【作者】 高岿然；

【导师】 史铁梅；

【作者基本信息】 中国医科大学 ， 影像医学与核医学， 2007， 博士

【摘要】 前言宫颈癌是女性生殖系统常见的肿瘤,其发病率居恶性肿瘤之首,是女性生殖系统恶性肿瘤死亡的主要原因之一,所以宫颈癌的早期诊断有着重要的意义。本实验应用瘤细胞悬液移植法,在具有正常免疫功能的大鼠体内建立大鼠宫颈癌移植瘤动物模型,为宫颈肿瘤的早期诊断提供前期的动物实验依据。探讨大鼠宫颈癌移植肿瘤组织中的有丝分裂原激活蛋白激酶P38的表达及其临床意义;研究P38的高表达与淋巴结转移的关系。同时应用新一代超声造影剂声诺维观察造影对实验兔肌肉内人宫颈癌移植肿瘤的血流灌注显像的影响,评价声诺维(SonoVue)在超声显像中的造影增强作用,探讨及检验不同造影剂剂量在二次谐波显像中对兔宫颈癌移植瘤造影效果的影响与变化规律,为临床肿瘤诊断和鉴别诊断提供依据。方法第一部分:体外培养HELA细胞,采用细胞记数法观察细胞生长情况。将对数生长期的HELA按2.5×10~7、2.5×10~6、2.5×10~5分别接种于大鼠的背部皮下、腹腔内、静脉注射,建立宫颈癌移植瘤动物模型,并于接种后观察成瘤率、肿瘤生长及播散情况及动物生存期,于种植后第7D、10D、14D、17D、21D行超声检查,然后等到实验鼠自然死亡,并对肿瘤进行病理学检查,确定肿瘤的存在及性质。第二部分:应用免疫组织化学方法和Western-blot方法检测人宫颈癌鼠移植瘤及其癌旁正常组织中P38及磷酸化P38的蛋白表达情况。(一)免疫组化法1.按照试剂盒说明书步骤抗p38免疫组化染色。切片常规脱蜡致水,高温高压抗原修复,一抗4℃孵育过夜,DAB显色,苏木素复染。PBS代替一抗作为阴性对照,用已知宫颈癌阳性片做阳性对照。2.结果判定:在双盲法下进行,以肿瘤细胞内出现黄色或棕黄色颗粒为阳性表达。每张切片在光、镜下随机选取5个视野,每个视野计数100个瘤细胞,着色强度分为无色(一)为0分,浅黄色(+)为1分,黄色及棕黄色(++)为2分;阳性细胞数＜10%为0分,10%—50%为1分,＞50%为2分。两项得分相乘结果≥2记为阳性。(二)Western blot法取-70°保存的移植瘤及癌旁正常组织约100mg加入预冷的蛋白裂解液,剪碎,匀浆超声处理,高速低温离心提取上清为细胞总蛋白。电泳(12%SDS-PAGE凝胶)、转印,5%脱脂奶粉封闭液封闭1h,一抗4℃孵育过夜,与相应二抗室温下孵育2h,曝光,双蒸水冲洗,干燥,避光保存,结果经自动电泳凝胶成像分析仪(chemilmager 5500,Alpha Innotech,USA)采集,进行灰度值测定。第三部分:建立兔宫颈癌移植瘤模型十只。彩超每周观察肿瘤生长情况,共10周,于1～4周测量血流动力学参数;同时,患有宫颈癌移植瘤的兔分别经耳缘静脉团注法注射声学造影剂0.2、0.4、0.6ml/kg,然后用二次谐波显像观察肿瘤造影效果;应用彩色多普勒、能量多普勒观察兔肌肉层内移植瘤血流灌注情况并定量分析。结果第一部分:在体外培养条件下的HELA细胞生长迅速,将不同数量的HELA细胞接种大鼠不同部位后,成瘤率为67%,迅速发生腹腔播散并产生大量腹水,其生物学行为与人宫颈肿瘤相似。肿瘤细胞负荷增加,动物生存期明显缩短(p＜0.01)。三组模型的组织病理学检查无明显差别。肿瘤的内部回声可分为高回声、等回声、低回声三类,随着肿瘤的生长其回声类型有所转变;鼠的人宫颈癌移植瘤的生长状况、回声类型及血供变化的动态演化大体符合宫颈癌的一般超声表现特点。该种植瘤的增长是进行性的,超声征象随肿瘤的进展而逐步体现,基本符合肿瘤侵袭的定义特征,该动物模型可用于超声对宫颈癌恶性生物学行为的观察。第二部分:P38在宫颈癌移植瘤组织中的表达阳性率为95.5%,在癌旁正常组织中的表达为33.1%。P38在宫颈癌移植瘤中的表达明显高于癌旁组织,有显著的统计学差异(P＜0.01)。P38的表达定位于胞质中,胞核中无表达;P38的表达与病程、肿瘤大小、肿瘤位置无明显相关。P38的表达与组织分化程度及有无淋巴结的转移有显著差异(P＜0.01)。第三部分:在兔的宫颈癌移植瘤模型中:PDI可探及星点状、短棒状和条状血流,各时期RI和PI值无显著性差异。造影后增强了肌肉内移植瘤的血管显示率及血流信号强度,肿瘤造影表现为快速增强,缓慢减退,持续时间较长的特点;新型声学造影方法可以明显增强肿瘤的二维超声影像。结论在具有正常免疫功能的大鼠体内成功建立的宫颈癌移植瘤模型能较好地模拟人自发宫颈癌的生物学特征,有助于了解宫颈癌生物学及免疫学特性,可作为良好的肿瘤模型用于宫颈癌的免疫治疗研究及进行影像学的研究。该种植瘤超声征象随肿瘤的进展而逐步体现,基本符合肿瘤侵袭的定义特征,该动物模型可用于超声对宫颈恶性肿瘤生物学行为的观察。本实验虽然属于临床前期的动物研究,但肿瘤直径基本≤3cm,其超声常规指标的动态观测将有助于临床早期宫颈肿瘤的诊断与鉴别。声学造影提高了兔肌肉内宫颈癌移植瘤血流灌注的显示,增强其血流信号,造影显像优于彩色多普勒显像。P38在宫颈肿瘤的细胞中呈高表达,并与病程进展密切相关,与宫颈癌的发生、发展及转移密切相关。更多还原

