【作者】 李墨林；

【导师】 贾玉杰；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2008， 博士

【摘要】 目的:恶性肿瘤已经成为严重危害全球人类健康的常见致命性疾病之一。据资料统计,全世界每年有约700万人患癌症,大约有500万人死于癌症。在我国,恶性肿瘤的发病率和死亡率都呈增加的趋势。由于恶性肿瘤的病因至今尚未完全清楚,因此,目前临床上恶性肿瘤的治疗主要是采用手术、放疗、化疗、生物治疗（包括免疫治疗）等综合治疗手段,其中化疗已经成为各种晚期恶性肿瘤有效治疗的基础。一般认为细胞毒性化疗药物杀伤或抑制肿瘤细胞的疗效与剂量成正比,呈剂量依赖性关系。根据药理学研究成果制定的肿瘤大剂量化疗方案虽然有成功治愈绒毛膜上皮癌等恶性肿瘤的临床报道,但大剂量化疗对多数恶性肿瘤的五年存活率却无明显提高。一般认为肿瘤患者大剂量化疗后可获得较好的瘤体缩小的短期疗效,同时剂量依赖性的毒副作用也明显,往往不利于恶性肿瘤患者生存质量的改善和远期疗效的提高。随着医学模式的转变,学术界已逐渐认识到生存质量和生存期的延长对肿瘤病人有着决定性意义,而瘤体缩小只是治疗评价中的次要结局指标。而在目前的抗恶性肿瘤药物中,无论是细胞周期特异性药物还是细胞周期非特异性药物,其对癌细胞的杀伤作用都服从一级动力学原理即只能按比例而不能全部杀伤肿瘤细胞。因此,与化疗治愈肿瘤密切相关的机体因素还不十分清楚,化疗药物治愈肿瘤的作用机制尚有待于进一步研究。为深入研究化疗治愈肿瘤的作用机制,本研究利用荷膀胱癌小鼠模型,建立单一剂量化疗药物治愈荷瘤小鼠的动物模型。在此基础上,检测在单一剂量化疗药物治愈肿瘤过程中荷瘤小鼠外周血象的动态变化,分析外周血象的变化与荷瘤小鼠化疗疗效之间的关系,了解血小板包括外周血血小板计数和血浆血小板激活因子（platelet activating factor, PAF）水平在化疗治愈肿瘤中的变化及其意义。利用该化疗治愈荷瘤小鼠的动物模型,进一步通过透射电镜观察化疗药物体内治疗72小时后荷瘤小鼠肿瘤组织细胞的形态学变化,明确该化疗药物对肿瘤细胞及肿瘤间质细胞（特别是肿瘤血管内皮细胞）的作用机制;并且分析药物体内治疗72 h内,荷瘤小鼠肿瘤组织内过氧化脂质降解产物丙二醛（malondialdehyde, MDA）含量的变化,明确肿瘤组织内肿瘤细胞的损伤或死亡与肿瘤局部脂质过氧化损伤的关系;同时,检测药物体内治疗72 h内,荷瘤小鼠血浆内毒素含量和一氧化氮（Nitric Oxide, NO）含量的变化,为进一步明确小剂量化疗治愈肿瘤的作用机制奠定基础。方法:给正常T739小鼠皮下接种小鼠可移植性膀胱移行细胞癌组织,建立荷膀胱癌小鼠模型。取肿瘤接种后第7天的荷瘤小鼠,称重后随机分两组:治疗组和对照组。治疗组小鼠单次腹腔内注射不同剂量的化疗药物,包括环磷酰胺（cyclophosphamide,CTX）、丝裂霉素C（Mitomycin C, MMC）;对照组小鼠单次腹腔内注射等量生理盐水。用药后每1～3天观察一次各组荷瘤小鼠,并记录小鼠的体重和肿瘤结节的直径。当肿瘤直径接近或等于15 mm,视为治疗无效、并将小鼠处死。存活小鼠的肿瘤结节应小于15 mm或无瘤。找出能使T739荷瘤小鼠肿瘤消退所需化疗药物的最小使用剂量。如果治疗后荷瘤小鼠肿瘤结节渐缩小甚至消失,继续观察至少60天。肿瘤治愈的标准为:用单一剂量化疗药物治疗后,荷瘤小鼠肿瘤消退,并在随后的2个月内无复发。然后,再取12只肿瘤接种后第7天的荷瘤小鼠,随机分成两组,治疗组小鼠腹腔内注射能使荷瘤小鼠肿瘤消退所需最小剂量的化疗药物,对照组小鼠腹腔内注射等量生理盐水。分别在用药后6 h、用药后第2、4、9、14 d内眦静脉取血,肝素抗凝后立即进行血常规检测,比较两组荷瘤小鼠外周血红细胞（red blood cell, RBC）数、血红蛋白（hemoglobin, Hb）浓度、白细胞数和血小板（Platelet,PLT）参数的动态变化,分析荷瘤小鼠外周血象的变化与化疗疗效的相互关系。同时,建立液相色谱-串联质谱（Liquid Chromatography -Mass Spectrometer, LC/MS/MS）测定小鼠血浆PAF的方法,检测单剂量化疗药物用药后6 h血浆PAF水平的变化。最后,再取18只肿瘤接种后第7天的荷瘤小鼠,随机分组,治疗组小鼠腹腔内注射能使荷瘤小鼠肿瘤消退所需最小剂量的化疗药物,对照组小鼠腹腔内注射等量生理盐水。CTX用药后6、24、48、72 h荷瘤小鼠内眦静脉取血,肝素抗凝后取血浆。应用BET-16细菌内毒素测定仪,采用动态浊度鲎试剂法检测荷瘤小鼠血浆内毒素水平;采用硝酸还原酶法测定荷瘤小鼠血浆NO浓度。随后将小鼠处死,肿瘤组织制成10%肿瘤组织匀浆,硫代巴比妥酸法检测肿瘤组织中MDA的含量。CTX用药后72 h,荷瘤小鼠处死后快速取肿瘤组织切成1.5 mm×2 mm大小,4%戊二醛固定后透射电镜观察肿瘤组织细胞的形态学变化。结果:1.小剂量CTX治愈荷瘤T739小鼠动物模型的建立接种2 mm3的小鼠膀胱癌肿瘤组织可使T739小鼠皮下肿瘤结节进行性生长。接种后第7天肿瘤结节直径约为2～4 mm。取肿瘤接种后第7天的荷瘤小鼠,1～16 mg/kg的MMC或15～400 mg/kg的CTX单次腹腔内注射。结果发现:（1）不同剂量的MMC用药后1 w,随MMC使用剂量的增加荷瘤小鼠化疗的毒副作用加大、肿瘤结节缩小的程度增加;MMC用药后2月,8～16 mg/Kg MMC治疗组和对照组荷瘤小鼠全部死亡,4 mg/kg和1 mg/kg MMC治疗组各有25%的荷瘤小鼠存活。（2）不同剂量的CTX用药后2 w,随CTX用药剂量的增加荷瘤小鼠化疗的毒副作用加大、肿瘤结节缩小的程度增加;CTX用药后2月,100～200 mg/kg CTX治疗组和对照组的荷瘤小鼠全部死亡,40 mg/kg CTX治疗后20%的荷瘤小鼠存活,15 mg/kg CTX治疗组60%的荷瘤小鼠存活,其与对照组比较差异具有统计学意义（P<0.01）。结果提示:15 mg/kg CTX是可治愈多数荷瘤T739小鼠所需CTX的最小有效剂量。2.外周血象和血小板激活因子在单剂量CTX治愈荷瘤小鼠过程中的变化及其意义15 mg/kg CTX对荷瘤小鼠外周血象无明显抑制作用。