【作者】 秦娟；

【导师】 王智彪； 陈文直；

【作者基本信息】 重庆医科大学 ， 妇产科学， 2012， 博士

【摘要】 研究背景外科手术发展趋势是采用对人体损伤更小的技术治疗疾病，高强度聚焦超声是近年来新兴的一种非侵入性肿瘤治疗方式,提供了一个可精确定位治疗却没有治疗通道切开暴露或穿刺通道的完全非侵入性的治疗方法。子宫肌瘤是女性生殖系统最常见的良性肿瘤，高强度聚焦超声治疗子宫肌瘤通过体外超声能量聚焦体内，使子宫肌瘤出现凝固性坏死，逐渐吸收，达到改善症状，恢复子宫形态的目的。随着高强度聚焦超声（High Intensity Focused Ultrasound，HIFU）广泛应用于子宫肌瘤的治疗，目前已有各个中心关于治疗安全性的临床观察报道，但尚无基于超声消融“无创性”特点，与传统“有创”治疗的安全性比较评价。超声消融子宫肌瘤的特点是实现了保留子宫、不开刀、不穿刺、不麻醉治疗子宫肌瘤方法的“无创”治疗，以及为避免影响邻近组织而实行瘤内治疗的原则。而关于超声消融技术的安全性研究就是考虑在HIFU“无创”特点的前提下，对比传统手术治疗，评价治疗本身对机体的创伤，来研究新技术的安全性。以及探讨HIFU治疗后残余未治疗肿瘤（非靶区瘤组织）的转归及相关机制，研究残余瘤对机体的影响。本研究旨在通过前瞻性同期对照临床试验研究HIFU消融子宫肌瘤对机体的创伤，评估技术的“无创性”。同时，通过对HIFU消融后非靶区子宫肌瘤的影像学数据的随访及分析，探讨影响非靶区瘤组织转归的相关影响因素，获得肿瘤局部安全性评价的临床证据并建立预测非靶区肌瘤转归的模型。研究拟基于临床资料，初步研究肿瘤超声消融的局部效应及机制，获得肿瘤局部效应评价的实验室依据。研究拟为系统评价HIFU消融子宫肌瘤安全性奠定了临床及实验室基础，为HIFU消融子宫肌瘤的临床治疗方案的制定及进一步优化提供了科学依据。目的1.综合评价HIFU消融子宫肌瘤与经腹子宫肌瘤剔除手术对机体的创伤程度；2．分析临床资料，研究HIFU消融后残余部分肿瘤（非靶区肌瘤）的转归及影响因素，探讨超声消融子宫肌瘤对非靶区瘤组织的局部效应，评价HIFU消融后的肿瘤安全性；3．初步探讨超声消融子宫肌瘤对非靶区瘤组织作用机制。研究内容1．设计同期随机对照试验（RCT），基于临床观察项目及抽样检测创伤相关血清学指标，研究HIFU消融子宫肌瘤与经腹子宫肌瘤剔除手术比较对机体的创伤程度。2．分析HIFU消融子宫肌瘤对非靶区瘤组织效应的临床资料，研究HIFU治疗后非靶区肌瘤的转归及相关影响因素，建立预测非靶区肌瘤转归的回归模型。3．基于回归模型，探讨不同肌瘤体积消融率与非靶区肌瘤转归的相关关系，初步提出可以获得残余部分肿瘤良好转归效应的超声消融方案。4．模拟临床超声消融方案，建立HIFU消融人离体子宫肌瘤模型，探讨HIFU对非靶区瘤组织生长调控因子表达的影响及机制。5．培养人原代子宫肌瘤细胞模型，建立HIFU辐照声场内子宫肌瘤细胞模型，探讨HIFU对声场内靶点外子宫肌瘤细胞的效应及相关机制。资料和方法第一部分1．研究对象2010年9月-2011年12月在重庆医科大学附属第一医院妇科初步诊断为子宫肌瘤并拟行手术治疗患者，共253例。按纳入标准入组共188人。2．研究设备JC型聚焦超声肿瘤治疗系统（重庆海扶（HIFU）技术有限公司）核磁共振成像系统Magnetom Symphony1.5T MR Tim（德国西门子公司）3．研究方法1）采用前瞻性同期随机对照分组方法（区组随机法），将符合纳入标准的188人作为研究对象随机分配到超声消融组(HIFU组)及经腹子宫肌瘤剔除手术组（肌瘤剔除组），观察临床安全性指标，包括生命体征、术后恢复时间手术不良反应。2）从188患者中随机抽样每组31例，检测患者手术前后创伤相关血清学指标变化情况，包括：（1）皮质醇、肾上腺素及去甲肾上腺素；（2）IL6、IL2、IL10的水平；（3）外周血白细胞值、中性粒细胞比例及血红蛋白值；（4）外周血中CD4+、CD8+比例及NK细胞的比例。4.统计方法应用SPSS16.0软件包作统计分析。第二部分1．研究对象2007年1月至2010年1月96名行HIFU治疗子宫肌瘤患者的临床资料。2．研究设备JC型聚焦超声肿瘤治疗系统（重庆海扶（HIFU）技术有限公司）hifu-3DTPS Microsoft基础类应用软件（重庆（HIFU）海扶技术有限公司）3．研究方法1）软件自动三维重建并计算非靶区肌瘤体积缩小率，分析影响非靶区肌瘤缩小率的相关因素，建立非靶区肌瘤转归的预测回归模型。2）将96名行HIFU治疗子宫肌瘤患者按照不同的消融率进行分组，比较不同组的非靶区瘤组织术后3、6、12月的影像学转归，寻找影响非靶区肌瘤的转归趋势的消融率“拐点”。4．统计方法应用SPSS16.0软件包作统计分析。第三部分1．研究对象1）人离体子宫肌瘤：2010年4月-2010年11月因子宫肌瘤行子宫次全切除术或子宫全切术患者8例，年龄40.2±3.9（35-47）岁，手术前行核磁共振检查均为T2WI均匀低信号，子宫肌瘤平均体积为48.35±12.02cm3（37.1-65cm3），最大直径为4.38±0.33cm（4.2-5.1cm）术后均经病理证实为子宫肌瘤。2）人原代子宫肌瘤细胞：取自2010年4月至2011年10月，重庆医科附属第一医院妇科的人子宫肌瘤标本，患者年龄在39-45岁，术前3个月未服用激素治疗。病理学诊断为子宫肌瘤，术中取出新鲜离体子宫肌瘤后立即送实验室培养原代细胞。2．研究设备JC型聚焦超声肿瘤治疗系统（重庆海扶（HIFU）技术有限公司）核磁共振成像系统Magnetom Symphony1.5T MR Tim（德国西门子公司）酶联免疫检测仪（宁波自动化仪表研究所）3．研究方法1）建立HIFU消融人离体子宫肌瘤模型，研究HIFU消融对非靶区瘤组织雌激素受体、孕激素受体及芳香化酶细胞色素P450表达的影响。2）培养人原代子宫肌瘤细胞，筛选全部靶区子宫肌瘤细胞致死的最小超声能量参数，建立靶区外声场内子宫肌瘤细胞的HIFU辐照模型，分析聚焦超声对声场内距离焦点不同层面细胞的影响，研究超声对声场内细胞的作用机制。4．统计方法应用SPSS16.0软件包作统计分析。结果第一部分1.对比观察HIFU组与肌瘤剔除组临床安全性指标，结果显示：HIFU组与肌瘤剔除组比较，HIFU组对术后的体温、收缩压影响较小，不良反应发生率低，术后恢复时间短，HIFU组术后麻醉类镇痛药使用率明显低于肌瘤剔除组，而两组术后疼痛分值比较差异无统计学意义。2. HIFU组与肌瘤剔除组对创伤相关的血清学指标的影响：（1）两组术后血清皮质醇、肾上腺素及去甲肾上腺素浓度变化不明显；（2）肌瘤剔除组对机体术后血清IL-6、IL-2、IL-10的浓度影响明显，主要是发生在术后24小时；（3）肌瘤剔除组患者术后外周血WBC计数、中性粒细胞比率高于HIFU组，而术后血红蛋白值明显低于HIFU组；（4）肌瘤剔除组术后外周血CD4+、CD8+及NK细胞受抑制程度比HIFU组明显。第二部分1．