Caspase 3 siRNA inhibits chondrocytes apoptosis / 中国组织工程研究

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~(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.