【作者】 孙莹；

【导师】 赵雪俭；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 目的:观察siRNA-Stat3真核重组质粒对胃癌生长抑制及诱导细胞凋亡的作用并探讨其机制。方法:以基因重组技术构建siRNA-Stat3真核重组质粒;应用真核细胞转染技术转染SGC-7901细胞株进行体外研究;建立胃癌裸鼠移植瘤进行体内研究;采用RT-PCR、Western blot、MTT、流式细胞仪、免疫组化染色、TUNEL法等技术观察其抑瘤作用及细胞凋亡。结果:(1)Stat3及相关因子在胃癌中高表达;(2)体外实验证实,构建的siRNA-Stat3能特异性沉默SGC-7901细胞系中Stat3及相关因子表达,抑制肿瘤细胞生长,诱导凋亡;(3)体内实验证实,构建的siRNA-Stat3能特异性沉默胃癌裸鼠移植瘤中Stat3表达,抑制肿瘤组织生长,促进凋亡。结论:成功构建的siRNA-Stat3重组质粒可以抑制胃癌中Stat3基因转录和蛋白表达,并诱导细胞凋亡。采用siRNA-Stat3对胃癌移植瘤进行研究国内外均未见报道。更多还原

【Abstract】 Gastric cancer is the fourth most common cancer worldwide with approximately 930 000 newly diagnosed cases annually. Early gastric cancer is considered a curable disease, with a more than 90% 5-year survival rate. In cases of advanced gastric cancer, results of treatment and the quality of life are less favorable, and the rate of recurrence and survival are quite different, even among patients with the same stage. The treatment of gastric cancer including operation, radiotherapy, chemotherapy and other complex treatment are not perfect. With the improvement of molecular biology, researchers have been trying to use gene therapy. Lots of achievements obtained in fundamental research, and then would be the clinical practice.Signal transducers and activators of transcription are molecules that forward signals in the cytoplasm. Actived Stat translocates to the nucleus bindind with DNA to regulate signal transduction and transcription. Of the STAT family members, STAT3 is most commonly activated in human cancer. Numerous in vitro studies have shown that STAT3 promotes proliferation and invasion of gastric cancer cells, upregulates expression of potent angiogenic factors, and desensitizes cancer cells to anti-proliferative and apoptotic stimuli. As a transcription factor, STAT3 promotes cancer cell growth by upregulating the transcription of many well characterized genes and suppress expression of Fas reducing activity of apoptotic pathways . The function of Stat3 like a hub in the pathway. It works with cell cycle regulators, apoptosis inhibitors, angiogenesis inducers. STAT3 is actively being considered as a novel molecular target for cancer drug.RNA interference (RNAi) is an important regulating mechanism of gene expression. Specific short interfering double-stranded RNAs serve as the effector molecules for sequence-specific gene silencing. RNAi has no effect to DNA replicating and transduction. The phenomenon exists in mammal cells to silence target gene naturally and can be used as an useful tool to gene knock out. RNAi-mediated gene therapy may against tumor development by reversing malignant phenotype, inducing apoptosis and reducing the drug tolerance of tomor cells.The purpose of the research is to evaluate the inhibition of specific SiRNA-Stat3 to the SGC-7901 cell line and nude mice gastric cancer xenografts.Methods: 1. The expression of Stat3, Survivin and Bcl-2 in gastric cancer were detected by immunohistochemical staining.2. Accroding to the design principles of shRNA, eukaryotic expression vector SiRNA-Stat3 and SiRNA-Scramble were synthesized. Restriction enzyme digestion (BamH I and Hind III) and sequence analysis were used to confirm the sequences of vectors.3. In vitro study: SGC-7901 cells were divided into three groups casually including liposome control, SiRNA-Scramble and SiRNA-Stat3. Semi-quantitative RT-PCR was performed to analyze Stat3 mRNA in gastric cancer cells. Western Blotting was used to analyze the expression of Stat3 protein. MTT was used to detect the inhibition of cell proliferation. The apoptosis was assessed by FACS. Western Blotting was performed to analyze the expression of Stat3-related proteins including Bcl-2, Survivin and Cyclin D1. 4. In vivo study: SGC-7901 cells were injected into subcutaneous of the nude mice to establish xenograft animal models. Mice were divided into three groups casually: mock (n=6), SiRNA-Scramble (n=6) and SiRNA-Stat3 (n=6). Electric transfection was performed to enhance transfect efficiency after plasmid injected. The plasmids were injected again after the first treatment eight days later. The life of nude mice was observed every day. The length and short diameters were measured to calculate the volume of tumor every six day. The curve of tumor growth was draw. we dissect the nude mice and peel off the tumor after 18 days of plasmid injected. Semi-quantitive RT-PCR was used to detect Stat3 mRNA in tumor