具体表现在:15 mg/kg CTX单次腹腔内注射后6 h,荷瘤小鼠外周血RBC计数（red blood cell count, RBC count）、血红蛋白（hemoglobin, Hb）浓度和WBC计数（white blood cell count, WBC count）分别为（9.86±0.64）×10¹²/L、166.6±15.2 g/L和（12.38±3.96）×10⁹/L,与对照组荷瘤小鼠（9.0±0.6）×10¹²/L、148±10 g/L和（10.54±1.74）×10⁹/L比较,差异无统计学意义（P>0.05）;CTX治疗后6 h荷瘤小鼠外周血血小板计数（ platelet count, PLT count）为（1483.4±184.4）×10⁹/L,与对照组（1086.6±81.0）×10⁹/L比较,差异具有统计学意义（P<0.01）;而血小板平均容积（mean platelet volume, MPV）、血小板分布宽度（platelet volume distribution width, PDW）和大血小板比率（platelet-large-cell ratio, P-LCR）与治疗组荷瘤小鼠比较差异无统计学意义（P>0.05）。用药后第2、4、9、14天治疗组荷瘤小鼠外周血RBC、WBC计数和PLT参数的变化与对照组小鼠比较差异无统计学意义（P>0.05）。进一步通过建立液相色谱-串联质谱测定小鼠血浆PAF的方法,检测了15 mg/kg CTX用药后6 h荷瘤小鼠血浆PAF水平的变化,结果发现:CTX治疗组荷瘤小鼠血浆PAF质量浓度为113.98±14.38 ng/ml,而对照组荷瘤小鼠血浆PAF质量浓度为58.34±13.89 ng/ml,两组数据比较差异具有统计学意义（P<0.01）。由此可以看出:在小剂量CTX用药后6 h,伴随荷瘤小鼠血小板计数的增加血浆PAF水平也明显提高,提示血小板在小剂量CTX治愈肿瘤过程中发挥重要作用。3.小剂量CTX抗肿瘤作用机制的研究小剂量CTX治疗后72 h,透射电镜下荷瘤小鼠肿瘤组织中肿瘤细胞以“凋亡”改变为主,具体表现在:肿瘤细胞体积变小,胞膜起泡或出“芽”,细胞表面突起减少、微绒毛减少甚至消失,相邻细胞间的细胞连接消失,染色质团块状散布核内并边集于核膜下,细胞核固缩呈不均一的块状结构,胞质内可见脂肪堆积,空泡变性等凋亡的特征性变化。同时,肿瘤组织中血管内皮细胞也出现典型的凋亡形态学变化,并伴随肿瘤毛细血管管腔的缩小。此外,肿瘤组织中还见部分肿瘤细胞细胞膜的完整性破坏,出现细胞坏死的形态学变化。电镜结果表明:小剂量CTX治疗后72 h,荷瘤小鼠肿瘤组织中肿瘤细胞凋亡和肿瘤细胞坏死同时存在;同时,肿瘤血管内皮细胞也出现典型的凋亡改变。通过检测荷瘤小鼠肿瘤组织匀浆中脂质过氧化产物MDA的含量,结果发现:小剂量CTX用药后6 h,荷瘤小鼠肿瘤组织匀浆中MDA的浓度为14.69±3.86 nmol/mgprot,与对照组荷瘤小鼠16.1±1.35 nmol/mgprot比较差异无统计学意义（p>0.05）, CTX用药后24、48、72 h荷瘤小鼠肿瘤组织匀浆中MDA浓度较对照组明显降低,分别为8.87±2.70、5.15±2.13和8.92±2.94 nmol/mgprot,提示小剂量CTX治疗后72 h内,荷瘤小鼠肿瘤组织内过氧化脂质降解产物MDA的含量无明显升高,说明肿瘤细胞的损伤或死亡与肿瘤局部的脂质过氧化反应无关。通过检测15 mg/kg CTX用药后荷瘤小鼠血浆NO含量,结果发现:CTX用药后6 h,荷瘤小鼠血浆NO含量为60.93±5.59μmol/ml,略高于对照组小鼠34.88±19.08μmol/ml,CTX用药后72 h,血浆NO含量升至81.86±17.33μmol/ml,与对照组荷瘤小鼠比较差异具有统计学意义（p<0.01）。提示小剂量CTX用药后6～72 h血浆NO含量逐渐升高,说明血浆NO在小剂量CTX治愈肿瘤过程中发挥重要作用。最后我们检测了小剂量CTX用药后6 h荷瘤小鼠血浆内毒素含量,结果发现CTX治疗组荷瘤小鼠血浆内毒素含量为0.29±0.38 Eu/ml,明显低于对照组荷瘤小鼠1.20±0.72 Eu/ml（p<0.05）,提示15 mg/kg CTX用药后6 h荷瘤小鼠未出现内毒素血症。结论:1. 15 mg/kg CTX单次腹腔内注射可治愈多数荷膀胱癌T739小鼠,提示单一剂量CTX治愈荷瘤T739小鼠模型成功建立。2. 15 mg/kg CTX单次腹腔内注射在治愈多数荷瘤小鼠的同时对荷瘤小鼠的外周血象无明显抑制作用。CTX用药后6 h,荷瘤小鼠外周血血小板计数和血浆PAF水平明显高于对照组,提示血小板可能在化疗治愈肿瘤发挥重要作用。3.小剂量CTX体内治疗可诱导荷瘤小鼠肿瘤细胞和肿瘤血管内皮细胞的凋亡,而肿瘤细胞的坏死可能与肿瘤血管内皮细胞的损伤有关。小剂量CTX用药后72 h内,荷瘤小鼠肿瘤组织内MDA含量的逐渐下降,提示肿瘤细胞的死亡与氧化应激损伤无关。小剂量CTX用药后6 h,荷瘤小鼠体内未出现内毒素血症,CTX用药后72 h内血浆NO含量的持续增高与可能CTX用药后血浆PAF水平的升高和肿瘤血管内皮细胞的损伤有关。更多还原

【Abstract】 Objective:With the great changes in human lifestyle and environment, the malignant tumor has become one of the most common diseases endangering human health in modern society. According to statistics, about 7 million people are diagnosed and nearly 5 million people die of cancer worldwide each year. In recent years there has been an increasing trend in the incidence and mortality of malignant tumors in china. Because the cause of malignant tumors is not completely clear, at the present time combined treatment methods including surgery, chemotherapy, radiotherapy, treatment with biological agents and so on had been adopted in clinic, and systematic treatment with cytotoxic drugs has been the basis of most effective treatments for patients with disseminated cancers. It is thought that cytotoxic drugs inhibit or kill tumor cells in a dose dependent manner. It