【作者】 易小平；

【导师】 李宜雄；

【作者基本信息】 中南大学 ， 外科学， 2010， 博士

【摘要】 目的胰腺癌是恶性程度最高的人类肿瘤之一,根治性手术仍然是其唯一可能治愈的手段。然而,胰腺癌手术切除率低,仅有<15%的患者可能手术切除。即使如此,根治性切除(R0)的患者术后5年生存率仅为15-20%。手术切除率低和术后高复发的特点使辅助治疗对胰腺癌的治疗具有重要的价值。放射治疗是胰腺癌治疗的重要辅助治疗方法,单独或配合化疗有可能提高切除率及改善预后,然而,胰腺癌对放、化疗高度抵抗。因此,寻找一种科学可行的途径来增强放、化疗疗效,改善预后成为了胰腺癌治疗领域研究的热点。XIAP和Survivin是IAPs家族中凋亡抑制作用最强的两种因子,参与了包括胰腺癌在内的多种恶性肿瘤的增殖和转化,并与其放、化疗抵抗密切相关。本研究的主要内容包括：①探讨胰腺癌细胞系中XIAP和Survivin的表达与放疗抵抗之间的关系。②探讨运用慢病毒载体介导RNAi稳定抑制胰腺癌细胞系中XIAP和／或Survivin表达的可行性及对胰腺癌放疗敏感性的影响。方法①体外培养人胰腺癌细胞系Panc-1、Sw1990、Bxpc-3和Miapaca-2，克隆形成实验检测放疗敏感性；实时定量RT-PCR和Western Blot检测亚致死剂量(1Gy)高能X线体外照射处理前后XIAP和Survivin的表达变化；对亚致死剂量X线处理后的胰腺癌细胞和原始胰腺癌细胞行致死剂量X线照射后,检测Caspase-3/7活性和细胞死亡率变化。②针对XIAP和Survivin,分别构建shRNA慢病毒载体,包装病毒后,感染胰腺癌细胞株Swl990,检测对XIAP和Survivin的表达抑制效率；筛选稳定克隆并扩大培养,检测稳定抑制XIAP和Survivin的效率及XIAP或Survivin稳定抑制后对细胞增殖、凋亡及放疗敏感性的影响。同时选择抑制效率最好的稳定克隆,应用直接细胞悬液接种法建立胰腺癌细胞裸鼠皮下成瘤模型,比较XIAP或Survivin稳定抑制后细胞的成瘤能力。③以XIAP-shRNA和Survivin-shRNA慢病毒同时感染胰腺癌细胞株Sw1990,筛选稳定克隆,检测XIAP和Survivin同时稳定抑制的效率及XIAP和Survivin同时稳定抑制后对细胞增殖、凋亡及放疗敏感性的影响,并与单独抑制XIAP和Survivin的策略进行对比。结果①4种胰腺癌细胞株中均有XIAP和Survivin表达,且表达水平最高的Panc-1放疗抵抗性最高,表达居中的Sw1990放疗中度抵抗,而表达最低的Miapaca-2对放疗最敏感。亚致死剂量X线可诱导4种胰腺癌细胞株中XIAP和Survivin表达升高,且能显著降低致死剂量X线照射诱导的Caspase-3/7活性升高和细胞死亡率(Panc-1、Sw1990、Miapaca-2)。②我们分别针对XIAP和Survivin,分别成功构建3对shRNA慢病毒载体：Lv-shRNA-XIAP-1(LV-X1)、Lv-shRNA-XIAP-2(LV-X2)、Lv-shRNA-XIAP-3(LV-X3)和Lv-shRNA-Survivin-1(LV-S1)、Lv-shRNA-Survivin-2(LV-S2)、Lv-shRNA-Survivin-3(LV-S3),并包装出较高滴度的慢病毒。感染结果显示,慢病毒载体能高效、稳定的感染Sw1990细胞。瞬转的结果表明：LV-X1、Lv-X2、Lv-X3对Sw1990细胞的XIAP mRNA抑制率均达70%以上蛋白抑制率大于95%(均P<0.05)；LV-S1对Sw1990细胞的Survivin mRNA抑制率(73.5±1.85)%,蛋白抑制率(87.64±5.57)%(均P<0.05),LV-S2、LV-S3无显著抑制效果。③我们成功筛选出XIAP表达稳定抑制的胰腺癌细胞株Sw1990-X1、X2、X3, XIAP的mRNA抑制率分别为(81.79±6.59)%、(71.89±3.14)%、(73.67±0.36)%,蛋白抑制率分别为(98.54±0.82)%、(97.33±0.49)%和(97.62±0.40)%(均P<0.05)。MTT检测显示Xl、X3细胞增殖显著受到抑制,且X1、X3放疗后的Caspase-3/7活性、细胞死亡率及细胞凋亡率(流式细胞仪)均显著增加。各组细胞克隆形成率随X线照射剂量(0、2、3、4、5Gy)升高而下降；同一剂量X线照射下X1的克隆形成率均显著低于对照组,细胞存活曲线显示：X1平均致死剂量下降至1.32Gy(多靶单击模型),放射增敏比(D0比)为1.65,X1的放疗敏感性显著增加。我们成功建立3组胰腺癌皮下成瘤模型(Sw1990、Sw1990-Xnc、Sw1990-X1), X1组成瘤较晚,且肿瘤生长缓慢,除第1个观察点外,肿瘤体积在每个观察点都明显小于Xnc组和Sw1990组；观察结束时,X1瘤重小于Xnc组和Swl990组,肿瘤生长抑制率44.14%。④我们成功筛选出Survivin表达稳定抑制的胰腺癌细胞株Sw1990-S1,Survivin mRNA表达抑制率为(76.51±0.99)%,蛋白表达抑制率为(49.32±2.20)%,放疗后的Caspase-3/7活性、细胞死亡率及细胞凋亡率(流式细胞仪)均显著增加。各组细胞克隆形成率随X线照射剂量(O、2、3、4、5Gy)升高而下降；同一剂量X线照射下S1的克隆形成率均显著低于对照组,细胞存活曲线显示：平均致死剂量下降至1.11Gy(多靶单击模型),放射增敏比(D0比)1.82,S1放疗敏感性显著增加。我们成功建立3组胰腺癌皮下成瘤模型(Sw1990、Sw1990-Snc、Sw1990-S1), S1组成瘤时间显著延长,且肿瘤生长缓慢,肿瘤体积在每个观察点都明显小于Snc组和Sw1990组；观察结束时,S1瘤重显著小于Xnc组和Sw1990组,肿瘤生长抑制率达78.95%。⑤我们对Sw1990细胞同时共感染Lv-X1和Lv-S1,并成功筛选出XIAP和Survivin同时稳定抑制的稳定感染株X1S1。XIAP和Survivin mRNA表达抑制率分别为(56.53±3.38)%和(56.19±3.31)%,蛋白表达抑制率分别为(56.26±5.18)%和(30.72±8.16)%(均P<0.05)。与对照相比,X1S1的生长曲线显示其生长显著受到抑制。放射治疗结果表明,X1S1放疗后的Caspase-3/7活性、细胞死亡率均显著增加；与X1和S1相比,Caspase-3/7活性显著增加,但细胞死亡率无显著差别。细胞克隆形成率随X线照射剂量(O、2、3、4、5Gy)升高而下降；同一剂量X线照射下X1S1的克隆形成率均显著低于对照组,且小剂量X线(2Gy和3Gy)照射后的克隆形成率显著低于S1和X1。细胞存活曲线显示：平均致死剂量下降至0.90Gy(多靶单击模型),放射增敏比(D0比)2.26,x1S1放疗敏感性显著增加。X1S1的放疗敏感性高于于X1和S1。结论①XIAP和Survivin在胰腺癌细胞系Panc-1、Sw1990、Bxpc-3和Miapaca-2中均有表达,表达高低与放疗敏感性有关,是胰腺癌细胞系可诱导的、组成性的放疗抵抗因子。②稳定抑制XIAP或Survivin表达,能显著抑制Sw1990的细胞增殖和长期存活,减弱成瘤能力,提高放疗敏感性。③同时稳定抑制XIAP和Survivin表达,与单独稳定抑制XIAP或Survivin表达相比,能更有效增加Sw1990的放疗敏感性。更多还原

