转染CD40L基因对结肠癌细胞增殖和侵袭的影响

Influence of transfection of CD40L gene on cell proliferation and invasion of colon cancer cells

目的:本研究通过将CD40L基因转染小鼠结肠癌colon26细胞,观察其对小鼠结肠癌细胞生物学特性的影响,以期探讨CD40L作为结肠癌基因治疗靶点的可行性。方法:经脂质体将CD40L基因转染小鼠结肠癌colon26细胞后,采用半定量RT-PCR法、Western印迹法和激光共聚焦显微镜检测CD40L mRNA及蛋白的表达情况,新型四氮唑盐MTS比色法检测CD40L基因转染对细胞生长的影响,FCM法检测其对细胞表面分子表达的影响,半定量RT-PCR法检测细胞中基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)mRNA的表达情况,以及Transwell法检测细胞的侵袭能力变化。结果:RT-PCR检测结果显示,转染CD40L基因的小鼠结肠癌colon26细胞中CD40L mRNA的表达量为1.09±0.12,与转染空质粒细胞和未转染细胞相比,分别上调了59.21%和58.33%,差异有统计学意义(P<0.05);而基因转染后colon26/CD40L细胞中MMP-2 mRNA水平降低。MTS法检测结果显示,colon26/CD40L细胞的生长速度与未转染细胞无明显变化。FCM法检测结果显示,colon26/CD40L细胞表面共刺激分子CD80和CD86水平升高。同时,Transwell实验结果显示,colon26/CD40L细胞侵袭能力下降(P<0.05)。结论:CD40L基因提高了结肠癌colon26细胞的免疫原性,降低了结肠癌细胞的侵袭能力,抑制了结肠癌细胞的恶性表型。

Objective:CD40/CD40 ligand(CD40L) interaction plays an essential role in cell-mediated anti-tumor immune response.However,the role of CD40L in mouse colon cancer gene therapy is not clear.We aimed to investigate the impacts of CD40L gene on the biological features such as proliferation and invasion capabilities of colon26 cells and discuss the feasibility of using CD40L as a molecular target for colon cancer gene therapy.Methods:The pMKITneo-CD40L plasmids were transfected into mouse colon26 cells mediated by LipofactamineTM2000.The mRNA transcription of CD40L was analyzed by RT-PCR.Western blot and laser scanning confocal microscopy were used to detect the CD40L protein expression.MTS method was used to determine the effect of CD40L gene transfection on cell growth;the phenotype of CD40L cells was assessed using flow cytometry;the expression of matrix metalloproteinase 2(MMP-2) mRNA was analyzed by RT-PCR;the invasion of cells was detected with Transwell chamber invasive assay.Results:RT-PCR indicated that the level of CD40L mRNA expression was(1.09±0.12) after transfection with CD40L gene,which was increased by 59.21% and 58.33% compared with the cells transfected with empty vector(0.69±0.08) and the non-transfection group(0.69±0.11,P<0.05).The level of MMP-2 mRNA was significantly decreased in colon26/CD40L cells.MTS showed that there was no significant difference in the growth speed between colon26/CD40L cells and parent colon26 cells.The expression levels of CD80 and CD86 were higher in colon26/CD40L cells than those on parent cells.The result of Transwell assays suggested that the invasion ability of colon26/CD40L cells decreased significantly(P<0.05).Conclusion:The immunogenicity of colon26/CD40L cells are enhanced and the invasion capability is decreased.The malignant phenotype of colon26 cells is inhibited after CD40L gene transfection.