FasLmRNA在椎间盘细胞中表达分布.docVIP

FasL mRNA在椎间盘细胞中表达的分布 　　作者：司春祥,陈伯华,吕振华　　作者单位：胜利石油管理局临盘医院骨科，山东 临邑 251507;青岛大学医学院附属医院骨科 　　【摘要】目的 观察凋亡相关基因FasL mRNA在椎间盘细胞中表达的分布。方法 收集胎儿及成人病理腰椎间盘髓核标本，PHAP激活单个核细胞诱导FasL表达，提取总RNA，RTPCR制备FasL基因片段。定向克隆入质粒载体pSPT19多克隆位点构建重组质粒，体外转录制备DigFasL cRNA探针。cRNA探针原位杂交进行FasL mRNA表达分布的观察。结果 克隆化FasL序列经DNA测序准确无误，体外转录cRNA探针标记效果满意;胎儿腰椎间盘组织脊索细胞、软骨样细胞中可检测到FasL mRNA的较高杂交信号，成人突出椎间盘细胞中罕见正常细胞，未检出FasL mRNA信号。结论 FasL基因在胎儿期腰椎间盘细胞内表达，细胞凋亡发生早。成年时期，细胞数急剧减少，代谢功能低下，无法检出凋亡基因表达。 　　【关键词】 基因 FasL RNA 互补 椎间盘 原位杂交 　　THE DISTRIBUTION OF Fasl mRNA IN HUMAN LUMBAR DISC TISSUE　SI CHUNXIANG, CHEN BOHUA, L ZHENHUA　(Department of Orthopaedics, Shengli Oil Field Linpan Hospital, Linyi 251507, China); 　　[ABSTRACT] Objective To observe the distribution of FasL mRNA in intervertebral disc. Methods Nucleus gelatinosus specimens were collected from fetus and pathologic disc. PBMC was treated by PHAP, and total RNA extracted from the actived PBMC. A fragment of FasL gene was reversely transcripted and amplified from total RNA using onestep RTPCR. PCR product was then inserted into the MCS of pSPT19. After verification of DNA sequencing, the recombinant plasmid pSPT19 was linearized to synthesize the digcRNA probe in vitro. The distribution of FasL mRNA was observed on lumbar intervertebral disc specimens using in situ hybridization. Results The inserted FasL cDNA fragment in pSPT19FasL was identified by DNA sequencing. The label efficiency of cRNA probe was satisfied; The FasL mRNA signals were emerged in both notochord cells and chondrocytelike cells in the nucleus of fetal discs. Conclusion The FasL mRNA was detected in the fetal lumbar intervertebral disc cells, and apoptosis occurred early. In adult, the quantity of intervertebral disc cells reduced, the metabolism of them decreased, and the expression of FasL mRNA could not be detected. 　　[KEY WORDS] Gene, FasL; RNA, complementary; Intervertebral disc; In situ hybridization 　　FasL基因是目前已知的最重要的凋亡调控基因之一，属于肿瘤坏死因子家族成员的凋亡促进因子，是Fas的配体。体内外多种