【作者】 王媛媛；

【导师】 张天夫；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2019， 硕士

【摘要】 研究背景:细菌治疗癌症起于19世纪,Coley医生观察到一些感染丹毒的肉瘤患者病情缓解的病例,由于当时抗生素未出现,丹毒不容易控制,且不容易诱导,Coley医生不能有效使用活菌,因此他使用灭活的细菌产物去治疗病人,然而这种方法有效性仅局限于少数病人,即不能用其他方法治疗的肉瘤患者。后来随着放化疗的出现,有极大局限性的细菌治疗的方法被逐渐忽视。数年后,人们发现癌症患者长期生存率及控制率并没有较好改善,于是逐渐又对细菌治疗癌症燃起了希望。为了扩大细菌治疗疫苗的适应症,人们逐渐改造细菌疫苗,例如通过基因工程改造细菌,使其具有特殊的性状,或是使用减毒的活细菌疫苗。改良后的细菌疗法治疗癌症拥有其他治疗不能企及的优点:特异性靶向肿瘤。铜绿假单胞菌-甘露糖敏感血凝菌(PA-MSHA)是牟希亚建立的经过改造的铜绿假单胞菌,菌体周边布满Ⅰ型菌毛。其借助免疫源性强的Ⅰ型菌毛,在体内能提高细胞免疫和体液免疫:包括增强NK细胞的杀伤性,提高T淋巴细胞数量及改变T淋巴细胞亚群比例关系,增加抗PA-MSHA抗体。PA-MSHA在临床上主要用作免疫调节剂,并作为肿瘤病人的辅助治疗,激活免疫系统杀伤肿瘤。PA-MSHA具有较强的对于肿瘤细胞的细胞毒性,实验发现PA-MSHA细胞毒性的原因是通过抑制EGFR信号通路实现的,而参阅文献发现PA-MSHA电镜下存在于细胞内部,因此本实验探讨PA-MSHA对舌鳞癌细胞杀伤作用及机制,并探讨PA-MSHA对舌鳞癌细胞系自噬的影响。目的:1.研究PA-MSHA对舌鳞癌细胞系CAL-27、SCC-15的杀伤作用及机制。2.探讨PA-MSHA对舌鳞癌细胞系CAL-27、SCC-15自噬的影响。3.探讨PA-MSHA对舌鳞癌细胞系CAL-27、SCC-15 TLR通路的影响。方法:1.用PA-MSHA处理舌鳞癌细胞株,观察细胞形态改变,CCK8、LDH检测药物对舌鳞癌细胞的活性及毒性影响。2.PA-MSHA处理舌鳞癌细胞株,用Hoechst33258染色检测核形态。3.PA-MSHA处理舌鳞癌细胞株,Annexin-V/PI染色,流式细胞术检测凋亡。4.RT-q PCR检测PA-MSHA处理后的舌鳞癌细胞株Caspase相关蛋白酶m RNA表达变化5.western blot检测PA-MSHA处理后的舌鳞癌细胞株Caspase相关蛋白酶表达变化。6.用氯喹、巴弗洛霉素A1处理舌鳞癌细胞株,CCK8检测药物的安全剂量。7.RT-q PCR检测PA-MSHA处理后的舌鳞癌细胞株TLR9、IFR7 m RNA表达变化。8.western blot检测PA-MSHA处理后的舌鳞癌细胞株LC3、p62相关蛋白的表达量的变化。结果:1.PA-MSHA杀伤舌鳞癌细胞株,并呈剂量依赖性。2.PA-MSHA诱导舌鳞癌细胞凋亡。3.PA-MSHA促使舌鳞癌细胞株Bcl-2家族中Bax表达升高,增加Caspase被剪切。4.对于本实验中的舌鳞癌细胞株,BAF用于抑制自噬溶酶体的饱和工作浓度为25n M。5.PA-MSHA能影响舌鳞癌细胞自噬的水平。6.舌鳞癌细胞株CAL-27、SCC-15表达TLR-9,PA-MSHA能激活SCC-15细胞株TLR9受体的My D88依赖的干扰素产生途径,且能促进CAL-27、SCC-15EGFR m RNA表达升高。结论:PA-MSHA通过线粒体-Caspase机制杀伤舌鳞癌细胞株CAL-27、SCC-15,并且能改变细胞自噬水平,激活TLR9受体。更多还原

