【作者】 魏红梅；

【导师】 郭坤元；

【作者基本信息】 第一军医大学 ， 内科学， 2007， 博士

【摘要】 背景肿瘤是严重威胁人类健康的恶性疾病之一，近年来其发病率与死亡率均呈上升趋势。尽管部分肿瘤利用放化疗结合有较高的治愈率，但是仍有部分患者出现局部复发和远处转移，这很大程度归因于癌细胞产生多药耐药性。耐药肿瘤细胞的存在，直接影响患者的预后，是肿瘤治疗失败的主要原因，为此临床需要寻找治疗肿瘤的新措施，特别是寻找逆转耐药的新方法。热疗作为一种新的肿瘤治疗方法引起了广泛的关注，但理论研究的滞后严重影响了肿瘤热疗的临床实践的深入。随着对高温生物学作用的深入研究以及热疗技术的提高，热疗作为放疗、化疗的辅助治疗方法，已在肿瘤的综合治疗中发挥了重要作用，被成为第五种肿瘤治疗的新方法。不同类型的肿瘤细胞对加热的敏感性及其可能的分子生物学机制是值得研究和探讨的问题之一。盐酸阿霉素(adriamycin，ADM)作为一种临床广泛应用的化疗药物，其抗肿瘤的效果显著，为细胞周期非特异性药物，主要用于多种实体瘤、白血病、淋巴瘤及多发性骨髓瘤的全身治疗，均有较好的疗效。目前阿霉素的临床应用主要是与其他治疗方式联合治疗实体肿瘤，而其与热疗联合治疗造血系统恶性肿瘤，目前为止报道较少，为此，本研究以人慢性髓系白血病敏感细胞株K562、耐药细胞株K562／ADM、人B细胞淋巴瘤细胞株Raji及多发性骨髓瘤细胞株ARH-77为研究对象，用细胞增殖实验和流式细胞仪多种实验方法研究热—药联合在体内外的抗瘤活性和诱导细胞凋亡的能力，并初步探讨其作用机制，对加热与阿霉素的联合作用效果进行初步评价，并对全身热疗治疗恶性血液病安全性的评价等，期望为开发高效安全的临床联合治疗方案提供科学的实验依据，为造血系统恶性肿瘤热疗的临床实践提供理论依据及探索新的治疗方法。第一章热疗联合阿霉素对恶性血液病细胞株体外抑制作用的实验研究目的观察热疗联合阿霉素对人慢性髓系白血病敏感细胞株K562、耐药细胞株K562／ADM、人B细胞淋巴瘤细胞株Raji及人多发性骨髓瘤细胞株ARH-77的体外增殖抑制作用及对肿瘤细胞凋亡的影响。方法1．选用K562、K562／ADM、Raji及ARH-77为研究对象，用含10％胎牛血清的RPMI-1640培养液常规培养。2．采用MTT法确定阿霉素的工作浓度，并以该浓度进行化疗或与热疗的联合。3．选用不同温度(40℃、41℃和42℃)和阿霉素为处理因素，采用组内分组设计，共分为8个组，并设37℃为对照组。4．对K562、K562／ADM、Raji及ARH-77细胞株分组处理后，台盼蓝拒染法检测细胞的存活率，MTT比色法检测细胞生长抑制率，并计算细胞数、克隆数及克隆形成抑制率，以及绘制细胞生长曲线等。5．流式细胞仪(FCM)检测各细胞株不同处理组细胞凋亡率的变化。6．应用Veleriore公式判断药物联合热疗的相互作用类型。7．实验数据用SPSS 11.0软件处理，结果采用均数±标准差((?)±s)表示及单向方差分析(One-way ANOVA)，组间比较采用LSD法和Tamhane’s T2法。第二章热疗体外增敏作用的实验研究目的观察热疗联合阿霉素对人慢性髓系白血病敏感细胞株K562、耐药细胞株K562／ADM、人B细胞淋巴瘤细胞株Raji及人多发性骨髓瘤细胞株ARH-77细胞内的药物浓度变化以及对抗凋亡蛋白Bcl-2、耐药蛋白P-gp表达的影响。方法1．选用K562、K562／ADM、Raji及ARH-77为研究对象，用含10％胎牛血清的RPMI-1640培养液常规培养。2．采用MTT法确定阿霉素的工作浓度，并以该浓度进行化疗或与热疗的联合。3．流式细胞仪(FCM)检测不同处理组细胞抗凋亡蛋白Bcl-2、蛋白耐药P-gp和细胞内药物浓度的变化。4．实验数据用SPSS 11.0软件处理，结果采用均数±标准差((?)±s)表示及单向方差分析(One-way ANOVA)，组间比较采用LSD法和Tamhane’s T2法。第三章阿霉素与热疗联合作用动物实验的初步研究目的研究阿霉素与热疗联合作用对人B细胞淋巴瘤细胞株Raji裸鼠成瘤及肿瘤生长的影响。方法将24只BALB／C裸鼠分为4组：对照组(37℃)、化疗组(37℃+ADM)、热疗组(42℃)、热化疗组(42℃+ADM)，每组6只。将对数生长期的Raji细胞，配制成每0.2ml含5×10~6个Raji细胞的悬液，每只裸鼠皮下接种5×10~6的Raji细胞，观察各组裸鼠成瘤时间、成瘤率、肿瘤体积变化，并绘制肿瘤生长曲线。成瘤后3周，处死老鼠，取肿块称重，计算抑瘤率，肿瘤组织行常规病理检查。实验数据采用SPSS 11.0软件处理，结果采用均数±标准差((?)±s)表示及单向方差分析(One-way ANOVA)，组间比较采用LSD和Tamhane’s T2法。第四章全身热疗系统治疗恶性血液病15例：加温、测温和控温技术的安全、顺应及有效性观察目的探讨恶性血液病患者对于全身加温治疗的耐受性，验证全身加温治疗的顺应性、安全性及有效性。方法实验于2005-04～2005-7在南方医科大学珠江医院血液科完成。选择恶性血液病患者15例，均自愿参加观察。应用广东威尔医学科技股份有限公司开发研制的WELLRAY光子热疗智能系统，对患者进行全身加温治疗。治疗前保留导尿，外周静脉插管，联接心电监护，固定体表等温度感受器，关舱加温至直肠内温度为40～41℃，保持恒温2h，同时监测多部位体表、空气、治疗床的温度变化，同时行心电监护，加温治疗前及治疗后24h行血液常规和生化等各项指标检查。于全身热疗前及治疗后24h分别应用Karnofsky(KPS)评分评价患者的生活质量，评分范围0～100分，100分正常，无症状及体征；10分病危，临近死亡；0分死亡。结果第一章热疗联合阿霉素对恶性血液病细胞株体外抑制作用的实验研究1．阿霉素工作浓度的确定：MTT法测定阿霉素的工作浓度。阿霉素对K562、K562／ADM、Raji及ARH-77的It50分别为1.1μg／ml、53.42μg／ml、6.06μg／ml及8.16μg／ml，并以此浓度进行各项试验。2．对细胞增殖和凋亡率的影响：各细胞株分组处理后48h，与对照组及热疗组、疗组相比，热化疗组的细胞增殖抑制率及凋亡率均明显增加(P＜0.05)。3．与对照组及热疗组、化疗组相比，各细胞株的热化疗组的克隆形成数和克隆形成抑制率等指标均有统计学意义(F＜0.05)。第二章热疗体外增敏作用的实验研究1．阿霉素工作浓度的确定：MTT法测定阿霉素的工作浓度。阿霉素对K562、K562／ADM、Raji及ARH-77的IC50分别为1.19μg／ml、53.42μg／ml、6.06μg／ml及8.16μg／ml，并以此浓度进行各项试验。2．热化疗组耐药蛋白P-gp和抗凋亡蛋白Bcl-2的表达均有下调，与对照组及热疗组、化疗组相比，差异有统计学意义(P＜0.01)。3．热化疗组能明显增加细胞内阿霉素的药物浓度(P＜0.01)。第三章阿霉素与热疗联合作用动物实验的初步研究1．成瘤时间：对照组(37℃)、化疗组(37℃+ADM)、热疗组(42℃)、热化疗组(42℃+ADM)的肿瘤出现时间分别为(11.2±1.7)、(20.2±2.3)、(15.3±1.6)及(23.8±1.7)天，热化疗组成瘤时间明显延长(P＜0.01)。2．生长曲线显示：热化疗能明显减缓Raji细胞BALB／C裸鼠体内肿瘤的生长速度。治疗结束，热化疗组的肿块较对照组肿块明显缩小。肿块称重结果显示：对照组(37℃)、化疗组(37℃+ADM)、热疗组(42℃)、热化疗组(42℃+ADM)裸鼠的肿瘤重量分别为(6.04±1.06)g、(2.75±0.32)g、(3.90±0.32)g及(1.48±0.21)g，热化疗组肿瘤抑制率为75.5％。3．