非靶区肌瘤缩小率的多变量相关关系结果提示：影响非靶区肌瘤转归的因素包括：子宫肌瘤血流分级、T2WI信号强度、超声消融率。建立回归模型为：[=非靶区肌瘤转归趋势（非靶区肌瘤体积缩小率）,χ1=超声消融率（%），χ2=子宫肌瘤血流分级，χ3=子宫肌瘤T2WI信号转化值]2．通过研究不同体积消融比例后的非靶区瘤组织的影像学转归。结果初步显示超声消融后残留的非靶区瘤组织体积小于子宫肌瘤体积的30%是可以得到满意的影像学转归的。第三部分1．靶区周围肌瘤组织温度也有不同程度的温升，并且靶区外组织温度呈逐渐下降。与假照组（对照组）比较，靶区外瘤组织雌、孕激素受体表达无明显差异。免疫组织化学法及免疫印迹发半定量检测P450arom蛋白在靶区外0.5cm及1.0cm处子宫肌瘤组织的表达明显低于对照组，差异有统计学意义。2．成功培育人原代子宫肌瘤细胞，选择导致靶点人子宫肌瘤细胞死亡的最小超声参数为声功率200W辐照时间为5秒。高强度聚焦超声可以诱导声场内子宫肌瘤细胞发生凋亡，细胞凋亡发生的时间和凋亡率与声场内距离焦点的位置相关，距焦点距离越近凋亡发生越明显。声场内细胞caspase-3也有不同程度的激活，其表达强度及活化情况与声场内距离焦点的位置相关。结论1. HIFU组与肌瘤剔除组比较，对机体的创伤更小。2.非靶区肌瘤的转归与子宫肌瘤的性质及超声消融率相关。超声消融率达到70%以上，可以观察到满意的非靶区子宫肌瘤转归。3．超声消融部分子宫肌瘤后在一定距离范围内非靶区的子宫肌瘤的生长会被抑制，其机制与温度影响P450酶的蛋白表达有关。4.高强度聚焦超声可以诱导声场内细胞凋亡，凋亡的发生及凋亡率与距离焦点的声场层面有关，其机制可能与激活Caspase-3有关。更多还原

【Abstract】 BackgroundSurgical trend is the development of surgical treatment based on less damage tohuman. High intensity focused ultrasound (HIFU) is a non-invasive therapy of tumouremerged in recent years, it provides non-invasive treatment with precise positioning andno incision. Uterine fibroid is the most common benign tumor of reproductive system.Uterine fibroids are treated with HIFU by focusing ultrasound energy to local fibroidtissue and cause coagulation necrosis and prevent the growth of uterine fibroids.Along with HIFU therapy is widely applied in the treatment of uterine fibroids,clinical observation reports on safety of HIFU represented by many centers. However,there is no safety evaluation based on the characteristic of HIFU,"no invasive",especially compared with traditional treatment. Ultrasound ablation of uterine fibroids isa noninvasive treatment characterized by retaining uterus, no surgery and no anesthesiamethods, as well as the principle of the treatment is practicing within tumor to avoidaffect neighboring tissue. Considered under the non-invasive characteristic, safetystudies on HIFU ablation is to evaluate the trauma on human compared with traditionalsurgery. The purpose of study was to evaluate the safety of the new technology, and toinvestigate the security of residual untreated tumor (non-target tumor tissue) after HIFUablation and related mechanisms.This study was designed to study the trauma of HIFU ablation with controlledclinical trials for assessment of noninvasive techniques. Meanwhile, we explored therelated factors affected non-target tumor tissue, evaluated clinical safety evidence of local tumor and built the prediction model of non-target fibroid tissue by follow-up andanalysis of clinical imaging data on HIFU ablation target of uterine fibroids. The studywas to explore the possible mechanisms of local tumor based on clinical data, andconcluded laboratory evidence of local tumor effects. The study provides a clinical andlaboratory basis to evaluate the safety of HIFU ablation for uterine fibroids. It alsoprovides a scientific basis of HIFU therapy for planning and optimizing treatmentprogram.Objective1. To evaluate trauma on the body of HIFU ablation for uterine