改良消减杂交法克隆大鼠脊髓损伤修复相关基因

Rat spinal cord injury-related genes cloned by improved subtractive hybridization

目的 克隆大鼠脊髓损伤修复相关基因,从分子水平了解中枢神经系统损伤修复的机制。方法 建立大鼠脊髓损伤模型,运用基于聚合酶链反应(PCR)的改良消减杂交技术,克隆与脊髓损伤修复相关的基因。结果 随机选取110个克隆进行研究,排除假阳性克隆和重复序列后,筛选到47个差异表达序列,其中39个已知序列,8个未知序列。39个已知序列中,包括synuclein、clusterin等与神经系统发育及退行性病变密切相关的蛋白编码序列;8个未知序列中,69号末端含有Leu拉链结构,包含该序列的基因可能编码DNA结合蛋白。结论 运用基于PCR的改良消减杂交技术成功克隆到损伤脊髓上调表达的基因序列,为探讨中枢神经系统损伤修复的分子机制奠定了基础。

Objective To clone regeneration-related genes after injury of rat spinal cord, and further to investigate the mechanism of CNS regeneration in molecular level, so as to obtain references for curing paralysis caused by spinal cord injury. Methods A rat spinal cord injury model was constructed, then the rat spinal cord regeneration-related genes after injury were obtained by using improved subtractive hybridization. Results Among more than 100 isolated fragments, approximately half were determined to identify the known sequences, and only 8 did not exhibit homology to any known sequences. Some known sequences, such as Gaa, Synuclein, Vimentin and so on had already been reported to be closely related with CNS injury or nerve degenerative diseases. One of the novel sequences (69 ) is supposed to code for a DNA binding protein. Conclusions Regeneration-related genes can be successfully cloned by improved subtractive hybridization. Characterization of these differentially expressed genes should provide useful information in understanding the molecular mechanism of CNS regeneration.