肿瘤组织病理学改变：成瘤组织经HE病理切片及免疫组化鉴定为人B细胞淋巴瘤，细胞呈卵圆形，细胞核呈卵圆形或不规则形，核仁较明显，核分离相多见。第四章全身热疗系统治疗恶性血液病15例：加温、测温和控温技术的安全、顺应及有效性观察纳入患者15例，全部进入结果分析，无脱落。1．15例恶性血液病患者均能耐受40℃、120min的加热过程，具有良好的顺应性。2．全身加温治疗24h，患者的临床症状、体征明显好转，KPS评分平均提高10分。3．全身加温治疗前及治疗24h，患者的血常规和生化等检查指标无变化(P＞0.05)。结论1．对细胞增殖和抑制率的影响：通过观察肿瘤细胞的生长曲线、克隆形成、存活率及抑制率等指标，热疗联合化疗对耐药及敏感细胞株均有体外抑制作用，对敏感细胞的抑制作用较强。2．对细胞凋亡率的影响：热疗与化疗联合后，细胞的凋亡率较对照组、热疗组及化疗组明显增强，并且随着加热温度的增加，细胞凋亡率也有增加趋势。3．根据Veleriote法判定热疗联合化疗的相互作用类型：在本实验条件下，热化疗对细胞的作用是协同作用和次加作用，可见热疗与化疗联合可以降低化疗药物的使用剂量。4．加热可提高敏感细胞和耐药细胞内的阿霉素药物浓度，下调耐药蛋白P-gp及抗凋亡蛋白Bcl-2的表达。5．本研究动物实验表明：热化疗组裸鼠成瘤时间较各组明显延长，肿瘤体积较对照组明显减小，抑瘤率较其他各组均明显增高。7．全身热疗系统可以用于恶性血液病的全身加热治疗，治疗过程是安全的，值得在临床上进步推广应用；而且能显著提高恶性血液病患者的生活质量及临床症状体征，初步显示其有效性，但长期疗效评价正在进行中。更多还原

【Abstract】 BackgroundCancer is one of the malignant diseases that threatens human health and the incidence and mortality kept on rising in recent years. Despite the high cure rate for patients treated by combination of radiotherapy and chemotherapy at early stage, the prognosis of patients with distant metastases or at late stage is still disappointing. With the emergence of cancer cells with multi-drug resistance,thus it is necessary for us to seek effective methods for the treatment of tumor patients,especially for the multi-drug resistant patients.Thermotherapy as a new therapeutic strategy has drawn many researchers’ attention to tumor therapy, but the laggard theoretical research has been being the obstacle of the progression in clinical practice. With further investigation into biological function to high temperature(hyperthermia) and improvement of thermotherapy technique, thermotherapy has played an important role in the complex treatments of tumor as an auxiliary method of radiotherapy and chemotherapy.The tumor thermotherapy (hyperthermia) has become a fifth treatment method for tumor.Thermal sensitivity of different tumor cells and possible molecular mechanisms were worth investigating. Adriamycin is an effective chemotherapeutic agent for tumor treatment,however the effects of combining with hyperthermia is not known. Adriamycin is a kind of peculiar medicine independing on cell cycle and has been applied into clinic with remarkable antitumor effect,and is one of the most commonly used and effective chemotherapeutic drugs and exhibits a wide spectrum of activity agaist many solid tumors,leukaemias,lymphomas and multiple myeloma in system chemotherapy in recent years.At present, adriamycin is applicated to mainly treat solid tumors in combination with other therapeutic modalities,but it was less reported that it was used for treating the tumors in the hematopoietic system in combination with hyperthermia.For the reason,in this study, tumors of the hematologic malignancies cell lines such as human chronic myelocytic leukaemia cell line K562, adriamycin-resistant subline K562/ADM, human B-cell lymphoma cell line Raji and human multiple myeloma cell line ARH-77 were investigated for the in vitro and in vivo antitumor activity and capacity of adriamycin and hyperthermia in inducing apoptosis and the sensitivities of these cell lines to hyperthermia and thermochemotherapy, as well as the possible mechanism by proliferation assay of cells and FCM and so on.Furthermore the combined effects could be evaluated.We expect to provide an experimental basis