人成骨样细胞机械牵拉力敏感基因cDNA扣除文库的构建

Substractive cDNA library construction of genes sensitive to mechanical stretch in human osteoblast like cells

目的 :构建人成骨样细胞机械牵拉力敏感基因cDNA扣除文库。方法 :对培养的人成骨样细胞Saos -2进行二维方向上的加载 ,细胞平均变形幅度为 12 % ,牵拉频率为 12次 /min ,加载 12h后提取应力作用后细胞及正常对照组细胞的mRNA ,制备各自的cDNA ,用基于PCR的消减杂交技术构建人成骨样细胞机械牵拉力作用下差异表达基因文库 ,即机械牵拉力敏感基因cDNA扣除文库 ,并进行初步的序列测定。结果 :成功地构建出人成骨样细胞机械牵拉力敏感基因cDNA文库 ,库容量约为 2 0 0 ;初步的测序结果表明文库内的基因多与应力作用下细胞发生的生物学变化有关 ,并成功地得到一个新的基因片段。结论

Objective: To reconstruct a substractive complementary deoxyribornucleic acid (cDNA) library of genes sensitive to mechanical stretch in human osteoblast like cells.Methods: Mechanical stretch at 12 cycles per minute was applied to human osteoblast like cells Saos-2 and the deformation of the stretched cells was 12%. Twelve hours after loading, mRNAs were isolated from both stretched and unstretched cells. Substractive cDNA library of the genes sensitive to stretch was constructed with the technique based on polymerase chain reaction (PCR) and substractive hybridization. Primary sequencing of clones in the library was carried out. Results: A substractive cDNA library of genes sensitive to stretch was constructed with a capacity of about 200 clones. According to the results of sequencing, most genes in the library were related to the mechanical stimulation. One novel gene fragment was obtained. Conclusion: The method used in the experiment is effective in cloning genes sensitive to mechanical stretch.