【作者】 钱伟峰；

【导师】 夏春林；

【作者基本信息】 苏州大学 ， 人体解剖与组织胚胎学， 2010， 博士

【摘要】 目的(1)探讨大肠癌组织中STAT3表达,并研究STAT3表达与肿瘤血管生成的相关性。(2)观察STAT3基因沉默对大肠癌HT-29细胞的增殖能力及细胞周期变化的影响。(3)观察STAT3基因沉默对大肠癌HT-29细胞裸鼠皮下种植瘤生长的影响。(4)在裸鼠体内观察STAT3信号对肿瘤血管生成的影响,并初步探讨其作用机理。方法(1)取临床大肠癌石蜡标本,免疫组化检测STAT3、CD34的表达情况,通过CD34的表达情况计数MVD,应用统计学方法分析STAT3表达与MVD的相关性。(2)设计合成3对靶向STAT3基因的siRNA序列,通过筛选获得最佳siRNA序列,构建shRNA表达序列并连接到慢病毒载体pRNAT-U6.2/Lenti中,获得pRNAT-shSTAT3重组慢病毒质粒,经酶切和测序鉴定正确后,进行包装产生病毒液,测定其滴度。将包装获得的病毒液感染大肠癌HT-29细胞,筛选扩增获得阳性细胞株HT-29-shSTAT3。同法,用慢病毒空质粒包装后产生的病毒液感染大肠癌HT-29细胞,筛选获得阳性克隆细胞株HT-29-GFP。Real-time PCR和Western blot检测shRNA对STAT3基因的沉默效率,MTT法检测细胞生长情况,流式细胞仪检测细胞周期变化。(3)分别将HT-29、HT-29-GFP、HT-29-shSTAT3细胞注射入裸鼠皮下成瘤,定期测量各组肿瘤体积变化。(4) 30天后处死裸鼠,取各组瘤组织通过免疫组化检测STAT3和CD34的表达情况,并计数MVD。血管生成基因芯片检测肿瘤组织内血管生成和抑制因子的表达情况。结果(1)临床大肠癌组织中STAT3蛋白表达阳性率为63.6%,显著高于正常大肠组织;大肠癌组织中MVD为47.55±12.15,而正常大肠组织中MVD为11.67±1.08,两者相比具有显著差异(P<0.01);大肠癌组织中STAT3的表达和MVD与肿瘤的恶性程度、Duke’s分期及淋巴结转移有关(P<0.05)。相关分析表明STAT3表达与MVD呈显著正相关(r=0.788)。(2)酶切鉴定和测序结果证实STAT3shRNA核苷酸序列插入正确,成功包装后产生病毒悬液的滴度为2×107TU/ml。病毒感染大肠癌HT-29细胞,经G418筛选获得稳定细胞株,Real-time PCR和Western blot分别显示HT-29-shSTAT3细胞STAT3 mRNA的表达和蛋白明显减弱,表达量分别为16.9±2.1%、18.8±2.4%(P<0.01)。MTT结果显示STAT3基因沉默后的大肠癌HT-29细胞生长明显减慢,G0/G1期细胞占68.73±2.88%,S期细胞占22.93±1.10%,与对照组相比差异显著(P<0.01)。(3)各组裸鼠在接种肿瘤细胞后均成瘤,至接种后第15天,HT-29-shSTAT3细胞接种组肿瘤体积明显小于HT-29和HT-29-GFP细胞接种组(P<0.05);至接种后第30天差异更为显著(P<0.01),肿瘤生长抑制率为57.46%。(4)取肿瘤组织行免疫组化检查,见HT-29和HT-29-GFP组肿瘤内有多量的微血管分布,MVD分别为28.73±5.11,27.60±4.27,两者无显著差异(P>0.05);而HT-29-shSTAT3组微血管明显减少,MVD为9.80±3.02,与前两组相比,差异显著(P<0.01)。(5)血管生成基因芯片结果提示在HT-29-shSTAT3裸鼠组大肠癌组织中,表达下调的基因有15条,上调的基因有7条。结论(1)临床大肠癌组织中存在STAT3的高表达,STAT3的表达可能在大肠癌的病程发展及转移过程中发挥重要的作用。大肠癌的发生过程中STAT3信号与肿瘤血管的生成密切相关。(2) STAT3信号通路对于大肠癌HT-29细胞生长起着重要的作用,沉默STAT3基因表达的大肠癌HT-29细胞生长速度明显减慢,细胞阻滞于G0/G1期。(3)抑制STAT3信号可以抑制大肠癌HT-29细胞裸鼠皮下种植瘤的生长。(4)在大肠癌组织中,STAT3信号可以促进肿瘤血管生成。(5)在大肠癌肿瘤血管生成过程中,STAT3信号可能通过调控VEGF-A、MMP-2、ECGF1、EPHB4、IGF1、NRP1、NRP2、STAB1、TNF、VEGFR-1、BAI1的表达来促进新生血管的生成。更多还原

