RANK基因干扰序列对小鼠骨髓巨噬细胞向破骨细胞分化及其活性的影响

The effect of retrovirus-mediated shRNA targeting mouse RANK on osteoclastogenesis of bone marrow-derived macrophages

目的建立逆转录病毒介导的沉默小鼠RANK基因RNA干扰表达体系,并观察其对小鼠骨髓巨噬细胞向破骨细胞分化的影响。方法将沉默小鼠RANK基因短发夹RNA(shRNA)片段重组到pSuper-Retro质粒中,构建沉默小鼠RANK基因RNA干扰逆转录病毒载体pSUPER-shRANK,与PIK包装质粒经293FT细胞包装后,产生的重组逆转录病毒感染小鼠骨髓巨噬细胞,并用嘌呤霉素筛选产生稳定的细胞克隆,用Western blot检测细胞中RANK蛋白表达的变化。对病毒感染的小鼠BMM和未经感染的小鼠BMM分别用100ng/ml RANKL和30ng/mlM-CSF诱导,9d后TRAP染色及扫描电镜观察破骨细胞形成及骨溶解情况。结果重组逆转录病毒质粒经测序鉴定正确;逆转录病毒感染小鼠骨髓巨噬细胞后用嘌呤霉素筛选出稳定的细胞克隆;对经病毒感染的细胞进行RANK蛋白检测发现,RANK表达较未感染BMM RANK下降了80.7%(P<0.01);与未感染BMM相比较,感染BMM在培养9d后,TRAP染色发现核≥3个的破骨细胞数量明显减少,分别为(82.00±4.64)和(6.80±1.64)(P<0.05);扫描电镜的结果显示骨陷窝面积明显减小,分别为(16.60±2.70)%和(2.80±0.84)%(P<0.05)。结论携带沉默小鼠RANK基因的逆转录病毒感染小鼠骨髓巨噬细胞能明显抑制RANK蛋白表达,沉默RANK抑制小鼠BMM向破骨细胞的分化及其活性。

Objective To construct a retrovirus-mediated expression system containing short hairpin RNA（shRNA） targeting mouse RANK gene, mouse bone marrow-derived macrophages. Methods and to study its influence on the osteoclastogenesis of A recombinant retrovirus vector pSUPER-shRANK was generated by cloning shRNA targeting mouse RANK gene into pSuper-Retro vector. To obtain retrovirus supernatant, pSUPER-retro-shRANK and packaging plasmid vector （PIK） were transfected into 293 FT packaging cells by calcium phosphate transfection method. BMMs were repeatedly infected by the retrovirus supernatant, and stable BMM clones were generated after screening with puromycin. RANK proteins were then analyzed by Western blot. BMMs and infected BMMs were induced by 100 ng/ml RANKL and 30 ng/ml M-CSF for nine days. Osteoclasts and bone resorption pits were detected by TRAP staining and Scanning Electron Microscopy. Results The pSUPER-shRANK recombinant retrovirus vector had been constructed correctly by restriction enzyme digestion and sequencing methods assays. Stable infected BMMs clones were generated after screening with puromycin. The expression of RANK protein in infected BMMs reduced significantly （80. 7% ）, compared with uninfected BMMs （P 〈0. 01 ）. Nine days later, there was a reduction in the number of TRAP ＋ multinucleated cells in infected BMMs （6. 80 ± 1.64） compared with uninfected BMMs （82.00 ± 4. 64） （P 〈 0. 05 ）. Furthermore there was significant reduction in resorption areas on bone slices in infected BMMs （2. 80% ±0. 84% ） compared with uninfected BMMs （ 16. 60% ± 2. 70% ） （P 〈 0. 05 ）, indicating profound inhibition of osteoelastie bone resorption. Conclusions The pSUPER-shRANK retrovirus vector showed effective inhibition on the expression of RANK at protein levels ; the osteoelastogenesis of BMMs was inhibited by RNAi targeting RANK.