以减毒沙门氏菌运载共表达质粒Stat3-SiRNA-Stat1靶向治疗脑胶质瘤的分子机制

Glioma is the most common tumor of nervous system, and also one of the diseases that seriously threaten human health. At present, surgery is still the first therapeutic method, radiotherapy and chemotherapy are assisted following surgery, but the overall effect is not very satisfactory, the median survival time after surgery is 12-18 months, five-year survival rate is less than 10%. Accordingly, the remarkable gene therapy is considered as the “4th mode” besides surgery, chemotherapy and radiotherapy. This project is based on the previous findings that signal transducer and activator of transcription 1 (stat 1) is highly expressed in normal brain tissue and lowly expressed in brain glioma tissue; on the contrary, stat 3 is highly expressed in brain glioma tissue and lowly expressed in normal brain tissue. We plan to constitute co-expressed plasmid Stat3-siRNA-Stat1, up-regulate stat1 and down-regulate stat3, explore the molecular mechanisms of apoptosis of glioma cell line U87MG. Furthermore, we select the new-style attenuated Salmonella as transport vector for the co-expressed plasmid, observe the targeted therapy effect for brain glioma in vivo; Simultaneously, we will also analyze the local micro-environment around tumor tissue, investigate the molecular mechanisms for the plasmid and plasmid expression product release, as well as how they escape from the clearance of immune system, all of these would provide theoretical basis for brain glioma gene therapy.

脑胶质瘤是神经系统疾病中最常见的恶性肿瘤，是严重威胁人类健康的疾病之一。目前，手术切除肿瘤仍是恶性胶质瘤首选的治疗方式，术后辅助放射治疗和化学药物治疗，但总体疗效不佳，手术后中位生存时间12-18个月，5年生存率不足10%。因此，受人瞩目的基因治疗被认为是除手术、放疗、化疗之外的“第四模式”。本课题基于前期发现信号传导与转录激活因子1（Stat1）在正常脑组织中高表达而在脑胶质瘤组织中低表达，而Stat3表达与其恰好相反这一事实，构建共表达质粒Stat3-siRNA-Stat1，使之上调Stat1而下调Stat3，探讨其诱导胶质瘤细胞系U87MG细胞凋亡的分子机制。并且利用减毒沙门氏菌这一新型载体作为共表达质粒的运载体，体内观察其对脑胶质瘤的靶向治疗作用。同时，对肿瘤组织局部微环境进行分析，探讨质粒及其表达产物的释放、逃逸免疫系统清除等作用的分子机制，从而为胶质瘤的基因治疗提供理论依据。

脑胶质瘤是神经系统疾病中最常见的恶性肿瘤。手术切除肿瘤仍是恶性胶质瘤首选的治疗方式，术后辅助放疗化疗，但总体疗效不佳，手术后中位生存时间12-18个月。因此，受人瞩目的基因治疗被认为是除手术、放疗、化疗之外的“第四模式”。本课题基于前期发现信号传导与转录激活因子1（Stat1）在正常脑组织中高表达而在脑胶质瘤组织中低表达，而Stat3表达与其恰好相反这一事实，构建共表达质粒Stat3-siRNA-Stat1，使之上调Stat1而下调stat3，体外实验探讨其诱导胶质瘤细胞系U251细胞凋亡的分子机制。体内实验研究对人U87-MG脑胶质瘤裸鼠皮下移植瘤模型的靶向治疗作用。体外实验结果：感染lenti-stat1和lenti-stat1/stat3-siRNA后，U251细胞STAT1的 mRNA和蛋白表达水平均明显升高，感染lenti-stat3-siRNA和lenti-stat1/stat3-siRNA后，U251细胞STAT3的mRNA和蛋白表达水平均明显降低，结果表明成功建立Stat1、Stat3-siRNA及其共表达Stat3-siRNA-Stat1载体。与对照组比较，在敲除STAT3和过表达STAT1后，各实验组U251细胞增殖和迁移能力明显下降，凋亡率和G0-G1期细胞数明显升高，其中以重组慢病毒共表达组对U251影响最显著。转录组测序结果显示重组慢病毒lenti-stat1、lenti-stat3-siRNA和lenti-stat1/stat3-siRNA可引起U251细胞转录水平差异表达，其中共表达基因组独有的差异基因KEGG分析提示主要富集到FOXO信号通路中。体内实验结果：重组腺病毒载体介导的Ad-stat1、Ad-Stat3-siRNA及共表达组Ad-stat1/stat3-siRNA均可显著抑制U87裸鼠皮下移植瘤生长共表达组治疗效果优于单基因治疗组。各治疗组的心、肝、脾、肺、肾及脑与PBS相比无明显组织形态差异。Western blotting、RT-PCR 方法结果显示，共表达质粒治疗后，STAT1 mRNA和蛋白水平表达显著增强，STAT3 mRNA和蛋白水平显著下降。Annexin V和TUNEL的的实验表明，共表达质粒联合治疗能够显著促进肿瘤细胞凋亡坏死。为共表达对胶质瘤的基因治疗提供理论及实验基础，为进一步开展临床实验提供数据。