RNA干扰技术抑制成骨细胞核激活因子受体配体基因对破骨细胞生成的影响

Effect of silencing ligand of receptor activator of NF-kappaB expression by RNA interference on osteoclastogenesis induced by osteoblast

目的:利用RNA干扰技术抑制大鼠成骨细胞核激活因子受体配体(Ligand of receptor activator of NF-κB,RANKL)的表达,观察成骨细胞RANKL/骨保护素(osteoprotegerin,OPG)比值变化,探讨成骨细胞与破骨细胞生成的相关性。方法:实验于2006-05/10在四川大学华西医院移植免疫实验室(国家重点实验室)完成。①实验材料:体外化学合成4条RANKL序列特异性小干扰RNA(small interfering RNA,siRNA),用Lipofectin2000TM转染成骨细胞。②实验方法:采用荧光实时定量聚合酶链反应检测RANKL mRNA的表达,筛选出最有效的干扰序列。③实验分组:分为最有效的siRNA转染组、阴性对照组和空白对照组,每组设5个平行样本。采用免疫组织化学染色检测成骨细胞中RANKL、OPG蛋白的表达,观察RANKL/OPG比值变化。④实验评估:采用成骨细胞、破骨细胞共培养技术观察以上3组转染后的成骨细胞对破骨细胞生成的影响。结果:①4种siRNA敲除RANKL基因后mRNA的表达:4种siRNA转染成骨细胞后,相对空白对照组,siRNA4分子对RANKLmRNA的抑制作用最为明显,达到89%。siRNA1,siRNA3对RANKLmRNA的抑制作用大于50%,而siRNA2对RANKLmRNA的也有相当的抑制作用。②各组成骨细胞中RANKL和OPG蛋白水平:RANKL和OPG的阳性表达均显示为棕黄色颗粒,RANKL主要分布于成骨细胞的胞浆和胞膜,OPG主要分布于成骨细胞的胞浆。siRNA4转染组RANKL表达(平均积分光密度值)低于空白对照组(分别为3.05±2.17,11.31±1.26),差异有显著性意义(P<0.01);阴性对照组与空白对照相比,差异无显著性意义(P>0.05)。siRNA4转染组、阴性对照组OPG表达与空白对照组比较,差异均无显著性意义(P>0.05)siRNA4转染组RANKL/OPG比值低于空白对照组(分别为0.12±0.03,0.45±0.04),差异有显著性意义(P<0.01)。③转染的成骨细胞对破骨细胞生成的影响:各组共培养形成的破骨细胞形态与分离的成熟破骨细胞相似,为不规则形,胞浆红染,并有伪足形成。siRNA4转染组破骨细胞形成低于空白对照组、阴性对照组[分别为(12±2),(31±7),(28±5)个/孔],差异有显著性意义(P<0.01);阴性对照组与空白对照相比,差异无显著性意义(P>0.05)。结论:RNA干扰沉默RANKL表达可下调成骨细胞RANKL/OPG比值,抑制破骨细胞的生成。

AIM： To observe the change of the ratio of ligand of receptor activator of NF-κB （RANKL） and osteoprotegerin （OPG） by inhibiting the expression RANKL with RNA interference （RNAi）, and explore the relationship between osteoblast and osteoclastogenesis. METHODS： The experiment was conducted in the laboratory of Transplantation Immunity of West China Hospital, Sichuan University from May to October 2006. （1）Four chemically synthesized small interfering RNA （siRNA） targeting RANKL were transfected into osteoblast using Lipofectamin 2000^TM. （2）RANKL mRNA level was determined by real-time quantitative reverse transcriptase polymerase chain reaction （RT-PCR）, and the most effective siRNA was screened. （3） Three groups including the most effective siRNA transfecting group, negative control group, and blank control group were established and each group involved 5 specimens. Meanwhile, the immunohistochemistry was employed to analyze the expression of RANKL and OPG in osteoblasts, and then the change of ratio of RANKL and OPG was observed. （4） The transfected cells of the above three groups and primary rat bone marrow cells were co-cultured to explore the osteoclastogenesis induced by osteoblast. RESULTS： （1）The most effective sequence was found out among the 4 candidates. Single dose of siRNA 4 caused neady 89% loss of RANKL mRNA compared with the blank control group, siRNA1 and siRNA3 reduced the expression of RANKL mRNA to below 50%, and siRNA2 could also efficiently inhibit the expression of RANKL mRNA. （2）The positive expressions of RANKL and OPG protein displayed buffy granules which of RANKL were distributed at endochylema and membrane and which of OPG at endochyiema. The expression of RANKL protein of siRNA4 transfecting group was lower than that of the blank control group （average integral optical density： 3.05±2.17, 11.31±1.26, respectively）, and the differences were significant statistical （P 〈 0.01）; There was no significant difference in the expression of RANKL protein between the negative control group and the blank control group （P 〉 0.05）. And there was no significant difference in the expression of OPG protein among three groups （P 〉 0.05）. The ratio of RANKL/OPG of siRNA4 transfecting group was lower than that of the blank control group （0.12±0.03, 0.45±0.04）, and there were obvious differences. （3） The morphous of co-cultured osteoclasts were similar to that of the isolated mature osteoclasts, irregular shape, red-dyed kytoplasm, and pseudopod. The osteoclast number of siRNA4 transfecting group was significantly lower than that of the blank and negative control groups [（12±2）, （31±7）, （28±5） cells per well, P 〈 0.01], and there was no significant difference between the negative control group and the blank control group （P 〉 0.05）. CONCLUSION： RANKL silenced by RNAi could reduce the ratio of RANKL/OPG and inhibit the formation of osteoclast.