fibroids and abdominalmyomectomy.2. To study the outcome of residual tumor (non-target fibroid) and influencing factorsby analysis of clinical data, and explore the local effect of non-target fibroid after highintensity focused ultrasound ablation for evaluation the safety of HIFU therapy.3. To explore the preliminary mechanism of high intensity focused ultrasound ablationon non-target fibroid.Research content1. Based on clinical observation and trauma-related serum markers, the study wasdesigned to evaluate the trauma of HIFU on patients compare with abdominalmyomectomy through randomized controlled trials (RCT).2. To explore the related factors affected non-target tumor tissue, evaluated clinicalsafety evidence of local tumor and build the prediction model of non-target fibroidtissue by analysis of clinical imaging data.3. To explore the relationship of the outcome of non-target fibroids and ablation ratebased on the regression model. It can be initially proposed the treatment program toacquire clinical outcome of non target fibroids.4. To explore the effect of HIFU on the expression of regulatory factors on growth ofnon-target fibroid tissue and related mechanisms by building HIFU ablation isolated human uterine fibroids model.5. To explore the he effect and mechanisms of HIFU on human fibroid cells within thesound field of ultrasound with cultured primary human fibroid cell model.Material and MethodsPart one1. ObjectiveFrom September2010to December2011,253patients diagnosed with uterine fibroidand scheduled for surgery were enrolled in the1st Affiliated Hospital of ChongqingMedical University2. EquipmentsJC focused ultrasound tumor therapy system (Chongqing HIFU Technology Co., Ltd.)Magnetic resonance imaging system Magnetom Symphony1.5T MR Tim (Siemens,Germany)3. Methods1)188patients who meet the inclusion criteria were randomly assigned to high-intensityfocused ultrasound ablation group (HIFU group) and abdominal myomectomy group(myomectomy group) with RCT (block randomized method). The indicators for clinicalsafety were observed, including vital signs, postoperative recovery time, surgicaladverse events and complications.2)31cases were randomly selected from188patients. All patients was detected withsurgical trauma indicators of peripheral blood before and after therapy, including:(1)cortisol, epinephrine and norepinephrine;(2) IL6, IL2, and IL10;(3) peripheral bloodwhite cell counts, neutrophil percentage and hemoglobin values;(4) CD4+, CD8+andNK ratio of peripheral blood.4. Statistical methodsSPSS16.0package was applied for statistical analysis.Part two1. Objective Clinical data of96patients treated with HIFU of uterine fibroids from January2007toJanuary2010.2. EquipmentsJC focused ultrasound tumor therapy system (Chongqing HIFU Technology Co., Ltd.)hifu-3DTPS Microsoft Foundation Classes application software (Chongqing HIFUTechnology Co., Ltd.)3. Methods1) Automatic3D reconstruction and calculation of