was reported that a high dose of chemotherapy could cure gestational trophoblastic tumor in clinic, but five years survival rate of most patients with malignant tumors was not elevated significantly with a high dose chemotherapy scheme. It was found that a high dose of chemotherapy could increase not only the short-term effects on tumor, but also the dose- dependent side effects. And it was not favorable to improving the patients’quality of life and elevating the long-term therapeutic efficacy. With the medical model changes, it was realized living quality and prolonged survival time are of decisive significance for patients with malignant tumors, and tumor-shrinkage is not the primary goal of treatment. Of all anti-cancer drugs used in clinic, they inhibited or killed tumor cells only by ratio. Therefore, factors related to tumor rejection induced by chemotherapy are not clearly understood, and the mechanisms of tumor rejection induced by chemotherapy should be studied further. In order to understand the mechanisms of tumor rejection induced by chemotherapy much better, a tumor-bearing mice model was used and an animal model cured by a single dose of chemotherapy was established. Further, dynamic changes of peripherial blood cells were measured during tumor rejection, and the relationship between chemotherapeutic effects and mouse peripherial blood cells was analyzed. The changes and significance of platelets including platelet count and platelet activating factor （PAF） in chemotherapy-induced tumor rejection was ensured. Using this tumor rejection model induced by chemotherapy, the ultrastructural morphology of tumor tissues 72 hours after chemotherapy treatment was studied by transmission electron microscopy. The mechanisms of chemotherapeutic drugs on tumor cells and tumor stroma cells （especially tumor vascular endothelial cell） will be detected. In order to know whether the injury or death of tumor cells was related to oxidative injury, the concentration of malondialdehyde （MDA）, the product of metabolism of lipid peroxidation was measured in tumor tissue within 72 h after chemotherapy. The content of endotoxin and nitric oxide （NO） in plasma of tumor bearing mice within 72 h after chemotherapy were also tested. Possible mechanisms of tumor rejection induced by low-dose CTX chemotherapy would be explained.Methods:Mouse bladder carcinoma tissue was inoculated subcutaneously into BTT739 mice. 7 days later, different doses of cyclophosphamide （CTX） or Mitomycin C （MMC） were used intraperitoneally to treat these tumor- bearing mice and the same volume of NS was used as control. Mice were observed and tumor sizes and body weight were measured and recordered everyday or every 2 to 3 days. Animals were sacrificed once tumors reached a maximal size of 15 mm in diameter. Mice with tumors less than 12 mm in diameter or