for clinical therapeutic alliance of the hematologic malignancies,a theoretical basis for clinical practice and a novel therapeutic approach for malignant hematopoietic system tumors.ChapterⅠThe experimental study of the inhibitory effect of adriamycin combined with hyperthermia on human the hematologic malignancies cell lines in vitroObjectiveTo investigate the inhibitory effects of adriamycin in combination with hyperthermia on proliferation of human cell lines K562, K562/ADM,Raji and ARH-77 and the influence on apoptosis of these cell lines in vitro.Methods 1. K562, K562/ADM, Raji and ARH-77 cell lines were cultivated using RPMI-1640 culture medium containing 10% fetal cattle serum.2. The working concentrations of adriamycin against K562, K562/ADM, Raji and ARH-77 cell lines were determined by MTT assay.3. Based on the different temperature of heating(40℃,41℃,42℃)and adriamycin, the tests were assigned to 8 groups, and 37~C hyperthermia was used as the control group.4. After K562, K562/ADM, Raji and ARH-77 cells were treated, the survival rate of cell lines were analysed with trypan blue exclusion method and the growth inhibitory rates were analysed with MTT assay. The numbers of the cells and colonies were counted, the rate of the colony formation and inhibition calculated and drawing of the cell survival curve of different groups.5. The cell apoptosis was determined by flow cytometry(FCM) in the different management groups.6. The correct formula of Veleriote was used to evaluate the interaction of thermochemotherapy.7. The data were processed with SPSS 11.0 software package. Results were analyzed with one-way ANOVA and expressed as mean + standard deviation. LSD and Tamhane’s T2 were used for multiple comparisons.ChapterⅡExperimental study in vitro sensitization effect of thermochemotherapy on human the hematologic malignancies cell linesObjectiveTo observe the sensitization effect of treatment with adriamycin in combination with hyperthermia in vitro on human cell lines K562, K562/ADM, Raji and ARH-77, the expressions of Bcl-2 and P-gp,and the intracellular concentration of adriamycin.Methods1. K562, K562/ADM, Raji and ARH-77 cell lines were cultivated using RPMI-1640 culture medium containing 10% fetal cattle serum.2. The working concentrations of adriamycin against K562, K562/ADM, Raji and ARH-77 cell lines were determined by MTT assay.3. The expressions of Bcl-2, P-gp and the intracellular concentration of adriamycin were determined by flow cytometric.4. The data were processed with SPSS 11.0 software package. Results were analyzed with one-way ANOVA and expressed as mean±standard deviation. LSD and Tamhane’s T2 were used for multiple comparisons.ChapterⅢAn initial study of adriamycin combined with hyperthermia in nude miceObjectiveTo study the influence of adriamycin in combination with hyperthermia on tumor formation and tumor growth of human B lymphoma cell line(Raji) in nude mice in vivo.Methods24 BALB/C nude mice were divided into four groups:the control group (37℃), the chemotherapy group (37℃+ADM), the hyperthermia group (42℃) and the thermochemotherapy group (42℃+ADM). Every nude mouse was injected 5×10~6 Raji cells subcutaneously.The time and the rate of tumor formation were observed,the perpencicular tumor