【作者】 陈俭云；

【导师】 李宜雄；

【作者基本信息】 中南大学 ， 普通外科学， 2009， 博士

【摘要】 [目的]胰腺癌对绝大部分化疗药物耐药。存活素（survivin）基因是凋亡抑制蛋白家族成员中抑制凋亡作用最强的一种,在大部分恶性肿瘤组织和细胞中高表达,是肿瘤增殖和化疗耐药的重要因子。本研究的主要目的有:（1）探讨survivin在胰腺癌组织中的表达及其与临床病理的关系。（2）观察胰腺癌细胞株PANC-1中survivin的表达及其在化疗药物吉西他滨作用后的变化。（3）运用RNAi载体抑制survivin的表达对诱导PANC-1细胞凋亡的影响。（4） RNAi抑制survivin的表达对增加化疗药物吉西他滨化疗敏感性的影响。为探索胰腺癌新的治疗方法奠定实验基础。[方法]（1）运用免疫组化法检测35例胰腺癌手术切除标本和10例癌旁胰腺组织,分析survivin的表达和临床病理特征的关系。（2）培养胰腺癌细胞PANC-1,加入吉西他滨,运用RT-PCR和Western blot检测survivin的表达。（3）构建以U6为启动子的RNAi载体并转染PANC-1,运用RT-PCR和Western blot检测survivin的表达,FCM检测凋亡指数和Hoechest 33258检测凋亡形态。（4） MTT、FCM和Hoechest 33258检测PANC-1对化疗药物吉西他滨的敏感性。[结果]（1）免疫组化显示胰腺癌组织中有survivin的阳性表达,而癌旁组织中无survivin的表达。胰腺癌组织中其表达的阳性率（26/35,74.29%）明显高于癌旁组织（0/10,0%）,差异有统计学意义（P＜0.05）。survivin在胰腺癌组织中的阳性率与肿瘤的临床分期和病理分级有关;两病理因素分组间差异有统计学意义（P＜0.05）,而与肿瘤的远处转移无明显关系;远处转移分组间差异无统计学意义（P＞0.05）。（2）用1μg/ml及10μg/ml浓度的吉西他滨作用胰腺癌细胞株24h和48h,survivin mRNA水平分别上升了（1.34±0.12）倍、（2.40±0.17）倍和（3.33±0.20）倍、（4.41±0.18）倍,蛋白水平分别上升了（1.20±0.07）倍、（1.48±0.19）倍和（2.90±0.04）倍、（4.50±0.20）倍,差异有统计学意义（p＜0.05）。（3）转染survivin的RNAi载体后24h、48h,survivin mRNA和蛋白的瞬时抑制率分别为52.67%±3.51%、75.33%±3.06%和58.00%±3.61%、76.67%±4.73%;流式细胞仪检测的凋亡指数分别为9.33%±1.53%和14.57%±1.50%;Hoechst33258染色发现,PANC-1细胞出现核皱缩、浓染和碎裂等典型的凋亡形态。（4） Survivin抑制后,PANC-1对吉西他滨的敏感性增加,流式细胞仪检测转染后48h凋亡指数由15.67%±1.16%增加到35.67%±1.53%,差异有统计学意义（p＜0.05）,吉西他滨的IC₅₀由1.24±0.11μg/ml下降到0.39±0.07μg/ml,差异有统计学意义（p＜0.05）。[结论]（1）Survivin在胰腺癌组织中的阳性率高于癌旁组织,其与病理分级和临床分期的关系表明survivin可能在胰腺癌的增殖和预后中起重要作用。（2）胰腺癌细胞株的化疗耐药可能通过吉西他滨诱导survivin的表达上调而增强。（3）以U6为启动子的survivin的RNAi载体,能有效地抑制胰腺癌细胞株PANC-1中survivin的表达,并且诱导细胞凋亡。（4）胰腺癌细胞株PANC-1对吉西他滨的化疗敏感性能够通过运用RNAi技术抑制survivin的表达而增强。更多还原