【Abstract】 ObjectiveCervical carcinoma is a common malignant tumor.To establish an animal model of cervical cancer in immunocompetent rat and to study its biological character.Based on pervious study,the purpose of this study is to found Hela mice tumor xenograft model and study the,through which to provide a way to prevent and cure the cervical cancer in clinic.To explore the regularity of the ultrasonographic dynamic changes such as the size,echo-intensity and edge echo-pattern of cervical tumor during its growth. To offer the animal experimental proves of the preclinical phase for the early diagnosis and treatment of cervical carcinoma.To investigate P38MAPK protein expression and its clinical significance in the cervical transplanted carcinoma of rat.In the meanwhile, to assess the blood perfusion of tumors in rabbit muscles with perfluorocarbon contrast flow imaging and high-frequency ultrasound.MethodsPart one:Hela was frozen in liquid nitrogen were revived in routine method and inoculated in RPMI-1640 Blended with 10%foetus cattle serum.HELA cells 2×10~7,2×10~6,2×10~5 was injected intraperitoneally,under skin,and vein.Human cervical cancer Hela cells were implanted into the mice to get the tumor model of HELA cell line.The transplanted tumors were observed every two days.The ultrasonographic observation was performed on the 7,10,14,21 and 30 day after implantation.The survivals were put to death and necropsy.The section preparations were stained with HE method.Part two:Hela was frozen in liquid nitrogen were revived in routine method and inoculated in RPMI-1640 Blended with 10%foetus cattle serum.2×10~5 was injected under skin. Immunohistochemical assay and Westem Blot was performed to study the expression and location of P38.Subcutaneously injected with PBS as a control group in 5 mice.Part Three:Ten rabbits model with cervical transplanted tumors in muscle were examined, and they were assessed by high-frequency color Doppler ultrasound every week for ten weeks.SonoUve was administered respectively to ten rabbits through ear veins at 0.2, 0.4,0.6 ml/kg,and then imaged with second harmonic imaging.After intravenous administration of contrast agent,Color Doppler imaging or Power Doppler imaging were used to evaluate the blood perfusion of tumors in muscle.ResultsHela cells grew progressively after injected into rats intraperitioneally,formed gross peritoneal dissemination and malignant hemorrhagicascit-es in a manner typical for human cervical carcinomas.Animal survival time decreased significantly with large Hela burden(p＜0.01).Pathological analysis and tissue culture confirmed the existence of the carcinoma tissue.The implant,The eho-intensity of the tumor can be divided into three patterns such as hyperecho,isoecho and hypoecho.The observed rate of abnormal vessels in the tumor increased as the implantation time elongated and the tumor enlarged.The observed time of arterial blood was earlier than that of venous blood.The effective factors of histopathology mostly were necrosis,fiber and sinusoid. In two -dimensional images,tumors were small and poor echo one week after implantation.Necrosis and liquefaction appeared could be found later at the 6-7 week cavum formed.Color Doppler flowing showed dot,branch,or line flow signals and the degree of blood abundance was from 0-Ⅲgrades,while PI and RI between phrases had no significant difference,there were significant improvements in detecting neovascularity of tumor in muscle at all contrast agent dosage levels and the intensity lasted a long period.P38 was mainly expressed in the cell cytoplasm and none nuclei of cervical cells and neutrophil and lymphcytes.Positive rate of p38MAPK protein expression was 70%in carcinoma tissues,33.1%in its paraneoplastic tissue,and there is a significant correlation between the expression of P38 with the stages of cervical cancer,histology grade,and local lymph node ivolvement,but not with time,site or size of tumor.ConclusionsA positive cervical cancer model has been successfully established in rats which have immunocompetent function that simulates preferably the biological behavior of human spontaneous cervical carcinoma.And is an optimal animal model for cervical cancer immunological therapy research.The ultrasonographic signs such as the size, echo -intensity,edge echo-pattern and blood flow changes of tumor during its growth were coincident to clinical cervical tumor.The gradual growth and gradual behavior of ultrasonography were essentially coincident to the defintive characteristics of tumor invasion.This animal model can be used to observe the malignant biological behaviors. The sizes of most tumors were less than 3cm,therefore the dynamic observation of the ultrasonographic roution parameters is useful to the diagnosis and odentification of the clinical little tumor.P38 is overexpressed in cervical cancer cells.The expression of P38 has a significant correlation with the stages of cervical cancer,p38 may play an important role in the occurrence,development and metastasis of human cervical carcinoma.The contrast agent is able to detect clearly the tumor vessels which cannot be shown with conventional color flow imaging.Moreover,the perfluorocarbon contrast agent can maintain the visuality of the vessels for a longer period.更多还原