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Effects of aggrecanase-2 shRNA transfection on chondrocytes of rheumatoid arthritis patient leaded by lentivirus / 中华骨科杂志
Chinese Journal of Orthopaedics ; (12): 936-944, 2014.
Artigo em Chinês | WPRIM | ID: wpr-455645
ABSTRACT
Objective To investigate the effects of aggrecanase-2 knockdown in chondrocytes from rheumatoid arthritis patient by shRNA infection.Methods Cartilage harvested from rheumatoid arthritis patients who underwent total knee arthroplasty was digested by pancreatin and type Ⅱ collagen enzyme to obtain chondrocytes.Then chondrocytes were cultured and passaged to second or third generation.After aggrecanase-2 shRNA5,aggrecanase-2 shRNA6,aggrecanase-2 shRNA7,aggrecanase-2 shRNA8 infection,growth and morphological changes of the chondrocytes were examined.To select the best target sequence,mRNA expression of aggrecanase-2 and aggrecan was detected by RT-qPCR assay on day 2,5,10,represented with Ct (Cycle threshold) value.Expression of aggrecan protein was detected by immunocytochemistry,represented with IOD (integral optical density) value.Results aggrecanase-2 knockdown had no obvious effects on the morphology and growth of the chondrocytes.MOI (multiplicity of infection) was 100,50,25,and infection efficiency was 90%,60%,30% with the corresponding viral load of 200 μl,100 μl,50 μl after transfection.mRNA expression of aggrecanase-2 was suppressed significantly,especially the group of shRNA5 which suppressed aggrecanase-2 expression from 0.876 3±0.115 6 to 0.069 9±0.015 1 (P < 0.05).mRNA and protein expression of aggrecan were significantly upregulated after infection.mRNA expression of aggrecan increased from 0.992 1±0.201 3 to 3.049 2±0.278 2 (P < 0.05) and protein expression of aggrecan increased from 496.160 5± 225.673 7 to 4 525.433 0±1 131.813 0 (P < 0.05).Conclusion aggrecanase-2 suppression in chondrocytes by lentivirius infection is an effective method to protect the expression of aggrecan.

Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Idioma: Chinês Revista: Chinese Journal of Orthopaedics Ano de publicação: 2014 Tipo de documento: Artigo

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Texto completo: DisponíveL Índice: WPRIM (Pacífico Ocidental) Idioma: Chinês Revista: Chinese Journal of Orthopaedics Ano de publicação: 2014 Tipo de documento: Artigo