凋亡素修饰的骨髓间充质干细胞靶向抑制肝癌的作用研究

Live cancer is one of the most malignant tumors, which is the third killer of the cancers all over the world. Patients with hepatocellular carcinoma in our country win the first place in the world. The traditional therapy for liver cancer is hepatectomy. There is a high recurrence rate and metastasis rate after hepatectomy, which becomes the problem for clinical therapy of liver cancer. Currently, stem cells therapy is one of exploring and novel biological treatment method for tumors. Mesenchymal stem cells are easy to obtain, have low immunogenicity and are able to migrate to the tumor, and have potential to become ideal vehicle for targeted delivery of anti-tumor factors. Apoptin has broad-spectrum anti-tumor property; moreover, it doesn't have toxic effects on normal cells. Helper-dependent adenovirus vector（HDAd）has the advantages of low toxicity, large transfer capacity and sustainable expression of transgene. Now it has not been reported that combining MSCs, HDAd with apoptin is used in liver cancer treatment. This project intends to construct an adenovirus vector which expresses apoptin and then infect human bone marrow MSCs, and explore the migration of MSCs to the liver cancer cells and inhibitory effect of MSCs as a vehicle for targeted delivery of apoptin on liver cancer by in vitro co-culture test and in vivo transplantation in mice model with hepatocellular carcinoma. This study tries to search for a stem cells therapy method for cancers, which will provide a new approach and experimental basis for the clinical treatment of cancer. At the same time, this research will also probe into a novel in vivo drug delivery system through stem cells as vehicle, which will resolve immune rejection induced by repeated applications of drug directly delivered by adenovirus vectors and reduce the high cost of a large scale of production and purification of exogenous drugs. This study has an important theoretical and practical value.

肝癌是全球癌症第三号杀手，术后复发转移率很高，这成为肝癌临床治疗的难题。干细胞治疗是目前正在探索的肿瘤新型生物治疗方法之一。间充质干细胞（MSCs）由于免疫原性低、能够向肿瘤迁移等特性有望成为抗肿瘤因子靶向治疗的理想载体；凋亡素具有对正常细胞无毒性的广谱抗肿瘤特性；辅助病毒依赖的腺病毒载体（HDAd）具有毒性低、转基因持续表达等优点。但目前将MSCs、HDAd及凋亡素联合应用于肝癌治疗的研究尚未见报道。本项目拟构建表达凋亡素的腺病毒载体并感染人骨髓MSCs，通过体外MSCs与肝癌细胞的共培养以及小鼠肝癌模型体内移植实验，探索MSCs向肝癌细胞的迁移及其腺病毒载体介导的凋亡素修饰的MSCs对肝癌细胞的靶向抑制作用。本项目的开展可以探讨癌症的干细胞疗法，为癌症的临床治疗提供新的途径与实验依据；同时可以探讨以干细胞为载体的新型体内给药系统，有望解决腺病毒载体直接递送药物引起的免疫排斥问题。

肝癌是全球癌症第三号杀手，术后复发转移率很高，这成为肝癌临床治疗的难题。干细胞治疗是目前正在探索的肿瘤新型生物治疗方法之一。间充质干细胞（Mesenchymal stem cells，MSCs）由于能够向肿瘤迁移、免疫原性低等特性有望成为抗肿瘤因子靶向治疗的理想载体；凋亡素具有对正常细胞无毒性的广谱抗肿瘤特性；腺病毒载体具有毒性低、转基因持续表达等优点。但目前将MSCs、腺病毒及凋亡素联合应用于肝癌治疗的研究未见报道。本论文拟构建表达凋亡素的腺病毒载体并感染人骨髓MSCs，通过一系列实验，探索MSCs向肝癌细胞的迁移及其腺病毒载体介导的凋亡素修饰的MSCs对肝癌细胞的体内外抑制作用。.通过迁移实验证明，MSCs具有明显向肝癌细胞HepG2迁移的能力（P＜0.01）；成功构建了可表达凋亡素Apoptin蛋白的重组腺病毒Ad-vp3，感染293细胞，获得了高滴度（1.0×1012 pfu/mL）的病毒颗粒，提取病毒基因组，检测有凋亡素基因的存在，证明符合构建目的；高滴度的病毒颗粒感染MSCs后，利用Western Blot检测发现感染后的MSCs培养上清中有凋亡素的表达，从而获得表达凋亡素蛋白的MSCs；体外抑制实验表明，稳定表达凋亡素的MSCs和HepG2共培养后，表达凋亡素的MSCs可以显著抑制HepG2的增殖（P＜0.01）。同时，表达凋亡素的MSCs的条件培养基也可以明显抑制HepG2的增殖（P＜0.05），但对正常细胞没有抑制作用。动物实验表明，表达凋亡素的MSCs对HepG2肿瘤有明显的抑制作用（P＜0.001）。.本研究的开展可以探讨癌症的干细胞疗法，为癌症的临床治疗提供新的途径与实验依据。同时为干细胞为载体的新型体内给药系统的研究奠定基础，有望解决腺病毒载体直接递送药物引起的免疫排斥问题。