目的 观察靶向过氧化物酶体增殖物激活受体γ(PPARγ)基因小干扰RNA(siRNA)阻断激素导致活体兔股骨头细胞内成脂基因表达的作用.方法 48只家兔随机分为4组,每组12只.正常组(N):臀肌注射生理盐水2 ml.模型组(M):每次臀肌注射地塞米松20 mg/kg,连续注射3d.干扰组(S):同M组给予地塞米松,并于1、3、6周穿刺注入一侧股骨头内siRNA腺病毒滴液25μl.无关序列组(Con):同M组给予地塞米松,注入家兔股骨头内无关序列siRNA病毒滴液.实验第4、8周末分批处死家兔,观察兔股骨头细胞内成脂基因与成骨基因的表达.结果 实验4周和8周时,S、Con、M、N组中的PPARγ mRNA表达量分别为0.40±0.05、0.69±0.09、0.71±0.08、0.38±0.06和0.43±0.05、0.71±0.08、0.71±0.08、0.42±0.08;Runx2 mRNA相对表达量分别为0.0035±0.0006、0.0019±0.0008、0.0019±0.0007、0.0036±0.0007和0.0034±0.0006、0.0017±0.0005、0.0020±0.0004、0.0035±0.0006;骨钙素(Osteocalcin) mRNA相对表达量分别为0.034±0.006、0.015±0.006、0.014±0.005、0.035±0.005和0.032±0.007、0.016±0.006、0.015±0.005、0.033±0.005.PPARγ蛋白表达量分别为0.18±0.02、0.85±0.14、0.87±0.18、0.24±0.02和0.17±0.02、0.90±0.15、0.90±0.18、0.22±0.02;核心结合蛋白因子2(Runx2)蛋白表达量分别为0.82±0.19、0.22±0.03、0.19±0.03、0.84±0.17和0.83±0.19、0.21 ±0.03、0.20±0.03、0.86±0.15;Osteocalcin蛋白表达量分别为0.83±0.17、0.19±0.02、0.20±0.02、0.83±0.15和0.82±0.17、0.18±0.02、0.20±0.03、0.80±0.14.S组中PPARγmRNA与其蛋白表达量均明显低于M、Con组(P＜0.05),与N组比较差异无统计学意义(P＞0.05).S组中Runx2、OsteocalcinmRNA与其蛋白表达量均明显高于M、Con组(P＜0.05),与N组比较差异无统计学意义(P＞0.05).结论 靶向PPARγ的腺病毒载体介导的小干扰RNA能够有效阻断激素诱导的活体兔股骨头细胞内成脂基因PPARγ表达,保持其成骨基因Runx2与Osteocalcin表达.%Objective To observe suppressive effects of the small interfering RNA (siRNA) targeting PPARγon the expression of adipogenic gene in cells of the femoral head of rabbit steroid-induced osteonecrosis model.Methods Forty-eight rabbits were randomly divided into 4 groups (n =12 each).In normal group (group N),2 ml normal saline was injected.In model group (group M),dexamethasone 20 mg/kg was injected for 3 days.In interference group (group S),dexamethasone 20 mg/kg was admin-istered for 3 dqays,and siRNA adenovirus (25 μ1) was injected into one side femoral head at 1st,3rd,and 6th week.In irrelated sequence group (group Con),dexamethasone 20 mg/kg was administered for 3days,and a vector carrying irrelative sequence was injected into one side femoral head at 1 st,3 rd,and 6thweek.Experimental animals were sacrificed at 4th and 8th week to observe the expression of the adipogenic gene and osteogenic gene in the cells of rabbit femoral head.Results At 4th and 8th week,the gene expression levels in groups S,Con,M and N were shown as below:for PPARγ mRNA [(0.40 ± 0.05),(0.69 ±0.09),(0.71 ±0.08),(0.38 ±0.06) and (0.43 ±0.05),(0.71 ±0.08),(0.71 ±0.08),(0.42 ± 0.08)];for related transcription factor-2 (Runx2) mRNA [(0.003 5 ± 0.000 6),(0.0019±0.0008),(0.0019±0.0007),(0.0036±0.0007) and (0.0034±0.0006),(0.001 7 ± 0.000 5),(0.002 0 ± 0.000 4),(0.003 5 ± 0.000 6)];for Osteocalcin mRNA [(0.034 ±0.006),(0.015 ±0.006),(0.014 ±0.005),(0.035 ±0.005) and (0.032 ±0.007),(0.016±0.006),(0.015 ±0.005),(0.033 ±0.005)];for PPARγ protein [(0.18 ±0.02),(0.85 ±0.14),(0.87±0.18),(0.24±0.02) and (0.17±0.02),(0.90±0.15),(0.90±0.18),(0.22±0.02)];for Runx2 protein [(0.82 ±0.19),(9.22 ±0.03),(0.19 ±0.03),(0.84 ±0.17) and (0.83 ± 0.19),(0.21 ± 0.03),(0.20 ± 0.03),(0.86 ± 0.15)];for Osteocalcin protein [(0.83 ±0.17),(0.19 ±0.02),(0.20 ±0.02),(0.83 ±0.15) and (0.82 ±0.17),(0.18 ±0.02),(0.20 ±0.03),(0.80 ±0.14)] respectively.The expression levels of PPAR mRNA and protein in group S were significantly lower than those in groups M and Con (P ＜ 0.05),and there was no significant difference between the group S and group N (P ＞ 0.05).The expression levels of Runx2 and Osteocalcin mRNA and proteins in group S were significantly higher than those in groups M and Con (P ＜ 0.05),and there was no significant difference between group S and group N (P ＞ 0.05).Conclusion The siRNA adenovirus vectors targeting PPARγcan efficaciously suppress the steroid-induced expression of adipogenic gene PPARγ in cells of the femoral head of rabbit model.
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