纳米粒子介导E1A基因体外转染人甲状腺未分化癌细胞HTC／3

E1A gene transfection of human undifferentiated thyroid cancer cell line HTC /3 by nanoparticles

目的探讨纳米粒子介导E1A基因转染人甲状腺未分化癌细胞HTC/3的可行性和效率,并检测转染细胞对X线的敏感性和X线诱导转染细胞凋亡。方法应用聚乳酸聚乙醇酸共聚物和聚乙烯醇包载E1A基因,制备纳米级粒子混合物,检测其包埋率、体外释放情况及粒径大小。用制备的包裹DNA纳米粒子转染人甲状腺未分化癌细胞HTC/3,并以阳离子脂质体为对照,用逆转录-聚合酶链反应(RT-PER)方法检测转染细胞(HTC/3-E1A)的E1A基因mRNA表达。四甲基偶氮唑盐(MTT)法检测HTC/3-E1A细胞、对照细胞的体外生长率和不同剂量X射线对HTC/3-E1A细胞的生长抑制率。HTC/3-E1A细胞经一定剂量X线(2 Gy)照射后,用流式细胞仪进行细胞周期分析,DNA电泳观察细胞凋亡。结果制备的纳米粒子直径为150-280 nm,包埋率为0.78%,体外释放约为18 d。在转染相等质量的DNA情况下,纳米组所得克隆数多于脂质体组(P<0.05)。RT-PCR结果表明,纳米粒子和脂质体转染细胞均有E1A基因mRNA表达。HTC/3-E1A细胞群体生长缓慢,倍增时间延长(是亲本细胞的1.44倍)。HTC/3-E1A细胞对X线敏感性较转染空载体细胞(HTC/3-Vect)和HTC/3细胞分别提高约2.9倍和2.8倍。流式细胞仪分析显示,HTC/3-E1A细胞亚G0/G1期比例显著高于HTC/3-Vect和HTC/3细胞(P均<0.01),S期比例显著低于HTC/3-Vect细胞和HTC/3细胞(P均<0.01)。DNA电泳显示,HTC/3-E1A细胞出现典型的凋亡梯度变化,而HTC/3-Vect和HTC/3细胞无此变化。结论纳米粒子可携带外源基因进行基因转染并获得表达,转E1A基因HTC/3细胞对X线敏感性明显提高,且X线诱导了转E1A基因HTC/3细胞凋亡。

Objective To prepare nanoparticles containing E1A gene and observe the efficiency and feasibility of transfecting E1A gene into human undifferentiated thyroid cancer cell line HTC/3. To examine the sensitivity of transgene cells to X-ray and X-ray-induced apoptosis in those cells. Methods Nanoparticle-DNA complex was prepared with PLGA coating adenoviral early expression gene E1A, and the package efficiency, release progress in vitro, and size of the complex were determined. The nanoparticle-DNA was transfected into the HTC/3 cells. Iipofectamine was used to transfect E1A gene as a control. RT-PCR was used to examine E1A gene mRNA expression in the transfected cells. The survival ratio of HTC/3-E1A and control cells, and the growth inhibition ratio induced by different doses of X-ray in HTC/3-E1A cells were examined by MTT assay. The apoptosis in HTC/3-E1A cells induced by 2 Gy X-ray iradiation was examined by flow cytometry and DNA electrophoresis. Results The package efficiency, release progress in vitro, and size of the nanoparticle-DNA complex were 0. 78% , 18 days, and 150-280 nm, respectively, when transfected the plasmid at the same level, the nanoparticle group got more positive transgene cell clones than that in Iipofectamine group, with a statistically significant difference （P〈0. 05）. RT-PCR showed that transgenic cells from both nanoparticle-DNA and lipofectamine groups had E1A gene mRNA expression. The HTC/3-El A cells grew slowly, and their doubling time was prolongated （1. 44 times in comparison with that in parental cells）. According to IC50 , the sensitivity of HTC/3-E1A cells to X-ray was improved 2.9 and 2. 8 times, respectively, in comparison with that in HTC/3-Vect and HTC/3 cells. The ratio of subG0/G1 phase of HTC/3-E1A cells was significantly higher than that in HTC/3-Vect and HTC/3 cells （P 〈 0. 01）, The ratio of S phase of HTC/3-E1A cells was significantly lower than that in HTC/3-Vect and HTC/3 cells （P 〈 0.01）. A typical DNA ladder pattern of apoptosis in HTC/3-E1A cells was observed by electrophoresis, but not found in HTC/3-Vect and HTC/3 cells. Conclusion A nanoparticle-DNA complex has been successfully prepared, and it may carry a foreign gene into cells. The sensitivity of HTC/3-E1A cells to X-ray is significantly improved. Moreover, apoptosis is induced by x-ray in the E1A gene-transfected cells.