【Abstract】 Objective(1) To explore the expressing of STAT3 in human colorectal carcinoma and study the impact on angiogenesis.(2) To observe the impact of STAT3 gene silencing on HT-29 cell growth and cell cycle distribution.(3) To observe the effect of STAT3 signal on tumor growth of human colorectal carcinoma in nude mice.(4) To investigate the effect of STAT3 signaling pathway on tumor angiogenesis of human colorectal carcinoma in nude mice, and probe the primary mechanism.Methods(1) STAT3 and CD34 expressing in the clinical colorectal carcinomas were studied by the way of immunohistochemistry. MVD were counted according to CD34 expression in tumor tissues. The correlation between the expression of STAT3 and MVD was statistically analyzed.(2) Three different siRNAs of STAT3 were designed and synthesized. After being screened, the most effective siRNA was found. According to this sequence, the short hairpin DNA of STAT3 was constructed. The shRNA duplex was ligated into the recombinant vector pRNAT-U6.2/Lenti. The recombinant vector was conformed by the restriction map and DNA sequencing. The correct recombinant lentiviral vectors were packaged in 293T cells. Viruses in the supernatant were collected and the titer was measured. HT-29 cells transfected with viruses were selected by G418 and transfected cells were gained and harvested. Real-time PCR and Western blot were used to detect the interference effects. Cell growth was assessed by MTT assay and cell cycle distribution was detected by flow cytometry.(3) HT-29, HT-29-GFP and HT-29-shSTAT3 cells were respectively injected subcutaneously into the back of the nude mice of each group. During the tumor growth, the appearance and size of each tumor were examined.(4) On 30th day all mice were killed. Tumor tissues were removed for histopatho- logical and CD34 immunohistochemical analysis and taken to extract total RNA for angiogenesis superarray assay.Results(1) The positive rate of STAT3 expressing in colorectal carcinomas was 63.6%, which was significantly higher than that in the normal group(P<0.01). MVD in colorectal carcinoma tissus was 47.55±12.15, which was significantly higher than that in the normal group(P<0.01). In colorectal carcinoma, the positive rate of STAT3 expression and MVD were closely related to malignancy degree、Duke’s stage and lymph node metastasis (P<0.05), and MVD was closely correlated to the expression of STAT3(r=0.788).(2) The restriction map and DNA sequencing demonstrated that the recombinant lentiviral vector of RNA interference of STAT3 gene was constructed successfully. Virus particles were packaged in 293T cells, and the title of viruses was 2×107 TU/ml. In HT-29 cells transfected with viruses effectively, the expressing level of STAT3 was down-regulated as Real-time PCR and Western blot analyses demonasrated. In transfected group, MTT assay showed the growth of HT-29 cells was suppressed and FCM assay indicated the proportion of cells at G0/G1 and S phase was 68.73±2.88% and 22.93±1.10% respectively, which was great different from that of the control group (P<0.01).(3) A tumor was formed in every nude mouse. On day 15 after inoculating, tumor volums of the HT-29-shSTAT3 group were less than those of HT-29 group and HT-29-GFP group (P<0.05). On day 30 after inoculating, the difference was more obvious (P<0.01), the inhibitory rate of tumor growth was 57.46%.(4) MVD in the HT-29-shSTAT3 group was less than that in the other two groups (P<0.01).(5) The result of angiogenesis superarray indicated that there were 15 down- regulated genes and 7 up-regulated genes in tumor tissues of the HT-29-shSTAT3 mice group.Conclusions(1) STAT3 expressing in colorectal carcinoma is significantly higher. It may play an important role in the development and metastasis of colorectal carcinoma. It is suggested that angiogenesis is closely correlated to STAT3 signaling pathway in colorectal carcinoma.(2) STAT3 singnaling pathway plays an important role in HT-29 cells growth. STAT3 targeting shRNA can silence the STAT3 gene remarkably, induce HT-29 cell growth inhibition and block cell cycle at G0/G1 phase.(3) In nude mice, STAT3 singnal is important to the growth of human colorectal carcinoma.(4) In human colorectal carcinoma, STAT3 signal can promote angiogenesis.(5) In human colorectal carcinoma, STAT3 singnal may participate in regulating angiogenesis through modulating expression of VEGF-A, MMP-2, ECGF1, EPHB4, IGF1, NRP1, NRP2, STAB1, TNF,VEGFR-1 and BAI1.更多还原