【Abstract】 Objective:Pancreatic cancer is one of the most aggressive human tumors,with surgical resection is the only curative treatment, but less than 15% of patients are candidates for potentially curative resection. Even among those patients with curative resection(R0), survival rates after surgical resection remain poor, with less than 20% survive 5 years, the high incidence of tumor recurrence both locally and at distant sites,and the low resection rate make the adjuvant therapy a rational treatment.Over the years, radiotherapy receives more attention, thus in conjunction to surgery or chemotherapy, radiotherapy may achieve better clinical results. However, pancreatic cancer is striking resistance to radiotherapy and chemotherapy. So,it has become a hotspot in the field of pancreatic cancer therapy to seek a scientific and applicable way to enhance the curative effect and improve the prognosis of pancreatic cancer. XIAP and Survivin,both as the most potent one of inhibitor of apoptosis (IAP)families,up-regulated in many cacer tissues and cells, including pancreatic cancer,is thought as an important factor comtributed to carcinoma proliferation transformation and radioresistance.The main object of our study include①To evaluate the expression of XIAP and S(?)(?)vivin in pancreatic carcinoma cell lines and further investigate the relationship between their expression and the radioresistance.②To explore the possibility of XIAP or Survivin (and combined inhibition of XIAP and survivin) inhibited by Lentiviral vector-mediad RNA interference technique, and to investigate the sensitivity to radiation changed of pancreatic carcinoma cells Sw1990 after XIAP and/or survivin being inhibited.Methods:①Expression of XIAP and Survivin in 4 pancreatic carcinoma cell lines (Panc-1、Sw1990、Bxpc-3 and Miapaca-2) before or after exposure to sublethal dose (1 Gy) of X-irradiation was evaluated by quantitative real-time RT-PCR and Western-blot;Cell survival was determined using a clonogenic assay to evaluate radiosensitivity of cells; After being exposured to a lethal dose of X-irradiation,Enzymatic activity of Caspase-3/7 and percent cytotoxicity determined by trypan blue staining were measured in 3 pancreatic carcinoma cell lines(Panc-1、sw1990 and Miapaca-2) which pretreated with sublethal dose of X-irradiation or not.②The Lentiviral vector of SiRNA targeted against XIAP(LV-shRNA-XIAP-1, LV-shRNA-XIAP-2,LV-shRNA-XIAP-3) and Survivin (Lv-shRNA-Survivin-1、Lv-shRNA-Survivin-2、Lv-shRNA-Survivin-3) were constructed, then virus was packaged for transfecting Sw1990 cells and the inhibition rate of XIAP and Survivin was measured respectively; stable transfective clones was selected by puromycin (for XIAP-shRNA) or G418(for Survivin-shRNA),after then, in Sw1990 cell lines in which XIAP or survivin expression was stably inhibited,the influence on the cell proliferation, apoptosis and radiosensitivity was measured. Inoculated Sw1990、Sw1990-Xnc、Sw1990-X1 and Sw1990-Snc、Sw1990-S1 respectively in flank subcutaneous tissue of nude mice to establish xenograft models of human pancreatic carcinoma,observed the tumor growth status and compared the tumorigenesis ability of these cells.