摘要
血管内皮生长因子(VEGF)是血管新生的中枢介质,在白血病的病理机制中具有重要作用,也是血液肿瘤患者一种独立的预后因素,但VEGF在白血病细胞分化或凋亡中的重要性尚待阐明。为了查明VEGF在白血病细胞分化或凋亡中的作用,构建了VEGF基因竞争模板(T-VEGFΔ)并建立竞争性定量逆转录聚合酶链反应(cQRT-PCR),以监测全反式维甲酸(ATRA)诱导NB4细胞分化过程中VEGF基因表达的改变;通过将系列稀释的靶分子与恒量的竞争模板共扩增,获得标准曲线,用于计算待测样本中靶基因的分子数;在不同时间点分别检测NB4细胞的CD11b抗原表达和NBT还原率。结果显示:cQRT-PCR可作为定量分析VEGF基因表达的有效工具,其测定范围约为1×104-2×105分子。NB4细胞经ATRA处理0、12、24和48小时后,其VEGF基因转录本的数量分别为42.3×105、12.6×105、3.6×105,低于1.0×105/μg总RNA,并伴有CD11b表达上调和NBT还原率增加。结论:成功地建立了cQRT-PCR方法,证明ATRA显著抑制VEGF表达并可能通过抑制血管新生发挥抗白血病的功效。
Vascular endothelial growth factor （VEGF）, a central mediator of angiogenesis, not only plays an important role in the pathogenesis of leukemia, but also is an independent prognostic factor in patients with hematologic malignancies, like those in solid tumors. However, the importance of VEGF during differentiation or apoptosis of leukemia cells remains to be elucidated. In order to assess the alternation of VEGF gene expression in the process of alltrans retinoic acid （ ATRA ）-induced differentiation of NIM acute promyelocytic leukemia cell line, and a competitor DNA fragment, VEGF gene competative template （T-VEGF△） was constructed by using gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction （cQRT-PCR） method was developed. A standard curve was obtained by co-amplification of serial dilutions of the target nulecules with constant amount of competitive template and this curve was used to detect molecular number of target gene in measuring sample.The surface expression of CD1 lb antigen and nitroblue tetrazolium （NBT） reduction rate of NB4 cells were also assayed at different time points. The results showed that cQRT-PCR was a sensitive, reliable tool for analysis of VEGF gene expression with a detectable range from 1 × 10^4 to 2 × 10^5 molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3 × 10%5, 12.6 × 10^5, 3.6 × 10^5, and less than 1.0 × 10^5/μg total RNA at 0,12, 24 and 48 hours after ATRA treatment, respectively. This rapid down-regulation of VEGF gene expression, during ATRA-induced NB4 cell differentiation, was accompanied by the up-regulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR method was successtully constructed, confirming that ATRA efficiently repressed VEGF, at the same time, the ATRA might exert an antileukemic effect, other than induction of differentiation via inhibition of angiogenesis.
出处
《中国实验血液学杂志》
CAS
CSCD
2005年第4期548-552,共5页
Journal of Experimental Hematology
基金
江苏省科技厅社会发展项目(BS2001070)
江苏省教育厅自然科学研究基金(02KJB320002)部分资助
