【作者】 王海茹；

【导师】 李晓明；

【作者基本信息】 吉林大学 ， 耳鼻咽喉科学， 2009， 博士

【摘要】 探讨携带短发夹RNA的质粒抑制STAT3基因表达联合放射对人喉鳞状细胞癌的作用,为喉癌的基因联合放射治疗提供理论支持。以基因重组技术构建pGPU6/GFP/Neo-shRNA重组质粒;应用真核细胞转染技术转染人喉癌Hep-2细胞进行体外研究;建立裸鼠人喉鳞状细胞癌皮下移植瘤模型,采用瘤周注射将重组质粒导入裸鼠移植瘤内并联合放疗观察抑瘤作用。(1)成功构建pGPU6/GFP/Neo-shRNA重组质粒。(2)体外实验证明pGPU6/GFP/Neo-shRNA重组质粒联合放疗对人喉鳞癌细胞具有增殖抑制及促进凋亡的作用。(3)体内实验证明pGPU6/GFP/Neo-shRNA重组质粒联合放疗具有抑制人喉癌裸鼠皮下移植瘤的生长作用。STAT3基因可作为喉癌基因治疗的有效靶点,应用RNAi技术沉默STAT3基因联合放疗对人喉鳞状细胞癌具有显著的治疗效果。更多还原