non-target fibroid volume withsoftware. Analysis of the impact factors on shrink rate of non-target fibroids forestablishing predictive regression model of outcome.2)96patients with HIFU were assigned to each group according to different ablationratio. We compared the imaging outcome of each group to find out the “inflection point”of ablation ratio affect the trend of non-target fibroid.4. Statistical methodsSPSS16.0package was applied for statistical analysis.Part three1. Objective1) Human uterine fibroid: From April2010to November2010,8cases were appliedwith hysterectomy and subtotal hysterectomy due to uterine fibroid. The age was40.2±3.9(35-47) years. The signals of pre-operative T2WI in all patients were uniformly lowsignal. Average volume of uterine fibroids were48.35±12.02cm3(37.1-65cm3), andmaximum diameter was4.38±0.33cm (4.2-5.1cm). The fibroids were confirmed bypathology after surgery, and fresh uterine fibroids were received with ChongqingMedical University Ethics Committee approval and patient consents.2) Primary uterine fibroid cell: From April2010to October2011, human uterine fibroidspecimens were taken from patients of39-45years in gynecological department of the1st Affiliated Hospital of Chongqing Medical College. All patients were not takinghormone treatment within three months before surgery. The fibroids were confirmed bypathology after surgery, and the fresh fibroids were immediately sent to the laboratory for culturing primary cell.2. EquipmentsJC focused ultrasound tumor therapy system (Chongqing HIFU Technology Co., Ltd.)Magnetic resonance imaging system Magnetom Symphony1.5T MR Tim (Siemens,Germany)ELISA analyzer (Ningbo Institute of Automation Instrumentation)3. Methods1) To establish the model of isolated human uterine fibroids with HIFU ablation, andstudy the effect of HIFU on expression of estrogen receptor, progesterone receptor andP450in non-target fibroid tissue.2) To build HIFU sonication model of cultured primary human uterine fibroid cellwithin the sound field by filtering the smallest ultrasound parameters which can causetarget uterine fibroid cell death. To study the mechanism of the effect of ultrasound oncells within the sound field by analysis of different impact of focused ultrasound on thecells represented within different distance to ultrasonic focus.4. Statistical methodsSPSS16.0package was applied for statistical analysis.ResultsPart one1. Clinical safety indicators were observed in HIFU group and myomectomy group. Theresults showed: Compared with myomectomy group, HIFU group had less impact onpostoperative body temperature, systolic blood pressure. Less surgical complicationsand adverse events, shorter recovery time can be found in patients of HIFU group. Nosignificant difference of the postoperative pain score was found in two groups.2. The effect of HIFU and myomectomy group on the levels of trauma-relatedserological markers:(1) No significant changes of postoperative