JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES) ›› 2014, Vol. 52 ›› Issue (8): 27-33.doi: 10.6040/j.issn.1671-7554.0.2014.060

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

ZHANG Yingzhe, WU Dongjin, PENG Changliang, LI Bohan, SONG Yang, ZHANG Cheng, ZHAO Jie, LI Dequan, YANG Zhongyan, LIU Peng, ZHAO Kun, MA Shengzhong, GAO Chunzheng   

  1. Department of Spinal Surgery, Second Hospital of Shandong University, Jinan 250033, Shandong, China
  • Received:2014-02-06 Revised:2014-06-10 Online:2014-08-10 Published:2014-08-10

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Abstract: Objective Transforming growth factor-β1 (TGF-β1) expression in fibroblasts of mice ligamentum flavum was inhibited by RNA interference (RNAi) technique, 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 rhBMP-2 (recombinant human bone morphogenetic protein-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 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-β1 mRNA 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 OC (osteocalcin) 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 siRNA-pSilencer2.0U6-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-β1 mRNA 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) could effectively inhibit the expression of TGF-β1 in osteoblasts and endogenous BMP-2, thus suppressing spinal ossification of ligamentum flavum.

Key words: Ossification, Bone morphogenetic protein-2, Osteocalcin, Alkaline phosphatase, Transforming growth factor-β1, Small interfering RNA

CLC Number: 

  • R686.5
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