碱性成纤维细胞生长因子小分子干扰RNA对胶质瘤细胞株U251增殖与凋亡的影响

Effects of Small Interfering RNA Targeting Basic Fibroblast Growth Factor on Proliferation and Apoptosis of Glioma Cell Line U251

背景与目的:碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)是成纤维细胞生长因子(fibroblast growth factors,FGFs)家族中的重要一员,bFGF是多功能细胞因子,参与创伤愈合、组织修复、未成熟神经细胞的增殖和分化过程。本课题组前期对bFGF在胶质瘤中的表达做过研究,证实bFGF在胶质瘤细胞中过表达,并刺激胶质瘤细胞的增殖和新生血管生成。本研究检测bFGF小分子干扰RNA对胶质瘤细胞的增殖和凋亡的影响。方法:用化学法合成四条针对bFGF的siRNA和一条阴性对照siRNA,并用脂质体法转染胶质瘤细胞系U251;以Opti-MEMI无血清培养基培养的U251细胞作为正常对照。通过RT-PCR和免疫组化的方法检测bFGF的表达,并用流式细胞术、MTT法检测转染后U251细胞的凋亡和增殖活性的变化。结果:转染48h后,正常对照、阴性对照、siRNA-1、siRNA-2、siRNA-3和siRNA-4组U251细胞中的bFGFmRNA水平分别为0.95±0.02、0.95±0.02、0.85±0.02、0.76±0.04、0.65±0.04和0.56±0.03;转染72h后,分别为0.95±0.04、0.89±0.05、0.81±0.05、0.80±0.05、0.77±0.04和0.53±0.05。四条bFGFsiRNA中,siRNA-4作用最显著。转染48h后,siRNA-4和阴性对照组细胞增殖抑制率分别为(66.4±1.2)%和(56.3±1.4)%;转染72h后,分别为(40.0±2.6)%和(14.7±0.6)%,siRNA-4与阴性对照组相比差异具有统计学意义(P<0.05)。结论:理想的bFGF小分子干扰RNA能够抑制该基因的表达并抑制胶质瘤细胞的增殖活性。

BACKGROUND ＆ OBJECTIVE： Basic fibroblast growth factor （bFGF）, a member of the fibroblast growth factor （FGF） family, is a multifunctional cytokine participating in the process of wound healing, tissue repairing, proliferation and differentiation of immature neural cells. We previously found that bFGF is overexpressed in glioma cells, and could stimulate the vascularization and cell proliferation of glioma. This study was to explore the effects of bFGF small interfering RNA （siRNA） on the proliferation and apoptosis of glioma cell line U251. METHODS： Four bFGF siRNAs （siRNA1-4） and one random siRNA were synthesized using chemical method and transfected into U251 cells. The U251 cells cultured with Opti-MEM Ⅰ （no serum media） were used as normal control. The expression of bFGF in transfected U251 cells was detected by reverse transcription-polymerase chain reaction （RT-PCR） and SP immunohistochemistry. Cell proliferation was detected by MTT assay; cell apoptosis was detected by flow cytometry. RESULTS： At 48 h after transfection, the mRNA levels of bFGF in U251 cells were 0.95±0.02 in normal control group, 0.95±0.02 in negative control group, 0.85±0.02 in siRNA-1 group, 0.76±0.04 in siRNA-2 group, 0.65±0.04 in siRNA- 3 group, and 0.56±0.03 in siRNA-4 group; at 72 h after transfection, the mRNA levels of bFGF were 0.95±0.04, 0.84±0.05, 0.81±0.05, 0.80±0.05, 0.77±0.04, and 0.53±0.05, respectively. Among the 4 siRNAs, siRNA-4 showed the strongest effect. The proliferation inhibition rate of U251 cells was significantly higher in siRNA-4 group than in negative control group [（66.39± 1.23）% vs. （56.3±1.45）% at 48 h after transfection, （40±2.6）% vs. （14.74± 0.62）% at 72 h, P 〈0.05]. CONCLUSION： The optimal bFGF siRNA can inhibit bFGF expression and proliferation of glioma U251 cells.