serum cortisol,epinephrine and norepinephrine concentrations were found in two groups significantly;(2) Patients in myomectomy group affected the levels of serum IL-6, IL-2, IL-10 significantly, especially within24hours after surgery;(3) WBC counts, neutrophil ratioin myomectomy group were higher than HIFU group, postoperative hemoglobin valuewas significantly lower than HIFU group;(4) The ratio of CD4+, CD8+and NK cellswas inhibited in myomectomy group significantly compared with HIFU group.Part two1. Correlation results of shrink rate of non-target fibroids with multivariate factorssuggest that: The factors which influence the outcome of non-target fibroids includedblood flow grade of uterine fibroid, signal of T2WI, non perfused volume ratio. Theregression model was:[=Non-target fibroid volume reduction ratio，Χ1=ultrasound ablation rate (%), χ2=uterine fibroids flow grade, χ3=uterine fibroids T2WI signal conversion value]2. The result of observing on the imaging outcomes of groups with different non-perfused volume ratio (NPV) showed satisfied imaging outcome can be get withnon-target volume less than30%of target fibroid.Part three1. Different degrees of temperature rise in non target fibroid tissue outside target fibroid,and temperature decreased gradually along with the distance outside target area.Compared with the control group, no significant difference was found in the expressionof estrogen receptor and progesterone receptor in non target fibroid.Immunohistochemical staining and Western blot semi-quantitative detection ofP450arom protein represented at0.5cm and1.0cm outside target area showed lowerlevel significantly than the control group, the difference was statistically significant.2. Primary human uterine fibroid cell was successfully cultivated. Smallest ultrasonicparameters (power200W,5seconds) lead to cell death was screened ultrasoundparameters of cells positioned in focus. High intensity focused ultrasound can induceuterine fibroid cell apoptosis positioned within sound field. The occurrence of apoptosisand apoptosis rate was related with the distance from focus. Different degree of caspase-3activation appeared in cells within sound field, and the expression ofcaspase-3the strength was related with the distance from focus.Conclusions1. Compared with myomectomy group, the result of HIFU group showed smallertrauma on patients.2. Outcome of non-target fibroids was related to NPV. NPV of more than70%can beobserved to get satisfactory outcome of non-target uterine fibroids.3. The growth of non-target fibroid within a certain range will be suppressed after HIFUablation part of uterine fibroid, and the mechanism was related to P450protein affectedby temperature.4. HIFU can induce uterine fibroid cell apoptosis positioned within the sound field. Andthe occurrence of cell apoptosis was related with the distance from focus, themechanism of apoptosis may be associated with activation of caspase-3.更多还原