【作者】 李勇；

【导师】 孟宪瑛；

【作者基本信息】 吉林大学 ， 外科学（专业学位）， 2019， 硕士

【摘要】 背景与目的:甲状腺癌是最常见的内分泌恶性肿瘤,约占所有癌症的1.7%,其发病率在近三十年中逐年增加,截止到2017年,全球甲状腺癌患者人数高达到320万。其中,女性甲状腺癌患者数量约为男性患者的三倍,甲状腺癌成为女性第五大常见恶性肿瘤。分化型甲状腺癌较未分化癌和髓样癌预后好,但对于发生远处转移、放射性碘治疗抵抗者,治疗方式的选择非常有限。未分化癌、髓样癌患者确诊时多为肿瘤晚期并出现明显的肿瘤远处转移,失去了手术最佳时机,且患者自身状态较差,无法耐受放化疗,给治疗带来了难度和挑战。因此,我们急需寻求一种更加有效的治疗方式来治疗难治性甲状腺癌。肿瘤免疫治疗是通过改变肿瘤免疫微环境,恢复机体正常的抗肿瘤免疫反应,从而控制和清除肿瘤的一种治疗方法。本课题首先探究了JQ1处理后的肿瘤细胞表达PD-L1和CD47的情况,设计并合成了BRD4小分子抑制剂JQ1与CD47siRNA分子相结合的纳米药物载体,用于荷瘤小鼠肿瘤模型的免疫治疗和化疗联合治疗。该载药纳米颗粒不仅可以在体外细胞系发挥抗肿瘤作用,还可以在荷瘤小鼠体内富集于肿瘤组织中,进入肿瘤细胞,并在肿瘤细胞内快速崩解,释放抗肿瘤药物,从而增强抗肿瘤作用,减少化疗药物的使用剂量,节约成本及减轻其毒副作用。方法:1.采用流式细胞分析仪检测JQ1对肿瘤细胞PD-L1和CD47表达的影响。2.采用粒度仪对合成的载药纳米颗粒表面电势和粒径进行测定。3.采用实时定量PCR检测纳米颗粒对肿瘤细胞PD-L1和CD47表达的影响。4.采用流式细胞分析仪和激光共聚焦显微镜观察检测载药纳米颗粒在肿瘤细胞内的分布情况。5.采用小动物活体成像仪检测载药纳米颗粒在荷瘤小鼠体内的分布情况,用激光共聚焦显微镜检测载药纳米颗粒在肿瘤组织内的分布情况。6.通过尾静脉注射不同载药纳米颗粒对荷瘤小鼠抗肿瘤治疗,观察肿瘤大小并绘制生长曲线。结果:1.JQ1可以下调肿瘤细胞PD-L1的表达。2.成功制备同时包载JQ1和CD47siRNA分子的纳米颗粒,_JQ1NPs ^CD47siRNA的粒径约为123.8±1.1 nm,电势约为10.05±0.66mV。3._JQ1NPs ^CD47siRNA纳米颗粒在体外可以下调肿瘤细胞PD-L1和CD47的表达。4._JQ1NPs ^CD47siRNA纳米颗粒在体外可将药物递送入肿瘤细胞系,在体内可以富集在荷瘤小鼠的肿瘤组织中并进入肿瘤细胞发挥抗肿瘤作用。5._JQ1NPs ^CD47siRNA纳米颗粒可显著抑制荷瘤小鼠的肿瘤生长。结论:本课题设计并合成了JQ1与CD47siRNA分子相结合的纳米药物载体,用于荷瘤小鼠肿瘤模型的免疫治疗和化疗联合治疗。该载药纳米颗粒可以富集于荷瘤小鼠的肿瘤组织中,进入肿瘤细胞,并在肿瘤细胞内崩解,释放抗肿瘤药物,发挥肿瘤免疫治疗,从而增强抗肿瘤作用,减少化疗药物的使用剂量,节约成本及减轻其毒副作用。更多还原