【Abstract】 Backgrounds:Bacterial treatment of cancer began in the 19 th century,Dr.Coley observed some sarcoma patients infected with erysipelas remission.Because antibiotics did not appear at the time,erysipelas is not easy to control,and is not easy to induce,Dr.Coley uses inactivated bacterial products to treat Patient instead of viral bacteria,however,the effectiveness of this method is limited to a small number of patients,ie sarcoma patients who cannot be treated by other methods.Later,with the advent of radiotherapy and chemotherapy,the method of bacterial treatment with great limitations was gradually ignored.A few years later,people found that the long-term survival rate and control rate of cancer patients did not improve,so gradually ignited hope for bacterial treatment of cancer.In order to expand the indications for bacterial therapeutic vaccines,bacterial vaccines have been gradually modified,for example by genetically engineering bacteria to have specific traits,or to use attenuated live bacterial vaccines.Improved bacterial therapy for cancer has the advantage that other treatments cannot match: specific targeting of tumors.Pseudomonas aeruginosa-mannose-sensitive blood coagulum(PA-MSHA)is a modified Pseudomonas aeruginosa established by Mou Xiya,which is surrounded with type I pili.It can improve cellular and humoral immunity in vivo by means of immunogenic I type pili: including enhancing the killing of NK cells,increasing the number of T lymphocytes and changing the proportion of T lymphocyte subsets,and increasing anti-PA-MSHA antibody.PA-MSHA is mainly used as an immunomodulator in clinical practice and as an adjuvant treatment for cancer patients,it activates the immune system to kill tumors.PA-MSHA has strong cytotoxicity to tumor cells.It was found that the cause of PA-MSHA cytotoxicity is achieved by inhibiting the EGFR signaling pathway,and the literature found that PA-MSHA could enter the cell under electron microscope,Therefore,this experiment explored the killing effect and mechanism of PA-MSHA on tongue squamous cell carcinoma,and autophagy flux level changes of tongue squamous cell carcinoma affected by PA-MSHA.Objectives:1.To study the killing effect and mechanism of PA-MSHA on tongue squamous cell carcinoma cell lines CAL-27 and SCC-15.2.To investigate the effect of PA-MSHA on autophagy of tongue squamous cell carcinoma cell lines CAL-27 and SCC-15.3.To investigate the effect of PA-MSHA on the TLR pathway of tongue squamous cell carcinoma cell lines CAL-27 and SCC-15.Methods:1.The tongue squamous cell carcinoma cell line was processed with PA-MSHA,and the cell morphology was observed.The effects of CCK8 and LDH on the activity and toxicity of tongue squamous cell carcinoma were detected.2.PA-MSHA was used to treat tongue squamous cell carcinoma cells,and nuclear morphology was detected by Hoechst 33258 staining.3.Annexin-V/PI staining,and flow cytometry were used to detect the apoptosis cells of tongue squamous cell carcinoma cell lines treated by PA-MSHA.4.RT-qPCR detected mRNA expression of Caspase-associated protease in tongue squamous cell carcinoma cells treated with PA-MSHA5.western blot was used to detect the expression of Caspase-associated protease in tongue squamous cell carcinoma cells treated with PA-MSHA.6.The squamous cell carcinoma cell line was treated with chloroquine and bafilomycin A1,and the safe dose of CCK8 was detected.7.RT-qPCR was used to detect the expression changes of TLR9 and IFR7 mRNA in tongue squamous cell carcinoma cells treated with PA-MSHA.8.western blot was used to detect the expression of LC3 and p62-related proteins in tongue squamous cell carcinoma cells treated with PA-MSHA.Results:1.PA-MSHA kills tongue cancer cell lines in a dose-dependent manner.2.PA-MSHA induces apoptosis in tongue squamous cell carcinoma.3.PA-MSHA promotes the increase of expression of Bax,one of the Bcl-2 family,,and increases the level of cleved Caspase9 in tongue squamous cell carcinoma cells.4.For the tongue squamous cell carcinoma cell line in this experiment,the saturation working concentration of BAF used to inhibit the autolysosome was 25 nM.5.PA-MSHA can affect autophagy of tongue squamous cell carcinoma.6.Tongue squamous cell carcinoma strains CAL-27 and SCC-15 express TLR-9,and PA-MSHA can activate TLR9 receptor through MyD88-dependent pathway of SCC-15 cell line,which produces interferon.PA-MSHA can promote the expression of EGFR mRNA in tongue squamous cell carcinoma cells.Conclusions:PA-MSHA killed tongue squamous cell carcinoma cells CAL-27 and SCC-15 through mitochondria-Caspase mechanism,and could change the level of autophagy and activate TLR9 receptor.更多还原