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目的探讨转染血管内皮生长因子(VEGF)-C cDNA 对急性早幼粒细胞白血病细胞系NB4细胞增殖、全反式维甲酸(ATRA)诱导分化及化疗药物介导的细胞凋亡作用的影响。方法采用脂质体法将重组表达载体(pcDNA3.1-VEGF-C)和 pcDNA3.1转染 NB4细胞建立稳定细胞株;采用MTT 法及集落形成实验分析转染 VEGF-C cDNA 对 NB4细胞增殖的影响;NB4/VEGF-C 细胞经 ATRA诱导后涂片经瑞特-姬姆萨染色作形态学分析,实时荧光定量 PCR 检测调控粘细胞分化基因 C/EBPα相对表达水平,同时以流式细胞术检测细胞表面的 CD11b 表达量;Annexin V/PI 双染流式细胞术检测NB4/VEGF-C 细胞经鬼闩乙叉甙(Vp16)诱导的细胞凋亡;并以实时荧光定量 PCR 检测 bcl-2基因相对表达水平,均以 NB4/pcDNA3.1细胞为对照。结果成功转染获得稳定表达 VEGF-C 的细胞株 NB4/VEGF-C,其增殖能力显著高于 NB4/pcDNA3.1细胞;NB4/VEGF-C细胞对抗 ATRA 介导的早幼粒细胞分化成熟,CD11b 表达量低于对照组,C/EBPα基因含量仅为对照组的1/32;NB4/VEGF-C 细胞经Vp16介导后 bcl-2基因表达约为 NB4/pcDNA3.1细胞的2.28倍,而凋亡的 NB4/VEGF-C 细胞百分率(7.20±2.52)%明显低于凋亡的 NB4/pcDNA3.1细胞的(16.07±3.58)%(P=0.005)。结论 VEGF-C 通过 VEGF 受体3(VEGFR-3)信号途径促进白血病细胞增殖,抑制分化诱导剂介导的 NB4 细胞分化及化疗药物介导的细胞凋亡,推测 VEGF-C/VEGFR-3信号途径在白血病疾病进展中发挥重要作用并可望作为白血病治疗的靶点。
Objective To investigate the effect of vascular endothelial growth factor (VEGF) -C cDNA transfection on the proliferation of acute promyelocytic leukemia NB4 cells, the induction and differentiation of all-trans retinoic acid (ATRA) and the chemotherapeutic drug-induced apoptosis. Methods NB4 cells were transfected with pcDNA3.1-VEGF-C and pcDNA3.1 by lipofectamine. Stable cell lines were established by MTT assay and colony formation assay. . The morphology of NB4 / VEGF-C cells induced by ATRA was analyzed by Reiter-Giemsa staining, and the relative expression of C / EBPα was detected by real-time fluorescence quantitative PCR. The flow cytometry The expression of CD11b on the cell surface was detected by flow cytometry. The apoptosis of NB4 / VEGF-C cells induced by Vp16 was detected by Annexin V / PI double staining flow cytometry. The bcl- 2 gene relative expression levels were NB4 / pcDNA3.1 cells as a control. Results The NB4 / VEGF-C cell line stably expressing VEGF-C was successfully transfected with NB4 / VEGF-C and its proliferation ability was significantly higher than that of NB4 / pcDNA3.1 cells. NB4 / VEGF-C cells were resistant to ATRA-mediated promyelocytic differentiation and maturation, The expression of CD11b was lower than that of the control group, and the content of C / EBPα gene was only 1/32 of the control group. The expression of bcl-2 gene was 2.28 times that of NB4 / pcDNA3.1 cells after NB4 / VEGF-C cells were transduced with Vp16, However, the percentage of apoptotic NB4 / VEGF-C cells (7.20 ± 2.52)% was significantly lower than that of apoptotic NB4 / pcDNA3.1 cells (16.07 ± 3.58)% (P = 0.005). Conclusion VEGF-C can promote the proliferation of leukemic cells through the VEGFR-3 signaling pathway and inhibit the differentiation-induced NB4 cell differentiation and chemotherapeutic drug-induced apoptosis. It is speculated that VEGF-C / VEGFR-3 signal Pathway plays an important role in the progression of leukemia and is expected to be the target of leukemia treatment.