siRNA抑制转化生长因子-β1对小鼠黄韧带骨化的影响

Effect of silencing TGF-β1 expression by small interfering RNA on ossification of the ligamentum flavum in mice

目的运用RNA干扰(RNAi)技术抑制转化生长因子-β1(TGF-β1)在小鼠黄韧带成纤维细胞中的表达,探讨其对脊柱黄韧带骨化的影响。方法培养小鼠黄韧带成纤维细胞,经人重组骨形态发生蛋白-2(rhBMP-2)诱导骨化,对骨化成功的黄韧带细胞(成骨细胞)进行形态学观察,并对其进行碱性磷酸酶(ALP)染色及钙结节茜素红染色鉴定;构建靶向TGF-β1基因的小干扰RNA(siRNA)真核表达载体(siRNA-pSilencer2.0U6-TGFβ1)并转染成骨细胞,分为3组:真核表达载体转染后细胞为实验组,空载体转染后细胞为阴性对照组,未转染细胞为空白对照组。应用免疫荧光技术检测转染前后细胞中TGF-β1、BMP-2的表达;RT-PCR检测TGF-β1 mRNA在细胞内表达变化;Western blotting检测TGF-β1和BMP-2蛋白在细胞内表达变化;酶联免疫吸附试验(ELISA)检测细胞中ALP、骨钙素(OC)水平的变化。结果经诱导骨化成功后,小鼠黄韧带细胞形态学观察可见细胞ALP染色、钙结节茜素红染色均呈阳性,具有典型成骨细胞生物学特征。构建的siRNA表达载体对细胞转染后,免疫荧光检测显示TGF-β1和BMP-2荧光强度下降;RT-PCR检测显示实验组较阴性对照组和空白对照组TGF-β1 mRNA表达分别下降41.94%、47.82%,差异有统计学意义(P<0.01);Western blotting检测显示实验组较阴性对照组和空白对照组TGF-β1/β-action蛋白表达比值分别下降35.88%、44.75%,差异有统计学意义(P<0.01);BMP-2/β-action蛋白表达比值分别下降81.79%、86.06%,差异有统计学意义(P<0.01);ELISA检测显示实验组较阴性对照组和空白对照组ALP分别下降24.14%、32.30%,差异有统计学意义(P<0.01);OC分别下降17.01%、21.63%,差异有统计学意义(P<0.01)。结论构建靶向TGF-β1基因的siRNA真核表达载体能够有效抑制成骨细胞中TGF-β1以及内源性BMP-2表达,达到抑制脊柱黄韧带骨化的目的。

Objective To inhibit the transforming growth factor-β1（ TGF-β1） expression in fibroblasts of mice ligamentum flavum by RNA interference（ RNAi） technique,and to investigate the effect of TGF-β1 on the ossification of ligamentum flavum. Methods Fibroblasts of mice ligamentum flavum were cultivated and ossification was induced with recombinant human bone morphogenetic protein-2（ rhBMP-2）. After that,the osteoblasts were identified with morphologicalobservation,alkaline phosphatase staining and alizarin red staining of calcified nodules. Eukaryotic expression vector（ siRNA-pSilencer2. 0U6-TGFβ1） was constructed to transfect the osteoblasts,which were then divided into three groups. The experiment group was transfected with eukaryotic expression vector,the negative control group was transfected with vacant plasmid and the blank control group was not treated. After that,the expressions of TGF-β1 and BMP-2 were detected by immunofluorescence technique before and after transfection; the expression change of TGF-β1mRNA in osteoblasts was determined by RT-PCR; the expression change of protein of TGF-β1 and BMP-2 was measured with Western blotting; the expression change of ALP and osteocalcin（ OC） was assessed with enzyme linked immunosorbent assay（ ELISA）. Results After ossification had been induced successfully,ligamentum flavum cell morphological observation showed that alkaline phosphatase staining and alizarin redstaining of calcified nodules were both positive,and those cells had typical biological features of osteoblasts. After the osteoblasts were transfected by siRNApSilencer2. 0U6-TGFβ1,immunofluorescence detection displayed decline in the fluorescence intensity of TGF-β1 and BMP-2. Compared with the negative control and bland control,RT-PCR showed that the expression of the TGF-β1mRNA in experiment group decreased significantly by 41. 94% and 47. 82%,respectively（ P〈0. 01）. Western blotting showed that the ratio of TGF-β1 /β-action in experiment group decreased remarkably by 35. 88% and 44. 75%,respectively（ P〈0. 01）,and BMP-2 /β-action decreased significantly by 81. 79% and 86. 06%,respectively（ P〈0. 01）. ELISA displayed that ALP in experiment group decreased notably by 24. 14% and 32. 30%,respectively（ P〈0. 01）,and OC decreased remarkably by 17. 01% and 21. 63%,respectively（ P〈0. 01）. Conclusion Eukaryotic expression vector（ siRNA-pSilencer2. 0U6-TGFβ1） can effectively inhibit the expression of TGF-β1 in osteoblasts and endogenous BMP-2,thus suppressing spinal ossification of ligamentum flavum.