without tumors were classified as tumor free mice. The minimal effective dose of chemotherapy that could cure most of the tumor-bearing mice was found. Tumor rejection was defined as complete tumor regression after chemotherapy and the absence of recurrent tumor for the entire follow-up period （at least 60 days）.Then another 12 tumor-bearing mice were randomly divided into two groups: treatment group and control group. The minimal effective dose of chemotherapy that could cure most of the tumor-bearing mice was given intraperitoneally and the same volume of NS was used as control. Blood samples were obtained from orbital venous sinus at different times after chemotherapy. Complete blood counts were performed and dynamic changes of peripherial blood RBC, Hb, WBC, platelet parameters were obtained. The relationships between the changes of peripheral blood count and tumor shrinkage induced by chemotherapy was analyzed. Establishing a method for testing the level of PAF in plasma by Liquid Chromatography - Mass Spectromete（rLC/MS/MS）Device, the level of plasma PAF in tumor bearing mice was measured 6 h after chemotherapy treatment.Finally, another 18 tumor-bearing mice were randomly divided into two groups: treatment group and control group. The minimal effective dose of chemotherapy that could cure most of the tumor-bearing mice was given intraperitoneally and the same volume of NS was used as control. Blood samples were obtained from orbital venous sinus at 6, 24, 48, 72 h after chemotherapy. Using the BET-16 bacterium endotoxin cryoscope, endo- toxinemia was measured in tumor bearing mice 6 h after CTX treatment by the kinetic turbidimetric limulus assay. Serum nitric oxide （NO） was measured by nitrate reductase method in tumor bearing mice within 72 h after CTX treatment. The mice were killed at different time after CTX treatment. And the content of malondialdehyde （MDA） was measured by the thiobarbituric acid reaction assay （TBARS） in tumor tissue before and different time after CTX treatment. Morphological changes of apoptosis and/or necrosis in tumor tissue 72 h after CTX treatment were observed with transmission electron microscope （TEM）.Results:1. Establishment of an animal model of tumor-bearing mice cured by a low dose of CTX Inoculation of 2 mm3 bladder carcinoma tissue could make tumor cell progressive growth in T739 mice. The tumor sizes were 2～4 mm on day 7 after inoculation. Tumor bearing mice were treated with different doses of CTX （15～400 mg/kg） or MMC （1～16 mg/kg）. It was found: （1） the higher dose of MMC used in the study could increase not only the speed of tumor shrinkage but also the toxic side effects on tumor bearing mice 1 week after treatment. 