Effect of TNF- alpha -shRNA on osteoclast formation induced by tuberculin

English Keywords:Gene knockout technique  Tumor necrosis factor  Tuberculin  Osteoclast

English Abstract:

  【Abstract】 Objectives: By constructing a short hairpin RNA (shRNA) vector for targeted tumor necrosis factor α (TNF-α) gene, to observe its effects on osteoclast formation induced by tuberculin. Methods: TNF-α-shRNA was constructed by double enzyme digestion and transfected with liposome to mouse mononuclear macrophages (RAW264.7 cells). The transfection of TNF-α-shRNA was observed by fluorescence microscopy. The expression of TNF-α gene before transfection and at the first, third, fifth and seventh day after transfection was observed by reverse transcription-polymerase chain reaction (RT-PCR). The RAW264.7 cells were regareded as the blank group, the RAW264.7 cells+1IU·ml-1 PPD as the control group, the RAW264.7 cells+TNF-α-shRNA +1IU·ml-1 PPD as the transfection group and the RAW264.7 cells+empty plasmid+1IU·ml-1 PPD as the negative transfection group, real-time quantitative PCR (qPCR) and Western blotting (WB) were used to detect the relative expressions of TNF-α gene and protein on the third day of each group. Tartrate resistant acid phosphatase (TRAP) was used to count the number of osteoclasts on the seventh day of each group. Results: TNF-α-shRNA was successfully transfected into RAW264.7 cells via liposomes, the transfection efficiency was 70%-80%. The results of RT-PCR showed that the expression of TNF-α gene was the least on the third day after transfection, was less on the fifth day, but presented no significant difference on the first day and seventh day compared with that before transfection. The relative expression of TNF-α gene on the third day was 1.00±0.00 in the blank group, 1.43±0.09 in the control group, 0.46±0.03 in the transfected observation group, and 1.38±0.06 in the negative transfection group, respectively. The relative expression of TNF-α protein on the third day was 59.13±1.43 in the blank group, 82.72±1.84 in the control group, 55.34±0.82 in the transfected observation group, and 84.62±0.97 in the negative transfection group. The number of osteoclast formation on the seventh day was 5.67±1.53 in the blank group, 56.67±3.79 in the control group, 19.33±1.53 in the transfection group, and 59.67±3.51 in the negative transfection group. The relative expression of TNF-α gene and protein and the number of osteoclasts in the observation group were compared with those in the blank group, the control group and the negative transfection group (P<0.05), and the differences were statistically significant. The difference between the blank group and the control group was statistically significant(P<0.05). There was no significant difference between the control group and the negative transfection group in P>0.05. Conclusions: TNF-α-shRNA can specifically silent the expression of TNF-α gene in RAW264.7 cells, thereby reduce the production of TNF-α and inhibit the formation of osteoclasts induced by tuberculin.