtain the peak intensity (peak intensity, PI), time to peak (time to, peak, TTP) and time of arrival (arrival time, AT) and other parameters of quantitative analysis ultrasound contrast.
After the end of ultrasound examination, the mice were killed by cervical dislocation, and the tumor was removed completely. The expression of CD31 and CD34 microvessel density was detected by immunohistochemistry, and the microvessel density (microvessel density, MVD) was calculated.
Result
1., 7 days after inoculation, the tumor was visible to the naked eye. The model was successfully established. The average diameter of the tumor was about 0.9CM, the largest diameter was 1.3cm. Most of the tumors were oval and the tumor formation rate was 100%, and no case was repeated.
2.25 cases of ultrasound contrast enhancement pattern of LLC mice showed ring enhancement, in which the tumor periphery was significantly enhanced, while the central part showed low enhancement or no enhancement. The time intensity curve on the LLC tumor showed "fast, slow out" performance, increases rapidly to peak, a steep, descending relatively slow.
3. in the LLC tumor of mice, there are two kinds of vascular markers representing different degree of differentiation of vascular endothelial cells: CD31 and CD34.
The peak intensity of perfusion parameters (PI) was positively correlated with CD31 microvascular density (r=0.428; P0.05), and there was no linear correlation with CD34 microvascular density (r=0.428).
5. in the LLC tumor of mice, the CD31 microvessel density was negatively correlated with the CD34 microvascular density (r=-0.474; P0.05).
conclusion
1., we successfully established a tumor model of Lewis lung cancer in mice, which further proved that this model is a reliable and practical animal experimental tool for studying tissue tumors, and has good repeatability.
2., in the mouse Lewis lung cancer model, there are two kinds of differentiated tumor vessels: CD31 microvessel density representing undifferentiated and differentiated vascular endothelial cells, and CD34 microvessel density representing only mature vascular endothelial cells.
3. correlation analysis of ultrasonic quantitative parameters and microvessel density of tumor tip: ultrasound contrast (bolus) can effectively evaluate tumor angiogenesis through the perfusion parameters monitoring tumor angiogenesis; the peak intensity in the parameters of CEUS can to some extent in the evaluation of tumor differentiation, clinical classification and prognosis guidance anti angiogenesis therapy and evaluation of tumor has important clinical value.
Therapeutic effect evaluation of endostatin on mouse Lewis lung carcinoma ultrasound contrast quantitative analysis of the second part
Research background and significance
Anti angiogenesis therapy is currently the research hotspot in tumor therapy. By targeting angiogenesis within the tumor abnormal or immature vascular antiangiogenesis therapy, the treatment will cause the tumor blood volume, blood perfusion rate and other hemodynamic index changes, monitoring the hemodynamic parameters change becomes effective index for clinical evaluation of anti angiogenesis therapy.
Check the previous method of multiple imaging (such as CT, MRI and ultrasound) have to evaluate the curative effect of tumor angiogenesis and anti angiogenesis therapy. Especially the ultrasound color Doppler ultrasound with real-time, low cost, good repeatability and can directly evaluate the tumor perfusion and other advantages, in the generation and the important role of anti angiogenesis therapy in evaluation of tumor blood vessels. But the blood perfusion of the evaluation of color Doppler ultrasound on microvascular angiogenesis have certain limitations, in recent years more for the clinical application of contrast-enhanced ultrasound, the evaluation of blood flow within the tumor in the tiny blood vessels become possible. Within a certain concentration range the signal intensity and contrast enhanced ultrasound microbubble concentration in the blood has a linear relationship, it can use the time intensity curve software for the quantitative analysis of the process of contrast-enhanced ultrasound.
Endostar (Endostar) recombinant human endostatin, is a kind of anti angiogenesis drugs, the possible mechanism is the inhibition of endothelial cell proliferation, migration to inhibition of tumor angiogenesis, blocking the nutrition supply of tumor cells, so as to achieve the purpose of suppressing tumor. Anti angiogenesis therapy in clinical application in non small cell lung cancer.
Due to the abnormal angiogenesis anti angiogenesis drugs mainly contributes to the tumor, after the treatment will not only cause the change of the number of blood vessels within the organization, more important is to cause tumor vascular morphology and function changes, these changes often secondary to abnormal tumor blood flow dynamics, so the changes can become the clinical monitoring on the evaluation of a reliable method for anti angiogenesis therapy.
research objective
This study used Lewis lung carcinoma bearing mice model by intraperitoneal injection of ultrasound contrast perfusion parameters of anti angiogenic drugs after Endostar treatment and control groups compared, and the parameters of contrast-enhanced ultrasound and microvessel density of tumor were compared, to explore the ultrasound contrast effect on the efficacy of anti angiogenic drugs.
research method
The establishment of 30 mice transplanted LLC tumor models were randomly divided into 2 groups, each model since the establishment of the next day respectively by intraperitoneal injection of saline sodium (normal saline, NS), Endostar (5mg/kg body weight), daily administration, continuous administration for 14 days. To observe the general situation of two groups of mice every day after administration. And every other day were measured tumor size.
At the dose of 24h after contrast-enhanced ultrasound, and the application of ultrasound contrast quantitative analysis software for off-line analysis, gets the peak intensity (PI), time to peak (TTP) and time of arrival (AT) and other parameters. Ultrasonography after cervical dislocation mice were sacrificed, and complete resection of the tumor, the expression of immunohistochemical detection of CD31 tumor microvessel density, and calculate the tumor (MVD).
1., 7 days after inoculation, the tumor was visible to the naked eye. The success of the model was established. The average diameter of the tumor was about 0.9CM, the largest diameter was 1.3cm. Most of the tumors were oval and the tumor formation rate was 100%, and no case was repeated.
The effect of 2. on the general condition of mice
After administration of saline group of mice with transplanted tumornodules gradually grew up, eating, drinking, eating activities were decreased; Endostar group mice, compared with the previous drinking water had no obvious change, the activity is normal, until the end of the experiment.
After the experiment, Endostar drug intervention LLC mice subcutaneous transplantation tumor volume (1762.72 + 53.08mm3) was significantly lower than the normal saline control group (2503.72 + 64.78mm3), there were significant differences between the two groups (P0.05).
Effect of 3. on microvascular density of transplanted tumor
Using CD31 as a marker of endothelium, vascular endothelial cells, the cytoplasm was stained brown.NS group and Endostar group CD31MVD count average value were 98.50 + 13.53,56.21 + 5.10. and NS group, Endostar drug intervention group MVD value decreased and the difference was significant (P0.05).
Quantitative analysis of 4. ultrasound contrast software
After the end of the experiment, the peak intensity of Endostar in the treatment group (PI) was significantly lower than the normal saline control group (P0.05), the peak intensities were 50.74 + 5.82 and 88.56 + 25.99.
The relationship between quantitative analysis parameters of 5. contrast-enhanced ultrasound and tumor microvessel density
After the experiment, Endostar group the parameters of CEUS peak intensity (PI) was positively correlated with tumor microvessel density CD31 (r=0.532, P0.05).
conclusion
1. recombinant human endostatin (endostatin) can effectively inhibit the growth of subcutaneous transplanted tumor of Lewis lung cancer in mice.
Analysis of 2. CEUS (bolus) can monitor tumor perfusion parameters to evaluate the change of Endostar antiangiogenic therapy.
3. ultrasound contrast quantitative analysis of the peak intensity in the parameters can effectively reflect the tumor perfusion caused by Endostar anti angiogenic treatment changes.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R445.1;R734.2

【参考文献】