结缔组织生长因子反义寡核苷酸对胰腺癌细胞基质金属蛋白酶-9、血管内皮生长因子表达及细胞增殖与侵袭的影响

Effect of CTGF antisense oligodeoxynucleotide on the expression of MMP-9,VEGF and proliferation,invasion in pancreatic cancer cells

目的通过研究结缔组织生长因子(CTGF)反义寡核苷酸(ASODN)对人胰腺癌细胞基质金属蛋白酶-9(MMP-9)、血管内皮生长因子(VEGF)表达及细胞增殖与侵袭力的影响,深入探讨CTGF在胰腺癌发生、发展中的作用机制,为CTGFASODN在胰腺癌基因治疗中的运用奠定理论基础。方法经脂质体介导将CTGFASODN转染人胰腺癌细胞株SW1990,荧光显微镜及流式细胞仪检测转染效率;逆转录-聚合酶链反应(RT-PCR)及Westernblot检测转染细胞CTGF、MMP-9与VEGFmRNA与蛋白表达;噻唑盐(MTT)比色法检测转染细胞的增殖活性;采用Transwell小室检测细胞侵袭能力。结果 (1)荧光显微镜及流式细胞仪检测显示CTGFASODN可成功转染SW1990细胞。(2)反义组、无义组和单加脂质体组细胞CTGFmRNA表达量分别为0.24±0.09、0.67±0.21、0.63±0.18,反义组较其他组的mRNA表达明显减少(P<0.01);相应的CTGF蛋白表达量分别为0.19±0.07、0.75±0.26、0.71±0.23;相应的MMP-9mRNA表达量分别为0.21±0.12、0.78±0.35、0.81±0.37;相应的VEGFmRNA表达量分别为0.16±0.06、0.42±0.22、0.43±0.28;相应的MMP-9蛋白表达量分别为0.68±0.22、1.97±0.46、1.92±0.39;相应的VEGF蛋白表达量分别为0.27±0.07、0.52±0.19、0.55±0.18,反义组CTGF、MMP-9和VEGF的表达较其他组显著降低(P<0.01)。(3)反义组、无义组、单加脂质体组细胞增殖活性分别为1.67±0.14、2.34±0.17、2.29±0.21,反义组较其他组细胞增殖被显著抑制(P<0.01)。(4)反义组、无义组、单加脂质体组微孔滤膜外侧细胞数分别为24.88±6.17、52.37±8.62、55.49±8.83,反义组较其他组微孔滤膜外侧细胞数显著减少(P<0.01)。结论 CTGFASODN转染可有效抑制SW1990细胞CTGF的表达,并降低其MMP-9、VEGF的表达及细胞增殖活性与细胞侵袭力。

Objective To investigate the effect of connective tissue growth factor antisense oligodeoxynucleotide(CTGF ASODN)on proliferation and invasion in human pancreatic cancer cell line SW1990,with a focus on the expression of matrix metalloproteinase-9(MMP-9)and vascular endothelial growth factor(VEGF).This study was designed to explore the underlying mechanism for the role of CTGF in the development of pancreatic cancer and the therapeutic value of gene therapy.Methods CTGF ASODN,nonsense oligodeoxynucleotide(NSODN)were transfected into SW1990 cells through lipofectamine.The transfection rate was accessed by fluorescence microscopy and flow cytometry.The expressions of CTGF,MMP-9 and VEGF in different groups of transfected cells were detected by RT-PCR and Western blot.Cell viability was assessed by MTT method.Results (1)Fluorescence microscopy and flow cytometry showed a successful transfection of CTGF ASODN in SW1990 cells.(2)The mRNA levels of CTGF,MMP-9 and VEGF in CTGF ASODN,NSODN and control groups were:CTGF 0.24±0.09,0.67±0.21,0.63±0.18;MMP-9 0.21±0.12,0.78±0.35,0.81±0.37;VEGF 0.16±0.06,0.42±0.22,0.43±0.28.Accordingly,protein levels were:CTGF 0.19±0.07,0.75±0.26,0.71±0.23;MMP-9 0.68±0.22,1.97±0.46,1.92±0.39;VEGF 0.27±0.07,0.52±0.19,0.55±0.18.The expressions of mRNA and protein of CTGF,MMP-9 and VEGF were significantly reduced in CTGF ASODN transfected group,compared with other two groups(P<0.01).(3)The A490 value of cells in CTGF ASODN,NSODN and control groups were 1.67±0.14,2.34±0.17,2.29±0.21.Proliferation of SW1990 cells was significantly decreased in CTGF ASODN transfected group,compared with other two groups(P<0.01).(4)The cells that invaded through the basement membrane filter in CTGF ASODN,NSODN and control groups were 24.88±6.17,52.37±8.62,55.49±8.83,the migration cells were significantly decreased in CTGF ASODN transfected group,compared with other two groups(P<0.01).Conclusions CTGF ASODN could effectively down-regulate the expressions of CTGF,MMP-9,VEGF and inhibit proliferation,invasion of SW1990 cells.