tissues. Western Blotting was used to analyse the expression of Stat3 protein. The tumor tissues were observed by HE staining. The expression of Stat3 and PCNA were observed by immunohistochemical staining. TUNEL method was used to analyse the apoptosis of gastric cancer tissues. SPSS 13.0 statistic software package was used to analyze the data. For enumeration data, nonparameter test (Mann-Whitney U method) was used to detected the difference between two independent samples. Parameter test was used for measurement data. ANOVA (Bonferroni method) was used to detected the difference between samples.Results: 1. In gastric cancer tissue, positive immunostaining of Stat3, Survivin and Bcl-2 was 74%, 69% 60% respectively. In normal gastric tissue, positive immunostaining of Stat3, Survivin and Bcl-2 was 11%, 0, 16% respectively. Significant difference was found between two groups in all three factors (P<0.01). 2. siRNA sequence and vectors were connected correctly, and it was confirmed by restriction enzyme digestion and sequence analysis. 3. In vitro study: Morphological changes were observed by phase contrast microscope 48h later. The growths of cells were normal in mock and SiRNA-Scramble control groups whereas the quantities of cells decreased and the fragments of cells increased in SiRNA-Stat3 group. Semi-quantitive RT-PCR: No significant difference was found between mock and SiRNA-Scramble control groups (P>0.05). The expression of Stat3 was significantly inhibited at mRNA level in SiRNA-Stat3 group comparing with the control groups (P<0.01). Western Blotting: No significant difference was found between mock and SiRNA-Scramble control groups (P>0.05). The expression of Stat3 was significantly inhibited at protein level (P<0.01) in SiRNA-Stat3 group comparing with the control groups. MTT: The inhibition rate of cell proliferation in mock was set 0. The inhibition rate of SiRNA-Scramble and SiRNA-Stat3 group was 7% and 41% respectively after 48 hours, while 8% and 63% respectively after 72 hours. No significant difference was found between mock and SiRNA-Scramble control groups (P>0.05). The proliferation of tumor cells was inhibited significantly in SiRNA-Stat3 group comparing with the control groups (P<0.01). FACS analysis: Significant cell apoptosis (P<0.01) was demonstrated between SiRNA-Stat3 group and control groups after 72h treatment. Western Blotting for Stat3-related proteins: The expression of Cyclin-D1, Survivin and Bcl-2 was significantly inhibited at protein level in SiRNA-Stat3 group comparing with the control groups (P<0.01). 4. In vivo study: Xenografts were established successfully in every nude mouse. No significant difference of tumor volumes was found among mock, SiRNA-Scramble and SiRNA-Stat3 groups in 6 days after the plasmid injection (P>0.05). The tumor volumes were almost same between mock and SiRNA-Scramble groups in 12 and 18 days respectively after the plasmid injection (P>0.05). But the tumor volumes in SiRNA-Stat3 group were significant lower than two control groups at two time points (P<0.01). According to the curve of tumor growth, mock group and SiRNA-Scramble group were almost same at each time point. The growth of tumor in SiRNA-Stat3 group became quite slowly after plasmid injection. Dissecting the nude mice and peeling off the tumor, we found the weight of tumor were significant lighter in SiRNA-Stat3 group comparing with two control groups (P<0.01). The expression of Stat3 was significantly inhibited at mRNA level comparing with the control groups (P<0.01), and no significant difference was found between two control groups (P>0.05) by semi-quantitive RT-PCR. Comparing with the control groups, the expression of Stat3 was significantly inhibited at protein level by Western Blotting (P<0.01), and no significant difference was found between two control groups (P>0.05). More dead cells were found in SiRNA-Stat3 group by HE staining. The expression of Stat3 and PCNA was inhibited significantly in SiRNA-Stat3 group (P<0.01) comparing with control groups. More apoptosis cells were observed in SiRNA-Stat3 group, and apoptosis index was significant higher in SiRNA-Stat3 group than in mock control and SiRNA-Scramble control groups by TUNEL (P<0.01).In our studies, SiRNA-Stat3 can interrupte the Stat3 gene transcription, down-regulate the Stat3 protein expression and induces the apoptosis in SGC-7901 cell line and gastric cancer tissues.更多还原