构建HSA-PEI-pcDNA-Apoptin复合物及对乳腺癌细胞增殖的影响

Preparation of HSA-PEI-pcDNA-Apoptin and its effect on the proliferation of breast cancer MCF-7 cells

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.