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背景:转化生长因子β1(transforminggrowthfactorβ1,TGFβ1)能通过自分泌和旁分泌机制促进肿瘤细胞的生长,最近有研究表明反义TGFβ1基因转染骨肉瘤细胞后可以降低肿瘤细胞的增殖活性。目的:观察反义TGFβ1基因对骨肉瘤细胞恶性表型的逆转作用,探讨其预防骨肉瘤的发生、发展的意义。设计:完全随机设计,对照实验研究。地点和材料:研究地点为华中科技大学同济医学院附属协和医院骨科实验室。TaqDNA聚合酶、TRIzolTM试剂、SuperscriptPreamplificationSystem、胎牛血清、DMEM培养基、Lipofectamine、无血清培养基、G418、PCR引物、反义TGFβ1表达质粒pcDNA3-TGFβ1(-)、人骨肉瘤细胞系MG-63、Balb/c裸鼠、流式细胞仪、CO2培养箱、相差显微镜。方法:将反义TGFβ1基因转染骨肉瘤细胞MG-63后,检测细胞周期、细胞凋亡和明胶酶A表达,并观察转染细胞软琼脂克隆形成数和裸鼠体内致瘤能力。主要观察指标:细胞周期和细胞凋亡检测;RT-PCR检测MMP-2基因表达;软琼脂培养克隆形成实验;裸鼠体内成瘤性的检测。结果:与MG-63和空载体转染细胞MG-pcDNA3相比,反义基因转染细胞MG-TGFβ1(-)的G0/G1期细胞从56.2%和60.1%增至71.6%,S期细胞从19.1%和17.8%降至12.9%,MMP-2mRNA表达明显减少。MG-TGFβ1(-)的软琼脂克隆形成数从48.
BACKGROUND: Transforming growth factor β1 (TGFβ1) can promote the growth of tumor cells through autocrine and paracrine mechanisms. Recently, it has been reported that the transfection of antisense TGFβ1 gene into osteosarcoma cells can reduce the proliferation activity of tumor cells. OBJECTIVE: To observe the reversal effect of antisense TGFβ1 gene on the malignant phenotype of osteosarcoma and to explore its significance in preventing the occurrence and development of osteosarcoma. Design: Completely randomized design, controlled experimental study. Location and Materials: The research site was the Orthopedic Laboratory of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. Taq DNA polymerase, TRIzolTM reagent, SuperscriptPreamplificationSystem, fetal bovine serum, DMEM medium, Lipofectamine, serum-free medium, G418, PCR primers, antisense TGFβ1 expression plasmid pcDNA3-TGFβ1 (- / c nude mice, flow cytometry, CO2 incubator, phase contrast microscope. Methods: The antisense TGFβ1 gene was transfected into osteosarcoma cells MG-63, and the cell cycle, apoptosis and gelatinase A expression were detected. The numbers of soft agar colony formation and tumorigenicity in nude mice were observed. MAIN OUTCOME MEASURES: Cell cycle and apoptosis assay; RT-PCR detection of MMP-2 gene expression; soft agar culture clonogenic assay; nude mice in vivo tumor detection. RESULTS: Compared with MG-63 and MG-63 cells transfected with empty vector, the number of cells in G0 / G1 phase of MG-TGFβ1 (-) transfected with antisense gene increased from 56.2% and 60.1% to 71.6% From 19.1% and 17.8% to 12.9%, MMP-2 mRNA expression was significantly reduced. Soft agar colony formation numbers of MG-TGFβ1 (-) ranged from 48.