【作者】 王宁；

【导师】 周云丽；

【作者基本信息】 天津医科大学 ， 临床医学（专业学位）， 2020， 硕士

【摘要】 研究目的:胰腺癌的恶性度高,且进展迅速,临床病例中极易发生转移,临床统计中仅有20%左右的患者在就诊时有手术机会,其他的患者则大多只能接受姑息化疗,胰腺癌的五年生存期仅5%左右。因此胰腺癌是目前最难以治疗的恶性肿瘤之一,且目前尚无任何有效治疗可以延长患者的的生存期和改善预后。近年来分子靶向治疗在某些肿瘤治疗中取得了一定的进步,在本课题中探究了miR-25-3p对胰腺癌细胞的作用和胰腺癌细胞对iRGD修饰的外泌体的靶向摄取作用,并将二者结合探索其在胰腺癌靶向治疗中的应用,以期日后可以通过此种治疗方式改善胰腺癌患者的生活质量,延长患者的生存时间。研究方法及结果:1.胰腺癌患者血清miR-25-3p血清表达情况及其与患者生存期相关性PCR对比临床样本后发现胰腺癌患者较体检正常人血清中miR-25-3p的表达量高,且胰腺癌患者血清中miR-25-3p的表达量与患者生存期呈负相关。2.Mi R-25-3p对胰腺癌细胞侵袭迁移能力的影响PCR比较不同胰腺癌细胞系miR-25-3p表达量后选取miR-25-3p表达量适中的panc-1和Bx PC-3细胞系进行后续实验,划痕愈合实验和transwell实验显示miR-25-3p可促进胰腺癌细胞的侵袭迁移能力。3.外泌体装载的anti-miR-25-3p对胰腺癌细胞侵袭迁移能力的影响对不同处理组胰腺癌细胞进行transwell和划痕愈合实验后显示外泌体装载的anti-miR-25-3p可被胰腺癌细胞摄取并抑制胰腺癌细胞的侵袭迁移。4.构建iRGD肽段修饰的外泌体运载anti-miR-25-3p免疫荧光和western blot结果显示过表达3×FLAG-iRGD-CD63融合蛋白的外泌体构建成功,PCR结果显示iRGD肽段修饰且装载anti-miR-25-3p的外泌体构建成功。荧光显微镜观察到体外胰腺癌细胞对iRGD修饰且装载anti-miR-25-3p的外泌体呈高摄取趋势,小动物成像结果显示胰腺癌细胞在体内对iRGD修饰且装载anti-miR-25-3p的外泌体呈特异性高摄取。5.iRGD修饰的外泌体包裹anti-miR25-3p的胰腺癌治疗效果对胰腺癌原位模型小鼠治疗过程中发现iRGD修饰且包裹anti-miR-25-3p外泌体的治疗可减慢小鼠体重的下降;对小鼠肝脾组织的HE染色结果显示iRGD修饰且其中包裹anti-miR-25-3p的外泌体治疗可以显著抑制小鼠胰腺癌的肝转移和脾转移;对小鼠的心肺肾HE染色结果结果显示外泌体注射治疗会一定程度上导致小鼠心脏、脾脏等产生炎症反应,但对于小鼠心脏纤维化、脾细胞坏死、脾出血、肺出血等情况有一定的改善作用。研究结论:本课题中,胰腺癌患者血清中miR-25-3p呈高表达趋势,且miR-25-3p的表达水平与患者的生存期呈负相关。Mi R-25-3p可促进胰腺癌细胞的侵袭迁移能力。胰腺癌细胞在体内和体外均可对iRGD修饰的外泌体快速靶向摄取且在小鼠胰腺癌原位模型的治疗中iRGD修饰且其中装载anti-miR-25-3p的外泌体可以抑制小鼠胰腺癌的肝转移和脾转移。更多还原

【Abstract】 Objectives:Pancreatic cancer is highly malignant and progresses rapidly.Only about 20%patientshave surgery opportunities at the time of treatment.Most of other patients can only receive palliative chemotherapy.The annual survival period is only about 5.Therefore,pancreatic cancer is one of the most difficult malignant tumors at present,and there is no effective treatment to prolong the survival time and improve the prognosis of patients.In recent years,molecular targeted therapy has made some progress in the treatment of certain tumors.In this topic,we explored the effect of miR-25-3p on pancreatic cancer cells and the targeted uptake of iRGD-modified exosomes by pancreatic cancer cells.Research methods and results:1.Serum miR-25-3p serum expression in patients with pancreatic cancer and its correlation with patient survival After using PCR comparing the clinical samples,it was found that the expression of miR-25-3p in the serum of patients with pancreatic cancer was higher than that of normal people,and the expression of miR-25-3p in the serum of patients with pancreatic cancer was negatively correlated with the survival time of patients.2.The selection of pancreatic cancer cell lines and the effect of miR-25-3p on the invasion and migration of pancreatic cancer cells After using PCR comparing the expression levels of miR-25-3p in different pancreatic cancer cell lines,panc-1 and Bx PC-3 cell lines were selected with moderate miR-25-3p expression for subsequent experiments.Scratch healing experiment and transwell experiment showed that miR-25-3p can promote the invasion and migration of pancreatic cancer cells.3.The effect of anti-miR-25-3p loaded with exosomes on the invasion and migration ability of pancreatic cancer cells Transwell and scratch healing experiments on pancreatic cancer cells in different treatment groups showed that anti-miR-25-3p loaded with exosomes can inhibit the invasion and migration of pancreatic cancer cells.4.Construction of iRGD peptide modified,anti-miR-25-3p carried exosomes Immunofluorescence and PCR results showed that the HEK-293 T overexpressing3 × FLAG-iRGD-CD63 fusion protein and overexpressing anti-miR-25-3p was successfully constructed.Western blot and PCR showed that iRGD peptide modified,ant-miR-25-3p carried exosomes was successfully modified.Fluorescence microscopy observed that pancreatic cancer cells showed a high uptake trend to iRGD peptide modified,ant-miR-25-3p carried exosomes in vivo and in vitro.Small animal imaging results showed that pancreatic cancer cells showed specific high uptake to iRGD peptide modified,ant-miR-25-3p carried exosomes in vivo.5.The therapeutic effect of iRGD modified,ant-miR-25-3p carried exosomes in pancreatic cancer IRGD modified,ant-miR-25-3p carried exosomes was found can slow down the weight loss of mice during the treatment of pancreatic cancer in situ model mice;HE staining results on liver and spleen tissues of mice showed that iRGD modified,ant-miR-25-3p carried exosomes can significantly inhibit liver metastasis and splenic metastasis of pancreatic cancer in mice;HE staining results of heart,lung and kidney of mice show that exosome injection treatment will causes inflammation in the heart and spleen of mice,but it has a certain improvement effect on cardiac fibrosis,spleen cell necrosis,splenic hemorrhage,and pulmonary hemorrhage in mice.Analysis conclusion:In this project,miR-25-3p in the serum of pancreatic cancer patients showed a high expression trend,and the expression level of miR-25-3p was negatively correlated with the survival time of patients.Mi R-25-3p can promote the invasion and migration ability of pancreatic cancer cells.IRGD modified exosomes can be rapidly targeted uptake in pancreatic cancer cells.IRGD modified,ant-miR-25-3p carried exosomes can inhibit liver metastasis and splenic metastasis of mouse pancreatic cancer.更多还原