2 months after MMC treatment, all mice died in 8～16 mg/kg MMC treatment group and control group; While 25% of mice were alive in 4 mg/kg and 1 mg/kg MMC treatment group respectivelyl; （2） and the higher dose of CTX used for the treatment of these tumor bearing mice could also increase the speed of tumor shrinkage and induce more severe toxic side effects 2 weeks after treatment. 2 months after CTX treatment. All mice died in 100～200 mg/kg CTX treatment group and control group; While 20% of mice were alive in 40 mg/kg CTX treatment group and 60% of mice were alive in 15 mg/kg CTX treatment group. Compared with control, there was a significantly difference in survival rate of tumor bearing mice between 15 mg/kg CTX group and control group （p<0.01）. The result indicated that 15 mg/kg CTX was the minimal effective dose of CTX that could cure most of the tumor-bearing T739 mice.2. Changes and the significance of peripheral blood cell count and plasma PAF level in tumor shrinkage induced by a low dose of CTX in T739 mice 15 mg/kg CTX had no obvious inhibitive effects on peripheral blood cell count of tumor bearing mice. The perpherial RBC count, concentration of Hb and WBC count was （9.86±0.64）×10¹²/L, 166.6±15.2 g/L and （12.38±3.96）×10⁹/L respectively at 6 h after 15 mg/kg CTX treatment. Compared with that in control mice （9.0±0.6）×10¹²/L, 148±10 g/L and （10.54±1.74）×10⁹/L, there was no significant difference between them（P>0.05）;The perpherial platelet count increased to （1483.4±184.4）×10⁹/L in mice 6 h after CTX treatment. There was significant difference compared with that in mice of control group （1086.6±81.0）×10⁹/L （P<0.01）; and there was no significant difference in MPV, PDW and P-LCR between the mice in control group and CTX treatment group （P>0.05）. During the 2nd to the 14th day, there was no obvious difference in peripheral RBC count, WBC count and platelet parameters in tumor bearing mice after CTX treatment （P>0.05）. Using LC/MS/MS, PAF level in plasma was measured. It was found that the concentration of plasma PAF was 113.98±14.38 ng/ml in tumor bearing mice 6 h after CTX treatment; while only 58.34±13.89 ng/ml in control mice. There was a significant difference in plasma PAF between CTX treatment and control groups （P<0.01）. Therefore, not only peripherial PLT count but also plasma PAF concentration increased in tumor bearing mice 6 h after 15 mg/kg CTX treatment. The results indicated that platelet played an important role in the tumor rejection induced by low-dose CTX chemotherapy.3. Possible mechanisms of tumor rejection induced by low-dose CTX chemotherapy 6 h after 15 mg/kg CTX treatment, most of tumor cells in tumor tissue seen under TEM were of classical morphological changes of apoptosis, including decreased cell volume, cytoplasmic condensation, cytoplasmic vacuoles, and condensed nuclear chromatin and so on. Vascular endothelial cells were also of classical morphological changes of apoptosis with decreased the diameter of capillary. Besides these, tumor cells also