The Experimental Study Of The Effect Of TNF-alpha Gene ShRNA On The Formation Of Osteoclasts In Spinal Tuberculosis

Objective Through constructing the shRNA vector of TNF-alpha gene and observing its effect on the formation of osteoclasts in spinal tuberculosis,we lay the foundation for further study on the bone destruction of spinal tuberculosis.Methods CCK was used to test the effect of PPD on cell proliferation and TRAP stain was used to observe the formation of osteoclasts after PPD induced mouse RAW264.7 cells to compare the discrepancy of the formation of osteoclasts under the condition of different dosages(0,2,20,50,100?L)and inducement time(1,4,7 days).The expression of TNF-alpha at different time(1,4,7 days)induced by PPD was detected by qPCR and Western Blotting.TNF-alpha gene shRNA was constructed by double enzyme digestion and transfected into RAW264.7 cells by liposome.Meanwhile,the transfected efficiency was showed under fluorescence microscope.The expression of TNF-alpha gene on 1,3,5,7 days after transfection was observed by RT-PCR.Collecting cells of the third day after transfection,the expression of TNF-alpha and RANK gene were tested by qPCR and the expression of TNFalpha protein was tested by Western Blotting.TRAP stain was performed to count the number of osteoclasts of the seventh day after transfection.Results PPD can inhibit the proliferation of RAW264.7 cells,and the formation of osteoclasts can be revealed by TRAP stain.The formation number of osteoclasts induced by different dosages of PPD is: PPD 0 ?L group(58.067.1 ?),2 ?L group(58.067.4 ?),20 ?L group(52.067.16 ?),50 ?L group(89.267.10 ?),100 ?L group(58.033.1 ?).The formation number of osteoclasts on 1,4,7day induced by 20?L PPD is: 1 day group(1.50±0.55),4 day group(7.50±1.87),7 day group(17.33±2.07).The expression of TNF-alpha gene on 1,4,7day induced by PPD is: Control group(1±0),1 day group(4.267±0.737),4 day group(13.60±0.648),7 day group(14.07±1.013).The expression of TNF-alpha protein is:Control group(0.066±0.004),1 day group(0.081±0.005),4 day group(0.162±0.003),7 day group(0.179±0.005).The expression of TNFalpha gene before and after transfection is:pre-transfection group(1.426±0.086),transfection group(0.464±0.029).The expression of RANK gene before and after transfection is :pre-transfection group(1.393±0.007),transfection group(1.154±0.006).The expression of TNF-alpha protein before and after transfection is: pretransfection group(82.72±1.843),transfection group(55.34±0.824).The number of osteoclasts before and after transfection is :pre-transfection group(56.67±3.786),transfection group(19.33±1.528).Conclusion PPD can induce osteoclasts formation and the formation of osteoclasts only dependents on the PPD stimulation time,rather than the PPD dosage.TNF-alpha plays a vital role in the formation of osteoclasts in spinal tuberculosis.Targeted TNF-alpha gene shRNA can regulate down the expression of TNF-alpha and inhibit the formation of osteoclasts.