③Sw1990 was transfected by XIAP-and Survivin-shRNA Lentivirus simultaneously, after selected by puromycin and G418, the inhibition rate of XIAP and Survivin was measured by quantitative real-time RT-PCR and Western-blot. After then, in Sw1990 cell lines in which XIAP and Survivin expression was stably combined inhibited,the influence on the cell proliferation, apoptosis and radiosensitivity was evaluated and compared with the result of single gene silence strategy used in Sw1990 targeted against XIAP or survivin.Results:①XIAP and Survivin were simultaneously existed in 4 pancreatic carcinoma cell lines (Panc-1,Sw1990,Bxpc-3 and Miapaca-2), and we found an inverse relationship between XIAP and Survivin mRNA expression and radiosensitivity. PANC-1 cells, which had the highest XIAP and Survivin mRNA levels, was most resistant to X-irradiation; Sw1990 cells, which had the moderate XIAP and Survivin mRNA levels,had the modest sencitivity to X-irradiation;MIAPaCa-2 cells, which showed the least XIAP and Survivin mRNA expression, showed the most sensitivity to X-irradiation. The mRNA and protein expression of XIAP and Survivin increased significantly in all four cell lines (Panc-1,Sw1990,Bxpc-3 and Miapaca-2) by treatment with a sublethal dose of X-irradiation. Three cells (Panc-1,Sw 1990 and Miapaca-2),were subjected to sublethal doses of X-irradiation followed by a lethal dose, result showed that the total cytotoxicity was decreased significantly and the caspase-3/7 activivty was significantly suppressed.②Three lentiviral vector-Xiap-shRNA (Lv-X1,Lv-X2,Lv-X3)and three lentiviral vector-Survivin-shRNA(Lv-S1,Lv-S2,Lv-S3) were constructed success-fully, and relative high-titer lentivirus were gain. The results of transfection showed that lentivirus can transfect Sw1990 cells efficiently and stablely. The results of instantaneous transfection show that Lv-X1,Lv-X2,Lv-X-3 all could decrease the XIAP mRNA expression level more than 70%, while the protein expression level was decreased more than 95%(P<0.05);Lv-S1 could effectively decrease the Survivin mRNA and protein expression level to (73.5±1.85)% and (87.64±5.57) % respectively(P<0.05), while Lv-S2 and Lv-S3 couldn’t inhibit Survivin in Sw1990 cells significantly(P>0.05).③Three pancreatic cancer cell line in which XIAP expression is stably suppressed was successfully set up(Sw1990-X1,X2,X3), the inhibition rate of XIAP mRNA was (81.79±6.59)%, (71.89±3.14)% and (73.67±0.36)% respectively, while the inhibition rate of XIAP protein was (98.54±0.82)%,(97.33±0.49)% and (97.62±0.40)% respectively(P<0.05)。 MTT showed that the cell number of X1 and X3 decreased significantly, and the radiation-induced Caspase-3/7 activity, total cytotoxicity and apoptotic index measurd by flow cytometry were increased significantly. With the rise of radiation dose, cell clone formation rate declined in all groups of cells and at the same dose of radiation, clone formation rate of X1 declined notably; the cell survival curve also showed a significant decrease of X1-Do and X1-SF2,were 1.32 and 21.4% respectively, and the radiation enhancement ratio of X1 was 1.65(a ratio of D0)(Multi-target click model).These data imply that the radiosensitivity of X1 was increased significantly.3 groups of xenograft models of human pancreatic carcinoma were established(Sw1990, Sw1990-Xnc, Sw1990-X1). compared with Xnc and Sw1990 group,the tumorigenesis time of X1 group delayed,tumor grew slowly,the tumor volume of X1 group was obviously less than that of Xnc and Sw1990 group at every checkpoint except the first one.At the terminal of observation,the tumor weight of X1 group was lower than that of Xnc group and Sw1990 group.The tumor growth inhibitive rate of X1 group is 44.14%.