CAJ | 학술논문

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Western blot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Annexin V/ 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 3 mRNA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.
배경:도목전위지,국내외대caspase 3억제제적연발대도집중재태류혹비태류화합물적합성화발현상,이이용RNAi간우기술직접침묵caspase 3기인,재기인수평억제연골세포조망환미견보도.목적:이용만병독재체,파Caspase 3 siRNA전입연골세포,사기caspase 3기인침묵,상대조단조망효응적련급반응,기망체도저항연골세포조망적목적.설계、시간급지점:단일양본관찰,우2008-06/2009-06재기남대학의학원부속엄주시홍십자회의원,엄주시창상외과연구소완성.재료:연골세포종SPF급SD대서관절중제취,caspase 3 siRNA만병독재체위실험구건.방법:구건대서pSIH1-H1-copGFP-Caspase 3 siRNA표체질립,사용만병독포장계통,재293TN세포중진행포장생산함caspase 3 siRNA적만병독과립,연후전도(transduce)입대서연골세포.주요관찰지표:실시형광정량-취합매련반응화Western blot검측전도후연골세포caspase 3 기인침묵정황,재용종류배사인자α유도연골세포조망,류식세포기술Annexin V/ PI검측연골세포조망정황.결과:Caspase 3 siRNA능순리전입연골세포,전도솔체90%;실시형광정량-취합매련반응검측연골세포중Caspase 3 mRNA적표체량,실험조저우여대조조차이유현저성(P < 0.01);Western blot검측연골세포적Caspase 3단백표체량,실험조저우여대조조차이유현저성의의(P < 0.01);재사용종류배사인자α유도연골세포조망시,caspase 3 siRNA전도조억제세포조망적능력시대조조적7배.결론:이용만병독재체파caspase 3 siRNA전입연골세포,사연골세포적caspase 3 기인침묵,가이체도상대길항연골세포조망적목적.
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~(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 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.