TNF-α-shRNA对结核菌素诱导破骨细胞形成的影响

Effect of TNF-alpha-sh RNA on osteoclast formation induced by tuberculin

目的 :构建靶向肿瘤坏死因子α(tumor necrosis factorα,TNF-α)基因的短发卡RNA(short hairpin RNA,sh RNA)表达载体,观察其对结核菌素(purified protein derivative,PPD)诱导破骨细胞形成的影响。方法 :双酶切法构建TNF-α-sh RNA,经脂质体转染小鼠单核巨噬细胞(RAW264.7细胞),通过荧光显微镜观察TNF-α-sh RNA的转染情况;采用逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)观察转染前及转染后第1、3、5、7天TNF-α基因的表达情况;以RAW264.7细胞为空白组、RAW264.7细胞+1IU·ml-1PPD为对照组、RAW264.7细胞+TNF-α-sh RNA+1IU·ml-1PPD为转染观察组、RAW264.7细胞+空载质粒+1IU·ml-1PPD为阴性转染组,采用实时荧光定量PCR(real-time quantitative PCR,q PCR)、蛋白免疫印迹法(Western blotting,WB)检测各组第3天TNF-α基因和蛋白的相对表达量;采用抗酒石酸酸性磷酸酶染色(tartrate resistant acid phosphatase,TRAP)计数各组第7天破骨细胞形成的数量。结果 :TNF-α-sh RNA经脂质体成功转染RAW264.7细胞,转染效率达70%~80%。RT-PCR显示转染后第3天TNF-α基因的表达最少,第5天次之,第1天、第7天和转染前比较差异不大。转染后第3天转染观察组TNF-α基因的相对表达量为0.46±0.03,同时间点空白组为1.00±0.00,对照组为1.43±0.09,阴性转染组为1.38±0.06。转染后第3天转染观察组TNF-α蛋白的相对表达量为55.34±0.82,同时间点空白组为59.13±1.43,对照组为82.72±1.84,阴性转染组为84.62±0.97。转染后第7天转染观察组破骨细胞形成的数量为19.33±1.53个,同时间点空白组为5.67±1.53个,对照组为56.67±3.79个,阴性转染组为59.67±3.51个。转染观察组的TNF-α基因和蛋白的相对表达量及破骨细胞数量与空白组、对照组、阴性转染组比较,差异有统计学意义(P<0.05);空白组与对照组比较,差异有统计学意义(P<0.05);对照组与阴性转染组比较,差异无统计学意义(P>0.05)。结论:TNF-α-sh RNA可以特异性地沉默RAW264.7细胞内TNF-α基因的表达,使TNF-α的产生减少,抑制PPD诱导的破骨细胞形成。

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.