【摘要】 背景:到目前为止,国内外对caspase 3抑制剂的研发大都集中在肽类或非肽类化合物的合成和发现上,而利用RNAi干扰技术直接沉默caspase 3基因,在基因水平抑制软骨细胞凋亡还未见报道。目的:利用慢病毒载体,把Caspase 3 siRNA转入软骨细胞,使其caspase 3基因沉默,相对阻断凋亡效应的联级反应,期望达到抵抗软骨细胞凋亡的目的。设计、时间及地点:单一样本观察,于2008-06/2009-06在暨南大学医学院附属广州市红十字会医院,广州市创伤外科研究所完成。材料:软骨细胞从SPF级SD大鼠关节中提取,caspase 3 siRNA慢病毒载体为实验构建。方法:构建大鼠pSIH1-H1-copGFP-Caspase 3 siRNA表达质粒,使用慢病毒包装系统,在293TN细胞中进行包装生产含caspase 3 siRNA的慢病毒颗粒,然后转导(transduce)入大鼠软骨细胞。主要观察指标:实时荧光定量-聚合酶链反应和Westernblot检测转导后软骨细胞caspase 3基因沉默情况,再用肿瘤坏死因子α诱导软骨细胞凋亡,流式细胞技术AnnexinV/PI检测软骨细胞凋亡情况。结果:Caspase 3 siRNA能顺利转入软骨细胞,转导率达90%;实时荧光定量-聚合酶链反应检测软骨细胞中Caspase 3mRNA的表达量,实验组低于与对照组差异有显著性(P<0.01);Western blot检测软骨细胞的Caspase 3蛋白表达量,实验组低于与对照组差异有显著性意义(P<0.01);在使用肿瘤坏死因子α诱导软骨细胞凋亡时,caspase 3 siRNA转导组抑制细胞凋亡的能力是对照组的7倍。结论:利用慢病毒载体把caspase 3 siRNA转入软骨细胞,使软骨细胞的caspase 3基因沉默,可以达到相对拮抗软骨细胞凋亡的目的。更多还原

【Abstract】 BACKGROUND:Up to date,studies concerning capspase 3 inhibitor mainly focus on peptide/non-peptide compounds synthesis and detection.Few reports addressing inhibits chondrocytes apoptosis using silenced caspase 3 gene.OBJECTIVE:To inhibit apoptosis of chondrocytes by blocking the apoptotic cascade reaction,gene silencing of caspase 3,and transduction of caspase 3 siRNA into chondrocytes with lentivirus vector.DESIGN,TIME AND SETTING:A single sample observation was performed at the Institute of Traumatic Surgery,Guangzhou Red Cross Hospital,Medical College of Jinan University from June 2008 to June 2009.MATERIALS:Chondrocytes were harvested from SD rats,and caspase 3 shRNA plasimid was constructed by our laboratory.METHODS:Rattus caspase 3 siRNA was synthesized and cloned into pSIH1-H1-copGFP plasmid.pSIH1-H1-copGFP-caspase 3 siRNA lentivirus was generated in 293TN cells by pPACKH1? Lentivector Packaging Kit and transducted into chondrocytes of rats.MAIN OUTCOME MEASURES:After the lentivirus was transducted into chondrocytes,the caspase 3 mRNA was tested by RT-PCR and the caspase 3 protein was tested by Western blot.Both the transducted cells and untransducted cells were induced apoptosis by tumor necrosis factor α (TNF-α).Cell apoptosis was assessed by flow cytometry,Annevin V/PI.RESULTS:The transduction rate of caspase 3 siRNA was about 90% by lentivirus vector.The expression of caspase 3 mRNA and caspase 3 protein in transducted chondrocytes was lower than the normal chondrocytes (P < 0.01).When the cells induced apoptosis by TNF-α,the apoptosis rate of the negative siRNA-chondrocytes was 7 times higher than that of caspase 3 siRNA-chondrocytes.CONCLUSION:The caspase 3 siRNA could inhibit caspase 3 expression and decrease drug-induced apoptosis of the chondrocytes.更多还原