diameter was measured with sliding caliper at a 3-day intervals and the tumor volume was calculated,then the growth curves of tumors were plotted. Three weeks after tumor formation,all the mice were killed and the histopathological changes of the tumor were observed by HE staining. The data were processed with SPSS 11.0 software package. Results were analyzed with one-way ANOVA and expressed as mean±standard deviation. LSD and Tamhane’s T2 were used for multiple comparisons. ChapterⅣWhole body hyperthermia in the treatment of 15 patients with malignant hematopathy: Observation of the safety, accustomization and efficacy in warming, temperature-measuring and temperature-controllingObjectiveTo probe into the toleration of patients with malignant hematopathy under whole-body hyperthermia(WBH),and test the compliance,safety and efficacy of WBH.MethodsThe experiment was conducted in the Department of Hematology, Zhujiang Hospital of Southern Medical University from April to July 2005. Fifteen patients with malignant hematopathy were selected, and all of them took part in the experiment voluntarily.Patients received WBH with WELLRAY photon thermotherapy intelligent system manufactured by Guangdong Weier Medicine and Sciences Co.Ltd. Retention catheterization and peripheral venous cannula were done before treatment, eletrocardio-monitoring was connected and the temperature receptor of body surface was fixed and so on. Then we warmed the intrarectal temperature until it reached 40～41℃, kept the homoiothermism for 2 hours,meanwhile,monitored the temperature changes of body surface,air and treatment couch, and electrocardio-monitoring was performed at the same time. Blood routine examination and biochemiscal indexes etc.were detected before treatment and 24 hours after treatment. The qualify of life in patients before and 24 hours after WBH were evaluated with Karnofsky(KPS) scale, the scale range was 0～100 points, 100 points as normal without symptoms and physical signs; 10 points as dangerous, close to death; Zero as death.ResultsChapterⅠThe experimental study of the inhibitory effect of adriamycin combined with hyperthermia on human the hematologic malignancies cell lines in vitro1. The concentration of adriamycin in the experiment was defined as its IC50.Then hyperthermia and chemotherapy were used singly or concurrently.2. The effect on cell proliferation and apoptosis rate: Compared with the control group, chemotherapy group, and hyperthermia group, the thermochemotherapy group showed suppression of cell growth, and the apoptosis rate was higher with statistical significance in the difference.3. Compared with the control group, the chemotherapy group and the hyperthermia group, the rates of colony formation and inhibition were decreased and the difference was of statistical significance in the thermochemotherapy group.ChapterⅡExperimental study in vitro sensitization effect of thermochemotherapy on human the hematologic malignancies cell lines1. The concentration of adriamycin in the experiment was defined as its IC50. Then hyperthermia and chemotherapy were used singly or concurrently.2. The FCM results indicated that the expression rates of Bcl-2 and P-gp proteins in hyperthermia, adriamycin chemotherapy and thermochemotherapy groups