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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 expression of transforming growth factor-β1 (TGF-β1) in the ligamentum flavum fibroblasts of mice by RNA interference (RNAi) technique and investigate its effect on the ossification of the ligamentum flavum. Methods Human yellow ligament fibroblasts were cultured and induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) to observe the morphological changes of ossification of the ligamentum flavum cells (osteoblasts) Alkaline phosphatase (ALP) staining and calcium nodulation alizarin red staining were performed. The siRNA-pSilencer2.0U6-TGFβ1 targeting TGF-β1 gene was constructed and transfected into bone The cells were divided into 3 groups: the cells transfected with the eukaryotic expression vector were the experimental group, the cells transfected with the empty vector were the negative control group, and the untransfected cells were the blank control group. The expression of TGF-β1 and BMP-2 in the cells before and after transfection was detected by immunofluorescence. The expression of TGF-β1 mRNA in the cells was detected by RT-PCR. The expression of TGF-β1 and BMP-2 in the cells was detected by Western blotting The changes of ALP and osteocalcin (OC) levels in the cells were detected by enzyme linked immunosorbent assay (ELISA). Results After the ossification was induced, the morphology of the cells in the ligamentum flavum of mice showed ALP staining and alizarin red staining of calcium nodules, which showed typical biological features of osteoblasts. The results of RT-PCR showed that the expression of TGF-β1 mRNA in the experimental group decreased by 41.94% compared with the negative control group and the blank control group, respectively , 47.82%, respectively (P <0.01). The results of Western blotting showed that the ratio of TGF-β1 / β-action protein in experimental group decreased 35.88% and 44.75% respectively compared with negative control group and blank control group (P <0.01). The ratio of BMP-2 / β-action protein decreased by 81.79% and 86.06% respectively (P <0.01). The results of ELISA showed that compared with the negative control group and the blank control group ALP decreased by 24.14% and 32.30% respectively, the difference was statistically significant (P <0.01); OC decreased by 17.01% and 21.63% respectively, the difference was statistically significant (P <0.01). Conclusion The construction of siRNA eukaryotic expression vector targeting TGF-β1 gene can effectively inhibit the expression of TGF-β1 and endogenous BMP-2 in osteoblasts and inhibit the ossification of the ligamentum flavum.