摘要
目的 构建人肝癌细胞中高效特异表达的反义c-fms真核表达载体，观察其对肝癌细胞生物学行为的影响。 方法 采用PCR法扩增人c-fms癌基因第571位酪氨酸为中心的DNA片段，将其反向克隆入pcDNA3载体（命名pAS）；将扩增的人甲胎蛋白（AFP）增强子核心区克隆入pAS（命名pAEAS）。磷酸钙法将空载体pcDNA3及反义真核表达载体pAS、pAEAS分别转导入HepG2肝癌细胞及HeLa宫颈癌细胞，观察细胞生长速度及凋亡。 结果 人反义c-fms基因片段及AFP增强子核心区片段，测序结果与Genbank中登录的序列一致。导入反义基因的HepG2肝癌细胞生长速度较对照细胞明显减慢(P＜0.05)，pAEAS抑制作用较pAS强(P＜0.05），pcDNA3组、pAS组、pAEAS组HepG2肝癌细胞凋亡率分别为5.25%、14.7%、31.2%（P＜0.01），pAEAS组细胞DNA出现梯状凋亡带。在HeLa宫颈癌细胞中，pAS及pAEAS组生长速度减慢，但二者差异无显著性（P＞0.05），pcDNA3组、pAS组、pAEAS组细胞凋亡率分别为3.99%、8.27%、8.86%（P＜0.05），DNA均未出现梯状凋亡带。 结论 携带AFP增强子的人反义c-fms真核表达载体对AFP阳性肝癌细胞生长有选择性抑制作用，可诱导凋亡，是一种新的肝癌基因治疗方法。
Objective To construct human c-fms antisense eukaryotic expressing vector that would express in human hepatoma cells efficiently and specifically and to observe its affection on biological behavior of hepatoma cells. Methods A c-fms fragment centered by TAC sequence coding for 571st tyrosine of CSF-1R was amplified with PCR. The fragment was cloned inversely into pcDNA3 vector (constructed plasmid was named as 'pAS'. Amplified human AFP enhancer core region fragment was cloned into pAS (constructed plasmid was named as pAEAS). pcDNA3, pAS and pAEAS were transfected into HepG2 hepatoma cell line and HeLa cervical carcinoma cell line with calcium phosphate method, respectively. Growth rate and apoptosis of transfected cells were observed. Results Detected sequences of human c-fms antisense fragment and AFP enhancer core region fragment were both consistent with the sequences registered in Genbank. The growth rates of HepG2 cells transfected with antisense gene were slower than control (P<0.05). The inhibitory effect of pAEAS was more remarkable than that of pAS (P<0.05). Apoptoic rates of pcDNA3, pAS and pAEAS group cells in HepG2 cells were 5.25%, 14.7% and 31.2%, respectively (P<0.01). Apoptoic DNA ladder was observed in pAEAS group. In HeLa cell groups, growth rates of cells in pAS and pAEAS groups were slower than control, while no difference was observed between them (P>0.05). Apoptoic rates of pcDNA3, pAS and pAEAS group cells in HeLa cells were 3.99%, 8.27% and 8.66%, respectively (P<0.05). No apoptoic DNA ladder was observed in HeLa cells. Conclusions Human c-fms antisense eukaryotic expressing vector bearing AFP enhancer selectively inhibit the growth of AFP positive hepatoma cells. It enables to induce apoptosis of hepatoma cells and is a new gene therapy method for hepatocellular carcinoma. [
出处
《中华肝脏病杂志》
CAS
CSCD
2001年第5期297-299,共3页
Chinese Journal of Hepatology
基金
广东省自然科学基金(990422)