were down-regulated. Compared with the control group, the difference had statistical significance(P＜0.01).3. Thermochemotherapy remarkably increased the intracellular concentration of adriamycin(P＜0.01).ChapterⅢAn initial study of adriamycin combined with hyperthermia in nude miceThe time of tumor formation in the control group (37℃), the chemotherapy group (37℃+ADM), the hyperthermia group (42℃) and the thermochemotherapy group (42℃+ADM) group were (11.2+1.7), (20.2+2.3), (15.3+1.6) and (23.8+1.7) days. Three weeks after tumor formation all the mice were killed, the average weight of tumors were (6.04+1.06)g, (2.75+0.32)g, (3.90+0.32)g and (1.48+0.21)g, the tumor growth inhibition rate was 75.5% and HE staining showed that there were more necrosis regions in the tumor tissue in the thermochemotherapy group.ChapterⅣWhole body hyperthermia in the treatment of 15 patients with malignant hematopathy:Observation of the safety, accustomization and efficacy in warming,temperature-measuring and temperature-controllingFifteen patients entered the final analysis and no subject withdrew from the experiment.1. All of the 15 patients with malignant hematopathy could endure being warmed to 40℃for 120 minutes, and had a good compliance.2. Clinical symptoms and physical signs of patients turned better 24 hours after WBH with an average increase of 10 points.3. There were no significant differences in blood routine and biochemical indexes etc. before WBH and 24 hours after WBH.Conclusion1. The effect on cell proliferation and inhibition rate: Thermotherapy could restrain the growth of both the drug-sensitive and drug-resistant cells with indexes of the clone formation, survival rate, and inhibition rate and so on. In the thermochemotherapy group, the ability of inhibition is stronger to the sensitive cells.2. The effect of apoptosis rate: These results indicated that with the increase of temperature, the apoptosis rate had the increasing trend. Compared with the control group,the chemotherapy group and the hyperthermia group, the apoptosis rate of the thermochemotherapy was higher.3. The correct formula of Veleriote was used to evaluate the interaction of thermochemotherapy: Under this experimental condition, the effect of the hyperthermia combined ADM is joint-action. The results also showed that themochemotherapy can reduce the doses of drugs and improve the effect.4. Thermochemotherapy remarkably increased the intracellular concentration of adriamycin both in the sensitive cells and in the drug resistant cells, and down-regulate the expression rates of Bcl-2 and P-gp proteins.5. The animal experiment showed that the time of tumor formation obviously prolonged, the tumor volume diminished, and the growth inhibition rate raised up in the thermochemotherapy group compared with the control group, the chemotherapy group and the hyperthermia group.7. WBH system can be used in whole body heating agaist malignant hematopathy, and the process of treatment is safe and worthy to be spread clinically. It can markedly increase the qualify of life as well as the clinical symptoms and physical signs of patients with malignant hematopathy, and has shown its effectiveness initially, but the evaluation of long-term curative effect is still in observation.更多还原