【作者】 王茂生；

【导师】 崔平； 贾伟； 李继梅；

【作者基本信息】 昆明医学院 ， 外科学， 2010， 硕士

【摘要】 目的：通过把X连锁凋亡抑制蛋白（X-chromosome-linked inhibitor of apoptosis,XIAP）基因的反义寡核苷酸（Antisense Oligodeoxynucleotide,ASODN）转染入人胆管癌QBC939裸鼠移植瘤细胞中,探讨ASODN对人胆管癌裸鼠移植瘤细胞中XIAP基因的抑制作用及对癌细胞增殖及凋亡的影响；观察反义寡核苷酸抑制XIAP基因表达后胆管癌细胞对化疗药物氟尿嘧啶（5-FU）敏感性的影响。方法：将处于对数期的QBC939细胞种植于裸鼠左前肢腋下,每只0.2m1（4×107）.待模型建立（5mm×5mm）以后随机分为5组：Control组、Lip组、5-FU组、ASODN/Lip组和ASODN/Lip-5FU组分别给予瘤周或瘤体直接注射细胞转染液（无血清无抗生素）600u1、浓度为10g/ml的Lip100μ1加细胞转染液至600u1、细胞转染液600u1、终浓度为1μM的ASODN和浓度为10μg/ml的Lip100μ1加转染液至600μ1与终浓度为1μM的ASODN和浓度为10μg/ml的Lip100μ1加转染液至600μ1进行转染,以后每周1次,共4次。转染后的第1、3、5天开始给予相关组腹腔注射5-FU10mg/Kg,其他组腹腔注射等量的生理盐水。观察肿瘤体积的变化及裸鼠体重变化,计算抑瘤率。4周后处死裸鼠,取肿瘤组织行RT-PCR检测XIAP基因mRNA表达情况,取肿瘤组织制成的细胞悬液行流式细胞术（FCM）检测细胞周期及凋亡情况,其余肿瘤组织行病理确认、免疫组织化学检测XIAP、PCNA蛋白表达及TUNEL原位凋亡检测,并计算细胞增殖指数及凋亡指数。结果：成功的建立了人胆管癌裸鼠皮下移植瘤模型。采用阳离子脂质体转染技术成功将ASODN转染入裸鼠体内QBC939细胞中。大体标本显示ASODN-Lip组及ASODN/Lip-5FU组的肿瘤生长受到抑制,体积较小,抑瘤率达54.55%和65.00%明显高于其他三组,差异有显著性（P<0.05）；而Control组、Lip组和5-FU组肿瘤体积逐渐增加；各组瘤体组织重量亦有显著差异(（P<0.05）,ASODN/Lip-5FU组及ASODN/Lip组重量抑瘤率分别为60.60%、39.02%,差异有显著性（P<0.05）；各组裸鼠体重变化无统计学意义。RT-PCR结果示ASODN/Lip组及ASODN/Lip-5FU组肿瘤的XIAP mRNA表达较其他组明显降低,差异有统计学意义（P<0.05）。流式细胞术表明ASODN/Lip组和ASODN/Lip-5FU组瘤体细胞均出现了代表凋亡的亚G₁峰（15.11±2.25）%、（60.36±2.17）%,且Go/G1期细胞均高于对照组,差异有统计学意义（P<0.05）。免疫组化结果示转染ASODN后ASODN/Lip-5FU组与ASODN/Lip组XIAP蛋白阳性表达率为（27.83±4.97）%、（31.62±6.85）%明显低于对照组（62.54±8.32）%,差异有统计学意义（P<0.05）。PCNA免疫组化显示联合化疗组增殖指数明显低于对照组,差异有统计学意义（P<0.05）。TUNEL结果示ASODN/Lip-5FU组、ASODN/Lip组与5-FU组凋亡指数分别为（18.93±0.21）%、（10.19±0.24）%、（10.22±0.18）%,与对照组比较差异有统计学意义（P<0.05）,ASODN/Lip-5FU组与5-FU组间也有统计学意义（P<0.05）。结论：转染ASODN能有效下调人胆管癌QBC939裸鼠移植瘤细胞XIAP基因表达,通过ASODN干扰XIAP基因关键区域能有效抑制胆管癌细胞的增殖并诱导其凋亡,从而抑制肿瘤瘤体生长；同时抑制XIAP基因的表达能显著增强胆管癌细胞对化疗药物5-FU的敏感性,可能为胆管癌基因治疗提供新途径。更多还原

