Caspase 3 siRNA抑制关节软骨细胞的凋亡

Caspase 3 siRNA inhibits chondrocytes apoptosis

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。

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 cespase 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^TM 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 cespase 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 ceils 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.