摘要
背景:凋亡素(Apoptin)能诱导50种以上不同类型的肿瘤细胞凋亡,有望成为一种极具应用前景的抗肿瘤制剂。如何安全、有效地将其应用于临床,是一个亟待解决的最具现实意义的问题。目的:拟制备人血清白蛋白(HSA)与Apoptin基因的复合物,通过体内外实验验证其抗瘤效果,为Apoptin尽早应用于基因治疗寻找一个简单有效的转移方式。方法:(1)构建含Apoptin基因的真核表达载体pcD NA-Apoptin。以水溶性碳二亚胺(EDC)为交联剂,将聚醚酰亚胺(PEI)与人血清白蛋白(HSA)连接,合成HSA-PEI复合物,然后经电荷中和效应将HSA-PEI复合物与pcD NA-Apoptin重组体及pcD NA空载体分别连接,形成相应HSA-PEI-pcDNA-Apoptin复合物;(2)将HSA-PEI-pcD NA-Apoptin复合物转染乳腺癌MCF-7细胞,Western blot检测Apoptin基因在细胞中的表达,MTT法和流式细胞术检测转染后细胞增殖情况;(3)建立裸鼠乳腺癌模型,使用尾静脉注射法,分别将0.5 mL HSA-PEI-pcD NA-Apoptin、HSA-PEI-pcD NA及生理盐水注射入裸鼠体内,4周后测量肿瘤体积。结果与结论:(1)成功构建并鉴定了HSA-PEI-pcDNA-Apoptin复合物,体外成功转染乳腺癌MCF-7细胞;(2)HSA-PEI-pcDNA-Apoptin转染MCF-7细胞24 h有Apoptin蛋白的表达,而转染HSA-PEI-pcD NA细胞及空白对照组细胞未见Apoptin蛋白的表达;(3)体外细胞增殖实验提示HSA-PEI-pcD NA-Apoptin组吸光度值明显低于HSA-PEI-pcDNA组及空白对照组(P<0.05);(4)裸鼠乳腺癌模型HSA-PEI-pcDNA-Apoptin组肿瘤体积小于HSA-PEI-pcDNA组及空白对照组(P<0.05);(5)结果表明,HSA-PEI-pcDNA-Apoptin复合物有明显的抑制肿瘤作用。
BACKGROUND: Apoptin is known to induce apoptosis of more than 50 kinds of tumor cells. However, efficient systems are required to deliver apoptin to the cancer cells for clinical use. OBJECTIVE: To construct human serum albumin(HSA) and Apoptin complex and transfect it to cancer cells in vitro and in vivo so as to find an efficient approach for apoptin delivery. METHODS: Polyethylenimine was used to react with N-hydroxysuccinimide(NHS)-HSA solution to synthesized HSA-PEI, and then react with pcDNA-Apoptin to construct recombinant HSA-PEI-pcDNA-Apoptin. HSA-PEI-pcDNA-Apoptin complex was transformed into MCF-7 breast cancer cell lines. Then the expression of apoptin was detected by western blot assay and the cell proliferation was detected by MTT assay and flow cytometry. The MCF-7 breast cancer cell xenograft model was used to detect the in vivo performance of HSA-PEI-pcDNA-Apoptin by measuring the tumor volume at 4 weeks, with saline and HSA-PEI-pcDNA as controls. RESULTS AND CONCLUSION:(1) We successfully prepared and confirmed the construction of HSA-PEI-pcDNA-Apoptin complex, which was successfully transformed into MCF-7 cells.(2) MCF-7 cells could express apoptin in the HSA-PEI-pcDNA-Apoptin group at 24 hours, but neither HSA-PEI-pcDNA group nor blank control group could express apoptin.(3) MTT assay for cell viability showed that HSA-PEI-pcDNA-Apoptin group had significantly lower optical density value than that in the other two groups(P 〈0.05).(4) The tumor volume in the HSA-PEI-pcDNA-Apoptin group was significantly less than that in the other two groups(P 〈0.05).(5) These findings indicate that the HSA-PEI-pcDNA-Apoptin complex markedly inhibits tumor growth in vitro and in vivo.
出处
《中国组织工程研究》
CAS
北大核心
2016年第49期7425-7430,共6页
Chinese Journal of Tissue Engineering Research
基金
陕西省自然科学基金面上项目(2013JM3010)~~