【Abstract】 Head and neck squamous cell carcinoma is a common human malignant tumor. Over the past 20 years, the incidence of the cancer has been increasing gradually and it is now the fifth of cancer incidence and the sixth of the cancer-related death causes in the world. According to incomplete statistics, there were about 645,000 new head and neck cancer cases occurred every year in the world. In Europe, new cases were about 100,800 and more than 40,000 cases dead at year 2002. Patients with head and neck carcinomas are treated with surgery or radiation therapy and adjuvant treatments. Radiation therapy effects are reliable and adverse reactions are clear.But simple radiation therapy only has relatively good efficacy to parts of the radiation-sensitive patients.Meanwhile, the side effect and injury to normal tissue caused by radiotherapy can not be ignored.Therefore, it is urgent to find radiation sensitizer to enhance tumor radiosensitivity and decrease injury in normal tissues. Gene therapy offers hope in this regard. Gene therapy involves the introduction of gene DNA into cells to exert effects by vector .Several stragies have developed including: replacement of tumor suppressor gene function, blockage of oncogene function, genetic immunomodulation and so on, which will selectively kill cancer cells with no toxicity to the surrounding non-malignant cells.STAT3 is also known as signal transduction and activator of transcription, and is the focus of EGFR, IL-6/JAK, Src and other tyrosine kinase signal carcinogenicity channel convergents and its abnormal activation can induce key genes downstreams activation. STAT3 transduction pathways downstreams have a variety of target genes including Bcl-xL, Mcl-l, c-Myc, VEGF, p21WAF\CIP1 cyclinD1, p53 and so on,which are related tightly to tumor cell proliferation, differentiation, apoptosis, etc. After blocking STAT3 signaling pathway,the tumor cell proliferation and apoptosis increases. Radiation factors are known to cause cell injury and death,which are related to many factors except with the DNA damage repair, but also with cell cycle regulation, apoptosis and so on. Therefore, in theory, we may improve the tumor radiosensitivity by blocking STAT3 pathway. RNA interference (RNAi) is a sequence-specific post-transcriptional gene silencing, resulting in the corresponding gene deletion phenotype. In this study, we transfected pGPU6/GFP/Neo-shSTAT3 to Hep-2 cell line and the cells in animal models with liposome to research the therapeutic value and possible mechanism of laryngeal carcinoma by blocking STAT3 signal transduction pathway combined with radiation. Therefore, a new method and theoretical basis may be provided about comprehensive treatments of head and neck malignant tumors.Objective: (1) To construct recombinant plasmid expressed short hairpin RNA and study the effects to proliferation and apoptosis of laryngeal carcinoma Hep-2 cells. (2) To explore the radiosensitivity of human laryngeal carcinoma cell line Hep-2 after inhibition of STAT3 gene by transfecting plasmid expressed short hair pin RNA (shRNA). (3) To study the inhibitory effect of STAT3 shRNA combined with radiotherapy on Hep-2 xenograft tumors.Methods:(1) Designed short hairpin RNA aiming to STAT3 gene mRNA and constructed pGPU6/GFP/Neo-shRNA expression vector by molecular cloning technique and identified by enzyme digestion and sequencing.(2) After transfecion of Hep-2 cells with recombinant plasmid, semi-quantitative reverse transcriptase-polymerase chain (RT-PCR) and Western blotting were used to detect mRNA and STAT3 protein expression. MTT assay was used to investigate the Hep-2 cells growth inhibition transfected with recombinant plasmid. Moreover, cell apoptosis and cell cycle were detected by flow cytometry (FCM).(3) In vitro studies: RNA interference plasmid that expresses shRNA targeting STAT3 gene and negative control plasmid were transfected into Hep-2 cells by Lipofectamine 2000.Twenty-four hours after the transfection, cells were observed under fluorescence microscopy and transfection efficiency was detected by FCM. Subsequently the cells were radiated by 60Coγray at 0、2、4、6、8、10Gy. Forty–eight hours later, cell survival was evaluated by MTT .FCM was used to detect cell apoptosis rate and STAT3, p-STAT3,Bcl-2 protein levels and cell proliferative activity as well as apoptosis rate at 12h, 24h and 48h after radiation with 6Gy.(4)The animal models of xenotransplanted human laryngeal carcinoma cell line Hep-2 were set up in 28 nude mice, which were distributed into 4 groups at random: Negative plasmid control group, the group that received pshSTAT3 transfecion; the radiation group; and the group of pshSTAT3 transfection combined with irradiation. Tumor volume was determined regularly. On the fifteenth day after termination of radiation treatment, the mice were sacrificed and the tumor weights were measured in all the groups. According to the data measured above, tumor growth curve was plotted and inhibition rate for tumor growth was calculated. Then HE staining was used to observe tumor morphology. Moreover the expression of STAT3、p-STAT3、Bcl-2、p53、VEGF protein and intratumor microvessel density (MVD) were investigated by immunohistochemistry and computer-assisted image analysis was used to obtain the results. Transmission electron microscope and flow cytometry were used to observe cellular ultrastructure and detect the cell apoptosis rate, respectively.Results:(1)The recombinant plasmid pGPU6/GFP/Neo-shSTAT3 was constructed successfully and confirmed by restiction enzyme digestion and sequencing results.(2)The recombinant plasmid can inhibit STAT3 mRNA and protein expression. After transfection with recombinant plasmid, green fluorescence were observed in Hep-2 cells under fluorescence microscopy. And efficiency deteced by FCM was 85.63% at 24 hour post-transfection .MTT assay demonstrated that cell growth was inhibited in transfected cells, and 24h, 48h or 72h growth inhibition rates of recombinant plasmid-transfection group were significantly lower than that of the other two control groups. FCM results showed obvious apoptotic peak in the cells transfected with pshSTAT3. Analysis of cell cycle showed that Hep-2 cells arrested in G1 stage.(3)In vitro study: The survival rate of Hep-2 cells exposed different dose of irradiation rays changed in a dose-dependent manner. MTT assay showed that STAT3 shRNA combined with 60Coγray significantly inhibited proliferation of Hep-2 cells compared with control (P <0.05). And flow cytometry detection showed that apoptosis rate of the STAT3 shRNA group was significantly higher than that of untransfected group at the same radiation dosage 0、2、4、6、8 and 10Gy( P < 0.05). After 6Gy radiation, cell proliferation and apoptosis rate variated in a time-dependent manner.Moreover, STAT3, p-STAT3, Bcl-2, VEGF and p53 expression in human laryngeal carcinoma Hep-2 cells of combined group were higher than those of control on 48h after irradiation(P<0.05). Meaninwhile, FI (fluorescence index) of p-STAT3 among all of groups showed a positive correlation with levels of Bcl-2, VEGF and p53 (r=0.974, r=0.988, r=0.976, P<0.05).(4)There was a significant difference in tumor volume among the groups (P<0.01). The rate of tumor inhibition in the radiation group, pshSTAT3 group and pshSTAT3 plus radiation group were 34.76%, 19.68% and 67.70%, respectively. The p-STAT3 protein expression decreased significantly in the combined treatment group. The intratumoral microvessel density (MVD) in the combined group decreased significantly compared to the negative plasmid control and the radiation group (P<0.01).Ultrastructure of cells observed by electron microscopy changed obviously in combined treatment group and the apoptosis rate was much higher than that of other groups (P<0.01). Moreover, there was a positive correlation between the expression of p-STAT3 and Bcl-2、p53、VEGF protein expression and MVD (r=0.727, r=0.738, r=0.735, r =0.691, P<0.01), and there was a negative correlation concerning cell apoptosis rate (r = -0.765, P<0.01).Conclusion: 1. Recombinant plasmid can decrease significantly STAT3 protein levels, inhibit proliferation and induce apoptosis in transfected Hep-2 cells.2. Recombinant plasmid targeting STAT3 gene can significantly enhance the sensitivity of human laryngeal carcinoma cells to radiation in vivo.3. Using STAT3 recombinant plasmid in conjunction with radiation can significantly inhibit the growth of xenograft tomor of human laryngeal carcinoma. The mechanism may be associated with down regulation expression of related protein located STAT3 signal transduction pathways downstream, therefore inhibiting tumor proliferation and induced apoptosis, reducing tumor angiogenesis.更多还原