Caspase 3 siRNA inhibits chondrocytes apoptosis
10.3969/j.issn.1673-8225.2009.50.006
- VernacularTitle:Caspase 3 siRNA抑制关节软骨细胞的凋亡
- Author:
Chunting YE
;
Yaoxiong HUANG
;
Xiaohong YANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2009;13(50):9832-9836
- CountryChina
- Language:Chinese
-
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.
