【作者】 任庆兰；

【导师】 李少林；

【作者基本信息】 重庆医科大学 ， 药理学， 2004， 博士

【摘要】 放射治疗在肿瘤治疗中占有重要地位。据统计，我国大约有70%的恶性肿瘤病人在病程的某一阶段需接受不同方式的放射治疗。在正常组织的耐受剂量范围内，肿瘤的平均治愈率仅为50%。卵巢癌、宫颈癌是严重威胁妇女健康的恶性疾患，直接影响患者的寿命及生存质量，而肿瘤细胞对放射治疗具有一定的放射抵抗性以及由于放疗副反应限制了有效治疗剂量的增加是疗效不佳的重要原因，所以如何降低肿瘤细胞的辐射抗性，增加辐射敏感性一直是放射肿瘤学者研究和关注的课题。第一部分目的：探讨c-erbB-2、c-raf-1基因反义寡核苷酸对受照后人卵巢癌SKOV3细胞增殖的抑制作用及其分子机制。方法：实验分为四组：正常对照组（空白对照组）、Lipofectin组（仅加入等量的脂质体）、正义寡核苷酸治疗组、反义寡核苷酸治疗组。卵巢癌细胞在寡核苷酸处理前后c-raf-1、c-erbB-2表达水平和细胞周期的变化用RT-PCR和FCM检测；接受60Coγ射线不同剂量照射前后肿瘤细胞的存活分数、集落形成率、凋亡情况分别用MTT试验、平板克隆形成试验、电镜、荧光显微镜观察。结果：c-raf-1与c-erbB-2反义寡核苷酸能明显抑制c-raf-1和c-erbB2癌基因的表达，但两者引起的细胞周期阻滞并不一<WP=10>致；反义寡核苷酸作用的卵巢癌细胞经60Coγ射线照射后其细胞存活分数、集落形成率和转染正义寡核苷酸+照射组及单纯照射组比较明显下降、凋亡率明显增加（p<0.01），而转染正义寡核苷酸+照射组与单纯照射组相比无明显差异（p>0.05）。结论：c-raf-1、c-erbB-2 反义寡核苷酸通过下调c-raf-1和c-erbB-2的表达抑制了人卵巢癌细胞株SKOV3的增殖，降低了辐射抗性。该作用可能与c-raf-1、c-erbB-2反义寡核苷酸抑制了引起细胞辐射抵抗的信号转导途径有关，而与改变受照前细胞周期分布关系不明确。第二部分目的：由于Ki-67反映肿瘤细胞的增殖能力，Bcl-2具有抗凋亡的作用，它们的表达程度可用于预测肿瘤细胞的放射疗效（即表达越高，辐射抗性越强），为此第二部分用免疫组织化学法检测与放射敏感性相关的Ki-67、Bcl-2蛋白，观察它们在c-erbB-2/ c-raf-1反义寡核苷酸作用后受照人卵巢癌SKOV3细胞的表达情况。结果：c-erbB-2反义寡核苷酸+照射组Ki-67、Bcl-2免疫组化阳性率分别为19.8%和20.7%（与照射组比较，p﹤0.01）；c-raf-1反义寡核苷酸+照射组两者的免疫组化阳性率分别为19.8%和21.7%（与照射组比较，p﹤0.01）。转染c-erbB-2/ c-raf-1正义寡核苷酸+照射组和照射组比较无明显差异(p>0.05)。结论：，脂质体介导的c-erbB-2/c-raf-1反义寡核苷酸能显著下调经60Coγ射线照射后卵巢癌细胞Ki-67、Bcl-2蛋白的表达。这可能是c-raf-1、c-erbB-2反义寡核苷酸对人卵巢癌细胞株SKOV3放射增敏的分子机制之一。 <WP=11>第三部分目的：体外测定实际上细胞脱离了其生长存在的体内微环境，许多决定其放射生物效应的因素将被改变，如氧合状态、周期分布、相关损伤修复的物理化学条件以及相关于增殖的细胞间接触效应等。临床一个肿瘤病灶的放疗疗效除取决于其本身固有的放射敏感性外，尚受到细胞内外，宿主情况等多种因素的影响，为此建立宫颈癌的皮下移植瘤动物模型，来研究c-erbB-2/c-raf-1基因反义寡核苷酸对小鼠宫颈癌皮下移植瘤放射治疗效应的影响。方法：将Balb/C小鼠随机分为六组：一组：对照组（腹腔注射等量脂质体），二组：腹腔注射正义寡核苷酸，三组：腹腔注射反义寡核苷酸，四组：照射组（腹腔注射等量脂质体），五组：腹腔注射正义寡核苷酸+照射，六组：腹腔注射反义寡核苷酸+照射。用原位杂交检测寡核苷酸作用前后的靶基因表达；通过观察肿瘤受60Coγ射线照射后肿瘤组织体积随时间的变化、肿瘤倍增时间、小鼠存活时间来研究c-erbB-2、c-raf-1基因反义寡核苷酸对小鼠宫颈癌皮下移植瘤放射治疗效应的影响。结果：c-erbB-2/c-raf-1基因反义寡核苷酸能明显抑制相应基因的表达。c-erbB-2/ c-raf-1反义寡核苷酸+照射组的抑瘤率、肿瘤的倍增时间、小鼠的存活时间比照射组明显增加(p﹤0.01)，而转染c-erbB-2/ c-raf-1正义寡核苷酸+照射组和照射组比较无明显差异(p>0.05)。结论：c-erbB-2/c-raf-1反义寡核苷酸作用后能使小鼠宫颈癌皮下移植瘤的生长明显受到抑制，小鼠生存期明显延长，提高了放疗疗效。第四部分 <WP=12>目的：由于PCNA表达一定程度上可以反映放疗后肿瘤的增殖情况，而c-Jun、c-Fos能被包括辐射在内的外界刺激激活，导致辐射抗性增加，因此本部分探讨c-erbB-2/c-raf-1反义寡核苷酸对小鼠宫颈癌皮下移植瘤照射前后c-Jun、c-Fos、PCNA表达的影响及其意义。方法：实验分为照射前和照射后各四组。采用免疫组化SABC法检测c-Jun、c-Fos、PCNA表达情况。结果：转染c-erbB-2反义寡核苷酸后受照小鼠宫颈癌皮下移植瘤c-Jun、c-Fos、PCNA的阳性表达率分别为19.76 %、19.47 %、16.49 %（与单纯照射组比较，P<0.01），而转染c-raf-1反义寡核苷酸组的阳性率分别为20.06 %、20.23 %、14.89 %（与单纯照射组比较，P<0.01）。转染c-erbB-2/c-raf-1正义寡核苷酸+照射组各指标与照射组无统计学显著性差异。结论：c-erbB-2、c-raf-1?更多还原