【Abstract】 Objective:Through transfected antisense oligonucleotide of x linked inhibitor of apoptosis protein into transplanted human cholangiocarcinoma QBC939 cells in nude mice by cationic liposomes, to explore the effect of ASODN on human cholangiocarcinoma cells inhibition of XIAP gene in nude mice, proliferation and induced apoptosis of cancer cells;To observate the sensitivity of cholangiocarcinoma cells to chemotherapeutical drugs fluorouracil（5-FU） after the expression of antisense oligonucleotides inhibit XIAP gene.Methods:planted the cells of the proliferative logarithmic phase of QBC939 into the area of left forlimb armpit of nude mice,each 0.2m1（4 X 10⁷）. after 10 days constructed tumor model（5mm X 5mm）,the mice were divided into 5 groups randomly:control group、liposomes （lip） group、5-FU group、ASODN/lip group and ASODN/lip5-FU group. Each group were injected transfer dye liquid 600μl （serum without antibiotics）、Lip 10μg/ml 100μl add transfer liquid to 600μl、transfer dye liquid 600μl、ASODN/Lip final concentration 1μM+Lipfectin 10μg/ml 100μl add transfer liquid to 600μl and ASODN/Lip final concentration 1μM+Lipfectin lOμg/ml 100μl add transfer liquid to 600μl into the tumor or around the tumor, respectively.4 times injections were given,one time per week. Each related group injected 5-FU 1Omg/Kg on the first、third、fifth day after transferred,the other group were injected normal saline. mice were killed after 4 weeks.The expression of XIAP mRNA was determined by RT-PCR; Apoptosis and cells cycle changes were determined by flow cytometry.Immunohistochemical SP staining method were used to detect the expression of XIAP and PCNA protein, TUNEL in situ apoptosis. Cell proliferation index and apoptosis index.were calculated.Results:The subcutaneous tumor model in nude mice was successfully established. Using cationic liposome transfected ASODN into nude mice successfully. Gross specimen shows the tumor volume of ASODN/Lip and ASODN/Lip-5FU decreased during treatment, the rate of tumor suppression reached 54.55% and 65.00%,which were significantly increased in the group of ASODN/Lip and ASODN/Lip-5FU compared with other 3 groups（p< 0.05）;while the tumor volume of the other three groups increase.The weight of tumor were significantly different in each group（p< 0.05）. The inhibition rates of tumor weight were 60.60%、39.02% in the group of ASODN/Lip-5FU and ASODN/Lip respectively, the different was significant（p< 0.05）.There are no statistical difference among the groups in body weight changes of 5 groups mice. RT-PCR results show that the expression of XIAP mRNA decreased significangtly in ASODN/Lip and ASODN/Lip-5FU than other groups（P<0.05）. Flow cytometry showed that ASODN/Lip group and ASODN/Lip-5FU group QBC939 cells were found to represent the sub-G₁ apoptotic peak （15.11±2.25）%, （60.36±2.17）%, and the Go/G₁ phase cells was higher than control group, the difference was significant （P<0.05）. Immunohistochemical results showed after transfection ASODN, ASODN/Lip-5FU group and ASODN/Lip group XIAP protein expression was （27.83±4.97）%、（31.62±6.85）%, significantly lower than the control group （62.54±8.32）%.The difference was statistically significant （P <0.05）. PCNA immunohistochemistry showed the proliferation index of the transfection ASODN and combined 5-FU group was significantly lower than the control group, the difference was statistically significant （P<0.05）. TUNEL results showed that the apoptosis index of ASODN/Lip-5FU group, ASODN/Lip group and 5-FU group were （18.93±0.21）%、（10.19±0.24）%、（10.22±0.18）%, compared with the control group the differences were statistically significant （P <0.05）,ASODN/Lip-5FU group and 5-FU group were statistically. significant（P <0.05）.Conlusion:The liposomal transfection of ASODN can down-regulate the expression of XIAP gene,Which can inhibit cells growth and induce apoptosis of transplanted cholangiocarcinoma QBC939 cells in nude mice, and increase the chemotherapeutic sensitivity of transplanted cholangiocarcinoma cells of QBC939 after XIAP inhibition. So it may be the new target for gene therapy in cholangiocarcinoma.更多还原