【Abstract】 [Objective]Pancreatic carcinoma is resist to most of all chemotherapeutical drugs.Survivin,as the most potent one of inhibitor of apoptosis（IAP） families,up-regulating in many cancer tissues and cells, is thought an important factor contributed to carcinoma proliferation and chemoresistance.The main aims of this study are as follows.（1） To discuss the expression of survivin in the pancreatic carcinoma tissue and the relationship between it’s expression and the clinicopathologic parameters.（2） To observe the change of the expression of survivin in the pancreatic carcinoma cell line PANC-1 induced by chemotherapeutical drug gemcitabine.（3） To investigate survivin inhibited by RNA interference（RNAi） vectors,and induce apoptosis.（4） To investigate the sensitivity to chemotherapeutical drug gemcitabine enhanced after survivin inhibited by RNAi.Lay an experimental foundation for further exploration of new therapy of pancreatic carcinoma.[Method]（1） 35 samples of resected pancreatic carcinoma tissues and 10 normal tissues from the carcinoma besides examined by immunohistochemistry and the relationship of survivin expression to the clinicopathologic parameters analyzed.（2） The pancreatic carcinoma cell line PANC-1 cultured with the gemcitabine,and survivin expression examined by RT-PCR and Western blot.（3） RNA interference vectors with U6 promoter against survivin constructed and transfected into the pancreatic carcinoma cell line PANC-1,the expression of survivin examined by RT-PCR and Western blot,FCM employed to examine the apoptosis index of PANC-1 and apoptotic morphology was examined by Hoechst 33258.（4） The sensitivity of PANC-1 to chemotherapeutical drug gemcitabine further examined by MTT,FCM and Hoechst 33258.[Result]（1） Survivin expressed in pancreatic carcinoma tissues, and none of carcinoma besides tissues;the survivin expression rate of carcinoma tissues（26/35,74.29%） higher than the carcinoma besides normal tissues（0/10,0%）,and the difference between them was statistically significant（P＜0.05）.The rate was related to the pathological grade and the clinical stage（P＜0.05）,not related to the metastasis （P＞0.05）.（2） 24 and 48 hours after cultured by gemcitabine with the concentration of 1μg/ml and 10μg/ml,survivin mRNA level was increased（1.34±0.12）、（2.40±0.17）、（3.33±0.20） and（4.41±0.18） folds,and the protein expression was enhanced（1.20±0.07）、（1.48±0.19）、（2.90±0.04） and（4.50±0.20） folds respectively,and the difference between them was statistically significant（P＜0.05）.（3） RNAi vectors against survivin could inhibite the survivin mRNA and protein expression level of 52.67%±3.51%、75.33%±3.06%and 58.00%±3.61%、76.67%±4.73%at 24h and 48h instantaneous effectively.The apoptosis index examined by FCM is 9.33%±1.53%and 14.57%±1.50%. The typical apoptotic morphology of condensed,shrinked and reptured nucleoli of PANC-1 found by Hoechst33258 stained.（4） The sensitivity of PANC-1 to gemcitabine increased after survivin was inhibited,the apoptosis index examined by FCM apparent increased from 15.67%±1.16%to 35.67%±1.53%by the action of gemcitabine（P＜0.05）, with the IC₅₀ to gemcitabine decreased greatly from 1.24±0.11μg/ml to 0.39±0.07μg/ml,and the difference between them was statistically significant（P＜0.05）.[Conclusion]（1） The positive rate of survivin higher than the carcinoma besides normal tissues,and which related to the differentiation and the clinical stage of pancreatic carcinoma indicate survivin is thought of an important factor contributed to carcinoma proliferation and prognosis.（2） The capacity of pancreatic carcinoma cell line chemoresistance could be enhanced by gemcitabine cultured with the survivin expression up-regulated.（3） The RNAi vectors with U6 promoter could effective suppress the expression of survivin in pancreatic carcionoma cell line PANC-1,and induce the PANC-1 apoptosis.（4） The chemosensitivity of gemcitabine in pancreatic carcinoma cell line PANC-1 could be enhanced by suppress the expression of survivin through RNAi technology.更多还原