抑制视网膜母细胞瘤结合蛋白2基因表达促进人脂肪基质细胞成骨向分化的研究

目的 探讨视网膜母细胞瘤结合蛋白2(retinoblastoma binding protein 2,RBP-2)在人脂肪基质细胞(human adipose-derived stromal cell,hASC)成骨向分化中的作用.方法 设计靶向RBP-2小干扰RNA(small interfering RNA,siRNA)序列4对,将相应寡核苷酸构建到慢病毒载体pLL 3.7中,于293T细胞进行病毒包装用于RBP-2基因敲低.采用RBP-2敲低效果好的两个siRNA序列进行病毒包装,并以非沉默siRNA的pLL 3.7质粒包装的慢病毒为对照组,感染hASC,培养第14天定量检测碱性磷酸酶(alkaline phosphatase,ALP)活性并行ALP染色及茜素红矿化结节染色,检测hASC成骨向分化的差异.结果 成功构建RBP-2基因RNA干扰慢病毒载体并获得相应慢病毒.慢病毒感染hASC后,RBP-2 mRNA及蛋白表达均被明显抑制.RBP-2 siRNA-1组和RBP-2 siRNA-4组的敲低效果较好,以此两组感染hASC.成骨诱导培养第14天RBP-2 siRNA-1组和RBP-2 siRNA-4组ALP活性(299.2±22.7)、(224.3±17.7)U/g]高于对照组(129.9±12.9)U/g,P＜0.05],RBP-2 siRNA-1组和RBP-2 siRNA-4组茜素红及ALP染色强于对照组.结论 成功构建的RBP-2 RNA干扰慢病毒能显著抑制RBP-2表达,并促进hASC向成骨细胞分化,即RBP-2可以抑制hASC的成骨向分化.

Abstract：

Objective To explore the effect of retinoblastoma binding protein 2 (RBP-2), a histone H3K4 demethylase, on osteogenic differentiation of human adipose-derived stromal cell(hASC).Methods According to the GenBank sequence information of RBP-2, four different small interfering RNAs (siRNA) targeting RBP-2 gene were designed and the corresponding short hairpin RNAs (shRNA) were cloned into pLL 3.7 lentivirus RNA interference vector. The lentivirus with RBP-2-siRNA was packaged in 293T cells. The effective sequence was examined and selected by Western blotting and real-time PCR. The lentiviruses with efficient knockdown effects were used to infect hASC. On the 14th day after osteogenic differentiation, alkaline phosphatase (ALP) activities of hASC were quantitatively tested and at the same time, ALP staining and alizarin red staining were performed to assess the difference of osteogenic differentiation between the knockdown group and the control group. Results The recombinant lentivirus siRNA targeting RBP-2 was successfully constructed and the expression of RBP-2 mRNA and protein were dramatically suppressed by infection with RBP-2-siRNA lentivirus. On the 14th day after osteogenic induction, ALP activity of hASC in the knockdown group(299.2 ±22.7), (224.3± 17.7) U/g]was much stronger than that in the control group( 129.9 ± 12.9) U/g, P ＜ 0. 05]and the same result was achieved for the ALP staining and alizarin red staining. Conclusions The constructed RBP-2-siRNA lentivirus could markedly decrease the expression of RBP-2 and promote osteogenic differentiation of hASC.It indicated that RBP-2 can repress the osteogenic differentiation of hASC.

Inhibition of retinoblastoma binding protein 2 promotes osteogenic differentiation of human adipose-derived stromal cells

Objective To explore the effect of retinoblastoma binding protein 2 (RBP-2), a histone H3K4 demethylase, on osteogenic differentiation of human adipose-derived stromal cell(hASC).Methods According to the GenBank sequence information of RBP-2, four different small interfering RNAs (siRNA) targeting RBP-2 gene were designed and the corresponding short hairpin RNAs (shRNA) were cloned into pLL 3.7 lentivirus RNA interference vector. The lentivirus with RBP-2-siRNA was packaged in 293T cells. The effective sequence was examined and selected by Western blotting and real-time PCR. The lentiviruses with efficient knockdown effects were used to infect hASC. On the 14th day after osteogenic differentiation, alkaline phosphatase (ALP) activities of hASC were quantitatively tested and at the same time, ALP staining and alizarin red staining were performed to assess the difference of osteogenic differentiation between the knockdown group and the control group. Results The recombinant lentivirus siRNA targeting RBP-2 was successfully constructed and the expression of RBP-2 mRNA and protein were dramatically suppressed by infection with RBP-2-siRNA lentivirus. On the 14th day after osteogenic induction, ALP activity of hASC in the knockdown group(299.2 ±22.7), (224.3± 17.7) U/g]was much stronger than that in the control group( 129.9 ± 12.9) U/g, P ＜ 0. 05]and the same result was achieved for the ALP staining and alizarin red staining. Conclusions The constructed RBP-2-siRNA lentivirus could markedly decrease the expression of RBP-2 and promote osteogenic differentiation of hASC.It indicated that RBP-2 can repress the osteogenic differentiation of hASC.