exhibited typical morphological changes of necrosis. TEM analysis displayed that not only tumor cells but also vascular endothelial cells exhibited typical apoptotic phenotype, and moreover, apoptosis and necrosis of tumor cells coexisted in tumor tissue after low-dose CTX chemotherapy. The MDA content in normal tumor tissue was 16.1±1.35 nmol/mgprot, and 14.69±3.86 nmol/mgprot in tumor tissue after 6 h of 15 mg/kg CTX treatment. There was no significant difference between them （p>0.05）. The MDA content decreased to 8.87±2.70, 5.15±2.13 and 8.92±2.94 nmol/mgprot in tumor tissue 24, 48, 72 h after CTX treatment, obviously lower than that in control. The results indicated that there was no lipid peroxidation damage of tumor cell membrane in tumor bearing mice within 72 h after CTX treatment. The plasma concentration of NO in tumor bearing mice 6 h after 15 mg/kg CTX treatment was 60.93±5.59μmol/ml, obviously higher than that 34.88±19.08μmol/ml in control mice （p<0.05）. And plasma concentration of NO increased to 81.86±17.33μmol/ml 72 h after CTX treatment, significantly higher that in control mice （p<0.01）. The increasing concentration of plasma NO may be related to the increased level of plasma PAF and the injured vascular endothelial cells in tumor bearing mice after CTX treatment. The level of plasma endotoxin was also measured. It was found that level of plasma endotoxin was 0.29±0.38 Eu/ml in mice 6 h after low-dose CTX chemotherapy; significantly lower than that 1.20±0.72 Eu/ml in control mice （p<0.05）. The results indicated that there was no endotoxinemia in tumor bearing mice at this time.Conclusions:1. 15 mg/kg CTX administration intraperitoneally could cure most of the tumor-bearing T739 mice. A new mouse tumor rejection model induced by low-dose CTX had been established successfully.2. 15 mg/kg CTX had on obvious inhibitive effects on peripherial blood cells. Peripherial platelet count and the level of plasma PAF was increased significantly in tumor bearing mice 6 h after CTX treatment. The results indicated that platelets played an important role in tumor rejection induced by chemotherapy.3. TEM analysis displayed that low-dose CTX treatment could induce apoptosis not only in tumor cells but also in vascular endothelial cells. The necrosis of tumor cells may be related to the injured vascular endothelial cells in tumors after low-dose CTX chemotherapy. The decreasing content of MDA in tumor tissue within 72 h after CTX treatment indicated that the death of tumor cells had nothing to do with oxidative injury in tumor tissue in vivo. 6 h after CTX treatment,there was no endotoxinemia in tumor bearing mice. The increasing level of plasma NO during 6～72 h after CTX treatment was probably related to the increased plasma PAF level and the injured vascular endothelial cells in tumor bearing mice after CTX treatment更多还原