抑制葡萄糖转运蛋白1表达对骨肉瘤MG63细胞体内外功能的影响

Inhibition of glucose transporter protein 1 in MG63 osteosarcoma cells effects cells function in vitro and in vivo

目的利用RNA干扰（RNAi）技术抑制骨肉瘤MG63细胞中葡萄糖转运蛋白（Glut-1）表达，观察对MG63细胞在体内外功能的影响。方法克隆Glut-1基因，通过慢病毒感染293T细胞，构建成干扰、原始及对照组的3种慢病毒载体后分别转染到人骨肉瘤MG63细胞，形成干扰、原始及对照3个稳转细胞株。应用反转录-聚合酶链反应（RT—PCR）技术鉴定细胞RNAi后Glut-1 mRNA的表达。噻唑蓝（MTT）检测RNAi后细胞增殖的改变；根据人工基底膜的细胞穿过数量检测RNAi后细胞侵袭功能改变；流式细胞术检测RNAi后细胞周期的改变。利用异种移植瘤模型观察Glut-1干扰后对肿瘤生长的影响。结果RNAi成功后，Glut-1的mRNA水平明显降低（P〈0．05）。干扰组细胞吸光度值的均值较原始组及对照组明显降低（P〈0．05）；细胞侵袭实验显示干扰组细胞穿过人工基底膜的数量明显减少（P〈0．05）；细胞周期中干扰组细胞G1相61．77％被阻断。裸鼠皮下成瘤实验表明，抑制Glut-1的表达可抑制肿瘤的生长。结论RNAi抑制MG63细胞Glut-1的表达，可通过糖代谢过程来发挥肿瘤抑制作用。

Objective To explore the influence of inhibition of Glucose transporter 1 （ Glut - 1 ） by RNA interference （RNAi） on tumorigenesis of human osteosarcoma cell line （MG63 cells） in vitro and in vivo. Methods Glut - 1 gene was cloned and 293T cells was infected by lentiviral to constructed interference, the original and blank control group of three lentiviral vector which were transfected into human osteosarcoma MG63 cells and the formation of three stable transfeeted cell lines including interference, original and blank control cell lines were build up. Reverse transcription - polymerase chain reaction （ RT - PCR） were taken to determine GLU - 1 levels of mRNA of siRNA - MG63 cells. Cell proliferation, cell invasion and cell cycle were studied to understand the various effects of osteosareoma cells of Glut - 1 - siRNA gene interference. Tumorigenesis was observed after transfected cells were injected subcutaneously in nude mice. Results After interference successfully, expression of Glut - 1 decreased significantly at mRNA level （P 〈 0. 05 ）. Absorbance at A490 nm demonstrate that transfection of siRNA - Glut - 1 decreased MG63cell proliferation over time compared with non - or mock - transfected cells （ P 〈 0.05 ）. Cell invasion experiment showed that the number of cells of siRNA - Glut - 1 infection of MG63 cells was significantly reduced through artificial basement membrane a normal and green fluorescent protein （GFP） MG63cells with 200 times more light microscopy after48 hours （P 〈 0.05 ）. Cells transfected with siRNA - Glut - 1 had an increased number of cells in the G1 phase （61.77%）. The growth rate of Glut - 1 inhibition tumors in subcutaneous xenograft model decreased drastically. Conclusion RNAi inhibits Glut- 1 expression. Reduced tumor growth by Glut - 1 inhibition may through inhibition glyeolysis metabolism.