【Abstract】 Background and objective:Thyroid cancer is the most common endocrine malignancy,accounting for 1.7%of all cancers.In the past 30 years,its incidence has increased year by year.By 2017,the number of patients with thyroid cancer worldwide has reached 3.2 million.Among them,the number of female thyroid cancer patients is about three times that of male patients,and thyroid cancer has become the fifth most common malignant tumor in women.Differentiated thyroid cancer has a better prognosis than undifferentiated carcinoma and medullary carcinoma,but for patients with distant metastasis and radioactive iodine resistance,treatment options are very limited.Patients with undifferentiated carcinoma and medullary carcinoma are mostly diagnosed at the advanced stage of the tumor and have obvious distant metastasis of the tumor.They lose the best time for surgery,and the patient’s own state is poor,and can not tolerate radiotherapy and chemotherapy,which brings difficulty to the treatment.Therefore,we urgently need to find a more effective treatment to treat refractory thyroid cancer.Tumor immunotherapy is a treatment method for controlling and clearing tumors by changing the tumor immune microenvironment and restoring the normal anti-tumor immune response of the body.This study firstly explored the expression of PD-L1 and CD47 in tumor cells treated with JQ1,and designed and synthesized a nano drug carrier combining BRD4 small molecule inhibitor JQ1 and CD47 siRNA molecules for the treatment of tumor-bearing mouse tumor models.The drug-loaded nanoparticles can not only exert anti-tumor effects in cell lines in vitro,but also can be enriched in tumor tissues in tumor-bearing mice.It enters tumor cells and rapidly disintegrates in tumor cells,releasing anti-tumor drugs,thereby enhancing anti-tumor effects,reducing the dosage of chemotherapeutic drugs,saving costs and reducing its side effects.Method:1.The effect of JQ1 on the expression of PD-L1 and CD47 in tumor cells was detected by flow cytometry.2.The surface potential and particle size of the synthesized drug-loaded nanoparticles were determined by a particle size analyzer.3.Real-time quantitative PCR was used to detect the effect of nanoparticles on the expression of PD-L1 and CD47 in tumor cells.4.Flow cytometry and laser confocal microscopy were used to observe the distribution of drug-loaded nanoparticles in tumor cells.5.The distribution of drug-loaded nanoparticles in tumor-bearing mice was detected by small animal living imager.The distribution of drug-loaded nanoparticles in tumor tissues was detected by laser confocal microscopy.6.The tumor growth curve was observed by injecting different drug-loaded nanoparticles into the tumor-bearing mice by tail vein injection.Result:1.JQ1 can down-regulate the expression of PD-L1 in tumor cells.2.Nanoparticles containing both JQ1 and CD47 siRNA molecules were successfully prepared.The particle size of _JQ1NPs^CD47siRNAD47siRNA was about 123.8±1.1 nm,and the potential was about 10.05±0.66 mV.3._JQ1NPs^CD47siRNAD47siRNA can down-regulate the expression of PD-L1 and CD47 in tumor cells in vitro.4._JQ1NPs^CD47siRNAD47siRNA can deliver the drug into the tumor cell line in vitro,and can be enriched in the tumor tissue of the tumor-bearing mouse in vivo and enter the tumor cell to exert an anti-tumor effect.5._JQ1NPs^CD47siRNAD47siRNA can significantly inhibit tumor growth in tumor-bearing mice.Conclusion:We designed and synthesized a nanocarrier combined with JQ1 and CD47 siRNA molecules for immunotherapy and chemotherapy combination in tumor-bearing mouse tumor models.The drug-loaded nanoparticle can be enriched in the tumor tissue of the tumor-bearing mouse,and can enter the tumor cell,disintegrate in the tumor cell,release the anti-tumor drug,and exert the tumor immunotherapy,thereby enhancing the anti-tumor effect and reducing the chemotherapy.The dosage of the drug is used to save costs and reduce its side effects.更多还原