【Abstract】 Radiotherapy plays an important role in the treatment of malignant tumors. About 70 percent tumor patients would receive radiotherapy in the progress of disease. The average cure rate is only 50 percent when adopting the bearable dosage of normal tissue。 Ovarian cancer and cervical cancer are the most common malignant gynecological tumors, seriously threatening women’ lives. Owing to high frequency of abdominal cavity metastasis and the existence of side complications, the life span, quality and survival of patients have been affected directly, while the radioresistance of these diseases has a bad effect on the radiotherapy. So how to increase the radiosensitivity has attracted significant interest to radiobiological research in recent years. Part OneObjective: To explore the effect and the mechanism of lipofectin-c-erbB2 or c-raf-1 antisense oligodeoxynucleotides on radiosensitivity in human ovarian cancer cell line SKOV3. Methods: There were four groups in the study: normal control group, <WP=14>Lipofectin control group, c-erbB-2 SODN (sense oligodeoxynucleotides) or c-raf-1 SODN experimental group, c-erbB-2 ASODN (antisense oligodeoxynucleotides) or c-raf-1 ASODN experimental group. The expression of c-erbB2 or c-raf-1 was detected by means of RT-PCR; cellular response to irradiation was evaluated by the colony forming test and MTT assay; Apoptosis and cell cycle were observed by flow cytometry, electronic microscope, fluorescent microscope. Results: Lipofectin- c-erbB2 or c-raf-1 ASODN could suppress the expression of c-erbB2 or c-raf-1, leading to different cell cycle arrests. They significantly decreased the proliferation rate and statistically increased the apoptosis rate of human ovarian cancer cells after ionizing irradiation (vs control group, p<0.01), while non-treament and the SODNs groups did not decrease the radio-resistance level of SKOV3 cell line (vs control group, p>0.05). Conclusions: c-erbB2 or c-raf-1 antisense oligodeoxynucleotides sensitized the SKOV3 to ionizing irradiation through decreasing the expression of c-erbB2 or c-raf-1, which might be the result of the fact that antisense oligodeoxynucleotides inhibit the celluar signal transduction pathway relating to the radiation-resistant phenotype, but there was no clear relationship between cell cycle distribution led by c-erbB2 or c-raf-1ASODNs and the radiosensitivity. <WP=15>Part TwoObjective: It was reported that the expressions of Ki-67 and Bcl-2 protein are associated with the resistance of radiotherapy, that is to say, the higher overexpression of Ki-67 or Bcl-2, the more resistant tumor cells might be. In the second part of the study, we investigated the effect of c-erbB-2 or c-raf-1 antisense oligodeoxynucleotides on expression of Ki-67,Bcl-2 protein in irradiated human ovarian cancer cell line SKOV3. Methods: Immunohistochemical method was performed to analysis expression of Ki-67, Bcl-2 protein in pre-or post-irradiated human ovarian cancer cells. There were four groups in the study, normal control group, Lipofectin control group, c-erbB-2 or c-raf-1 SODN experimental group, c-erbB-2 or c-raf-1 ASODN experimental group. Results: In terms of the c-erbB-2 post-irradiation experimental groups, the positive rate of Ki-67 were 42.7% in SODN group (VS control group p>0.05) and 19.8% in ASODN group (VS control group, p<0.01). Among c-raf-1 post-irradiation experimental groups, positive rates were 43.9% in SODN group (VS control group, p>0.05) and 19.8% in ASODN group(VS control group, p>0.05), while among c-erbB-2 experimental groups, positive rates of Bcl-2 were the 39.4% in SODN group (VS control group, p>0.05) and 20.7% in ASODN group(VS control group , p<0.01). Among c-raf-1 <WP=16>experimental groups, positive rates of Bcl-2 were 41.8% in SODN group(VS control group, p>0.05) and 21.7% in ASODN group(VS control group , p>0.05) respectively. Results: c-erbB-2 ASODN or c-raf-1 ASODN significantly inhibited the expression of Ki-67 and Bcl-2 protein in human ovarian cancer cells after ionizing更多还原