④A pancreatic cancer cell line in which survivin expression is stably suppressed was successfully set up(Sw1990-S1), the inhibition rate of Survivin mRNA and protein was (76.51±0.99)% and(49.32±2.20)% respectively, and the radiation-induced Caspase-3/7 activity, total cytotoxicity and apoptotic index measurd by flow cytometry were increased significantly(P<0.05). With the rise of radiation dose, cell clone formation rate declined in all groups of cells and at the same dose of radiation, clone formation rate of X1 declined notably; the cell survivalcurve also showed asignificant decrease of S1-D0 and S1-SF2,were 1.11 and 16.35% respectively, and the radiation enhancement ratios of S1 was 1.82(a ratio of D0)(Multi-target click model).These data imply that the radiosensitivity of S1 was increased significantl.3 groups of xenograft models of human pancreatic carcinoma Sw1990,Sw1990-Snc,Sw1990-S1) were established successfully. Compared with Snc and Sw1990 group,the tumorigenesis time of S1 group delayed dramaticly, tumor grew slowly, the tumor volume of S1 group was obviously less than that of Snc and Sw1990 group at every checkpoint.At the terminal of observation,the tumor weight of S1 group was significantly lower than that of Snc group and Sw1990 group.The tumor growth inhibitive rate of S1 group is 78.95%.⑤Sw1990 was transfected by XIAP-and Survivin-shRNA Lentivirus simultaneously, after selected by puromycin and G418, a pancreatic cancer cell line in which XIAP and Survivin expression is stably suppressed was successfully set up(Sw1990-X1S1), the inhibition rate of XIAP-and survivin mRNA was (56.53±3.38)% and (56.19±3.31)% respectively, while the data was(56.26±5.18)% and(30.72±8.16)% in protein level. AS expected, the radiation-induced Caspase-3/7 activity and total cytotoxicity were both increased significantly in X1S1 cells. When compared with X1 cells and S1 cells, the radiation-induced Caspase-3/7 activity increased significantly in X1S1 cells, but there is no significant difference on total cytotoxicity after radiation among the three cell linces. With the rise of radiation dose, cell clone formation rate declined in all groups of cells and at the same dose of radiation, clone formation rate of X1S1 declined notably, besides, when exposure to low-dose X-irradiation (2 and 3 Gy), the clonogenic survival of X1S1 cells was significantly less than S1 cells and X1 cells; the cell survival curve also showed a significant decrease of X1S1-Do and X1S1-SF2,were 0.90 and 10.64% respectively, and the radiation enhancement ratios of X1 was 2.26 (a ratio of D0)(Multi-target click model).These data imply that the radiosensitivity of X1S1 was increased significantly, and the radiosensitivity of X1S1 is better than that of X1 and S1.Conclusion:①XIAP and Survivin were simultaneously existed in 4 pancreatic carcinoma cell lines (Panc-1,Sw1990,Bxpc-3 and Miapaca-2),and there was an inverse relationship between XIAP and Survivin mRNA expression and radiosensitivity. XIAP and Survivin both act as a constitutive and inducible radioresistance factor in pancreatic cancer cells.②Stablely inhibit XIAP or Survivin expression in Sw1990 cell lines could significant inhibit its cell proliferation and long-term survival, weaken the ability of turmorigenesis and enhance the rediosensitivity significantly.③Combined Stable-inhibition of XIAP and Survivin expression could enhance rediosensitivity of Sw1990 more effective than single stable-inhibition of XIAP or survivin expression in pancreatic carcinoma cell lines Sw1990.更多还原